WO2025207481A1 - Measuring viability in cell aggregates - Google Patents

Abstract

The present disclosure relates to a method for determining cell viability in a population comprising cell aggregates. The methods typically comprise contacting a sample prepared from a cell population with a first agent and a second agent, and determining the ratio of living cells to total cells, e.g., following three-dimensional imaging of cell aggregates contacted with the two agents.

Description

MEASURING VIABILITY IN CELL AGGREGATES

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application nos. 63/569,599, filed March 25, 2024, and 63/569,609, filed March 25, 2024, the contents of each of which are incorporated herein in their entireties by reference thereto.

2. BACKGROUND

[0002] Traditional cell counting and viability assessment methods typically rely on enumeration of single cells. Accurate assessment of cell viability without disrupting or damaging cells remains a challenge, especially when the cells are in aggregates. Determining the viability of cell aggregates is often necessary before they can be used in downstream procedures. For example, photoreceptor precursor cells are a cell type that may be useful as a cell therapy, but may be found in aggregate form. However, dissociation of cell aggregates (e.g., photoreceptor precursor (PRP) cell aggregates) can be harsh and lead to cell damage or loss, and consequently, result in errors in estimates of cell viability. Therefore, traditional cell counting and viability assessment methods that use preparations of single cells pose a challenge with cell populations comprising aggregates. Further, an imprecise measurement can lead to variability in downstream applications of cell populations comprising cell aggregates, e.g., in pharmaceutical preparations in the case of cell therapies.

[0003] There is need in the art for improved methods of determining the viability of cells in cell aggregates, for example cell aggregates comprising PRP cells.

3. SUMMARY

[0004] The present disclosure provides methods of determining cell viability in a population of cells comprising cell aggregates (e.g., a population of cells comprising cell aggregates in which photoreceptor precursor (PRP) cells are present). The methods typically entail detecting (e.g., by imaging) the total number of cells or the number of live cells, on the one hand, and the number of dead cells, on the other hand, and determining cell viability accordingly. Detecting (i) the total number of cells or the number of live cells and (ii) the number of dead cells can be achieved by using a combination of agents (e.g., two) that together are capable of differentially staining living and dead cells such that the staining of a living cell and the staining of a dead cell will be different. Cell viability is the ratio or percentage of living cells to the total number of cells among detected cells. In some embodiments, the cells detected represent a portion of a sample prepared from a population of cells comprising aggregates. [0005] Detecting total cells or living cells can be achieved using a “first agent,” typically an agent that can penetrate living cells as well as dead cells. Suitable agents for staining total cells (e.g., living cells and dead cells) include cell permeable dyes, e.g., cell-permeable nucleic acid binding dyes. An exemplary agent for staining total cells is acridine orange. Detecting living cells can also be achieved using a first agent that can penetrate and stain living cells but not stain dead cells. An exemplary agent for staining living cells is Calcein AM.

[0006] Detecting dead cells can generally be achieved using a “second agent,” typically an agent that can penetrate dead cells but not living cells. Suitable agents for staining dead cells include cell impermeable dyes, e.g., cell impermeable DNA binding or DNA intercalating agents. An exemplary agent for staining dead cells is propidium iodide. Another exemplary agent for staining dead cells is Incucyte™ Cytotox Red. A dying cell penetrated by such agents can be considered a dead cell for purposes of the methods described herein.

[0007] Depending on the type of first agent and the detection methods, a first agent may be used to detect total cells or living cells.

[0008] If total cells are detected using the first agent, the living cell count is typically the difference between (a) number of cells that have been stained with the first agent and (b) number of cells that have been stained with the second agent.

[0009] If live cells are detected using the first agent, the total cell count is the sum of the (a) number of cells that have been stained with the first agent and (b) number of cells that have been stained with the second agent.

[0010] In each case, cell viability is the ratio or percentage of live cells to total cells.

[0011] Exemplary methods of measuring cell viability are set forth in Section 5.2 and numbered embodiments 1 to 136 and 148 to 152 of Group I embodiments, 1 to 78 of Group II embodiments and 1 to 70 of Group III embodiments, including exemplary compositions comprising populations of cells with aggregates (e.g., iPSC cell cultures, photoreceptor precursor (PRP) cell cultures, or other cell cultures comprising population of cells with aggregates), methods of sample preparation, exemplary agents for cell visualization, and exemplary methods of live cell imaging. Further exemplary cell cultures and types whose viability can be measured according to the methods of the disclosure (e.g., exemplary populations of cells with aggregates comprising PRPs) are set forth in Section 5.3. Further exemplary methods of sample preparation for performing cell viability determinations according to the methods of the disclosure are set forth in Section 5.4. Further exemplary agents suitable for staining cells in the cell viability assays of the disclosure are set forth in Section 5.5. Further exemplary methods of live imaging of samples comprising cell aggregates in the cell viability assays of the disclosure are set forth in Section 5.6. Determining cell viability based on the method of detection of the first agent and the second agent are described in Section 5.7. Exemplary systems that can be used to perform the methods of measuring cell viability of the disclosure as set forth in Section 5.8 and Group I embodiments 137 to 147. Exemplary methods of downstream processing making use of cell viability measurements are described in Section 5.9 and Group I embodiments 153 to 159.

4. BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 displays the percent viability determined in a population of PSCs.

[0013] FIGS. 2A-2B are graphs displaying percent viability (FIG. 2A) and total numbers of cells per well (FIG. 2B) determined in four populations of iPSCs.

[0014] FIG. 3 shows phase-contrast images of photoreceptor precursor cells overlayed with fluorescent images of acridine orange (AO) or propidium iodide (PI) staining, without and with the analysis mask layer.

[0015] FIG. 4 shows fluorescent images of photoreceptor precursor cells stained with AO + PI, without and with analysis mask layer. The solid arrow points to a dead cell stained with PI and the dashed arrow points to a live cell stained with AO.

[0016] FIGS. 5A-5C are graphs showing the results of image-based counting of PRP cells in aggregates from six samples. FIG. 5A displays the number of total cells counted per image, FIG. 5B displays the number of dead cells counted per image, and FIG. 5C displays the percent viability calculated with the numbers shown in FIGS. 5A and 5B.

[0017] FIGS. 6A-6B are graphs that show the variations in percent viability across five compositions comprising PRP cell aggregates. FIG. 6A displays percent cell viability obtained using image-based counting of cells in aggregates and FIG. 6B displays percent viability of each sample in each PRP cell composition, demonstrating reproducibility of method, obtained using image-based counting of cells in aggregates.

[0018] FIGS. 7A-7C are graphs that show the effect of cell seeding density on cell counts obtained using image-based counting of PRP cells in aggregates. FIG. 7A displays percent viability of samples with different cell seeding densities with or without the presence of a coating matrix. FIG. 7B displays numbers of live and dead cells counted in samples with different cell seeding densities, FIG. 7C displays the numbers of cells per well in samples with different cell seeding densities calculated using image-based counting of cells in aggregates.

[0019] FIG. 8 is a graph that shows the effect of AOPI dilution ratio on the numbers of live and dead cells obtained using image-based counting of cells in aggregates. [0020] FIG. 9 is a graph that shows the effect of the composition dilution ratio on cell viability obtained from samples diluted 1 :10 to 1 :80 from a single PRP cell composition.

[0021] FIGS. 10A-10D are fluorescent images of photoreceptor precursor cells stained with a combination of AO + PI, AO alone, PI alone, a combination of Calcein and Incuctye® Cytotox Red, Calcein alone or Incuctye® Cytotox Red alone. FIG. 10A shows images of cells treated with 1% triton-X and untreated control cells that were stained with either a combination of AO + PI or AO only, captured in green channel. FIG. 10B shows images of cells treated with 1% triton-X and untreated control cells that were stained with either a combination of Calcein + I ncucyte® Cytotox Red or only with Calcein, captured in green channel. FIG. 10C shows images of cells treated with 1% triton-X and untreated control cells that were stained with either a combination of AO + PI or PI only, captured in orange channel. FIG. 10D shows images of cells treated with 1% triton-X and untreated control cells that were stained with either a combination of Calcein + Incucyte® Cytotox Red or only with Incucyte® Cytotox Red, captured in orange channel.

[0022] FIGS. 11A-11C show images with analysis mask layers of AO + PI stained cell preparations with high or low minimum threshold intensity analysis parameters. FIG. 11 A shows images with analysis markers, obtained using high minimum threshold intensity analysis parameters in green (top) and orange (middle) channels and PI staining overlaid with brightfield image layer (bottom). FIG. 11 B shows images with analysis markers, obtained using lower minimum threshold intensity analysis parameters in green (top), orange (middle) channels and PI staining overlaid with brightfield image layer (bottom). FIG. 11C shows PI staining images with analysis markers of untreated control cells (top) and triton-X- treated cells (bottom), obtained using high minimum threshold intensity analysis parameters (left) and low minimum threshold intensity analysis parameters (right).

5. DETAILED DESCRIPTION

5.1. Definitions

[0023] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0024] Aggregate, Cell Aggregate: As used herein, the terms “aggregate” and “cell aggregate” refer to multicellular assemblies of cultured cells. The cell aggregates used herein can be of various shapes, such as, for example, spherical, cylindrical, cuboidal, or elongated (e.g., rod-like, spindle-like, etc.), or can be of other regular (e.g., C-shaped, spiralshaped, etc.) or irregular shapes. The number of cells in cell aggregates can vary. For instance, cell aggregates may comprise a minimal number of cells (e.g., two to twenty cells, etc.) per aggregate or may comprise tens, hundreds, or thousands of cells per aggregate. Typically, cell aggregates comprise hundreds to thousands of cells per aggregate. In some embodiments, the cell aggregate is not an organoid.

[0025] Cell Count, Cell Counting: As used herein, the terms “cell count” and “cell counting” refer to quantification or enumeration of cells. In various embodiments, the cells counted are individual cells, cell aggregates, cell clusters, spheroids, or a combination of any two or more of the foregoing. The cells can be recombinant or non-recombinant, from any source (including but not limited to a cell or tissue culture) and in any state (including but not limited to a primary cell culture, cells differentiated from a primary cell culture, immortalized and/or thawed from a frozen state). In various embodiments, when counted, the cells are adhered to a surface (e.g., the surface of a cell culture vessel, dish, or a well), embedded within a matrix, or suspended in a medium.

[0026] Cell Population, Population: As used herein, the terms “cell population” and “population” refer to an in vitro or ex vivo collection of cells, e.g., cells in a cell culture or cells in a vial. The cells in the cell population can be derived from a common progenitor and/or may comprise more than one cell type. The cell population may be in the form of a composition comprising one or more additional components, e.g., culture media, storage solutions, nutrients, matrices suitable for cell growth, pharmaceutical excipients, carriers and/or diluents, etc. Thus, the term “composition” in relation to cell populations is intended to encompass cell populations in any form, including but not limited to cell cultures, frozen cell stock, and pharmaceutical preparations.

[0027] Cell Viability: As used herein, the terms “cell viability" and “viability” refer to the percentage or fraction of viable (e.g., living) cells in a cell population.

[0028] Hetero-cellular: As used herein, the term “hetero-cellular” in relation to a cell population refers to a cell population (e.g., a population of single cells, cell aggregates, cell clusters, spheroids, organoids, assembloids, or combinations thereof) that comprises cells of at least two cell types. In relation to a cell aggregate, the term hetero-cellular refers to an aggregate that comprises cells of at least two cell types. In relation to a cell culture, the term hetero-cellular refers to a culture that comprises cells of at least two different cell types. In relation to a cell sample (e.g., a sample prepared from a population as described in Section 5.3), the term hetero-cellular refers to a sample that comprises cells of at least two different cell types. In relation to a cell composition, the term hetero-cellular refers to a composition that comprises cells of at least two different cell types.

[0029] For instance, in the context of the present disclosure, a hetero-cellular PRP cell population may comprise PRP cells and one or more types of cells that are not PRP cells, e.g., PSCs (including but not limited to iPSCs), Muller glial cells, retinal amacrine cells, bipolar cells, neural retinal progenitor cells (NRPCs), retinal pigment epithelium (RPE), and/or cells that are in the process of differentiating (e.g., from PSCs) into PRP cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells in a hetero-cellular population are PRP cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells in a hetero- cellular population are PRP cells that are AIPL1+ and RCVRN+.

[0030] In some embodiments, in the context of the present disclosure, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells in a hetero-cellular aggregate are PRP cells. In some embodiments, in the context of the present disclosure, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells in a hetero-cellular aggregate are PRP cells that are AIPL1+ and RCVRN+.

[0031] Homo-cellular: As used herein, the term “homo-cellular” in relation to a cell population refers to a cell population (e.g., a population of single cells, cell aggregates, cell clusters, spheroids, organoids, assembloids, or combinations thereof) that comprises cells of a single cell type, e.g., iPSC cells, PRP cells, etc. In relation to a cell aggregate, the term homo-cellular refers to an aggregate that comprises cells of a single cell type. In relation to a cell culture, the term homo-cellular refers to a culture that comprises cells of a single cell type. In relation to a cell sample (e.g., a sample prepared from a population as described in Section 5.3), the term homo-cellular refers to a sample that comprises cells of a single cell type. In relation to a cell composition, the term homo-cellular refers to a composition that comprises cells of a single cell type.

[0032] iPSC: As used herein, the terms “induced pluripotent stem cell” and “iPSC” refer to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, such as an adult somatic cell, partially differentiated cell or terminally differentiated cell, such as a fibroblast, a cell of hematopoietic lineage, a myocyte, a neuron, an epidermal cell, or the like, by introducing or contacting the cell with one or more reprogramming factors. iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an embryonic stem (ES) cell-like morphology, growing as flat colonies with large nucleo- cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181 , TDGF 1 , Dnmt3b, Fox03, GDF3, Cyp26al, TERT, and zfp42.

[0033] Examples of methods of generating and characterizing iPSCs may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646 and PCT patent publication Nos. WO2013177133 and WO2022204567, the disclosures of each of which are incorporated herein by reference. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells.

[0034] Live Imaging, Live Cell Imaging: As used herein, the terms “live imaging” and “live cell imaging” refer to visualization of living cells (e.g., visualization of a single cell or a population comprising single cells, an individual or a plurality of multicellular assemblies comprising living cells, including but not limited to cell aggregates, cell clusters, spheroids, or combinations thereof), typically via microscopy.

[0035] Photoreceptor Precursor Cells or PRP Cells: The terms “photoreceptor precursor cells” and “PRP cells” refer to cells isolated from tissue and cultured, or differentiated from embryonic stem cells or induced pluripotent stem cells (iPSCs), which can differentiate into photoreceptor cells that expresses the cell marker rhodopsin or any of the three cone opsins, and optionally express the rod or cone cGMP. The PRP-derived photoreceptors may be rod and/or cone photoreceptors. In some embodiments, the PRP cells are generated using methods described in PCT Patent Publication No. WO2019/204817 A1 , the contents of which are incorporated by reference in their entireties herein. In some embodiments, PRP cells are AIPL1 + , NR2E3+, PPP4R4+, and/or RCVRN+. In some embodiments, PRPs are CHX10-, PAX6-, Ki67-, and/or TYRP1-.

