CN121358853A - Enzymes - Google Patents

Abstract

The present invention relates to methods and compositions for dissociating biological tissue with the aim of obtaining single cells for analysis. More specifically, the compositions of the present invention are enzyme compositions comprising proteinase K. The compositions of the invention are useful in methods for efficiently dissociating tissue at lower temperatures for use in high throughput single cell histology studies.

Description

Enzymes

Technical Field

The present invention relates to compositions comprising proteinase K and methods for tissue dissociation.

Background

Enzymes are useful tools for dissociating cells from solid tissue for various applications in research, industry, and medicine. REICHARD AND Astinghetal. Cytomet A2019 95 (2): 219-226 describe examples of enzymes that can be used for dissociation of solid tissue. Combinations of different enzymes (also known as enzyme "mixtures") may be used for tissue dissociation. However, the specific enzymes must be carefully selected, as not all enzymes are suitable when used alone or in combination. For example REICHARD AND Asosinghet al 2019 reports that trypsin is not suitable for preparing single cells due to the inherent risk of cleaving cell surface receptors.

Furthermore, due to the heat sensitive nature of many enzymes, tissue dissociation is typically performed at temperatures that result in maximum activity and efficiency of the enzyme. Because many enzymes have an optimal working temperature of about 37 ℃ (e.g., trypsin, trypLE ^TM, pronase, collagenase, liberase, and dispase, as discussed in Adamet al development.2017 144 (19): 3625-3632), methods for enzymatic tissue dissociation are routinely performed at this temperature.

There are a number of commercial products for dissociation of kidney tissue, such as the multi-tissue dissociation kit 2 of Miltenyi Biotec, which can be used in combination with a specific device, such as GENTLEMACSTM dissociator (Miltenyi Biotec multi-tissue dissociation kit 2 acquisition: 2023, 5 th day [ https:// static, miltenyibiotec, com/asset/150655405641/document_ phpdh7tnkl2it4bi11qp1l0o6 o)Content-disposition = inline ]; miltenyi Biotec obtained from mouse kidneys using the multi-tissue dissociation kit 2: 2023, 5 th day [ https:// www.miltenyibiotec.com/upload assets/IM0015569.PDF ]). The kidneys of mice can also be dissociated with more general tissue dissociation enzymes at 37 ℃, one such example being the protocol used in Adamet al 2017.

Recently, it has been found that dissociation of tissue at 37 ℃ results in artificial changes in cells, such as induction of stress response genes. Such variations are particularly problematic in high throughput single cell histology studies (genomics, proteomics, metabolomics). Thus, the method for dissociating tissue is desirably performed at a lower temperature. Adamet al 2017 describes one such method, which utilizes subtilisin a. Subtilisin A is a serine endopeptidase expressed in the soil bacterium Bacillus licheniformis (Bacillus licheniformis) isolated from Himalayan glacier, which is known to be effective at lower temperatures. O' FLANAGANET al genome biology 2019 20 (210) describes the use of subtilisin A as a protease in enzyme mixtures to reduce stress responses to kidney cells during dissociation.

In light of the foregoing, there remains a need for more enzymes that can efficiently dissociate tissues at lower temperatures for use in high throughput single cell histology studies.

Disclosure of Invention

In a first aspect, the invention provides a composition comprising proteinase K, a buffer, wherein the buffer comprises Hakks equilibrium salt solution (HBSS), DNase, and a source of Ca ²⁺.

In a second aspect, the invention provides the use of proteinase K for dissociating tissue at a temperature below 20 ℃.

In a third aspect, the invention provides an ex vivo method of dissociating tissue comprising a) contacting a tissue with a composition comprising proteinase K, b) incubating the tissue with the composition for a first period of time, and c) obtaining a cell suspension.

In a fourth aspect, the present invention provides a system configured to perform the method according to the third aspect of the present invention.

In a fifth aspect, the present invention provides a kit comprising a composition according to the first aspect of the invention and instructions for use.

The present invention relates to the unexpected finding that proteinase K can be used as a tool for efficient dissociation of tissue into single cells. Single cells show advantageous properties that make them suitable for use in high throughput single cell histology studies, in particular single cell RNA sequencing analysis such as single cell RNA-seq (scRNA-seq). Furthermore, it was found that tissue dissociation can be performed at lower temperatures (e.g., 4 ℃) to avoid artifacts associated with thermally induced stress reactions. Surprisingly, it has also been found that proteinase K can dissociate tissue at lower temperatures, at least as effectively as subtilisin A (an enzyme known to function at lower temperatures). This is particularly surprising since proteinase K is known to have an optimal temperature range of 20 to 65℃and maximum activity at 37 ℃ (BajorathBiochim Biophys acta 1988 954 (2): 176-182). Furthermore, it was found that the proteinase K-containing composition of the invention does not cleave certain cell surface markers compared to known enzyme compositions.

Proteinase K (EC 3.4.21.64), also known as peptidase K, endoprotease K and endopeptidase K, is a non-specific serine endopeptidase in family S8 (subfamily S8A). It is commonly used to break down keratins (free proteins that are not required in molecular biology) and nucleases (DNase and RNase). It is also used in prion studies (Petrotchenkoet al.molecular & Cellular proteomics 2012 11 (7)), endotoxin/Limulus Amebocyte Lysate (LAL) assays (Petschet al.analytical biochemistry 1998 259 (1): 42-47), protease footprinting (Hori and CareyJournal of Biological chemistry 1997 272 (2): 1180-1187A) and isolated nucleic acids (Hilzet al European Journal of Biochemistry 1975 56 (1): 103-108).

Proteinase K is stable over a pH range of about 4.0 to 12.5, most preferably pH 8.0.

Detailed Description

Terminology and definitions

Unless explicitly defined is provided, nomenclature used herein and the laboratory procedures and techniques described herein are those well known in the art. Standard chemical symbols are used interchangeably with the full name represented by such symbols. Thus, for example, the terms "hydrogen" and "H" are understood to have the same meaning. Standard techniques can be used to formulate and test compositions. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. As used herein, the use of the singular includes the plural unless specifically stated otherwise. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, "proteinase K" refers to the amino acid sequence of SEQ ID NO. 1 (UniProt reference number P06873 by day 5, month 16 of 2023), homologs in other species, or variants thereof. The amino acid residues of proteinase K used herein are numbered relative to SEQ ID NO. 1.