[0036] Pluripotent: As used herein, the term “pluripotent” or “pluripotency” refers to the capacity of a cell to self-renew and to differentiate into cells of any of the three germ layers: endoderm, mesoderm, or ectoderm. “Pluripotent stem cells” or“PSCs” include, for example, embryonic stem cells derived from the inner cell mass of a blastocyst or derived by somatic cell nuclear transfer, and iPSCs derived from non-pluripotent cells.

[0037] PRP (Cell) Aggregate, PRP (Cell) Population, PRP (Cell) Culture, PRP (Cell) Sample, PRP (Cell) Composition: As used herein, the terms (a) “PRP aggregate” and “PRP cell aggregate”; (b) “PRP population” and “PRP cell population”; (c) “PRP culture” and “PRP cell culture”; (d) “PRP sample” and “PRP cell sample”; and (e) “PRP composition” and “PRP cell composition” are used conveniently to refer to (a) aggregates, (b) populations, (c) cultures, (d) samples, and (e) compositions, respectively, comprising PRP cells and do not require that the cells in the aggregates, populations, cultures, samples, and compositions consist of PRP cells. Thus, a PRP (cell) aggregate, a PRP (cell) population, a PRP (cell) culture, a PRP (cell) sample, and a PRP (cell) composition can each be hetero-cellular or homo-cellular.

[0038] Seeding Density: As used herein, the terms “seeding density” or “cell seeding density” refer to the number of cells per unit area that are introduced to a container suitable for cell growth and/or imaging, such as, but not including, a well (e.g., in a multi-well plate), flask, petri dish, or roller bottle. Typically, the container is sterile upon seeding. The container may be empty or contain a substrate suitable for cell growth.

[0039] Spheroid: As used herein, the term “spheroid” refers to a self-assembling cell aggregate or cluster, which is able to form without requiring a scaffolding. Typically, spheroids are spherical in shape and formed in conditions where cell-cell interactions predominate over cell-substrate interactions. A spheroid may be highly organized with a well-defined morphology and mimic different in vivo cell interactions, or it may be a simple mass of cells that have clustered or adhered together with minimal organization that relates to the tissue of origin.

[0040] Three-Dimensional (Cell) Imaging: As used herein, the terms “three-dimensional imaging” and “three-dimensional cell imaging" refer to imaging cells in more than one focal plane. Three-dimensional (cell) imaging may include assembly of a three-dimensional image (e.g., using computational methods) from a series of two-dimensional images. The terms “three-dimensional imaging” and “three-dimensional cell imaging” encompass the Z-stacking methods described herein.

[0041] Total Cells: As used herein, the term “total cells” in reference to cells of a population, sample or portion thereof describes all cells that are present in the population, sample or portion. Typically, “total cells” is the sum of living cells and dead cells. Dead cells include cells that are undergoing or have undergone apoptosis, cells that have compromised and/or damaged (e.g., leaky) cell membranes, or any combination thereof. In some embodiments, a dead cell is represented by a naked nucleus.

[0042] Z-stacking, Focus-stacking, and Focal Plane Merging: As used herein, the terms “Z-stacking”, “focus-stacking” and “focal plane merging” refer to a digital image processing technique, which combines multiple images taken at different focus distances (e.g., focal planes or Z-planes) to obtain a resulting image with a combined depth of field (DOF), which is greater than the DOF of individual source images. For instance, Z-stacking can be used to generate three-dimensional (3D) images of cell aggregates that are stained with a first agent and a second agent. The resulting 3D images can be used to determine cell viability of cells in aggregates.

5.2. Cell Viability Assay

[0043] The present disclosure provides an assay for determination of viability of a cell population comprising cells in cell aggregates, for example a cell population comprising photoreceptor precursor (PRP) cells in cell aggregates. In general, the methods of assessing cell viability comprises contacting a sample prepared from a composition comprising the population (e.g., a cell population comprising PRP cells in cell aggregates) with a first agent and a second agent, quantifying the number of live and total cells in at least a portion of the sample, e.g., by imaging cells stained with the first agent and/or second agent, and determining cell viability based on the percentage of live cells in the sample.

[0044] In some embodiments, an assay as provided herein for determining viability in a population comprising cell aggregates comprises: a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells; b) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample; and c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates. [0045] The expression “differentially staining living and dead cells” in reference to a first agent and second agent pair means that the staining observed when staining with the first agent and second agent pair distinguishes living and dead cells. Thus, differential staining in reference to a first and second agent pair does not require that the first agent stain only living cells and the second agent stain only dead cells (or vice versa), although such embodiments are encompassed. For example, an exemplary first agent and second agent pair comprises Acridine orange (AO) and Propidium iodide (PI). AO stains total cells and PI stains dead cells. Thus, AO staining alone cannot distinguish living cells from dead cells. However, when used together, AO and PI differentially stain living and dead cells such that living and dead cells can be distinguished (AO⁺Pk cells can be considered living cells and Pl⁺ cells can be considered dead cells). Another example of a first agent and second agent pair that together are capable of differentially staining living and dead cells is Calcein AM (first agent) and Incucyte® Cytotox Red (second agent). Calcein AM stains live cells and Incucyte® Cytotox Red stains dead cells such that staining with both agents allows living cells and dead cells to be distinguished.

[0046] In some embodiments, an assay as provided herein for determining viability in a population comprising cell aggregates comprises: a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells; b) detecting cells stained with the first agent in at least a portion of the sample; and c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

[0047] In some embodiments, an assay as provided herein for determining viability in a population comprising cell aggregates comprises: a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells; b) detecting cells stained with the first agent in at least a portion of the sample; c) detecting cells stained with the second agent in the same portion of the sample; d) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample based on the staining with the first agent; and e) quantifying dead cells in the same portion of the sample based on the staining with the second agent; thereby determining cell viability in the population of cells comprising cell aggregates.

[0048] In some embodiments, an assay as provided herein for determining viability in a population comprising cell aggregates comprises: a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells; b) quantifying the total number of cells in at least a portion of the sample; and c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cell aggregates.

[0049] In some embodiments, an assay as provided herein for determining viability in a cell population comprising cell aggregates comprises: a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells; b) detecting cells stained with the first agent in at least a portion of the sample; and c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

[0050] In some embodiments, an assay as provided herein for determining viability in a cell population comprising PRP cell aggregates comprises: a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells; b) quantifying the total number of cells in at least a portion of the sample; and c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising PRP cell aggregates.

[0051] In some embodiments, an assay as provided herein for determining viability in a cell population comprising PRP cell aggregates comprises: a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells; b) detecting cells stained with the first agent in at least a portion of the sample; and c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising PRP cell aggregates.

[0052] In other embodiments, an assay as provided herein for determining viability in a cell population (e.g., a cell population comprising cell aggregates) comprises: a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells; b) quantifying the number of living cells in at least a portion of the sample; and c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cell aggregates. [0053] In some other embodiments, an assay as provided herein for determining viability in a cell population (e.g., a cell population comprising cell aggregates) comprises: a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells; b) detecting cells stained with the first agent in at least a portion of the sample; and c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

[0054] In other embodiments, an assay as provided herein for determining viability in a cell population comprising PRP aggregates comprises: a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells; b) quantifying the number of living cells in at least a portion of the sample; and c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising PRP aggregates.

[0055] In some other embodiments, an assay as provided herein for determining viability in a cell population comprising PRP cell aggregates comprises: a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells; b) detecting cells stained with the first agent in at least a portion of the sample; and c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising PRP cell aggregates.

[0056] The first agent in the assays provided herein is typically an agent (e.g., a dye) that can penetrate and stain living cells as well as dead cells. Agents that penetrate both living and dead cells but preferentially stain living cells can also be used. Accordingly, in some embodiments, the first agent stains both living and dead cells. In other embodiments, the first agent stains only living cells but not dead cells. Examples of agents that can be used as first agents are described in Section 5.5.1.

[0057] The second agent in the assays provided herein is typically an agent (e.g., a dye) that can penetrate and stain dead cells but not living cells. Examples of agents that can be used as second agents are described in Section 5.5.2. [0058] As explained in Section 5.5, depending on the detection method (e.g., a detection method involving Forster resonance energy transfer), a first agent may be used to detect total cells or living cells.

[0059] If total cells are detected using the first agent, the living cell count is typically the difference between (a) number of cells that have been stained with the first agent and (b) number of cells that have been stained with the second agent.

[0060] If live cells are detected using the first agent, the total cell count is the sum of the (a) number of cells that have been stained with the first agent and (b) number of cells that have been stained with the second agent.

[0061] Cell viability can be determined by calculating the percentage of living cells to total number of cells (e.g., the total number of all cells (e.g., living cells and dead cells) stained with the first agent or the sum of total number of living cells stained with the first agent and total number of dead cells stained with the second agent).

[0062] In some embodiments, the visualization of the cells (e.g., PRP cells in cell populations comprising PRP cell aggregates) is performed using an instrument capable of imaging live cells, e.g., in three dimensions (e.g., by generating a Z-stack). Live cell imaging of population of cells comprising cell aggregates is described in Section 5.6.

5.3. Cell Populations

[0063] The present disclosure provides an assay for determination of cell viability of cell populations comprising cell aggregates.

[0064] Suitable populations can be in the form of, or prepared from, cell cultures. Cell cultures can comprise multicellular assemblies (e.g., cell aggregates, etc.) or a mixture of multicellular assemblies and single (e.g., dissociated and/or non-aggregated) cells. Thus, in some embodiments, the cell population comprises multicellular assemblies. In some embodiments, the cell population comprises a mixture of single cells and multicellular assemblies.

[0065] In some embodiments, the multicellular assemblies are cell aggregates.

[0066] In some embodiments, the populations and/or aggregates within them are hetero- cellular. In some embodiments, the populations and/or aggregates within them are homo- cellular.

[0067] The number of cells in cell aggregates can vary. For instance, cell aggregates may comprise a minimal number of cells (e.g., two to twenty cells, etc.) per aggregate or may comprise tens, hundreds, or thousands of cells per aggregate. In some embodiments, cell aggregates comprise 50-400 cells per aggregate. In some embodiments, the average cell aggregate in a population from which a sample is prepared and/or in a sample comprises between 2 cells and 100 cells or between 2 cells and 50 cells per aggregate. In some embodiments, a cell aggregate on average comprises at most 100 cells (e.g., at most 100 cells, at most 80 cells, at most 60 cells, at most 50 cells, at most 40 cells, at most 30 cells, at most 25 cells, at most 20 cells, or at most 10 cells). In some embodiments, a cell aggregate on average comprises at least 2 cells (e.g., at least 2 cells, at least 5 cells, at least 10 cells, at least 15 cells, or at least 20 cells).

[0068] The cell aggregates in a cell population can be of various shapes, such as, for example, spherical, cylindrical, cuboidal, or elongated (e.g., rod-like, spindle-like, etc.), or can be of other regular (e.g., C-shaped, spiral-shaped, etc.) or irregular shapes.

[0069] In some embodiments, the cell aggregates are of spherical shapes.

[0070] In some embodiments, the spherical cell aggregates are spheroids.

[0071] In some embodiments, the average diameter of cell aggregates ranges between 20 and 300 pm (e.g., 20-300, 20-250, 20-200, 20-175, 20-150, 20-125, 20-100, 20-75, 20-50, 50-300, 50-250, 50-100, 50-175, 50-150, 50-125, 50-100, 50-75, 100-300, 100-250, 100- 200, 100-175, 100-150, 100-125, 100-250, 100-200, 100-175, or 100-150 pm). In some embodiments, the average diameter of cell aggregates ranges between 25 and 300 pm. In some embodiments, the average diameter of cell aggregates ranges between 50 and 250 pm. In some embodiments, the average diameter of aggregates in a cell population is no greater than 400 pm (e.g., no greater than 400 pm, no greater than 350 pm, no greater than 300 pm, no greater than 275 pm, no greater than 250 pm, no greater than 225 pm, no greater than 200 pm, no greater than 175 pm, no greater than 150 pm, or no greater than 100 pm). In some embodiments, the average diameter of aggregates in a cell population is at least 20 pm (e.g., at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, at least 100 pm, at least 110 pm, at least 120 pm, at least 130 pm, at least 140 pm, at least 150 pm, at least 160 pm, at least 170 pm, at least 180 pm, at least 190 pm, or at least 200 pm).

[0072] In some embodiments, the median diameter of cell aggregates ranges between 20 and 300 pm (e.g., 20-300, 20-250, 20-200, 20-175, 20-150, 20-125, 20-100, 20-75, 20-50, 50-300, 50-250, 50-100, 50-175, 50-150, 50-125, 50-100, 50-75, 100-300, 100-250, 100- 200, 100-175, 100-150, 100-125, 100-250, 100-200, 100-175, or 100-150 pm). In some embodiments, the median diameter of cell aggregates ranges between 25 and 300 pm. In some embodiments, the median diameter of cell aggregates ranges between 50 and 250 pm. In some embodiments, the median diameter of aggregates in a cell population is no greater than 400 m (e.g., no greater than 400 pm, no greater than 350 pm, no greater than 300 pm, no greater than 275 pm, no greater than 250 pm, no greater than 225 pm, no greater than 200 pm, no greater than 175 pm, no greater than 150 pm, or no greater than 100 pm).

[0073] In some embodiments, the median diameter of aggregates in a cell population is at least 20 pm (e.g., at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, at least 100 pm, at least 110 pm, at least 120 pm, at least 130 pm, at least 140 pm, at least 150 pm, at least 160 pm, at least 170 pm, at least 180 pm, at least 190 pm, or at least 200 pm).

[0074] The population of cultured cells (e g., cell aggregates in culture) can be homo-cellular (e.g., comprise a single type of cell) or hetero-cellular (e.g., comprise at least two types of cells). Suitable types of cells that can be used are described in Section 5.3.1.

[0075] In some embodiments, (a) the cell population is homo-cellular, e.g., consists of a single type of cells and/or (b) the cell population comprises homo-cellular cell aggregates, e.g., aggregates consisting of a single type of cells.

[0076] In some embodiments, (a) the cell population is hetero-cellular, e.g., comprises two or more types of cells and/or (b) the cell population comprises hetero-cellular cell aggregates, e.g., aggregates comprising two or more types of cells.

5.3.1. Types of Cells

[0077] The assay for determining viability in a population of cells comprising cell aggregates can be used with samples of any suitable type of cell including, but not limited to, stem cells (e.g., pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs)) and cells differentiated therefrom.

[0078] Stem cells are the starting point for the potential generation of large numbers of a specific cell type that can be delivered for regenerative medicine in patients with many different diseases.

[0079] In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, the population of cells are stem cells. In some embodiments, the stem cells are PSCs. In some embodiments, the stem cells are iPSCs.

[0080] The assays of the disclosure can be used to determine the viability of a population of cells differentiated from at least 99% of the cells in a undergoing differentiation from stem cells (e.g., PSCs, iPSCs, ESCs) into another type of cell. [0081] In some embodiments, the population of cells are differentiated or undergoing differentiation from PSCs.

[0082] In some embodiments, the population of cells are differentiated or undergoing differentiation from iPSCs.

[0083] In some embodiments, the population of cells are undergoing differentiation from PSCs or iPSCs (or have been differentiated from PSCs or iPSCs) into cells of the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof), ectoderm (e.g., skin, neuronal, or pigment cells, or progenitors thereof) and mesoderm (e.g., cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or progenitors or precursors thereof) lineages.

[0084] In some embodiments, the population of cells are cells comprising neural lineage cells.