SEQ ID NO. 1 (Parengyodontium album (Tritirachium album), 384 amino acids, uniProt reference P06873 by day 5, month 16 of 2023):

MRLSVLLSLLPLALGAPAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA

As used herein, "or" means "and/or" unless indicated otherwise. Furthermore, the use of the term "include" and other forms, such as "include" and "include (included)" is not limiting.

As used herein, "about" means that a number referred to as "about" includes 1-5% of the number plus or minus the number. For example, "about" 100 degrees may mean 95-105 degrees or as little as 99-101 degrees, depending on the context. When appearing herein, a numerical range such as "1 to 20" means each integer in the given range, i.e., means only 1, only 2, only 3, etc., up to and including only 20.

As used herein, the term "composition" includes products, formulations and mixtures, as well as devices, apparatuses, components, kits, and the like. Similarly, the term "method" includes procedures, programs, steps, and the like.

As used throughout this disclosure, the words "may" and "may" are used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). In addition, the terms "comprising," having, "" involving (involving) "," containing, "" characterized by (CHARACTERISED BY) ", variants thereof (e.g.," comprising, "" having, "" involving (involves) "," containing (containing), etc.), and the like, as used herein (including the claims), should be inclusive and/or open-ended, should have the same meaning as the term "comprising" and variants thereof (e.g., "comprising" and "comprising"), and should not exclude additional unrecited elements or method steps, for example.

It should be noted that embodiments of the present disclosure may include one or more combinations of two or more of the features described herein. As used herein, "feature" and similar terms may include, for example, composition, ingredient, component, element, member, part, portion, system, method, configuration, parameter, property, and the like. Embodiments may include elsewhere in the disclosure, including any of the features, options, and/or possibilities set forth in other aspects or embodiments of the disclosure. It should also be noted that each of the foregoing, following, and/or other features described herein represent different embodiments of the present disclosure. The features may also be combined with and/or combined with one or more other features, with or without inclusion or progression of one or more other features therebetween, in any suitable combination and/or order, to form a unique embodiment, each of which is contemplated in the present disclosure. Such a combination of any two or more such features represents different embodiments of the present disclosure. Thus, the present disclosure is not limited to a particular combination of the exemplary embodiments described in detail herein, and disclosure of certain features with respect to particular embodiments of the present disclosure should not be construed as limiting application or inclusion of such features to the particular embodiments.

Furthermore, unless a feature is described as requiring a particular embodiment, features described in the various embodiments may be optional and may not be included in other embodiments of the disclosure. Furthermore, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein, unless the feature is described as requiring another feature in combination therewith. Likewise, any steps recited in any methods described herein and/or recited in the claims may be performed in any suitable order and are not necessarily limited to the order described and/or recited unless otherwise indicated (explicitly or implicitly). However, such steps may also be required to be performed in a specific order in certain embodiments of the present disclosure.

It will also be appreciated that where two or more values or ranges of values are disclosed or recited (e.g., less than, greater than, at least, and/or up to a certain value, and/or between two such values), any particular value or range of values falling within the disclosed values or ranges of values is specifically disclosed and contemplated herein as such. Thus, disclosure of exemplary measurements (e.g., length, width, thickness, etc.) of less than or equal to about 10 units or between 0 and 10 units includes, for example, specific disclosures of (i) 9 units, 5 units, 1 unit, or any other value between 0 and 10 units, including 0 units and/or 10 units, and/or (ii) measurements of any other value range between 9 units and 1 units, between 8 units and 2 units, between 6 units and 4 units, and/or between 0 and 10 units.

Embodiments of the invention

The following description of embodiments includes disclosure related to one or more embodiments of the present disclosure. Accordingly, some embodiments may include the features disclosed in the examples that follow without departing from the scope of the disclosure. In other words, features disclosed in the examples that follow may be included in and/or incorporated into any one or more of the embodiments disclosed herein.

The composition of the invention comprises proteinase K, which is an active peptidase that cleaves amino acids present in proteins holding cells together.

Proteinase K is commercially available from a number of manufacturers, such as Qiagen ^TM (including Blirt ^TM). Preferably, proteinase K in the composition of the invention is Blirt ^TM catalog RP107B by day 2023, month 5 and 17. It is also possible to use host cells for the production of recombinant proteinase K.

The host cell may be selected from Pichia species, hansenula species such as Hansenula polymorpha, saccharomyces species, schizosaccharomyces species, yarrowia species such as yarrowia lipolytica, kluyveromyces species and Aspergillus species. Preferably, pichia pastoris is used as host cell.

Preferably, proteinase K in the composition of the invention comprises the amino acid sequence of SEQ ID NO. 1. More preferably, proteinase K in the composition of the invention consists of the amino acid sequence of SEQ ID NO. 1.

The amino acid sequence of a variant of proteinase K may share at least about 85% sequence identity with SEQ ID NO. 1 and thus may share at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with SEQ ID NO. 1. Preferably, the sequence identity is at least 90% or at least 95%. More preferably, the sequence identity is 100%. Preferably, the variant of proteinase K substantially retains the enzymatic activity associated with the sequence of SEQ ID NO. 1.

The amino acid sequence of the variant proteinase K may be altered by substitution, addition or deletion of a suitable number of amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 residues in the sequence of SEQ ID NO. 1. By "substitution, addition or deletion" is meant to include a combination of substitution, addition and deletion. Preferably, the variant of proteinase K substantially retains the enzymatic activity associated with the sequence of SEQ ID NO. 1.

When the sequence is modified by substitution of a particular amino acid residue, the substitution may be a conservative amino acid substitution. The term "conservative amino acid substitution" as used herein refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acids with similar side chains tend to have similar properties, so conservative substitutions of amino acids important to the structure or function of the polypeptide may be expected to affect the polypeptide structure/function less than non-conservative amino acid substitutions at the same position.

Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, conservative amino acid substitutions may be considered as substitutions in which a particular amino acid residue is replaced with a different amino acid in the same family. However, substitution of an epitope residue may be equivalent to a non-conservative substitution in which one amino acid is replaced with another amino acid having a side chain belonging to a different family.