[0085] In some embodiments, the population of cells are cells of the ocular system.

[0086] In some embodiments, the population of cells are photoreceptor progenitor (PRP) cells, e.g., as described in Section 5.3.2.

[0087] In some embodiments, the population of cells are immune cells.

[0088] In some embodiments, the population of cells are enteric progenitor cells or enteric cells.

[0089] In some embodiments, the population of cells are metabolic system cells (e.g., hepatocytes, cholangiocytes, pancreatic beta cells).

[0090] The assays of the disclosure can be used to determine the viability of a homo-cellular population (e.g., a population of cells comprising a single type of cell) or a hetero-cellular cell population and/or (e.g., a population of cells comprising at least two types of cells).

[0091] In some embodiments, the population of cells is homo-cellular.

[0092] In some embodiments, the population of cells is hetero-cellular.

[0093] In some embodiments, the hetero-cellular population of cells comprises two types of cells. In some embodiments, the hetero-cellular population of cells comprises more than two, e.g., three types of cells.

[0094] In some embodiments, the hetero-cellular population of cells comprises two types of cells.

[0095] In some embodiments, the hetero-cellular population of cells comprises three types of cells. [0096] In some embodiments, the hetero-cellular population of cells comprises more than three types of cells.

5.3.2. PRP Cell Populations

[0097] In some aspects, the present disclosure provides an assay for determination of cell viability of cell populations comprising aggregates that include PRP cells.

[0098] Suitable populations can be in the form of, or prepared from, PRP cell cultures. PRP cell cultures can comprise multicellular assemblies (e.g., cell aggregates, etc.) or a mixture of multicellular assemblies and single (e.g., dissociated and/or non-aggregated) cells. Thus, in some embodiments, the PRP cell population comprises multicellular assemblies. In some embodiments, the PRP cell population comprises a mixture of single cells and multicellular assemblies.

[0099] In some embodiments, the multicellular assemblies are cell aggregates.

[0100] In some embodiments, the populations and/or aggregates within them are hetero- cellular. In some embodiments, the populations and/or aggregates within them are homo- cellular.

[0101] The number of cells in cell aggregates can vary. For instance, cell aggregates may comprise a minimal number of cells (e.g., two to twenty cells, etc.) per aggregate or may comprise tens, hundreds, or thousands of cells per aggregate. In some embodiments, cell aggregates comprise 50-400 cells per aggregate. In some embodiments, the average cell aggregate in a PRP cell population from which a sample is prepared and/or in a PRP sample comprises between 2 cells and 100 cells or between 2 cells and 50 cells per aggregate. In some embodiments, a PRP cell aggregate on average comprises at most 100 cells (e.g., at most 100 cells, at most 80 cells, at most 60 cells, at most 50 cells, at most 40 cells, at most 30 cells, at most 25 cells, at most 20 cells, or at most 10 cells). In some embodiments, a PRP cell aggregate on average comprises at least 2 cells (e.g., at least 2 cells, at least 5 cells, at least 10 cells, at least 15 cells, or at least 20 cells).

[0102] The cell aggregates in a PRP cell population can be of various shapes, such as, for example, spherical, cylindrical, cuboidal, or elongated (e.g., rod-like, spindle-like, etc.), or can be of other regular (e.g., C-shaped, spiral-shaped, etc.) or irregular shapes.

[0103] In some embodiments, the cell aggregates are of spherical shapes.

[0104] In some embodiments, the spherical cell aggregates are spheroids.

[0105] In some embodiments, the average diameter of PRP cell aggregates ranges between 20 and 250 pm (e.g., 20-250, 20-200, 20-175, 20-150, 20-125, 20-100, 20-75, 20-50, 50- 250, 50-100, 50-175, 50-150, 50-125, 50-100, 50-75, 100-250, 100-200, 100-175, 100-150, 100-125, 100-250, 100-200, 100-175, or 100-150 pm). In some embodiments, the average diameter of PRP cell aggregates ranges between 30 and 200 pm. In some embodiments, the average diameter of PRP cell aggregates ranges between 50 and 150 pm. In some embodiments, the average diameter of PRP aggregates in a cell population is no greater than 400 pm (e.g., no greater than 400 pm, no greater than 350 pm, no greater than 300 pm, no greater than 275 pm, no greater than 250 pm, no greater than 225 pm, no greater than 200 pm, no greater than 175 pm, no greater than 150 pm, or no greater than 100 pm). In some embodiments, the average diameter of PRP aggregates in a cell population is at least 20 pm (e.g., at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, at least 100 pm, at least 110 pm, at least 120 pm, at least 130 pm, at least 140 pm, at least 150 pm, at least 160 pm, at least 170 pm, at least 180 pm, at least 190 pm, or at least 200 pm).

[0106] In some embodiments, the median diameter of PRP cell aggregates ranges between 20 and 250 pm (e.g., 20-250, 20-200, 20-175, 20-150, 20-125, 20-100, 20-75, 20-50, 50- 250, 50-100, 50-175, 50-150, 50-125, 50-100, 50-75, 100-250, 100-200, 100-125, 100-250, 100-200, 100-175, or 100-150 pm). In some embodiments, the median diameter of PRP cell aggregates ranges between 30 and 200 pm. In some embodiments, the median diameter of PRP cell aggregates ranges between 50 and 150 pm. In some embodiments, the median diameter of PRP aggregates in a cell population is no greater than 400 pm (e.g., no greater than 400 pm, no greater than 350 pm, no greater than 300 pm, no greater than 275 pm, no greater than 250 pm, no greater than 225 pm, no greater than 200 pm, no greater than 175 pm, no greater than 150 pm, or no greater than 100 pm).

[0107] In some embodiments, the median diameter of PRP aggregates in a cell population is at least 20 pm (e.g., at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, at least 100 pm, at least 110 pm, at least 120 pm, at least 130 pm, at least 140 pm, at least 150 pm, at least 160 pm, at least 170 pm, at least 180 pm, at least 190 pm, or at least 200 pm).

[0108] In some embodiments, (a) the PRP cell population is homo-cellular, e.g., consists of PRP cells and/or (b) the PRP cell population comprises homo-cellular PRP cell aggregates, e.g., aggregates consisting of PRP cells.

[0109] In some embodiments, (a) the PRP cell population is hetero-cellular, e.g., comprises PRP cells and one or more types of cells that are not PRP cells, e.g., PSCs (including but not limited to iPSCs), Muller glial cells, retinal amacrine cells, bipolar cells, neural retinal progenitor cells (NRPCs), retinal pigment epithelium (RPE), and/or cells that are in the process of differentiating into PRP cells and/or (b) the PRP cell population comprises hetero- cellular PRP cell aggregates, e.g., aggregates comprising PRP cells and one or more types of cells that are not PRP cells, e.g., PSCs (including but not limited to iPSCs), Muller glial cells, retinal amacrine cells, bipolar cells, neural retinal progenitor cells (NRPCs), retinal pigment epithelium (RPE), and/or cells that are in the process of differentiating into PRP cells.

[0110] In some embodiments, at least at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in a hetero-cellular PRP cell population and/or hetero- cellular cell population are PRP cells.

5.4. Sample Preparation

[0111] The assays of the disclosure can be used to determine viability of a cell population.

[0112] In some embodiments, the cell population is a pluripotent stem cell population, e.g., embryonic stem (ES) cell population, iPSC population, etc. In other embodiments, the cell population is a population of cells that are differentiated from pluripotent stem cells.

[0113] In some embodiments, the cell population is a PRP cell population isolated from a developing retina. In other embodiments, the PRP cell population is differentiated from pluripotent stem cells (e.g., embryonic stem (ES) cells, iPSCs, etc.). The population can be from any suitable species, e.g., a mammalian species, such as human. Exemplary methods of differentiating pluripotent stem cells into PRP cells may be found in PCT Patent Publication No. WO2019/204817 A1 , the disclosure of which is incorporated herein by reference.

[0114] In some embodiments, the PRP cell population is differentiated from mammalian pluripotent stem cells (PSCs). In some embodiments, the PRP cell population is differentiated from human pluripotent stem cells (PSCs), e.g., human induced pluripotent stem cells (iPSCs).

[0115] The assays of the disclosure can be used to determine viability of a population of cells of a non-frozen cell (e.g., PRP) culture (e.g., a PRP culture at one of various stages of differentiation from a PSC cell population), or a population of cells comprising cells (e.g., PRP cells) that were frozen (e.g. cryopreserved) and thawed, or a population of a cell (e.g., PRP cell) culture established from frozen (e.g., cryopreserved) cells (e.g., PRP cells) that were thawed in a manner as commonly known in the art for frozen cultured cells.

[0116] In some embodiments, a cell population is from a non-frozen cell culture. In some embodiments, a PRP cell population is from a non-frozen PRP cell culture. [0117] In some embodiments, a cell population is from a cell culture that was frozen and thawed. In some embodiments, a PRP cell population is from a PRP cell culture that was frozen and thawed.

[0118] The assays of the disclosure can be used with cell populations (e.g., PRP cell populations) from cultures that were allowed to grow for any number of passages, e.g., splittings. For example, cell populations (e.g., PRP cell populations) may be from cultures that may have been passaged 0 times, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7, times, 8 times, 9 times, or 10 or more times.

[0119] Typically, sample preparation excludes any manipulations of cells from a population in a way that is likely to change the viability of cells in the sample. Thus, when live imaging a sample, the viability determination closely reflects the viability of the cells in the population as a whole.

[0120] In some embodiments, an assay of the disclosure comprises preparing one or more samples (e.g., duplicate or triplicate samples) from a cell population (e.g., a PRP cell population). In some embodiments, a sample on which an assay of the disclosure is performed has the same cell concentration as the cell population (e.g., a PRP cell population) from which it is prepared. In some embodiments, a sample on which an assay of the disclosure is performed has a lower cell concentration than the cell population (e.g., a PRP cell population) from which it is prepared. Accordingly, in some embodiments, sample preparation comprises diluting cells from a cell population (e.g., a PRP cell population) to a suitable concentration. Cells from a population (e.g., a PRP cell population) can be diluted using a suitable cell culture medium, such as, but not limited to, Essential 8 (E8), MEM, DMEM F12, BSS+HSA, or Neurobasal medium.

[0121] In some embodiments, cells from a cell population (e.g., a PRP cell population) are diluted at a cell suspension:medium ratio between 1 :10 and 1 :160. In some embodiments, cells from a cell population (e.g., a PRP cell population) are diluted in medium at a cell suspension:medium ratio of between 1 :10 and 1 :80 or between 1 :20 and 1 :40.

[0122] In some embodiments, cells from a cell population (e.g., a PRP cell population) are diluted in medium at a cell suspension:medium ratio of 1 :20.

[0123] In some embodiments, cells from a cell population (e.g., a PRP cell population) are diluted in medium at a cell suspension:medium ratio of 1 :40.

[0124] In some embodiments, the concentration of cells in a sample range between 10,000 and 200,000 cells/pL (e.g., 10,000-25,000 cells/pL, 10,000-50,000 cells/pL, 10,000-75,000 cells/ L 10,000-100,000 cells/pL, 10,000-175,000 cells/pL 10,000-150,000 cells/pL, 10,000- 200,000 cells/pL, 25,000-50,000 cells/pL, 25,000-75,000 cells/pL, 25,000-100,000 cells/pL, 25,000-125,000 cells/pL, 25,000-150,000 cells/pL, 25,000-200,000 cells/pL, 50,000-75,000 cells/pL, 50,000-100,000 cells/pL, 50,000-125,000 cells/pL, 50,000-150,000 cells/pL, 50,000-175,000 cells/pL, 50,000-200,000 cells/pL, 75, 000-100, 000 cells/pL, 75,000-125,000 cells/pL, 75,000-150,000 cells/pL, 75,000-175,000 cells/pL, 75,000-200,000 cells/pL, 100,000-125,000 cells/pL, 100,000-150,000 cells/pL, 100,000-175,000 cells/pL, 100,000- 200,000 cells/ L, 125,000-150,000 cells/ L, 125,000-175,000 cells/pL, 125,000-200,000 cells/pL, 150,000-175,000 cells/pL, 150,000-200,000 cells/pL, 175,000-200,000 cells/pL). In some embodiments, the concentration of cells in a sample range between 20,000 and 175,000 cells/pL. In some embodiments, the concentration of cells in a sample range between 10,000 and 50,000 cells/pL.

[0125] Once prepared, the samples are transferred to a suitable container, e.g., for staining and imaging. In various embodiments, the samples are transferred into a solution as a suspension (e.g., culture medium) or deposited (in whole or in part) onto a suitable surface, e.g., a multi-well plate (e.g., 12-well plates, 24-well plates, 96-well plates), roller bottle, or a tissue culture dish (e.g., Petri dishes, Nunc trays). The samples can be deposited (in whole or in part) onto an uncoated surface, or they can be deposited (in whole or in part) onto a coated surface (e.g., a surface layered with a coating matrix, e.g., a matrix that supports the growth and/or organization of cells). Examples of suitable coating matrices include, but are not limited to, Matrigel™, Geltrex™, fibronectin, collagen I, collagen IV, vitronectin, laminin, poly-lysine, and poly-ornithine.

[0126] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the uncoated surface of a multi-well plate, e.g., a 96-well plate.

[0127] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with a coating matrix.

[0128] In some embodiments, cell (e.g., PRP) samples are combined with a coating matrix material and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96- well plate. The multi-well plate can be uncoated or coated with the same coating matrix or a different coating matrix.

[0129] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with Matrigel™. In some embodiments, cell (e.g., PRP) samples are combined with Matrigel™ and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with Matrigel™ or a different coating matrix. [0130] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with Geltrex™. In some embodiments, cell (e.g., PRP) samples are combined with Geltrex™ and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with Geltrex™ or a different coating matrix.

[0131] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with fibronectin. In some embodiments, cell (e.g., PRP) samples are combined with fibronectin and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with fibronectin or a different coating matrix.

[0132] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with collagen I. In some embodiments, cell (e.g., PRP) samples are combined with collagen I and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with collagen I or a different coating matrix.

[0133] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part the surface of a multi-well plate, e.g., a 96-well plate, coated with collagen IV. In some embodiments, cell (e.g., PRP) samples are combined with collagen IV and deposited in whole or in part onto the surface of a multi-well plate, e.g, a 96-well plate. The multi-well plate can be uncoated or coated with collagen IV or a different coating matrix.

[0134] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part the surface of a multi-well plate, e.g., a 96-well plate, coated with vitronectin. In some embodiments, cell (e.g., PRP) samples are combined with vitronectin and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with vitronectin or a different coating matrix.

[0135] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with laminin. In some embodiments, cell (e.g., PRP) samples are combined with laminin and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with laminin or a different coating matrix.

[0136] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with poly-lysine. In some embodiments, cell (e.g., PRP) samples are combined with poly-lysine and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with poly-lysine or a different coating matrix. [0137] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate, coated with poly-ornithine. In some embodiments, cell (e.g., PRP) samples are combined with poly-ornithine and deposited in whole or in part onto the surface of a multi-well plate, e.g., a 96-well plate. The multi-well plate can be uncoated or coated with poly-ornithine or a different coating matrix.

[0138] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the uncoated surface of a roller bottle.

[0139] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a roller bottle, coated with a coating matrix.