Proteinase K may be present in the compositions of the invention at a concentration of 1.+ -. 75% mg/mL, 1.+ -. 50% mg/mL, 1.+ -. 10% mg/mL, 1.+ -. 5% mg/mL or 1.+ -. 1% mg/mL. Proteinase K may be present in the compositions of the invention at a concentration of 10 mg/mL, 5 mg/mL or 2 mg/mL. Preferably, the concentration of proteinase K in the composition of the invention is 1.+ -. 1% mg/mL. When "±x%" is used herein, it means ± x% of the value.

Proteinase K may be present in the composition of the invention at a concentration of about 1 mg/mL. Preferably proteinase K is present in the composition of the invention at a concentration of 1 mg/mL.

Proteinase K may be present in the compositions of the invention at a concentration of 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, or 1.5 mg/mL.

Proteinase K may be present in the compositions of the invention at a concentration of 0.1 to 10mg/mL, 0.2 to 9mg/mL, 0.3 to 8mg/mL, 0.4 to 7mg/mL, 0.5 to 6mg/mL, 0.6 to 5mg/mL, 0.7 to 4mg/mL, 0.8 to 3mg/mL or 0.9 to 2 mg/mL. Preferably proteinase K is present in the composition of the invention in a concentration of 0.1 mg to 5 mg/mL.

The amount of proteinase K in the composition may be defined in terms of enzyme units. The enzyme unit definition can be used that proteinase K hydrolyzes urea denatured hemoglobin, producing a color equivalent of 1 μmol tyrosine per 1 minute at 37 ℃ and pH 7.5 (Folin & Ciocalteu method), 1U =1 mAnsonU. For example, when 20mg/mL (minimum) and 800U/mL (minimum) stock solutions are used, the final concentration is 1mg/mL and the activity in solution will be 40U/mL.

Proteinase K may be present in the compositions of the invention at a concentration of 40.+ -. 75% U/mL, 40.+ -. 50% U/mL, 40.+ -. 10% U/mL, 40.+ -. 5% U/mL or 40.+ -. 1% U/mL. Proteinase K may be present in the compositions of the invention at a concentration of 400U/mL, 200U/mL or 80U/mL. Preferably proteinase K is present in the composition of the invention at a concentration of 40.+ -. 1% U/mL.

Proteinase K may be present in the compositions of the present invention at a concentration of about 40U/mL. Proteinase K may be present in the compositions of the invention at a concentration of 40U/mL.

Proteinase K may be present in the compositions of the invention at concentrations of 4 to 400U/mL, 8 to 360U/mL, 12 to 320U/mL, 16 to 280U/mL, 20 to 240U/mL, 24 to 200U/mL, 28 to 160U/mL, 32 to 120U/mL, 36 to 80U/mL, 37 to 70U/mL, 38 to 60U/mL and 39 to 50U/mL.

The composition of the invention further comprises DNase. DNase, in particular dsDNase, has been described as useful in reducing cell aggregation/clumping caused by cell-free DNA, resulting in good quality samples for single cell RNA sequencing (REICHARD AND Asosinghet al 2019).

Although proteinase K is known to decompose nucleases, such as DNase, the presence of Ca2+ ions in solution is known to have a protective effect on DNase (Tullis and RubinAnalytical biochemistry. 1980 107 (1): 260-264).

DNase is commercially available from a number of manufacturers, such as Qiagen ^TM (including Blirt ^TM). DNase may be present in the compositions of the invention at a concentration of 4.+ -. 75% U/mL, 4.+ -. 50% U/mL, 4.+ -. 10% U/mL, 4.+ -. 5% U/mL or 4.+ -. 1% U/mL. DNase may be present in the compositions of the invention at a concentration of 40U/mL, 20U/mL or 8U/mL. Preferably, DNase is present in the compositions of the present invention at a concentration of 4.+ -. 1% U/mL.

DNase may be present in the compositions of the invention at a concentration of about 4U/mL. DNase may be present in the compositions of the invention at a concentration of 4U/mL.

DNase may be present in the compositions of the present invention at concentrations of 0.4 to 40U/mL, 0.8 to 36U/mL, 1.2 to 32U/mL, 1.6 to 28U/mL, 2 to 24U/mL, 2.4 to 20U/mL, 2.8 to 16U/mL, 3.2 to 12U/mL, 3.6 to 8U/mL, 3.7 to 7U/mL, 3.8 to 6U/mL and 3.9 to 5U/mL.

For DNase one unit may be defined as an increase in absorbance at 260 nm over 30 minutes at 37℃and pH 8.0 with herring sperm DNA as substrate of 1.0.

Preferably, DNase is double stranded DNase (dsDNase). Preferably dsDNase in the composition of the invention is Blirt ^TM catalog number EN33 by day 5, month 17 of 2023.

The DNase may be a member of the DNase I family selected from DNase I, DNase1L1, DNase1L 2 and DNase1L 3. Preferably, the DNase is DNase I.

The DNase may be a member of the DNase II family selected from DNase II α and DNase II β.

The composition of the invention comprises a source of Ca ²⁺, which may be calcium chloride. The purpose of the additional calcium ion is to protect DNase and its activity from degradation by proteinase K (tunelis and Rubin 1980). The source of Ca ²⁺ may comprise calcium chloride. The calcium chloride may be solid or aqueous calcium chloride. Preferably, the calcium chloride is aqueous calcium chloride.

Calcium chloride may be present in the compositions of the present invention at a concentration of 10.+ -. 75% mM, 10.+ -. 50% mM, 10.+ -. 10% mM, 10.+ -. 15% mM, 10.+ -. 5% mM or 10.+ -. 1% mM. Calcium chloride may be present in the compositions of the present invention at a concentration of 100 mM, 50mM, or 20 mM. Preferably, calcium chloride is present in the composition of the invention at a concentration of 10.+ -. 1% mM.

Preferably, calcium chloride is present in the composition of the invention in a concentration of 11 to 12 mM, more preferably 11.3 mM.

Calcium chloride may be present in the compositions of the present invention at a concentration of about 11 mM. Preferably, calcium chloride is present in the composition of the invention at a concentration of 11 mM.

Calcium chloride may be present in the compositions of the present invention at concentrations of 1 to 100mM, 2 to 90mM, 3 to 80mM, 4 to 70mM, 5 to 60mM, 6 to 50mM, 7 to 40mM, 8 to 30mM, and 9 to 20 mM.

The compositions of the present invention may comprise one or more buffers. One or more buffers may be required for the methods or uses of the present invention. Suitable buffers of the invention help support cell viability and yield, as well as reduce cell aggregation. The buffer may be selected from HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), hanks Balanced Salt Solution (HBSS), MOPS (3- (N-morpholino) propanesulfonic acid), MES (2- (N-morpholino) ethanesulfonic acid), minimal Essential Medium (MEM), du's Modified Eagle's Medium (DMEM), roswell Park Memorial Institute (RPMI) -1640, ai Sike Du's medium (IMDM), BES (NN-bis [ 2-hydroxyethyl ] -2-aminoethanesulfonic acid; 2- [ bis (2-hydroxyethyl) amino ] -ethanesulfonic acid), er Balanced Salt Solution (EBSS), MOPSO (2-hydroxy-3-morpholino propanesulfonic acid), ACES (N-2-aminoethanesulfonic acid), fetal Bovine Serum (FBS), TAPS (([ tri (hydroxymethyl) methylamino ] propanesulfonic acid), phosphate Buffered Saline (PBS), du's Phosphate Buffered Saline (DPBS), bicine, tricine, cell culture grade water, ethylenediamine tetraacetic acid (EDTA), bovine serum albumin (34) and ethylene glycol-bis (N, N-aminoethyl) -N, N, N32-tetraacetic acid (Tn-32).

Preferably, the buffer is HBSS. HBSS is a buffer commonly used in cell culture and contains the necessary balance of inorganic ions for cell thriving, as well as maintaining the correct pH and osmotic pressure. HBSS is commercially available from a number of manufacturers, such as Cytiva ^TM. Preferably, the HBSS in the composition of the present invention is Cytiva ^TM catalog No. SH30268.01 by day 2023, month 5, and day 17. The composition of Cytiva ^TM catalog No. SH30268.01 is shown in the table below. The components shown in the following table may vary by + -75%, + -50%, + -20%, + -10%, + -5% or + -1% of the values shown. Calcium chloride (anhydrous) and/or magnesium sulfate (anhydrous) may be omitted.

Description of the Components	mg/L	mmol/L
			Calcium chloride (Anhydrous)	140	1.2615
Potassium chloride	400	5.3655
			Potassium dihydrogen phosphate (Anhydrous)	60	0.4409
Magnesium sulfate (Anhydrous)	97.67	0.8112
			Sodium chloride	8000	136.8925
Disodium hydrogen phosphate (Anhydrous)	47.68	0.3359
			D-glucose (Anhydrous)	1000	5.5506
Sodium bicarbonate	350	4.1662

Preferably, the composition of the present invention comprises 1.+ -. 10% mg/mL proteinase K, 4.+ -. 10% U/ML DSDNASE, hakk equilibrium salt solution (HBSS), and 10.+ -. 15% mM calcium chloride.

More preferably, the composition of the invention comprises 1 mg/mL proteinase K, 4U/ML DSDNASE, hakks equilibrium salt solution (HBSS), and 10+ -15% mM calcium chloride.

In a further aspect, the invention provides a composition comprising a buffer, proteinase K, DNase, and a source of Ca ²⁺.

In a preferred embodiment, the ex vivo method of dissociating tissue of the present invention comprises a) contacting a tissue with a composition comprising 1 mg/mL proteinase K, 4U/ML DSDNASE, hakks equilibrium salt solution (HBSS), and 10+ -15% mM calcium chloride, b) incubating the tissue with the composition for a first period of time, and c) obtaining a cell suspension.

In a more preferred embodiment, the ex vivo method of dissociating tissue of the present invention comprises a) contacting a tissue with a composition comprising 1mg/mL proteinase K, 4U/ML DSDNASE, hakk Balanced Salt Solution (HBSS), and 10+ -15% mM calcium chloride, b) incubating the tissue with the composition for a first period of time, c) obtaining a cell suspension, d) passing the cell suspension through a first filter, e) quenching proteinase K, preferably with 10% Fetal Bovine Serum (FBS) in Phosphate Buffered Saline (PBS), and preferably f) passing the cell suspension through a second filter, preferably a 40 or 70 μm filter.

The compositions of the invention may also comprise one or more additional agents, such as other enzymes (in particular dissociating enzymes).

Alternatively, the compositions of the invention may lack one or more additional agents, such as other enzymes (particularly dissociating enzymes).

The one or more additional agents may be selected from accutase, trypLE ^TM, trypsin, chymotrypsin, pronase, papain, liberase, collagenase, elastase, dispase, thermolysin, hyaluronidase, clostripain and neutral proteases from Clostridium histolyticum (clostridium histolyticum), pronase, pepsin, lysozyme, chelators of divalent ions (e.g., EDTA or citrate), and combinations thereof.

The method of the invention may further comprise passing the cell suspension through a first filter and/or a second filter.

The size of the filter may be selected from 100, 95, 80, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, and 1 μm.

Preferably, the size of the filter is 40 or 70 μm.

The method of the invention may further comprise quenching proteinase K to terminate the dissociation reaction. Quenching may be performed with the buffers described herein. Quenching may be performed with Fetal Bovine Serum (FBS) in Phosphate Buffered Saline (PBS) or in another buffer described herein. The concentration of FBS can be 1 to 100% v/v, 2 to 90% v/v, 3 to 80% v/v, 4 to 70% v/v, 5 to 60% v/v, 6 to 50% v/v, 7 to 40% v/v, 8 to 30% v/v, 9 to 20% v/v. Preferably, the concentration of FBS is 10%.

Alternatively or additionally, quenching may be performed with a Cu ²⁺ source. The Cu ²⁺ source may be present in the compositions of the present invention in the buffers described herein.

Quenching with FBS is more suitable than quenching with Cu ²⁺ according to the examples below, which demonstrate that quenching with FBS significantly improves viability and yield of dissociated cells compared to quenching with Cu ²⁺.

After dissociation, cell yield, viability and aggregation may be assessed by any suitable method known in the art. Preferably, cell yield, viability and aggregation are quantified using an automated cell counter, preferably Nucleocounter ^® NC-200TM using Via2-CASSETTETM (CHEMOMETEC). Other automated cell counters (e.g., products of Nexcelom and Thermofisher) may also be used. Cell yield, viability and aggregation can also be quantified with trypan blue by using a hemocytometer. The yield can be calculated as 1000 ((viable cells per mL x dilution for counting x total volume per mL)/(mass per mg)) =viable cells per gram of tissue.