[0140] In some embodiments, cell (e.g., PRP) samples are combined with a coating matrix and deposited in whole or in part onto the surface of a roller bottle. The roller bottle can be uncoated or coated with the coating matrix combined with cell (e.g., PRP) samples or a different coating matrix.

[0141] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the uncoated surface of a tissue culture dish.

[0142] In some embodiments, cell (e.g., PRP) samples are deposited in whole or in part onto the surface of a tissue culture dish, coated with a coating matrix.

[0143] In some embodiments, cell (e.g., PRP) samples are combined with a coating matrix and deposited in whole or in part onto the surface of a culture dish. The culture dish can be uncoated or coated with the coating matrix combined with cell (e.g, PRP) samples or a different coating matrix.

[0144] For embodiments concerning cell (e.g., PRP) samples plated in 96-well plates, a suitable range of cell seeding density is 25,000 cells/well to 100,000 cells/well of a 96-well plate, or equivalent thereof in a container of a different size. In some embodiments, samples are plated at a seeding density of 25,000 cells/well of a 96-well plate, or equivalent thereof in a container of a different size. In some embodiments, samples are plated at a seeding density of 50,000 cells/well of a 96-well plate, or equivalent thereof in a container of a different size. In other embodiments, samples are plated at a seeding density of 100,000 cells/well of a 96-well plate, or equivalent thereof in a container of a different size. One of skill in the art would understand that cell seeding densities are often imprecise, and thus the cell densities and cell density ranges of the disclosure are intended to be approximations rather than precise counts. In some embodiments, the cell density ranges are within 10% of the foregoing ranges. In some embodiments, the cell density ranges are within 5% of the foregoing ranges. [0145] The assays of the disclosure can be run singly but are typically performed in replicates (e.g., replicates of wells in a multi-well plate). In some embodiments, samples of the disclosure are plated (e.g., seeded) in 2 replicates, 3 replicates, 4 replicates, 5 replicates, or 6 replicates. For instance, if the samples are plated on a multi-well plate (e.g., a 96-well plate), the sample replicates can be plated into 2 wells, 3 wells, 4 wells, 5 wells, or 6 wells. The term replicate encompasses multiple sample preparations from the same population or a sample preparation divided into multiple wells for imaging.

[0146] In some embodiments, samples are plated in two replicate wells of a multi-well plate. In some embodiments, samples are plated in three replicate wells of a multi-well plate. In some embodiments, samples are plated in four replicate wells of a multi-well plate. In some embodiments, samples are plated in five replicate wells of a multi-well plate. In some embodiments, samples are plated in six replicate wells of a multi-well plate.

5.5. Staining of Cells

[0147] The assays of the disclosure typically comprise contacting a sample from the cell (e.g., PRP) population with a first agent capable of staining living cells and/or total cells, and a second agent capable of staining dead cells.

[0148] In some embodiments, the first agent is a cell permeable agent capable of permeating both intact membranes, e.g., in living cells, and compromised and/or damaged membranes, e.g., in dead cells. In some embodiments, the first agent is a dye that is detectable in both living and dead cells. In other embodiments, the first agent is detectable in living cells but not dead cells.

[0149] In some embodiments, the second agent is a cell impermeable dye that stains cells with compromised and/or damaged membranes, e.g., dead cells, but not cells with intact membranes, e.g., living cells.

[0150] Examples of first agents are described in Section 5.5.1. Examples of second agents are described in Section 5.5.2.

[0151] The assays of the disclosure can be used with any suitable combination of a first agent and a second agent. Suitability of a combination of a first and second agent is determined by the excitation/emission wavelength profiles of each agent. For instance, a suitable combination of two agents would entail individual agents to emit fluorescence at wavelengths distinguishable from one another. Forster resonance energy transfer (FRET) may be present, where, for instance, the second agent absorbs the emission signal of the first agent, producing results without double positives or spill-over signals. The excitation/emission filters of the imaging instrument can be taken into account when selecting a combination of first and second agents, as FRET can depend on the excitation/emission filters of the instrument. Thus, for any given pair of first and second agents, FRET may absent under some measurement conditions but present to varying degrees under others.

[0152] The assays of the disclosure can be used with established combinations of fluorescent agents, such as AOPI staining mixture (Nexcelom; see www.nexcelom.com/applications/cellometer/viability/viability-using-ao-pi/), which comprises the fluorescent first agent acridine orange (AO) and the fluorescent second agent propidium iodide (PI). FRET can be present when analyzing a population of AOPI stained cells. For example, double positive cells (AO⁺PI⁺ cells) may not be detected due to FRET between AO and PI in dead cells. In such a situation, the AO⁺ cells correspond to live cells. Under different conditions, double positive cells (AO⁺PI⁺ cells) may be detected (e.g., due to relatively broad filters that capture spectral bleedthrough). Under such conditions, the A0⁺ cells correspond to total cells.

[0153] The samples are typically stained with the first and second agents by adding an appropriate volume of each agent or an appropriate volume of a first agent and second agent mixture (e.g., AOPI staining mixture) to cultured cell (e.g., PRP) samples in media. For instance, the agents or the agent mixture (e.g., AOPI staining mixture) can be added to cultured cell samples in media at a dye:medium ratio ranging between 1 :200 and 1 :1000, e.g., between 1 :500 and 1 :1000.

[0154] In some embodiments, a first and second agent mixture (e.g., AOPI staining mixture) is used at a dye:medium ratio of 1 :200 to 1 :500.

[0155] In some embodiments, a first and second agent mixture (e.g., AOPI staining mixture) is used at a dye:medium ratio of 1 :300.

[0156] In some embodiments, a first and second agent mixture (e.g., AOPI staining mixture) is used at a dye:medium ratio of 1 :500.

[0157] In some embodiments, a first and second agent mixture (e.g., AOPI staining mixture) is used a dye:medium ratio of 1 :750.

[0158] In some embodiments, a first and second agent mixture (e.g., AOPI staining mixture) is used at a dye:medium ratio of 1 :1000.

5.5.1. First Agents

[0159] Typically, a first agent is a cell membrane permeable agent (e.g., a fluorescent cell membrane permeable agent) that can penetrate into living cells. First agents typically also penetrate dead cells, and in some cases penetrate dead cells to a greater extent than living cells. Thus, the use of the term “first agent” encompasses an agent that is capable of permeating both living and dead cells.

[0160] In some embodiments, a first agent is a dye (e.g., fluorescent dye) that can stain the cytoplasm, organelles (e.g., mitochondria, lysosomes, etc.), cytoskeleton (e.g., actin filaments) or the nuclei (e.g., by binding to nucleic acids in nuclei) of both living and dead cells.

[0161] In some embodiments, the first agent is a cytoplasmic dye. A non-limiting example of a dye that can stain the cytoplasm is Calcein AM. Calcein AM is capable of being cleaved by cytoplasmic esterases after entering the cytoplasm of a living cell, yielding Calcein Green and/or Calcein Blue, which emit green and blue light, respectively. Calcein AM can penetrate the membrane of living and dead cells, but only becomes fluorescent when the molecule is cleaved by active enzymes in living cells. Thus, Calcein AM stains living cells but not dead cells.

[0162] In some embodiments, the first agent is a nucleic acid binding dye. Non-limiting examples of nucleic acid binding dyes are Acridine orange (AO) (3,6- Bis(dimethylamino)acridine), a Hoechst dye (e.g., Hoechst 33342 (Bisbenzimide ethoxide trihydrochloride) or Hoechst 33258 (Bisbenzimidazole trihydrochloride)), EasyProbe™ Green 488 live cell stain (e.g., ABP Catalog No.: FP029), NucRed™ live cell nucleic acid stain (e.g., ThermoFisher Catalog No.: R37106), NucGreen™ live cell nucleic acid stain (e.g., ABP Catalog No.: C013-1), DRAQ5™ (1 ,5-Bis{[2-(dimethylamino)ethyl]amino}-4,8-dihydroxy- 9,10-anthraquinone), SYTO™Red (e.g., ThermoFisher Catalog No.: S34900), SYTO™Orange (e.g., ThermoFisher Catalog No.: S11365), SYTO™Green (e.g., ThermoFisher Catalog No.: S34854), and SYTO™Blue (e.g., ThermoFisher Catalog No.: S11351). Another non-limiting example of nucleic acid dye is Incucyte® Nuclight Rapid Red (e.g., Sartorius Catalog No.: 4717).

[0163] In some embodiments, the first agent is Acridine orange (AO), which emits green light when bound to DNA.

[0164] In some embodiments, the first agent is Incucyte® Nuclight Rapid Red, which emits red light when bound to DNA.

[0165] In some embodiments, the first agent is a Hoechst dye (e.g., Hoechst 33342 or Hoechst 33258), which emits blue light when bound to DNA.

[0166] In some embodiments, the first agent is EasyProbe™ Green 488 live cell stain, which emits green light when bound to DNA. [0167] In some embodiments, the first agent is NucRed™ live cell nucleic acid stain, which emits red light when bound to DNA.

[0168] In some embodiments, the first agent is NucGreen™ live cell nucleic acid stain, which emits green light when bound to DNA.

[0169] In some embodiments, the first agent is DRAQ5, which emits red light when bound to DNA.

[0170] In some embodiments, the first agent is SYTO^TMRed, which emits red light when bound to DNA.

[0171] In some embodiments, the first agent is SYTO™Orange, which emits orange light when bound to DNA.

[0172] In some embodiments, the first agent is SYTO^TMGreen, which emits green light when bound to DNA.

[0173] In some embodiments, the first agent is SYTO^TMBlue, which emits blue light when bound to DNA.

5.5.2. Second Agents

[0174] Unlike living cells, dead cells have compromised and/or damaged membranes. Typically, a second agent is a membrane-impermeable agent (e.g., a fluorescent membrane- impermeable agent).

[0175] Second agents typically cannot penetrate into living cells but enter cells with compromised and/or damaged membranes. Thus, the use of the term “second agent” encompasses an agent that is capable of staining dead cells but not living cells.

[0176] In some embodiments, a second agent can stain various parts of cells, e.g., it can bind to cytoplasmic proteins or bind to DNA. Some second agents can intercalate with double-stranded DNA in nuclei.

[0177] In some embodiments, the second agent is a DNA binding dye. In some embodiments, the second agent is a DNA-intercalating dye. Non-limiting examples of DNA- binding and/or DNA-intercalating dyes are Propidium iodide (PI) (e.g., ThermoFisher Catalog No.: P1304MP), 7-aminoactinomycin D (7-AAD) (e.g., ThermoFisher Catalog No.: A1310), Ethidium Homodimer-1 (EthD-1) (e.g., ThermoFisher Catalog No.: E1169), Ethidium Homodimer-3 (EthD-3) (e.g., Fisher Scientific Catalog No.: 50-196-4647), Ethidium monoazide (EMA) (e.g., ThermoFisher Catalog No.: E1374), Oxazole Blue Homodimer (POPO^TM-1) (e.g., Biotium Catalog No.:40093), Oxazole Yellow Homodimer (YOYO^TM-1) (e.g., Biotium Catalog No.:40090), Thiazole Orange Homodimer (TOhD, TOTOO-1) (e.g., Biotium Catalog No.:40079), EasyProbe™ Green 488 Dead Cell Stain (e.g., ABP Catalog No.: FP030), NucGreen™ dead cell nucleic acid stain (e.g., ThermoFisher Catalog No.: R37109). Futher non-limiting examples include Incucyte® Cytotox Red (e.g., Sartorius Catalog No.: 4632) and Incucyte® Cytotox Green (e.g., Sartorius Catalog No.: 4633).

[0178] In some embodiments, the second agent is Propidium iodide (PI), which emits red light when bound to DNA.

[0179] In some embodiments, the second agent is Incucyte® Cytotox Red, which emits red light when bound to DNA.

[0180] In some embodiments, the second agent is Incucyte® Cytotox Green, which emits green light when bound to DNA.

[0181] In some embodiments, the second agent is 7-AAD, which emits red light when bound to DNA.

[0182] In some embodiments, the second agent is EthD-1 , which emits red light when bound to DNA.

[0183] In some embodiments, the second agent is EthD-3, which emits red light when bound to DNA.

[0184] In some embodiments, the second agent is EMA, which emits red light when bound to DNA.

[0185] In some embodiments, the second agent is Oxazole Blue Homodimer, which emits blue light when bound to DNA.

[0186] In some embodiments, the second agent is Oxazole Yellow Homodimer, which emits green light when bound to DNA.

[0187] In some embodiments, the second agent is Thiazole Orange Homodimer, which emits green light when bound to DNA.

[0188] In some embodiments, the second agent is EasyProbe™ Green 488 Dead Cell Stain, which emits green light when bound to DNA.

[0189] In some embodiments, the second agent is NucGreen™ dead cell nucleic acid stain, which emits green light when bound to DNA.

5.6. Live Imaging of Cells in Samples Comprising Aggregates

[0190] Cell detection in samples comprising cell aggregates (e.g., PRP cell aggregates) can be performed using standard live cell microscopy techniques well known in the art, e.g., live cell fluorescent microscopy, brightfield microscopy, phase contrast microscopy, etc. For instance, live cell fluorescent microscopy can be used to detect cells that are stained with fluorescent first agents or fluorescent second agents; brightfield microscopy can be used to identify the location and boundaries of cells and cell aggregates; and phase contrast microscopy can be used to visualize structures and boundaries at a greater detail than with brightfield microscopy.

[0191] In some embodiments, detection and/or quantification of cells in cell aggregates (e.g., PRP cell aggregates) comprises live cell fluorescent microscopy. Detection and/or quantification of the cells in cell aggregates (e.g., PRP cell aggregates) can comprise obtaining fluorescent images of a sample or a portion of a sample at the peak emission wavelength of the first agent and at the peak emission wavelength of the second agent simultaneously or separately.

[0192] In some embodiments, fluorescent images of a sample or a portion of a sample are obtained simultaneously at the peak emission wavelength of the first agent and at the peak emission wavelength of the second agent.

[0193] In some embodiments, fluorescent images of a sample or a portion of a sample are obtained individually at the peak emission wavelength of the first agent and at the peak emission wavelength of the second agent. In some embodiments, the fluorescent image of a portion of a sample obtained at the peak emission wavelength of the first agent and the fluorescent image of the same portion of the sample obtained at the peak emission wavelength of the second agent are assessed individually to determine via. In some embodiments, composite fluorescent images (e.g., fluorescent images that are generated by overlaying an image obtained at the peak emission wavelength of the first agent with an image of the same portion of the sample obtained at the peak emission wavelength of the second agent) are used to determine viability.

[0194] In some embodiments, detection and/or quantification of cells in cell aggregates (e.g., PRP cell aggregates) comprises combining live cell fluorescent microscopy with brightfield and/or phase contrast microscopy.

[0195] Detection and/or quantification of cells in cell aggregates (e.g., PRP cell aggregates) typically comprises obtaining individual images at different focal planes (e.g., Z-planes) and generating composite images (e.g., Z-stacks) using the individual images at different focal planes, e.g., by Z-stacking or focal plane merging of multiple images taken at different focus distances (e.g., different Z-planes or focal planes), which results in an image with a combined depth of field, extending from the bottom focal plane to the top focal plane of the evaluated portion of the sample. Generation of such images (e.g., Z-stacks) allows detection of cells located inside aggregates (e.g., aggregated cells in a sample prepared from a cell population, for example aggregated PRP cells in a sample prepared from a PRP cell population, for example). Fluorescent microscopy Z-stacks can be overlaid and/or analyzed simultaneously with brightfield and/or phase contrast Z-stacks, wherein the fluorescent Z- stacks allow detection of first agents and second agents and brightfield and/or phase contrast Z-stacks allow identification of boundaries of cells and cell aggregates.