The method of the invention may further comprise removing the supernatant. Removal of the supernatant may include centrifugation.

The methods of the invention may further comprise resuspending the cells in a buffer as described herein. The buffer may comprise Bovine Serum Albumin (BSA) and/or ethylenediamine tetraacetic acid (EDTA). The concentration of BSA may be selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1,2, 3, 4, and 5%. The concentration of BSA may be 0.04 to 4%, 0.08 to 3.6%, 0.12 to 3.2%, 0.16 to 2.8%, 0.2 to 2.4%, 0.24 to 2%, 0.28 to 1.6%, 0.32 to 1.2%, or 3.6 to 0.8%. The concentration of EDTA may be selected from 0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9 and 1mM. The concentration of EDTA may be 0.01 to 1mM, 0.02 to 0.9mM, 0.03 to 0.8mM, 0.04 to 0.7mM, 0.05 to 0.6mM, 0.06 to 0.5mM, 0.07 to 0.4mM, 0.08 to 0.3mM, or 0.09 to 0.2mM. Preferably, the buffer comprises 0.4% BSA and 0.1mM EDTA.

The method of the invention may further comprise mechanical dissociation, which may be performed by suitable means (preferably an automated tissue dissociation system), for example selected from VIA ExtractorTM tissue dissociation, GENTLEMACSTM dissociation, GENTLEMACSTM OCTO dissociation, singulatorTM and SingulatorTM 200. Preferably, the method of the present invention is performed using VIA ExtractorTM tissue disruptors.

The compositions, methods, or systems of the present invention may be adapted for use with VIA ExtractorTM tissue disruptors.

The first time period of the method of the invention may be at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 minutes. The first time period of the method of the invention may be no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24 or 25 minutes, preferably 20 minutes, more preferably 15 minutes. The first time period of the method of the present invention may be 1.5 to 150 minutes, 3 to 135 minutes, 4.5 to 120 minutes, 6 to 105 minutes, 7.5 to 90 minutes, 9 to 75 minutes, 10.5 to 60 minutes, 12 to 45 minutes, or 13.5 to 30 minutes. Preferably, the first period of time of the method of the invention is about 15 minutes, more preferably 15 minutes.

The method or use of the present invention may be performed at less than or about 50, 45, 40, 35, 30, 25, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 ℃. The process or use of the present invention may be carried out at 0.4 to 40 ℃,0.8 to 36 ℃, 1.2 to 32 ℃, 1.6 to 28 ℃,2 to 24 ℃, 2.4 to 20 ℃, 2.8 to 16 ℃, 3.2 to 12 ℃, or 3.6 to 8 ℃.

The tissue for dissociation is preferably solid tissue and may be selected from spleen, heart, liver, brain and other neural tissue, kidney, lung, pancreas, breast, umbilical cord, skin, placenta, ovary, fallopian tube, uterus, prostate, tonsil, thymus, stomach, testis, trachea, cartilage, tendon, bone, skeletal muscle, smooth muscle, digestive tract, colon, intestine, bladder, urethra, eye, gall bladder, organoids from cell culture and tumors. Preferably, the tissue is kidney tissue, more preferably intact kidney tissue, even more preferably intact kidney tissue weighing less than 300 mg.

The tissue may be obtained from a mammal, such as from the group consisting of mice, rats, birds such as chickens, ruminants such as cows, goats, deer, sheep, horses and other animals such as pigs, cats, dogs and primates such as humans, chimpanzees, gorillas and monkeys. Preferably, the tissue is obtained from a mouse, more preferably a female mouse.

In a further aspect, the invention provides a cell suspension obtainable by the method of the third aspect of the invention.

The compositions of the present invention may be administered to tissue using a device such as the device described in EP3171152 A1.

Kits of the invention may further comprise one or more containers. The various components of the compositions of the present invention may be present in the kit in one or more of the containers. For example, the components may all be present in one container or in separate containers.

Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The preferred features of the various aspects of the invention are equally applicable to each of the other aspects, with appropriate adaptations. The documents mentioned herein are incorporated by reference to the fullest extent permitted by law.

Examples

The invention will now be further described by reference to the following examples and figures, which are included for reference purposes only and are not to be construed as limiting the invention.

FIG. 1 visual cleavage of the kidney of mice was quenched with different concentrations of proteinase K (0.1 mg/mL, 1mg/mL and 5 mg/mL) followed by Cu ²⁺.

FIG. 2 quantification of yield, viability and aggregation after dissociation of the kidneys of mice with different concentrations of proteinase K (0.1 mg/mL, 1mg/mL and 5 mg/mL) followed by quenching with Cu ²⁺.

FIG. 3 quantification of yield, viability and aggregation after dissociation of the mouse kidneys with different concentrations of proteinase K (0.1 mg/mL, 0.5 mg/mL and 1 mg/mL) followed by quenching with FBS at 4 ℃ ("cold") and 37 ℃ ("hot").

FIG. 4 results of triplicate experiments with proteinase K incubation times of 15 and 20 minutes.

FIG. 5 comparison of results after dissociation using i) VIA ExtractorTM subtilisin A at 4 ℃ (left), ii) VIA ExtractorTM proteinase K at 4 ℃ (middle) and iii) Miltenyi Multitissue dissociation kit 2 with GENTLEMACSTM OCTO dissociator Miltenyi enzyme at 37 ℃ (right).

FIG. 6 is a UMAP chart comparing kidney cell clusters detected after dissociation of mouse kidneys with proteinase K and subtilisin A at 4℃using VIA ExtractorTM tissue dissociator.

FIG. 7 is a UMAP chart comparing cell clusters detected after dissociation of mouse kidneys with proteinase K at 4 ℃ ("cold") and Miltenyi Multi-tissue dissociation kit 2 using VIA ExtractorTM tissue dissociator at 37 ℃.

FIG. 8 comparison of stress markers of kidney cells dissociated with proteinase K at 4℃and Miltenyi Multi-tissue dissociation kit 2 using VIA ExtractorTM tissue dissociator.

Example 1

The following compositions were prepared for use as reagents for tissue dissociation.

1Mg/mL recombinant proteinase K (Blirt ^TM/QIAGEN^TM);

4U/ML DSDNASE (Blirt ^TM/QIAGEN^TM), and

HBSS buffer (Cytiva ^TM) with addition of 10mM CaCl ₂ (Thermofisher^TM).

Example 2-method for dissociating mouse kidney cells compatible with VIA ExtractorTM tissue dissociator and VIA FreezeTMUno

Methods of dissociating mouse kidney tissue were developed using the compositions prepared in example 1. The method was tested on <300mg intact kidneys from female mice. All steps are routinely performed on ice.

Criteria for evaluating the suitability of cells after dissociation, in particular for single cell sequencing, were performed according to 3 parameters, yield (> 1x10 ⁷ viable cells/g), viability (> 80% viable cells) and aggregation (< 10%). However, other parameters for evaluating such suitability may be used.

Viability and aggregation were assessed using Nucleocounter ^® NC-200TM and Via2-CASSETTETM (CHEMOMETEC). Yield was calculated as 1000 ((viable cells per mL x dilution for counting x total volume per mL)/(mass per mg)) =viable cells per gram of tissue.

VIA Freeze UnoTM was set to cool to 2 ℃ (to allow VIA ExtractorTM tissue disruptors to reach 4 ℃) and run at 200rpm for 60 minutes and held until ready for use. Kidney tissue was placed in an Omics bag using an Omics applicator. A composition was prepared according to example 1 and 5mL was applied per sample/kidney/pouch portion.

Then VIA ExtractorTM tissue dissociator settings were run for 15 minutes. Thereafter, the sample is mostly dissociated and may be further dissociated if necessary. The resulting cell suspension was then passed through a 100 μm cell filter and proteinase K was quenched with an excess of Phosphate Buffered Saline (PBS) containing 10% Fetal Bovine Serum (FBS). The samples were centrifuged at 300Xg for 10min, the supernatant removed, and the pellet resuspended in a buffer consisting of PBS+0.4% Bovine Serum Albumin (BSA) +0.1 mM ethylenediamine tetraacetic acid (EDTA).

Additional purification steps, such as further filtration using 70 μm and 40 μm cell filters and/or Red Blood Cell (RBC) lysis, may then be performed as desired.

The sample quality is then determined after the previous decontamination step by measuring the above mentioned parameters using a cell counter.

EXAMPLE 3 Pre-experiment to determine optimal proteinase K concentration

The first pre-experiment test dissociated mouse kidney tissue using three compositions containing proteinase K at different enzyme concentrations (0.1 mg/mL, 1mg/mL, 5 mg/mL). The Cu ²⁺ quenching step was used to quench protease activity. The results are shown in fig. 1. Kidney tissue is effectively dissociated.

The evaluation of the parameters discussed in example 2 is shown in fig. 2. 1mg/mL showed the optimal concentration.

Example 4-Pre-experiment to evaluate kidney cell aggregation, yield and viability of mice after dissociation with different proteinase K concentrations at 4 ℃ ("cold") or 37 ℃ ("hot") with FBS quenching

Alternative proteinase K quenching steps were tested by replacing Cu ²⁺ quench with FBS quench. The results are shown in fig. 3. Surprisingly, quenching proteinase K activity with FBS showed significantly improved viability and yield compared to quenching with Cu ²⁺.

EXAMPLE 5-Pre-experiment to determine optimal proteinase K incubation time for use with VIA ExtractorTM tissue disruptors

Experiments were performed in triplicate with 15 and 20 minute incubation times to confirm the optimal incubation time and to allow for deterministic statistical analysis to be performed. The results are shown in fig. 4.

The most effective incubation time with proteinase K was found to be 15 minutes.

Example 6-comparison of tissue dissociation between VIA ExtractorTM tissue dissociation kit 2 and Miltenyi multiple tissue dissociation kit with gentleMACSTMOcto dissociation

Experiments were performed to compare tissue dissociation of the composition prepared according to example 8 using a VIA ExtractorTM tissue dissociator with a commercially available kit, miltenyi multiple tissue dissociation kit 2 using a GENTLEMACSTM OCTO dissociator. The results are shown in fig. 5.

When the proteinase K composition was used with a VIA ExtractorTM tissue disruptor according to example 8, the viability was improved and the aggregates were significantly lower compared to Miltenyi multi-tissue dissociation kit 2 with a GENTLEMACSTM OCTO disruptor.

Although the nature of the DNase used was different from example 1, a comparison can be made between the dissociation capacity of the active protease for VIA ExtractorTM tissue dissociation, i.e. proteinase K, at 4 ℃ and Miltenyi multitissue dissociation kit 2 with GENTLEMACSTM OCTO dissociator at 37 ℃. Statistical comparisons were made as in example 8.

EXAMPLE 7 characterization of Single cell RNA-seq (scRNA-seq) data obtained by dissociation with proteinase K

After the dissociation and purification method according to example 2, the resulting cell suspension was used to perform the scRNA-seq in the same manner as in example 9 below. As a comparison, a composition comprising 5mM CaCl ₂, 125U/mL DNase I (Sigma), 5mg/mL and subtilisin A (known to be effective at low temperatures) in HBSS was used. It was confirmed that there was no difference between the identified clusters when samples were dissociated using subtilisin a or proteinase K as the main protease (see fig. 6).

Thus, the results indicate that proteinase K is at least as suitable as subtilisin A for dissociating tissue at lower temperatures.

Example 8-comparison of dissociation of kidney tissue when proteinase K and subtilisin A are used

The following experiment allows a comparison between dissociation of tissue by proteinase K and subtilisin A (a known to be effective dissociating enzyme at low temperatures). The experimental procedure was performed as follows.

For dissociation with subtilisin A, all reagents were prepared, including PBS+10% FBS, HBSS+5 mM CaCl ₂, 10mg/mL DNase I in HBSS with 5mM CaCl ₂, 1 XRBC lysis buffer (Miltenyi Biotec), DBPS +0.4% BSA+0.1 mM EDTA, and set VIA to 4℃200 RPM. All steps were completed on ice.

For dissociation with proteinase K, all reagents were prepared, including PBS+10% FBS, HBSS+10 mM CaCl ₂, 10mg/mL DNase I with 10mM CaCl ₂, 1 XRBC lysis buffer (Miltenyi Biotec), DBPS +0.4% BSA+0.1 mM EDTA, and set VIA to 4℃200 RPM. All steps were completed on ice.