[0196] Detection and/or quantification of the cells in populations comprising cell aggregates (e.g., PRP cell aggregates) can comprise imaging the entire sample of the cell population or a portion (e.g., a fraction of the area, e.g., field of view, within a well of a multi-well plate) of the sample and generation of Z-stacks from individual images at different focal planes.

[0197] In some embodiments, the entire sample of the cell (e.g., PRP) population (e.g., all cells in a well of a multi-well plate, such as a 96-well plate) is imaged and/or quantified.

[0198] In some embodiments, a portion of the sample of the cell (e.g., PRP) population (e.g., a fraction of the area, e.g., field of view, within a well of a multi-well plate, such as a 96-well plate) is imaged and/or quantified. In some embodiments, the imaged and/or quantified portion of the sample in a well of a multi-well plate (e.g., a 96-well plate) is the center portion of the well.

[0199] Detection and/or quantification of the cells in populations comprising cell aggregates (e.g., PRP cell aggregates) can in some embodiments utilize a minimum fluorescence intensity threshold (herein also referred to as minimum intensity threshold or intensity threshold) for a staining agent, which allows identification of specific objects or regions of interest based on their fluorescence intensity. Setting an appropriate minimum intensity threshold can, for example, help to distinguish the signal of interest from background noise, allowing for more accurate analysis and quantification, which is done by defining a minimum intensity value for a staining agent, with pixels at or above that value considered as foreground and those below as background. For instance, when a minimum fluorescence intensity threshold is utilized, each object with a fluorescence intensity at or above the intensity threshold is considered as an object (e.g., cell or cell aggregate) positive for that staining agent, whereas each object (e.g., cell or cell aggregate) with a fluorescence intensity below the intensity threshold is considered as an object (e.g., cell or cell aggregate) negative for that staining agent. Fluorescence intensity measurements can be influenced by object (e.g., cell or cell aggregate) size, with larger objects (e.g., cells or cell aggregates) typically exhibiting higher fluorescence intensity due to a greater volume containing the fluorescent molecule. Accordingly, a low minimum intensity threshold for a staining agent can allow consideration of small objects (e.g., single cells or small cell aggregates) as positive for that staining agent. [0200] The same minimum fluorescence intensity threshold can be used for the first agent and the second agent. Alternatively, the minimum fluorescence intensity threshold of the first agent can be different from the minimum fluorescence intensity threshold of the second agent.

[0201] Minimum fluorescence intensity thresholds can be selected, for example, by measuring the fluorescence intensity of an area that is known to not contain any cells (e.g., an empty well) and selecting a threshold corresponding to the fluorescence intensity of that area, such that all objects with a fluorescence intensity above the threshold are identified as an object for analysis.

[0202] Any live cell imaging system capable of detecting and/or quantifying aggregated cells (e.g., a sample of cell (e.g, PRP) culture comprising aggregated cells) and generating stacked images can be used. Non-limiting examples of live cell imaging system that can be used to detect and/or quantify are IncuCyte® (Sartorius), BioTek® Cytation cell imaging multimode reader (Agilent), BioTek® BioSpa live cell analysis system (Agilent), THUNDER® 3D Live Cell Imager (Leica), Opera Phenix® Plus High-Content Screening System (PerkinElmer), Operetta CLS^TM High-Content Analysis System (PerkinElmer), ImageXpress® Micro Widefield High Content Imaging System (Molecular Devices), and Celloger® Pro (Curiosis). Live cell imaging systems allow for identification and/or automated quantification of cells in a sample.

[0203] In some embodiments, the live cell imaging system is IncuCyte®.

[0204] In some embodiments, samples are imaged with the spheroid mode of IncuCyte®.

[0205] In some embodiments, the live cell imaging system is BioTek® Cytation cell imaging multimode reader.

[0206] In some embodiments, the live cell imaging system is BioTek® BioSpa live cell analysis system.

[0207] In some embodiments, the live cell imaging system is THUNDER® 3D Live Cell Imager.

[0208] In some embodiments, the live cell imaging system is Opera Phenix® Plus High- Content Screening System.

[0209] In some embodiments, the live cell imaging system is Operetta CLS^TM High-Content Analysis System.

[0210] In some embodiments, the live cell imaging system is ImageXpress® Micro Widefield High Content Imaging System. [0211] In some embodiments, the live cell imaging system is Celloger® Pro.

5.7. Determining Cell Viability

5.7.1. Determining Cell Viability Based on Detection of Total Cells and Dead Cells

[0212] When cell staining by a first agent capable of staining both live and dead cells is assessed simultaneously with cell staining by a second agent, e.g., when both the first agent and second agent fluorescence emissions are detected simultaneously for a first agent and second agent that emit fluorescence at distinguishable wavelengths without FRET interference, the number of cells stained with the first agent (e.g., the number of cells emitting the emission wavelength associated with the first agent) reflects the total number of cells.

[0213] When cell staining by a first agent capable of staining both live and dead cells and cell staining by a second agent are assessed separately, e.g., when the images of the same portion of the sample are obtained at the peak emission wavelengths of each of the first and second agent individually (e.g., by imaging for the first agent followed by imaging for the second agent; or imaging for the second agent followed by imaging for the first agent) without the generation of a composite image (e.g., an image that overlays two individual images obtained at different emission settings), the number of cells stained with the first agent (e.g., the number of cells emitting the emission wavelength associated with the first agent) reflects the number of total cells (e.g., living cells and dead cells).

[0214] In each case, the number of dead cells is the number of cells stained with the second agent.

[0215] If total cells are detected using the first agent, the living cell count is typically the difference between (a) number of cells that have been detected as stained with the first agent and (b) number of cells that have been detected as stained with the second agent.

[0216] Cell viability is the percentage or ratio of the number of live cells to the number of total cells.

5.7.2. Determining Cell Viability Based on Detection of Live Cells and Dead Cells

[0217] When cell staining by a first agent capable of staining both live and dead cells is assessed simultaneously with cell staining by a second agent, e.g., when both the first agent and second agent fluorescence emissions are detected simultaneously, and the second agent absorbs the emission signal of the first agent via FRET, the number of cells stained with the first agent (e.g., the number of cells emitting the emission wavelength associated with the first agent) reflects the number of live cells and the number of dead cells is the number of cells stained with the second agent.

[0218] If live cells are detected using the first agent, the total cell count is the sum of the (a) number of cells that have been detected as stained with the first agent and (b) number of cells that have been detected as stained with the second agent.

[0219] Cell viability is the percentage or ratio of the number of live cells to the number of total cells.

5.8. Systems

[0220] The present disclosure provides systems configured to perform one or more of the methods disclosed herein.

[0221] The systems can comprise an imaging device capable of imaging cells from a sample stained with a first agent and a second agent, and/or detecting the first agent and the second agent (the system can, in some embodiments, comprise the sample). The systems can further comprise a processor configured to receive one or more images from the imaging device and detect cells stained with the first agent and/or second agent, and/or quantify the cells in at least a portion of the sample from the one or more images (e.g., quantify total number of cells, living cells, dead cells, and/or combinations thereof). In some embodiments, a system of the disclosure comprises a live cell imaging system described in Section 5.6.

[0222] In some embodiments, the systems of the disclosure are configured to perform any of the methods of determining cell viability in a population of cells comprising cell aggregates disclosed herein.

[0223] The systems of the disclosure can include one or more processors coupled to a memory storing one or more computer readable instructions for execution by the one or more processors.

5.9. Downstream Processing

[0224] The cell viability of a cell population as determined by a method described herein can be used to inform subsequent processing of the cell population. For example, if a target concentration of viable cells is desired when passaging cells, the cell viability can be used to calculate the amount of new cell culture medium to combine with the cell population or a portion thereof.

[0225] If a target concentration of viable cells is desired, for example, when preparing cells for subsequent culture, storage or formulation as a therapeutic composition, the cell viability can be used to determine whether the cell population or a portion thereof should be concentrated, diluted (e.g., with a culture medium, storage medium or a pharmaceutical excipient, carrier, and/or diluent), or a combination thereof (e.g., concentrated and then diluted with a culture medium, storage medium or a pharmaceutical excipient, carrier, and/or diluent to obtain a population having the target concentration of viable cells). Concentration can include a step of separating cells from some or all of their medium, for example by centrifugation or filtration. Concentrated cells can be diluted or resuspended (e.g., if cells are pelleted during a concentration step) in a sufficient volume of liquid to provide the target concentration of viable cells. Exemplary liquids that can be used to dilute or resuspend the cells include culture media, storage media (e.g., containing a cryopreservative), and pharmaceutical excipients, carriers, and diluents. In some embodiments, concentrated cells are washed one or more times before being transferred to a container (e.g., vial or cell culture dish) and diluted with liquid to provide the target concentration of viable cells.

6. SPECIFIC EMBODIMENTS

[0226] The present disclosure is exemplified by the specific embodiments below.

Group I Embodiments:

1 . A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

2. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample;

(c) detecting cells stained with the second agent in the same portion of the sample;

(d) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample based on the staining with the first agent; and (e) quantifying dead cells in the same portion of the sample based on the staining with the second agent; thereby determining cell viability in the population of cells comprising cell aggregates.

3. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) quantifying the total number of cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

4. The method of any one of embodiments 1 to 3, wherein cells that are stained by the first agent and cells that are stained by the second agent are identified, e.g., using an algorithm that differentiates between cells and other material stained with the first agent and/or the second agent.

5. The method of any one of embodiments 1 to 4, wherein the first agent and the second agent are detected separately.

6. The method of embodiment 5, wherein the number of total cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

7. The method of embodiment 6, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

8. The method of embodiment 7, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

9. The method of any one of embodiments 1 to 4, wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

10. The method of embodiment 9, wherein the number of total cells is the number of cells detected as stained with the first agent and the number of dead cells is the number of cells detected as stained with the second agent. 11 . The method of embodiment 10, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

12. The method of embodiment 11 , wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

13. The method of any one of embodiments 1 to 4, wherein the first agent and the second agent are detected simultaneously in the presence of FRET between the first agent and the second agent.

14. The method of embodiment 13, wherein the number of total cells is sum of the number of cells detected as stained with the first agent and the number of cells detected as stained with the second agent.

15. The method of embodiment 13 or embodiment 14, wherein the number of dead cells is the number of cells detected as stained with the second agent.

16. The method of any one of embodiments 13 to 15, wherein the number of live cells is the number of cells detected as stained with the first agent.

17. The method of any one of embodiments 13 to 16, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

18. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

19. A method of determining cell viability in a population of cells comprising cell aggregates (e.g., cell aggregates in which photoreceptor precursor (PRP) cells are present), the method comprising: (a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates (e.g., PRP cell aggregates).

20. The method of embodiment 18 or embodiment 19, wherein cells that are stained by the first agent and cells that are stained by the second agent are identified, e.g., using an algorithm that differentiates between cells and other material stained with the first agent and/or the second agent.

21. The method of any one of embodiments 18 to 20, wherein the first agent and the second agent are detected separately.

22. The method of embodiment 21 , wherein the number of total cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

23. The method of embodiment 22, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

24. The method of embodiment 23, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

25. The method of any one of embodiments 18 to 20, wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

26. The method of embodiment 25, wherein the number of total cells is the number of cells detected as stained with the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

27. The method of embodiment 26, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells. 28. The method of embodiment 27, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

29. The method of any one of embodiments 18 to 20, wherein the first agent and the second agent are detected simultaneously in the presence of FRET between the first agent and the second agent.

30. The method of embodiment 29, wherein the number of total cells is sum of the number of cells detected as stained with the first agent and the number of cells detected as stained with the second agent.

31 . The method of embodiment 29 or embodiment 30, wherein the number of dead cells is the number of cells detected as stained with the second agent.

32. The method of any one of embodiments 29 to 31 , wherein the number of live cells is the number of cells detected as stained with the first agent.

33. The method of any one of embodiments 29 to 32, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

34. The method of any one of embodiments 1 to 33, wherein the first agent is a cell permeable agent.

35. The method of any one of embodiments 1 to 34, wherein the first agent is a nucleic acid binding agent.

36. The method of any one of embodiments 1 to 35, wherein the first agent is a nucleic acid dye.

37. The method of any one of embodiments 1 to 36, wherein the first agent is acridine orange.

38. The method of any one of embodiments 1 to 37, wherein the second agent is a cell impermeable agent.

39. The method of any one of embodiments 1 to 36, wherein the second agent is a DNA binding agent. 40. The method of any one of embodiments 1 to 39, wherein the second agent is a nucleic acid dye.

41 . The method of any one of embodiments 1 to 40, wherein the second agent is a DNA intercalating agent.

42. The method of any one of embodiments 1 to 41 , wherein the first agent and second agent emit fluorescence at different peak wavelengths when excited by light.

43. The method of any one of embodiments 1 to 42, wherein the second agent is propidium iodide.

44. The method of any one of embodiments 1 to 33, wherein the first agent is acridine orange and the second agent is propidium iodide.

45. The method of any one of embodiments 1 to 44, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging live cells.

46. The method of any one of embodiments 1 to 45, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells in three dimensions.

47. The method of any one of embodiments 1 to 46, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells at different planes of focus and/or generating a z-stack.

48. The method of any one of embodiments 1 to 47, wherein quantification of total and/or dead cells is performed on one or more images of the portion of the sample.

49. The method of any one of embodiments 1 to 48, wherein quantification of total and/or dead cells is performed on one or more images or z-stack of cells in the portion of the sample (e.g., one or more images detecting the first agent and/or one or more images detecting the second agent).

50. The method of any one of embodiments 1 to 49, wherein quantification of total and/or dead cells is performed by an instrument capable of counting cells. 51 . A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

52. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample;

(c) detecting cells stained with the second agent in the same portion of the sample;

(d) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample based on the staining with the first agent; and

(e) quantifying dead cells in the same portion of the sample based on the staining with the second agent; thereby determining cell viability in the population of cells comprising cell aggregates.

53. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) quantifying living cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

54. The method of any one of embodiments 51 to 53, wherein cells that are stained by the first agent and cells that are stained by the second agent are identified, e.g., using an algorithm that differentiates between cells and other material stained with the first agent and/or the second agent.

55. The method of any one of embodiments 51 to 54, wherein the first agent and the second agent are detected separately. 56. The method of any one of embodiments 51 to 55, wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

57. The method of any one of embodiments 51 to 56, wherein the number of living cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

58. The method of embodiment 57, wherein the number of total cells is obtained by adding the number of dead cells to the number of living cells.

59. The method of embodiment 58, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

60. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

61 . A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

62. The method of embodiment 60 or embodiment 61 , wherein the first agent and the second agent are detected separately. 63. The method of embodiment 60 or embodiment 61 , wherein cells that are stained by the first agent and cells that are stained by the second agent are identified, e.g., using an algorithm that differentiates between cells and other material stained with the first agent and/or the second agent.

64. The method of embodiment 60 or embodiment 61 , wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

65. The method of any one of embodiments 60 to 64, wherein the number of living cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

66. The method of embodiment 65, wherein the number of total cells is obtained by adding the number of dead cells to the number of total cells.