A comparison can be made between the dissociation functions of proteases, despite the difference in the concentration of calcium chloride. Statistical comparisons were made between two sets of Miltenyi results by performing a t-test, finding that the differences were not significant, then combining the data together and averaging the results, treating them as 6 replicates, performing a one-way ANOVA and multiple comparisons between dissociation results using Miltenyi procedure, proteinase K and subtilisin a.

1. Tissues were weighed and rinsed with PBS. The tissue is placed in the bag, heat sealed and a clamp is added to the bag.

2. For dissociation using proteinase K, an enzyme master mix (1 mg/mL proteinase K+125U/mL DNase I; total = 15 mL) was prepared 750. Mu.L proteinase K20 mg/mL, 201.9. Mu.L DNase I10 mg/mL, 14.22mL HBSS+10 mM Ca2+, and 5mL was added to each bag portion.

3. For cleavage using subtilisin, an enzyme master mix was prepared (5 mg/mL subtilisin A+125U/mL DNase I; total = 15 mL): 750. Mu.L subtilisin A100 mg/mL, 201.9. Mu.L DNase I10 mg/mL, 14.22mL HBSS+10 mM Ca2+, and 5mL was added to each bag portion.

4. The tissue was left to dissociate for 15 minutes.

5. The resulting cell suspension was transferred to a 50mL tube with 1mL of soaked 100 μm filter and the bag was rinsed with 5mL PBS + FBS.

6. The cell suspension was centrifuged at 300Xg for 10 minutes, the supernatant removed and resuspended in 1mL DPBS+BSA+EDTA. 14mL of RBC lysis solution was then added and the sample was inverted and incubated for 5 minutes.

7. The cell suspension was then centrifuged at 300Xg for 10 minutes to remove the supernatant. 3mL of DPBS+BSA+EDTA was added and mixed thoroughly.

8. Optionally, the above filtration step is repeated with a 70 μm+40 μm filter.

9. The resulting samples were then evaluated according to the parameters in example 2.

The results are shown in fig. 5. The results shown in the middle relate to the use of proteinase K and VIA ExtractorTM tissue dissociators. The left hand side results involved the use of subtilisin a and VIA ExtractorTM tissue dissociators. The right side results relate to the use of Miltenyi multiple tissue dissociation kit 2 and GENTLEMACSTM OCTO dissociator.

The viability scores can be found to be similar between all three test conditions from the data. Proteinase K performed at least as well as subtilisin a in the final sample and significantly better than Miltenyi multi-tissue dissociation kit 2 with GENTLEMACSTM OCTO dissociator in terms of aggregation.

The above experiments demonstrate that i) proteinase K can be used as a tool for efficiently dissociating tissue into single cells, ii) dissociation using proteinase K can be performed efficiently at lower temperatures, and iii) proteinase K can dissociate tissue at lower temperatures, at least as efficiently as subtilisin A.

Example 9-comparison of Single cell RNA-seq (scRNA-seq) spectra after dissociation with proteinase K at 4℃or Miltenyi Multi-tissue dissociation kit at 37℃using VIA ExtractorTM tissue dissociator

After dissociation of the mouse kidney using the composition outlined in example 1, scRNA-seq workflow was performed and summarized as follows.

For single cell sequencing, cells from the sample are quantified to determine an average count. The manufacturer's instructions for 10X Genomics Chromium Next GEM Single Cell 3' dual index kit v3.1 were followed in order to sequence 1000 cells for each sample. 10X Genomics Chromium Controller are used to capture cells into gel beads (GEMs) in an emulsion. The library was sequenced using NextSeq 550 high output kit v2.5 (Illumina inc.) on NextSeq 550 Base (Illumina inc.). Two sequencing runs were performed to obtain depth. scRNA matrix data were analyzed in Seurat (Hao et al cell. 2021 184 (13)) using UMAP (Bechtet al Nature Biotechnology 2018 37 (1): 38-44). Each sample was analyzed separately and filtered to remove duplicates and included cells with 200 to 4000 characteristic RNAs, as well as all cells that maintained a percentage of mitochondrial gene expression below 50%. Once all samples are filtered and clustered, the data from each sample is combined into a single dataset to allow comparison in Seurat (Haoet al.cell. 2021 184 (13)) using UMAP (Bechtet al.Nature Biotechnology 2018 37 (1): 38-44). Cell types representing each cluster were identified using Seurat and marker genes identified by Heet al. Nature communications 2021 9;12 (1) and Chunget al. J Am Soc Nephrol 2020 10;31 (10): 2341-54. The list of genes from the cell clusters with differential gene expression profiles was further analyzed using the gene ontology software package PANTHER (Muller (2017) PANTHER.db: A set of annotation maps describing the entire PANTHER Gene Ontology. R package version 1.0.4.). The results are shown in fig. 7 and 8.

FIG. 7 demonstrates that kidney tissue dissociated with proteinase K at 4℃exhibits a scRNA-seq profile similar to kidney tissue dissociated with Miltenyi Multi-tissue dissociation kit 2 at 37 ℃.

FIG. 8 shows the expression of stress marker genes between dissociation of kidney tissue with Miltenyi Multi-tissue dissociation kit 2 at 37℃and proteinase K at 4 ℃. The dot plot shows the percentage of cells in each cluster that express the "stress" genes listed in O' FLANAGANET al. The dark color dots represent Miltenyi multi-tissue dissociation kit 2 and the light color dots represent proteinase K. Dissociation with proteinase K at 4 ℃ demonstrated reduced stress gene expression, thus improving suitability for use in high-throughput single-cell histology studies.

It will be understood that certain embodiments (e.g., compositions, kits, methods, etc.) may include, incorporate, or otherwise comprise features (e.g., properties, components, ingredients, elements, parts, portions, steps, etc.) described in other embodiments disclosed and/or described herein. Thus, various features of one embodiment may be compatible with, combined with, included within, and/or incorporated in other embodiments of the present disclosure. The disclosure of certain features with respect to one embodiment of the present disclosure should not be construed as limiting the application or inclusion of such features to the particular embodiment. On the contrary, it will be understood that other embodiments may also include the features without necessarily departing from the scope of the disclosure. Furthermore, any feature described herein may be combined with any other feature of the same or different embodiments disclosed herein, unless the feature is described as requiring another feature in combination therewith.

The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. Various changes and/or modifications and additional applications of the features illustrated herein, which may occur to those skilled in the relevant art and having possession of this disclosure, may be made to the illustrated embodiments without departing from the spirit and scope of the invention, which is defined by the claims, and are to be considered within the scope of the disclosure. Although various features and embodiments have been disclosed herein, other features and embodiments are contemplated. For example, well-known features and embodiments have not been described in particular detail herein to avoid obscuring aspects of the described embodiments. However, such features and embodiments are also contemplated herein.

Claims (27)

1. A composition comprising:

A buffering agent;

proteinase K;

DNase, and

Ca ²⁺ source.

2. The composition of claim 1, wherein the buffer is selected from HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), hanks Balanced Salt Solution (HBSS), MOPS (3- (N-morpholino) propanesulfonic acid), MES (2- (N-morpholino) ethanesulfonic acid), minimal Essential Medium (MEM), dulex Modified Eagle Medium (DMEM), roswell Park Memorial Institute (RPMI) -1640, ai Sike f modified dulex medium (IMDM), BES (NN-bis [ 2-hydroxyethyl ] -2-aminoethanesulfonic acid; 2- [ bis (2-hydroxyethyl) amino ] -ethanesulfonic acid), herm balanced salt solution (EBSS), MOPSO (2-hydroxy-3-morpholino propansulfonic acid), ACES (N-2-aminoethanesulfonic acid), fetal Bovine Serum (FBS), TAPS (([ tris (hydroxymethyl) methylamino ] propanesulfonic acid), phosphate Buffered Saline (PBS), dulex Phosphate Buffered Saline (DPBS), bicine, tricine, cell culture grade water, ethylenediamine tetraacetic acid (EDTA), bovine Serum (BSA) and ethylene glycol (N-bis-ethyl) -N-32 ta (N- ʹ, N-tetraacetic acid).

3. The composition according to claim 1 or 2, wherein the proteinase K is present in the composition at a concentration of 0.1 mg to 5 mg/mL, preferably at a concentration of 1 ± 10% mg/mL.

4. The composition according to any of the preceding claims, wherein the DNase is double stranded DNase (dsDNase), optionally wherein the DNase is DNase I.

5. The composition according to any of the preceding claims, wherein the DNase is present in the composition at a concentration of 4±10% U/mL.

6. The composition of any one of the preceding claims, wherein the source of Ca ²⁺ comprises calcium chloride, optionally wherein the calcium chloride is present in the composition at a concentration of 10 ± 15% mM.

7. The composition of any one of the preceding claims, comprising:

1+ -10% mg/mL proteinase K;

4±10% U/mL dsDNase;

Hanks Balanced Salt Solution (HBSS), and

10.+ -. 15% MM calcium chloride.

8. Use of proteinase K for dissociating tissue at a temperature below 20 ℃, optionally below 10 ℃, optionally about 4 ℃.

9. The use according to claim 8, wherein the proteinase K is present in a composition, wherein the composition is according to any one of claims 1-7.

10. The use according to claim 8 or 9, wherein the proteinase K is incubated with the tissue for no more than 20 minutes, optionally no more than 15 minutes.

11. An ex vivo method of dissociating tissue, comprising:

a) Contacting the tissue with a composition comprising proteinase K;

b) Incubating the tissue with the composition for a first period of time, and

C) A cell suspension is obtained.

12. The method of claim 11, wherein the composition further comprises a DNase, optionally wherein the DNase is double stranded DNase (dsDNase), optionally wherein the DNase is DNase I.

13. The method of claim 11 or 12, wherein the composition further comprises a source of Ca ²⁺.

14. The method of any one of claims 11-13, wherein the composition further comprises Hanks Balanced Salt Solution (HBSS).

15. The method of any one of claims 11-14, wherein the composition is a composition according to any one of claims 1-7.

16. The method of any one of claims 11-15, wherein the method further comprises passing the cell suspension through a first filter, optionally a 100 μm filter.

17. The method according to any one of claims 11-16, wherein the method further comprises quenching the proteinase K, optionally with 10% Fetal Bovine Serum (FBS) in Phosphate Buffered Saline (PBS) and/or with a Cu ²⁺ source.

18. The method of any one of claims 11-17, wherein the method further comprises removing supernatant, optionally removing supernatant by centrifugation, and resuspending the cells in buffer, optionally in Bovine Serum Albumin (BSA) and ethylenediamine tetraacetic acid (EDTA), optionally in 0.4% Bovine Serum Albumin (BSA) and 0.1mM ethylenediamine tetraacetic acid (EDTA).

19. The method of any one of claims 11-18, wherein the method further comprises passing the cell suspension through a second filter, optionally a 40 or 70 μm filter.

20. The method of any one of claims 11-19, wherein the method further comprises mechanical dissociation, optionally wherein the mechanical dissociation is performed by a VIA ExtractorTM tissue dissociator.

21. The method of any one of claims 11-20, wherein the first period of time is at least 15 minutes.

22. The process of any one of claims 11-21, wherein the process is conducted at a temperature of less than 20 ℃, optionally less than 15 ℃, optionally less than 10 ℃, optionally less than 5 ℃, optionally about 4 ℃.

23. The use according to any one of claims 8-10 or the method according to any one of claims 11-22, wherein the tissue is selected from the group consisting of spleen, heart, liver, brain and other neural tissue, kidney, lung, pancreas, breast, umbilical cord, skin, placenta, ovary, fallopian tube, uterus, prostate, tonsil, thymus, stomach, testis, trachea, cartilage, tendon, bone, skeletal muscle, smooth muscle, alimentary canal, colon, intestine, bladder, urethra, eye, gall bladder, organoids and tumors from cell culture.

24. The use according to any one of claims 8-10 or claim 23 or the method according to any one of claims 10-22, wherein the tissue is obtained from the group consisting of mice, rats, birds such as chickens, ruminants such as cows, goats, deer, sheep, horses and other animals such as pigs, cats, dogs and primates, such as humans, chimpanzees, gorillas and monkeys, optionally mice, optionally female mice.

25. The use according to any one of claims 8-10, 23 or 24 or the method according to any one of claims 11-24, wherein the tissue is kidney tissue, optionally intact kidney tissue and/or weighed below 300mg.

26. A system configured to perform the method of any one of claims 11-25.

27. A kit comprising the composition of any one of claims 1-7 and instructions for use.