67. The method of embodiment 66, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

68. The method of any one of embodiments 51 to 67, wherein the first agent is a cell permeable agent.

69. The method of any one of embodiments 51 to 68, wherein the first agent is cleaved by one or more cytoplasmic enzymes after entering the cytoplasm of a living cell.

70. The method of embodiment 69, wherein the one or more cytoplasmic enzymes is an esterase.

71 . The method of any one of embodiments 51 to 70, wherein the first agent is Calcein AM.

72. The method of any one of embodiments 51 to 71 , wherein the second agent is a cell impermeable agent.

73. The method of any one of embodiments 51 to 72, wherein the second agent is a DNA binding agent. 74. The method of any one of embodiments 51 to 73, wherein the second agent is a nucleic acid dye.

75. The method of any one of embodiments 51 to 74, wherein the second agent is a DNA intercalating agent.

76. The method of any one of embodiments 51 to 75, wherein the first agent and second agent emit fluorescence at different peak wavelengths when excited by light.

77. The method of any one of embodiments 51 to 76, wherein the second agent is Incucyte™ Cytotox Red.

78. The method of any one of embodiments 51 to 67, wherein the first agent is Calcein AM and the second agent is Incucyte™ Cytotox Red.

79. The method of any one of embodiments 51 to 78, wherein quantification of living and/or dead cells in the portion of the sample is performed by an instrument capable of imaging live cells.

80. The method of any one of embodiments 51 to 79, wherein quantification of living and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells in three dimensions.

81 . The method of any one of embodiments 51 to 80, wherein quantification of living and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells at different planes of focus and/or generating a z-stack.

82. The method of any one of embodiments 51 to 81 wherein quantification of living and/or dead cells is performed on one or more images of the portion of the sample.

83. The method of any one of embodiments 51 to 82, wherein quantification of living and/or dead cells is performed on one or more images or z-stack of cells in the portion of the sample (e.g., one or more images detecting the first agent and/or one or more images detecting the second agent).

84. The method of any one of embodiments 51 to 83, wherein quantification of living and/or dead cells is performed by an instrument capable of counting cells. 85. The method of any one of embodiments 1 to 84, which further comprises imaging the portion of the sample after step (a).

86. The method of any one of embodiments 1 to 85, wherein quantification of total and/or live cells comprises detecting cells that exhibit fluorescence signal level at or above a minimum intensity threshold of the first agent in the portion of the sample.

87. The method of any one of embodiments 1 to 86, wherein quantification of dead cells comprises detecting cells that exhibit fluorescence signal level at or above a minimum intensity threshold for the second agent in the portion of the sample.

88. The method of any one of embodiments 1 to 87, wherein the portion of the sample is a fraction of the sample, e.g., a field of view within the sample.

89. The method of any one of embodiments 1 to 87, wherein the portion of the sample is the entire sample.

90. The method of any one of embodiments 1 to 89, further comprising repeating steps (a)-(c) on (i) one or more (e.g., two) additional samples of the population of cells comprising aggregates (e.g., PRP cell aggregates) and/or (ii) one or more additional portions of the sample.

91 . The method of any one of embodiments 1 to 90, which is performed in a multi-well plate.

92. The method of embodiment 91 , which further comprises, prior to step (a), transferring a sample from the population into the multi-well plate.

93. The method of any one of embodiments 1 to 92, wherein the sample is imaged on a matrix-coated surface, e.g., a Matrigel™ matrix-coated surface.

94. The method of any one of embodiments 91 to 93, wherein the cells are plated at a seeding density cell seeding density of 25,000 cells/well to 100,000 cells/well (+/-10% or +/- 5%) of a 96-well plate, or equivalent thereof in a container of a different size.

95. The method of any one of embodiments 1 to 94, wherein the concentration of cells in the sample is the same as the concentration of cells in the population. 96. The method of any one of embodiments 1 to 94, wherein the concentration of cells in the sample is more dilute than the concentration of cells in the population.

97. The method of any one of embodiments 1 to 96, further comprising preparing the sample.

98. The method of embodiment 97, wherein preparing the sample comprises diluting a portion of the population.

99. The method of embodiment 97 or embodiment 98, wherein preparing the sample does not comprise dissociating cells or lysing cells.

100. The method of any one of embodiments 97 to 99, wherein the sample is diluted 1 AOfold to 1 :40-fold.

101. The method of any one of embodiments 97 to 100, wherein preparing the sample comprises thawing the population or a portion thereof.

102. The method of any one of embodiments 1 to 101 , wherein the sample comprises aggregates having an average and/or median diameter of 25 m to 300 pm.

103. The method of any one of embodiments 1 to 101 , wherein the sample comprises aggregates having an average and/or median diameter of 50 pm to 250 pm.

104. The method of any one of embodiments 1 to 101 , wherein the sample comprises aggregates having an average and/or median diameter of 100 pm to 200 pm.

105. The method of any one of embodiments 1 to 104, wherein the sample does not comprise organoids.

106. The method of any one of embodiments 1 to 105, wherein the sample is not fixed.

107. The method of any one of embodiments 1 to 106, wherein the population comprises mammalian cells.

108. The method of any one of embodiments 1 to 107, wherein the population comprises human cells. 109. The method of any one of embodiments 1 to 108, wherein the population comprises stem cells or cells differentiated therefrom.

110. The method of any one of embodiments 1 to 109, wherein the stem cells are pluripotent stem cells.

111. The method of embodiment 110, wherein the pluripotent stem cells are induced pluripotent stem cells.

112. The method of any one of embodiments 1 to 111 , wherein the population comprises cells of endoderm lineage (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof).

113. The method of any one of embodiments 1 to 111 , wherein the population comprises cells of ectoderm lineage (e.g., skin, neuronal, or pigment cells, or progenitors thereof).

114. The method of any one of embodiments 1 to 111 , wherein the population comprises cells of mesoderm lineage (e.g., cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or progenitors or precursors thereof).

115. The method of any one of embodiments 1 to 111 , wherein the population comprises neural lineage cells.

116. The method of any one of embodiments 1 to 111 , wherein the population comprises cells of the ocular system (e.g., cells of the retina, or cells of cornea).

117. The method of any one of embodiments 1 to 111 , wherein the population comprises immune cells.

118. The method of any one of embodiments 1 to 111 , wherein the population comprises enteric progenitor cells or enteric cells.

119. The method of any one of embodiments 1 to 111 , wherein the population comprises metabolic system cells, optionally wherein the metabolic system cells comprise hepatocytes, cholangiocytes, or pancreatic beta cells.

120. The method of any of embodiments 1 to 119, wherein the cell population and/or the aggregate is homo-cellular. 121. The method of any of embodiments 1 to 119, wherein the cell population and/or the aggregate is hetero-cellular.

122. The method of any of the embodiments 1 to 111 , wherein the cell population comprises PRP cells and/or aggregates comprising PRP cells.

123. The method of embodiment 122, wherein the cell population comprises aggregates having an average and/or median diameter of less than 125 pm.

124. The method of embodiment 122 or embodiment 123, wherein the cell population comprises aggregates having an average and/or median diameter of 50 pm to 100 pm.

125. The method of any one of embodiments 122 to 124, wherein the aggregates in which PRP cells are present do not comprise organoids.

126. The method of any one of embodiments 122 to 125, wherein the sample is not fixed.

127. The method of any one of embodiments 122 to 126, wherein the PRP cells are mammalian cells.

128. The method of any one of embodiments 122 to 127, wherein the PRP cells are human cells.

129. The method of any one of embodiments 122 to 128, wherein the PRP cells are differentiated from stem cells.

130. The method of embodiment 129, wherein the stem cells are pluripotent stem cells.

131. The method of embodiment 130, wherein the pluripotent stem cells are induced pluripotent stem cells.

132. The method of embodiments 122 to 131 , wherein the cell population and/or the aggregate is homo-cellular.

133. The method of embodiments 122 to 132, wherein the cell population and/or the aggregate is hetero-cellular. 134. The method of embodiment 133, wherein at least 80% or at least 90% of the cells in the cell population and/or the aggregate are PRP cells.

135. The method of embodiment 134, wherein the first agent is acridine orange and the second agent is propidium iodide.

136. The method of embodiment 134, wherein the first agent is Calcein AM and the second agent is Incucyte™ Cytotox Red

137. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device;

(ii) optionally, identify cells based on staining with the first agent and/or second agent; and

(iii) quantify the total number of cells or living cells, and quantify dead cells in at least a portion of the sample from the one or more images.

138. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device; 4

(ii) optionally, identify cells based on staining with the first agent and/or second agent; and

(iii) quantify the total number of cells and dead cells in at least a portion of the sample from the one or more images.

139. A system comprising: (a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device; and

(ii) detect cells stained with the first agent and cells stained with the second agent in at least a portion of the sample from the one or more images.

140. The system of any one of embodiments 135 to 139, wherein the first and/or second agent are first and/or second agents as described in any one of embodiments 34 to 43.

141. The system of any one of embodiments 137 to 140, wherein the system comprises an instrument described in any one of embodiments 44 to 50.

142. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device;

(ii) optionally, identify cells based on staining with the first agent and/or second agent; and

(iii) quantify the total number of cells or living cells, and quantify dead cells in at least a portion of the sample from the one or more images.

143. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device; (ii) optionally, identify cells based on staining with the first agent and/or second agent; and

(iii) quantify living cells and dead cells in at least a portion of the sample from the one or more images.

144. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device; and

(ii) detect cells stained with the first agent and cells stained with the second agent in at least a portion of the sample from the one or more images.

145. The system of embodiment 143 or embodiment 144, wherein the first and/or second agent are first and/or second agents as described in any one of embodiments 68 to 77.

146. The system of any one of embodiments 143 to 145, wherein the system comprises an instrument described in any one of embodiments 78 to 84.

147. The system of any one of embodiments 137 to 146, wherein the population of cells is a population of cells as described in any one of embodiments 107 to 134.

148. The method of any one of embodiments 1 to 50 and 85 to 134 when depending directly or indirectly from embodiment 1 , 2, 3, 18, or 19, wherein steps (b) and (c) are performed with the system of any one of embodiments 137 to 141.

149. The method of any one of embodiments 51 to 84 and 85 to 134 when depending directly or indirectly from embodiment 51 , 52, 53, 60, or 61 , wherein steps (b) and (c) are performed with the system of any one of embodiments 142 to 146.

150. A method of determining cell viability in a population of cells comprising cell aggregates with the system of any one of embodiments 137 to 147, comprising imaging the sample with the imaging device and processing one or more images with the processor to (a) quantify the total number of cells or quantify living cells in at least a portion of the sample; and

(b) quantify dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

151. A method of determining cell viability in a population of cells comprising cell aggregates with the system of any one of embodiments 135 to 147, comprising imaging the sample with the imaging device and processing one or more images with the processor to:

(a) detect cells stained with the first agent in at least a portion of the sample; and

(b) detect cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

152. A method of determining cell viability in a population of cells comprising cell aggregates with the system of any one of embodiments 135 to 147, comprising imaging the sample with the imaging device and processing one or more images with the processor to:

(a) detect cells stained with the first agent in at least a portion of the sample;

(b) detect cells stained with the second agent in the same portion of the sample;

(c) quantify the total number of cells or quantify living cells in at least a portion of the sample based on the staining with the first agent; and

(d) quantify dead cells in the same portion of the sample based on the staining with the second agent; thereby determining cell viability in the population of cells comprising cell aggregates.

153. The method of any one of embodiments 1 to 134 and 148 to 152, further comprising combining the population of cells or a portion thereof with a cell culture medium to obtain a cell population having a target concentration of viable cells.

154. The method of embodiment 153, further comprising transferring the cell population having a target concentration of viable cells or a portion thereof to a cell culture container.

155. The method of any one of embodiments 1 to 134 and 148 to 152, further comprising adjusting the concentration of the population of cells or a portion thereof to a target concentration of viable cells.

156. The method of embodiment 155, wherein adjusting the concentration of the population of cells or a portion thereof to a target concentration of viable cells comprises diluting the population of cells or a portion thereof, e.g., with a cell culture medium, a storage medium, or a pharmaceutical excipient, carrier and/or diluent.

157. The method of embodiment 155, wherein adjusting the concentration of the population of cells or a portion thereof to a target concentration of viable cells comprises concentrating the population of cells or a portion thereof, e.g., by centrifugation or filtration.

158. The method of embodiment 155, wherein adjusting the concentration of the population of cells or a portion thereof to a target concentration of viable cells comprises concentrating the population of cells or a portion thereof followed by diluting the population of cells or a portion thereof, e.g., with a cell culture medium, a storage solution, or a pharmaceutical excipient, carrier and/or diluent.

159. The method of embodiment 157 or embodiment 158, further comprising washing the concentrated population of cells or a portion thereof one or more times.

Group II Embodiments

1 . A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) quantifying the total number of cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

2. The method of embodiment 1 , wherein the first agent and the second agent are detected separately.

3. The method of embodiment 2, wherein the number of total cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

4. The method of embodiment 3, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells. 5. The method of embodiment 4, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

6. The method of embodiment 1 , wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

7. The method of embodiment 6, wherein the number of total cells is the number of cells detected as stained with the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

8. The method of embodiment 7, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

9. The method of embodiment 8, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

10. The method of embodiment 1 , wherein the first agent and the second agent are detected simultaneously in the presence of FRET between the first agent and the second agent.

11 . The method of embodiment 10, wherein the number of total cells is sum of the number of cells detected as stained with the first agent and the number of cells detected as stained with the second agent.

12. The method of embodiment 10 or embodiment 11 , wherein the number of dead cells is the number of cells detected as stained with the second agent.

13. The method of any one of embodiments 10 to 12, wherein the number of live cells is the number of cells detected as stained with the first agent.

14. The method of any one of embodiments 10 to 13, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

15. A method of determining cell viability in a population of cells comprising aggregates in which photoreceptor precursor (PRP) cells are present, the method comprising: (a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising PRP cell aggregates.

16. The method of embodiment 15, wherein the first agent and the second agent are detected separately.

17. The method of embodiment 16, wherein the number of total cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

18. The method of embodiment 17, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

19. The method of embodiment 18, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

20. The method of embodiment 15, wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

21 . The method of embodiment 20, wherein the number of total cells is the number of cells detected as stained with the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

22. The method of embodiment 21 , wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

23. The method of embodiment 22, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells. 24. The method of embodiment 15, wherein the first agent and the second agent are detected simultaneously in the presence of FRET between the first agent and the second agent.

25. The method of embodiment 24, wherein the number of total cells is sum of the number of cells detected as stained with the first agent and the number of cells detected as stained with the second agent.

26. The method of embodiment 24 or embodiment 25, wherein the number of dead cells is the number of cells detected as stained with the second agent.

27. The method of any one of embodiments 24 to 26, wherein the number of live cells is the number of cells detected as stained with the first agent.

28. The method of any one of embodiments 24 to 27, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

29. The method of any one of embodiments 1 to 28, wherein the first agent is a cell permeable agent.

30. The method of any one of embodiments 1 to 29, wherein the first agent is a nucleic acid binding agent.

31 . The method of any one of embodiments 1 to 30, wherein the first agent is a nucleic acid dye.

32. The method of any one of embodiments 1 to 31 , wherein the first agent is acridine orange.

33. The method of any one of embodiments 1 to 32, wherein the second agent is a cell impermeable agent.

34. The method of any one of embodiments 1 to 33, wherein the second agent is a DNA binding agent.

35. The method of any one of embodiments 1 to 34, wherein the second agent is a nucleic acid dye. 36. The method of any one of embodiments 1 to 35, wherein the second agent is a DNA intercalating agent.

37. The method of any one of embodiments 1 to 36, wherein the first agent and second agent emit fluorescence at different peak wavelengths when excited by light.

38. The method of any one of embodiments 1 to 37, wherein the second agent is propidium iodide.

39. The method of any one of embodiments 1 to 38, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging live cells.

40. The method of any one of embodiments 1 to 39, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells in three dimensions.

41. The method of any one of embodiments 1 to 40, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells at different planes of focus and/or generating a z-stack.

42. The method of any one of embodiments 1 to 41 wherein quantification of total and/or dead cells is performed on one or more images of the portion of the sample.

43. The method of any one of embodiments 1 to 42, wherein quantification of total and/or dead cells on one or more images or z-stack of cells in the portion of the sample (e.g., one or more images detecting the first agent and/or one or more images detecting the second agent).

44. The method of any one of embodiments 1 to 43, which further comprises imaging the portion of the sample after step (a).

45. The method of any one of embodiments 1 to 44, wherein the portion of the sample is a fraction of the sample, e.g., a field of view within the sample.

46. The method of any one of embodiments 1 to 44, wherein the portion of the sample is the entire sample. 47. The method of any one of embodiments 1 to 46, further comprising repeating steps (a)-(c) on (i) one or more (e.g., two) additional samples of the population of cells comprising PRP aggregates and/or (ii) one or more additional portions of the sample.

48. The method of any one of embodiments 1 to 47, which is performed in a multi-well plate.

49. The method of embodiment 48, which further comprises, prior to step (a), transferring a sample from the population into the multi-well plate.

50. The method of any one of embodiments 1 to 49, wherein the sample is imaged on a matrix-coated surface, e.g., a Matrigel™ matrix-coated surface.

51 . The method of any one of embodiments 48 to 50, wherein the cells are plated at a seeding density cell seeding density of 25,000 cells/well to 100,000 cells/well (+/-10% or +/- 5%) of a 96-well plate, or equivalent thereof in a container of a different size.

52. The method of any one of embodiments 1 to 51 , wherein the concentration of cells in the sample is the same as the concentration of cells in the population.

53. The method of any one of embodiments 1 to 51 , wherein the concentration of cells in the sample is more dilute than the concentration of cells in the population.

54. The method of any one of embodiments 1 to 53, further comprising preparing the sample.

55. The method of embodiment 54, wherein preparing the sample comprises diluting a portion of the population.

56. The method of embodiment 54 or embodiment 55, wherein preparing the sample does not comprise dissociating cells or lysing cells.

57. The method of any one of embodiments 54 to 56, wherein the sample is diluted 1 AOfold to 1 :40-fold.

58. The method of any one of embodiments 54 to 57, wherein preparing the sample comprises thawing the population or a portion thereof. 59. The method of any one of embodiments 1 to 58, wherein the sample comprises aggregates having an average and/or median diameter of 25 pm to 300 pm.

60. The method of any one of embodiments 1 to 58, wherein the sample comprises aggregates having an average and/or median diameter of 50 pm to 250 pm.

61 . The method of any one of embodiments 1 to 58, wherein the sample comprises aggregates having an average and/or median diameter of 100 pm to 200 pm.

62. The method of any one of embodiments 1 to 61 , wherein the sample does not comprise organoids.

63. The method of any one of embodiments 1 to 62, wherein the sample is not fixed.

64. The method of any one of embodiments 1 to 63, wherein the population comprises mammalian cells.

65. The method of any one of embodiments 1 to 63, wherein the population comprises human cells.

66. The method of any one of embodiments 1 to 65, wherein the population comprises stem cells or cells differentiated therefrom.

67. The method of any one of embodiments 1 to 65, wherein the stem cells are pluripotent stem cells.

68. The method of embodiment 67, wherein the pluripotent stem cells are induced pluripotent stem cells.

69. The method of any one of embodiments 1 to 68, wherein the population comprises cells of endoderm lineage (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof).

70. The method of any one of embodiments 1 to 68, wherein the population comprises cells of ectoderm lineage (e.g., skin, neuronal, or pigment cells, or progenitors thereof). 71 . The method of any one of embodiments 1 to 68, wherein the population comprises cells of mesoderm lineage (e.g., cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or progenitors or precursors thereof).

72. The method of any one of embodiments 1 to 68, wherein the population comprises neural lineage cells.

73. The method of any one of embodiments 1 to 68, wherein the population comprises cells of the ocular system (e.g., cells of the retina, or cells of cornea).

74. The method of any one of embodiments 1 to 68, wherein the population comprises immune cells.

75. The method of any one of embodiments 1 to 68, wherein the population comprises enteric progenitor cells or enteric cells.

76. The method of any one of embodiments 1 to 68, wherein the population comprises metabolic system cells, optionally wherein the metabolic system cells comprise hepatocytes, cholangiocytes, or pancreatic beta cells.

77. The method of any of embodiments 1 to 76, wherein the cell population and/or the aggregate is homo-cellular.

78. The method of any of embodiments 1 to 76, wherein the cell population and/or the aggregate is hetero-cellular.

Group III Embodiments

1 . A method of determining cell viability in a population of cells comprising aggregates in which photoreceptor precursor (PRP) cells are present, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) quantifying the total number of cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising PRP cell aggregates. 2. The method of embodiment 1 , wherein the first agent and the second agent are detected separately.

3. The method of embodiment 2, wherein the number of total cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

4. The method of embodiment 3, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

5. The method of embodiment 4, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

6. The method of embodiment 1 , wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

7. The method of embodiment 6, wherein the number of total cells is the number of cells detected as stained with the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

8. The method of embodiment 7, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

9. The method of embodiment 8, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

10. The method of embodiment 1 , wherein the first agent and the second agent are detected simultaneously in the presence of FRET between the first agent and the second agent.

11 . The method of embodiment 10, wherein the number of total cells is sum of the number of cells detected as stained with the first agent and the number of cells detected as stained with the second agent.

12. The method of embodiment 10 or embodiment 11 , wherein the number of dead cells is the number of cells detected as stained with the second agent. 13. The method of any one of embodiments 10 to 12, wherein the number of live cells is the number of cells detected as stained with the first agent.

14. The method of any one of embodiments 10 to 13, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

15. A method of determining cell viability in a population of cells comprising aggregates in which photoreceptor precursor (PRP) cells are present, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising PRP cell aggregates.

16. The method of embodiment 15, wherein the first agent and the second agent are detected separately.

17. The method of embodiment 16, wherein the number of total cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

18. The method of embodiment 17, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

19. The method of embodiment 18, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

20. The method of embodiment 15, wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

21 . The method of embodiment 20, wherein the number of total cells is the number of cells detected as stained with the first agent and the number of dead cells is the number of cells detected as stained with the second agent. 22. The method of embodiment 21 , wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

23. The method of embodiment 22, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

24. The method of embodiment 15, wherein the first agent and the second agent are detected simultaneously in the presence of FRET between the first agent and the second agent.

25. The method of embodiment 24, wherein the number of total cells is sum of the number of cells detected as stained with the first agent and the number of cells detected as stained with the second agent.

26. The method of embodiment 24 or embodiment 25, wherein the number of dead cells is the number of cells detected as stained with the second agent.

27. The method of any one of embodiments 24 to 26, wherein the number of live cells is the number of cells detected as stained with the first agent.

28. The method of any one of embodiments 24 to 27, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

29. The method of any one of embodiments 1 to 28, wherein the first agent is a cell permeable agent.

30. The method of any one of embodiments 1 to 29, wherein the first agent is a nucleic acid binding agent.

31 . The method of any one of embodiments 1 to 30, wherein the first agent is a nucleic acid dye.

32. The method of any one of embodiments 1 to 31 , wherein the first agent is acridine orange.

33. The method of any one of embodiments 1 to 32, wherein the second agent is a cell impermeable agent. 34. The method of any one of embodiments 1 to 33, wherein the second agent is a DNA binding agent.

35. The method of any one of embodiments 1 to 34, wherein the second agent is a nucleic acid dye.

36. The method of any one of embodiments 1 to 35, wherein the second agent is a DNA intercalating agent.

37. The method of any one of embodiments 1 to 36, wherein the first agent and second agent emit fluorescence at different peak wavelengths when excited by light.

38. The method of any one of embodiments 1 to 37, wherein the second agent is propidium iodide.

39. The method of any one of embodiments 1 to 38, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging live cells.

40. The method of any one of embodiments 1 to 39, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells in three dimensions.

41. The method of any one of embodiments 1 to 40, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging cells at different planes of focus and/or generating a z-stack.

42. The method of any one of embodiments 1 to 41 wherein quantification of total and/or dead cells is performed on one or more images of the portion of the sample.

43. The method of any one of embodiments 1 to 42, wherein quantification of total and/or dead cells on one or more images or z-stack of cells in the portion of the sample (e.g., one or more images detecting the first agent and/or one or more images detecting the second agent).

44. The method of any one of embodiments 1 to 43, which further comprises imaging the portion of the sample after step (a). 45. The method of any one of embodiments 1 to 44, wherein the portion of the sample is a fraction of the sample, e.g., a field of view within the sample.

46. The method of any one of embodiments 1 to 44, wherein the portion of the sample is the entire sample.

47. The method of any one of embodiments 1 to 46, further comprising repeating steps (a)-(c) on (i) one or more (e.g., two) additional samples of the population of cells comprising PRP aggregates and/or (ii) one or more additional portions of the sample.

48. The method of any one of embodiments 1 to 47, which is performed in a multi-well plate.

49. The method of embodiment 48, which further comprises, prior to step (a), transferring a sample from the population into the multi-well plate.

50. The method of any one of embodiments 1 to 49, wherein the sample is imaged on a matrix-coated surface, e.g., a Matrigel™ matrix-coated surface.

51 . The method of any one of embodiments 48 to 50, wherein the cells are plated at a seeding density cell seeding density of 25,000 cells/well to 100,000 cells/well (+/-10% or +/- 5%) of a 96-well plate, or equivalent thereof in a container of a different size.

52. The method of any one of embodiments 1 to 51 , wherein the concentration of cells in the sample is the same as the concentration of cells in the population.

53. The method of any one of embodiments 1 to 51 , wherein the concentration of cells in the sample is more dilute than the concentration of cells in the population.

54. The method of any one of embodiments 1 to 53, further comprising preparing the sample.

55. The method of embodiment 54, wherein preparing the sample comprises diluting a portion of the population.

56. The method of embodiment 54 or embodiment 55, wherein preparing the sample does not comprise dissociating cells or lysing cells. 57. The method of any one of embodiments 54 to 56, wherein the sample is diluted 1 AOfold to 1 :40-fold.

58. The method of any one of embodiments 54 to 57, wherein preparing the sample comprises thawing the population or a portion thereof.

59. The method of any one of embodiments 1 to 58, wherein the sample comprises aggregates having an average and/or median diameter of less than 125 m.

60. The method of any one of embodiments 1 to 58, wherein the sample comprises aggregates having an average and/or median diameter of 50 pm to 100 pm.

61 . The method of any one of embodiments 1 to 60, wherein the aggregates in which PRP cells are present do not comprise organoids.

62. The method of any one of embodiments 1 to 61 , wherein the sample is not fixed.

63. The method of any one of embodiments 1 to 62, wherein the PRP cells are mammalian cells.

64. The method of any one of embodiments 1 to 62, wherein the PRP cells are human cells.

65. The method of any one of embodiments 1 to 64, wherein the PRP cells are differentiated from stem cells.

66. The method of embodiment 65, wherein the stem cells are pluripotent stem cells.

67. The method of embodiment 66, wherein the pluripotent stem cells are induced pluripotent stem cells.

68. The method of embodiments 1 to 67, wherein the cell population and/or the aggregate is homo-cellular.

69. The method of embodiments 1 to 67, wherein the cell population and/or the aggregate is hetero-cellular. 70. The method of embodiment 69, wherein at least 80% or at least 90% of the cells in the cell population and/or the aggregate are PRP cells.

7. EXAMPLES

7.1 . Example 1 : Image-Based Determination of Viability of a Cell Population Comprising Cell Aggregates

7.1.1. Methods

[0227] A population of pluripotent stem cells was thawed to room temperature. Cell suspensions were prepared by resuspending cell aggregates in balanced salt solution (BSS) with 0.1 % human serum albumin (HSA). Triplicate samples of 50 pL of cell suspension aliquots each, diluted 1 :100 in BSS+HSA were transferred into the wells of a 96-well plate (Corning). 50 pL of 2x AO+PI (AOPI) dye mixture (Nexcelom) in BSS+HSA was added to each well to obtain a final dilution of 1 :750 of AO+PI unless specified otherwise. After gentle mixing, the plates were placed in an IncuCyte® live cell imager (Sartorius) and single images at 10x magnification were acquired using the spheroid module, which allows visualization of cell aggregates at different focal planes along an axis perpendicular to the plate surface. AO staining was detected using the green channel (A_ex - 453-485 nm; A_em = 494-533 nm) and PI staining was detected using the orange channel (A_ex = 546-568 nm; A_em = 576-639 nm) of the IncuCyte® live cell imager. Each image was analyzed using the IncuCyte® software by counting the numbers of AO-positive cells (AO⁺ cells) and Pl-positive cells (Pl⁺ cells) per image. Viability was calculated as a percentage of cells stained by AO cells but not PI (AO⁺PI‘ cells) per total number of cells (AO⁺ cells or sum of AO⁺PI‘ and AO⁺PI⁺ cells).

7.1.2. Results

[0228] The results presented in FIG. 1 show that percent viability in a PSC population was consistent across different cell compositions.

7.2. Example 2: Cell Number and Viability Assessment of Cell Populations Comprising Cell Aggregates

7.2.1. Methods

[0229] Cell viability determination was performed using the live imaging protocol as described in Section 7.1.1. using four populations of induced pluripotent stem cells (iPSCs) comprising cell aggregates with an average diameter of 100-200 pM. Total cell counts were extrapolated from the number of cells in the imaged portion of the surface area of the well corrected for the total surface area of the well.

7.2.2. Results

[0230] Percent viability values were consistent across the four iPSC populations and within each iPSC population (FIG. 2A). [0231] The average values for the total number of cells per well were estimated to be between 12,000 and 18,000 cells/well for the four iPSC populations with comparable variations within each population (FIG. 2B), suggesting homogenous plating of samples. Taken together, these results suggest that percent viability does not seem to be dependent on the number of total cells assessed, at least for the ranges shown in FIG. 2B.

7.3. Example 3: Image-Based Detection of PRP Cells in Aggregates

7.3.1. Methods

[0232] Cell suspensions were prepared by resuspending a cell population comprising photoreceptor precursor cell aggregates in balanced salt solution (BSS) with 0.1% human serum albumin (HSA). Triplicate samples of 50 L of cell suspension aliquots each, diluted 1 :100 in BSS+HSA were transferred into the wells of a 96-well plate (Corning). 50 pL of 2x AO+PI (ACPI) dye mixture (Nexcelom) in BSS+HSA was added to each well to obtain a final dilution of 1 :750 of AO+PI unless specified otherwise. After gentle mixing, the plates were placed in an IncuCyte® live cell imager (Sartorius) and single images at 10x magnification were acquired using the spheroid module, which allows visualization of cell aggregates at different focal planes along an axis perpendicular to the plate surface. AO staining was detected using the green channel (A_ex = 453-485 nm; A_em = 494-533 nm) and PI staining was detected using the orange channel ( _ex = 546-568 nm; A_em = 576-639 nm) of the IncuCyte® live cell imager. Each image was analyzed using the IncuCyte® software by counting the numbers of AO-positive cells (AO⁺ cells) and Pl-positive cells (Pl⁺ cells) per image. Viability was calculated as a percentage of cells stained by AO cells but not PI (AO⁺PI' cells) per total number of cells (AO⁺ cells or sum of AO⁺PI- and AO⁺PI⁺ cells).

7.3.2. Results

[0233] Six samples were imaged. Exemplary images used for analysis are shown with and without analysis mask overlays in FIGS. 3 and 4. The number of total cells (AO⁺ cells) and dead cells (Pl⁺ cells) cells are shown in FIGS. 5A and 5B, respectively, and percent viability is shown in FIG. 5C.

7.4. Example 4: Assessment of Variability of Cell Viability

7.4.1. Methods

[0234] Cell viability determination was performed using the protocol as described in Section

7.1.1. using compositions diluted 1 :40 in BSS with HSA to prepare samples. Image analysis was carried out with samples thawed on different days.

7.4.2. Results

[0235] Percent viability values obtained with live imaging of a portion of each sample of two or three samples for each tested population comprising photoreceptor progenitor cell aggregates varied minimally between the different runs of each sample (FIG. 6A). The variability between individual samples within each PRP cell population was also low for each sample (FIG. 6B).

7.5. Example 5: Effect of Cell Density and Coating Matrix on Image-Based Viability Assessment in Cell Samples Comprising Aggregates

7.5.1. Methods

[0236] The effects of cell seeding density, coating matrix, AOPI dye dilution ratio, and sample dilution ratio on the cell numbers obtained with the counting protocol described in Section 7.3.1 were evaluated. The effect of cell seeding density was assessed using samples of a cell population with three different seeding densities per well in 96-well plates: 25,000 cells/well, 50,000 cells/well, and 100,000 cells/well. The effect of composition dilution was assessed using cell suspension:medium dilution ranges between 1 :10 to 1 :80. The effect of the AOPI dye dilution ratio was tested in samples with a seeding density of 1 :50,000 cells/well using dye:medium ratios between 1 :500-1 :1000.

7.5.2. Results

[0237] Cell viability detected in a cell population comprising photoreceptor progenitor cell aggregates was not affected by cell seeding density for the cell densities tested (FIG. 7A). Further, the coating of the well surface with a coating matrix did not result in any changes in viability (FIG. 7A). Total number of cells counted increased with each increment of cell seeding density for both live cells and dead cells (FIG. 7B). The numbers of cells per well in samples comprising aggregates generally correlated with cell seeding density. (FIG 7C).

[0238] All three AOPI dilution ratios enabled counting of cells in aggregated cell samples (FIG. 8). The cell count numbers were similar for assessments using the AOPI mixtures that were diluted 1 :500 and 1 :750 in BSS+HSA. Percent cell viability determined with samples comprising cell aggregates was not affected by sample dilution ratios evaluated (FIG. 9).

7.6. Example 6: Evaluation of Cell Staining with Two Different Dye Combinations

7.6.1. Methods

[0239] Two different dye combinations were evaluated using cells that were treated with triton-X, which is a treatment that results in cell death, in parallel to untreated control cells. The first combination included AO and PI dyes (AO+PI) described in Sections 7.1 to 7.5. The second combination included Calcein, which is a membrane-permeable live-cell dye, and Incucyte® Cytotox Red, which is a nucleic acid binding dye that stains dead or dying cells.

[0240] Control cell suspensions were prepared by resuspending a cell population comprising photoreceptor precursor cell aggregates in balanced salt solution (BSS) with 0.1% human serum albumin (HSA). Triton-X-treated cell suspensions were prepared by adding triton-Xto cell suspensions to achieve a final concentration of 1% triton-X. Cells were stained with AO+PI and Calcein+Cytotox Red dyes in singleplex and duplex. . Images were acquired after 30 minutes of incubation in the IncuCyte® SX5 live cell imager. AO and Calcein staining was detected using the green channel (A_ex = 453-485 nm; A_em - 494-533 nm) whereas PI and Incucyte® Cytotox Red staining was detected using the orange channel (A_ex = 546-568 nm; A_em = 576-639 nm). Each image was analyzed using the IncuCyte® software by counting the numbers of AO-positive cells (AO⁺ cells) and Pl-positive cells (Pl⁺ cells) per image for the AO+PI stained cells and by counting the numbers of Calcein-positive cells (Calcein* cells) and Incucyte® Cytotox Red-positive cells (CytotoxRed* cells) per image for the Calcein + Incucyte® Cytotox Red stained cells.

7.6.2. Results

[0241] Both triton-X-treated and untreated control cells were stained with AO, confirming that this dye stains live cells as well as dead cells (FIG. 10A). In contrast, only cells in the untreated control condition were stained with Calcein but not those treated with triton-X (FIG. 10B), confirming that Calcein stains only live cells. Staining patterns were similar for PI and Incucyte® Cytotox Red indicating that the dyes are accurately staining dead cells with minimal non-specific binding. Both dyes were associated with increased signal intensity in cells treated with triton-X, further confirming that the dyes accurately stain dead cells, as dead cells have more condensed nuclei and stain brighter due to increased dye accumulation (FIGS. 10C and 10D). Calcein staining intensity, which was ~600-1000 green calibrated units (GCU), was higher than the staining intensity of AO, which was ~10-60 GCU.

[0242] In sum, the combination of Calcein and Incucyte® Cytotox Red resulted in more distinct staining of live cells and dead cells compared to the AO+PI combination.

7.7. Example 7: The Effect of Analysis Parameters on Assessment of Cell Viability

7.7.1. Methods

[0243] Preliminary analyses led to the observation that single cells in cell suspension preparations were associated with lower staining intensities with PI relative to cells in aggregates. To further investigate, two different image analysis parameters were evaluated: a first parameter with high minimum intensity thresholds (AO minimum intensity threshold = 5 and PI minimum intensity threshold = 7) that captures cells with high intensity staining, and a second parameter with lower minimum intensity thresholds (AO minimum intensity threshold = 4 and PI minimum intensity threshold = 2) that captures cells with lower staining intensities. The first parameter was predicted to predominantly capture cell viability of cells in aggregates and the second parameter to capture cell viability of single cells in addition to those in aggregates. Cell preparations and image capturing and processing using IncuCyte® SX5 live cell imager were performed as described in Section 7.6.1. Each image was analyzed using the IncuCyte® software by counting the numbers of AO-positive cells (AO⁺ cells) and Pl-positive cells (Pl⁺ cells) per image. Viability was calculated as a percentage of cells stained by AO cells but not PI (AO⁺PI cells) per total number of cells (AO⁺). An additional set of assessments were carried out with Calcein and Incucyte® Cytotox Red. This time, each image was analyzed using the IncuCyte® software by counting the numbers of Calcein-positive cells (Calcein⁺ cells) and Incucyte® Cytotox Red-positive cells (CytotoxRed⁺ cells) per image for the Calcein + Incucyte® Cytotox Red stained cells and viability was calculated as a percentage of cells stained by Calcein (Calcein⁺ cells) per total number of cells (Calcein* cells + CytotoxRed⁺ cells).

7.7.2. Results

[0244] The number of dead cells was higher in assessments using the second parameter as cells with low intensities were included in these analyses, relative to assessments using the first parameter (FIGS. 11A and 11B). Therefore, the use of the first parameter was associated with higher values of percent viability values relative to when the second parameter was used, even when they were used in parallel for the same cell samples (Table E1).

[0245] Low viability values obtained with low minimum intensity threshold analyses suggested that most single cells in the evaluated preparations were dead or dying, and they were not captured with high minimum intensity threshold analyses. To determine whether this was the case, an additional set of assessment was carried out with triton-X-treated cells. This analysis showed that there were far fewer objects determined as PI* with the high minimum intensity threshold analysis relative to the number of objects that were Pl⁺ with the low minimum intensity threshold analysis (FIG. 11 C). 8. CITATION OF REFERENCES

[0246] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

Claims

WHAT IS CLAIMED IS:

1 . A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

2. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample;

(c) detecting cells stained with the second agent in the same portion of the sample;

(d) quantifying (i) the total number of cells or (ii) living cells in at least a portion of the sample based on the staining with the first agent; and

(e) quantifying dead cells in the same portion of the sample based on the staining with the second agent; thereby determining cell viability in the population of cells comprising cell aggregates.

3. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) quantifying the total number of cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

4. The method of any one of claims 1 to 3, wherein cells that are stained by the first agent and cells that are stained by the second agent are identified, e.g., using an algorithm that differentiates between cells and other material stained with the first agent and/or the second agent.

5. The method of any one of claims 1 to 4, wherein the first agent and the second agent are detected separately.

6. The method of claim 5, wherein the number of total cells is the number of cells detected as stained by the first agent and the number of dead cells is the number of cells detected as stained with the second agent.

7. The method of claim 6, wherein the number of live cells is obtained by subtracting the number of dead cells from the number of total cells.

8. The method of claim 7, wherein cell viability is the percentage or ratio of the number of live cells to the number of total cells.

9. The method of any one of claims 1 to 4, wherein the first agent and the second agent are detected simultaneously in the absence of FRET between the first agent and the second agent.

10. The method of any one of claims 1 to 4, wherein the first agent and the second agent are detected simultaneously in the presence of FRET between the first agent and the second agent.

11. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

12. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

13. The method of any one of claims 1 to 12, wherein the first agent is a cell permeable agent.

14. The method of any one of claims 1 to 13, wherein the first agent is acridine orange.

15. The method of any one of claims 1 to 14, wherein the second agent is a cell impermeable agent.

16. The method of any one of claims 1 to 15, wherein the first agent and second agent emit fluorescence at different peak wavelengths when excited by light.

17. The method of any one of claims 1 to 16, wherein the second agent is propidium iodide.

18. The method of any one of claims 1 to 16, wherein the first agent is acridine orange and the second second agent is propidium iodide.

19. The method of any one of claims 1 to 18, wherein quantification of total and/or dead cells in the portion of the sample is performed by an instrument capable of imaging live cells.

20. A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) quantifying living cells in at least a portion of the sample; and

(c) quantifying dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

21 . A method of determining cell viability in a population of cells comprising cell aggregates, the method comprising:

(a) contacting a sample from the population with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) detecting cells stained with the first agent in at least a portion of the sample; and

(c) detecting cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

22. The method of claim 20 or claim 21 , wherein the first agent is a cell permeable agent.

23. The method of any one of claims 20 to 22, wherein the first agent is cleaved by one or more cytoplasmic enzymes after entering the cytoplasm of a living cell.

24. The method of any one of claims 20 to 23, wherein the second agent is a cell impermeable agent.

25. The method of any one of claims 20 to 24, wherein the first agent is Calcein AM and the second agent is Incucyte™ Cytotox Red.

26. The method of any one of claims 1 to 25, wherein (a) quantification of total and/or live cells comprises detecting cells that exhibit fluorescence signal level at or above a minimum intensity threshold of the first agent in the portion of the sample and/or (b) quantification of dead cells comprises detecting cells that exhibit fluorescence signal level at or above a minimum intensity threshold for the second agent in the portion of the sample.

27. The method of any one of claims 1 to 26, wherein the population comprises mammalian cells, e.g., human cells.

28. The method of any one of claims 1 to 27, wherein the population comprises stem cells or cells differentiated therefrom.

29. The method of any one of claims 1 to 28, wherein the stem cells are pluripotent stem cells, e.g., induced pluripotent stem cells.

30. The method of any of the claims 1 to 29, wherein the cell population comprises PRP cells and/or aggregates comprising PRP cells.

31. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent and a second agent that together are capable of differentially staining living and dead cells;

(b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device;

(ii) optionally, identify cells based on staining with the first agent and/or second agent; and

(iii) quantify the total number of cells or living cells, and quantify dead cells in at least a portion of the sample from the one or more images.

32. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining total cells and a second agent capable of staining dead cells; (b) an imaging device capable of imaging cells from the sample and/or detecting the first agent and the second agent; and

(c) a processor configured to:

(i) receive one or more images from the imaging device;

(ii) optionally, identify cells based on staining with the first agent and/or second agent; and

(iii) quantify the total number of cells and dead cells in at least a portion of the sample from the one or more images.

33. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining total cells and a second agent capable of staining dead cells;

(b) an imaging device capable of imaging cells from the sample; and

(c) a processor configured to:

(i) receive one or more images from the imaging device; and

(ii) detect cells stained with the first agent and cells stained with the second agent in at least a portion of the sample from the one or more images.

34. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) an imaging device capable of imaging cells from the sample; and

(c) a processor configured to:

(i) receive one or more images from the imaging device;

(ii) optionally, identify cells based on staining with the first agent and/or second agent; and

(iii) quantify living cells and dead cells in at least a portion of the sample from the one or more images.

35. A system comprising:

(a) a sample from a population of cells comprising cell aggregates that has been contacted with a first agent capable of staining living cells and a second agent capable of staining dead cells;

(b) an imaging device capable of imaging cells from the sample; and

(c) a processor configured to:

(i) receive one or more images from the imaging device; and (ii) detect cells stained with the first agent and cells stained with the second agent in at least a portion of the sample from the one or more images.

36. A method of determining cell viability in a population of cells comprising cell aggregates with the system of any one of claims 31 to 35, comprising imaging the sample with the imaging device and processing one or more images with the processor to

(a) quantify the total number of cells or quantify living cells in at least a portion of the sample; and

(b) quantify dead cells in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

37. A method of determining cell viability in a population of cells comprising cell aggregates with the system of any one of claims 31 to 35, comprising imaging the sample with the imaging device and processing one or more images with the processor to:

(a) detect cells stained with the first agent in at least a portion of the sample; and

(b) detect cells stained with the second agent in the same portion of the sample; thereby determining cell viability in the population of cells comprising cell aggregates.

38. A method of determining cell viability in a population of cells comprising cell aggregates with the system of any one of claims 31 to 35, comprising imaging the sample with the imaging device and processing one or more images with the processor to:

(a) detect cells stained with the first agent in at least a portion of the sample;

(b) detect cells stained with the second agent in the same portion of the sample;

(c) quantify the total number of cells or quantify living cells in at least a portion of the sample based on the staining with the first agent; and

(d) quantify dead cells in the same portion of the sample based on the staining with the second agent; thereby determining cell viability in the population of cells comprising cell aggregates.

39. The method of any one of claims 1 to 29 and 36 to 38, further comprising adjusting the concentration of the population of cells or a portion thereof to a target concentration of viable cells.

40. The method of claim 39, wherein adjusting the concentration of the population of cells or a portion thereof to a target concentration of viable cells comprises (a) diluting the population of cells or a portion thereof, (b) concentrating the population of cells or a portion thereof, or (c) concentrating the population of cells or a portion thereof followed by diluting the population of cells or a portion thereof.