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WO2020077244A1 - Procédés d'évaluation de la viabilité cellulaire et de perforation de membrane cellulaire - Google Patents

Procédés d'évaluation de la viabilité cellulaire et de perforation de membrane cellulaire Download PDF

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Publication number
WO2020077244A1
WO2020077244A1 PCT/US2019/055906 US2019055906W WO2020077244A1 WO 2020077244 A1 WO2020077244 A1 WO 2020077244A1 US 2019055906 W US2019055906 W US 2019055906W WO 2020077244 A1 WO2020077244 A1 WO 2020077244A1
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WIPO (PCT)
Prior art keywords
cell
injection pipette
tip
injection
electrical
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PCT/US2019/055906
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English (en)
Inventor
Amir Mor
Emre SELI
Xiao-Bing Gao
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Yale University
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Publication of WO2020077244A1 publication Critical patent/WO2020077244A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

  • Determination of cell viability is important in many fields relating to cell biology and medicine, and there are several accepted assays of viability that utilize such diverse parameters as the redox potential of the cell population, the integrity of cell membranes, or the activity of cellular enzymes such as esterases. Each assay may or may not be suitable for the particular applications relating for cell viability, cytotoxicity, or drug efficacy with several integrated components.
  • the viability of eggs is typically assessed by visual inspection. The embryologist examines the zona pellucida, plasmatic membrane, and cell’s cytoplasm. For example, abnormal cytoplasm appearance and/or fragmented cytoplasmic membrane (i.e., no evidence of intact smooth plasmatic membrane) are consistent with a non-viable cell/egg.
  • Intracytoplasmic sperm injection is an in vitro fertilization procedure in which a single sperm cell (spermatozoid) is injected directly into the cytoplasm of an oocyte (egg). This technique is used to obtain embryos that may be subsequently transferred to a maternal uterus.
  • the ICSI procedure has been found to be an effective method of achieving fertilization and treating male factor infertility, although it may also be used where oocytes cannot be easily penetrated by sperm cells, and occasionally in addition to sperm donation. ICSI may be used, e.g., for males with teratospermia, whose sperm has abnormal
  • oocyte viability is not considered prior to the injection.
  • Sperm cells are injected into all available oocytes that have been aspirated from the ovaries. It is not certain at the time of injection which oocytes are viable, and which oocytes are dying. The practitioners count on the viable sperm cells to fertilize the viable eggs. When considering male partners with azoospermia, this lack of information becomes significant. These male partners usually undergo a testicular biopsy for sperm cell retrieval. If sperm cells are retrieved, their number is oftentimes low (a single digit number). When sperm cells number is the limiting factor for fertilization, the selection of the most viable eggs is critical. In these cases, injection of the few harvested sperm cells into the most viable eggs is the desired option.
  • ICSI intracytoplasmic sperm injection
  • Light microscopy has another limitation often encountered when attempting cell membrane penetration.
  • the egg is penetrated with a sharp micropipette, which is used to inject a sperm cell directly into the egg.
  • the actual cell penetration cannot always be clearly visualized by simple light microscopy. Therefore, the embryologist tries to aspirate cytoplasm contents into the pipette, prior to sperm injection, to confirm cell membrane penetration.
  • This aspiration of cytoplasm into the pipette has the potential to damage the cell. Thus, this is an additional reason to seek for alternative techniques to confirm cell membrane penetration that lessens the likelihood of damage to the cell.
  • Embryologists who perform ICSI procedures relatively infrequently may not be comfortable with visual confirmation of sperm injection pipette advancement into the egg.
  • robotic injection systems rely on complex computer vision algorithms and pressure sensing technologies in order to confirm pipette advancement into the cell. These methods still require the confirmation of cell membrane penetration by skilled human personnel.
  • a highly reliable and reproducible technique confirming membrane penetration of a cell before attempting intracellular injection of substance such as, e.g., a peptide, a protein, a fatty acid, a carbohydrate, a metal, a basic element, a nucleic acid (e.g., DNA, RNA), a vector (e.g., a viral vector), a microparticle (e.g., a virus, nanoparticle, liposome), a cell (e.g., a sperm cell), or a molecule (e.g., a small molecule, a dye, a fluorescent molecule, a reporter, a pharmaceutical).
  • a reliable technique independent of human monitoring
  • the robotic injection systems can become fully automated.
  • the method provides a method of determining viability of a cell.
  • the invention further provides a method of determining cell membrane piercing.
  • the method comprises (a) providing an electrical resistance meter and an injection pipette having a tip.
  • the tip is configured to penetrate a membrane of the cell.
  • the electrical resistance meter is connected directly or indirectly to the injection pipette tip.
  • the method comprises (b) measuring electrical resistance of the injection pipette tip outside the cell.
  • the method comprises (c) inserting the tip of the injection pipette into the cell and measuring electrical resistance of the injection pipette tip inside the cell.
  • the method comprises (d) calculating resistance of the cell by subtracting the electrical resistance outside the cell of step (b) from the electrical resistance inside the cell of step (c).
  • the method comprises determining viability of the cell by comparing the resistance of the cell of step (d) with a control.
  • the method comprises (a) providing an electrical capacitance meter and an injection pipette having a tip.
  • the tip is designed for cell penetration and injection of a material.
  • the electrical capacitance meter is connected directly or indirectly to the injection pipette tip.
  • the method comprises (b) measuring electrical capacitance of the injection pipette tip outside the cell.
  • the method comprises (c) inserting the tip of the injection pipette into the cell and measuring electrical capacitance of the injection pipette tip inside the cell.
  • the method comprises (d) calculating capacitance of the cell by subtracting the capacitance outside the cell of step (b) from the capacitance inside the cell of step (c). In certain embodiments, the method comprises (d) determining viability of the cell by comparing the capacitance of the cell of step (d) with a control.
  • the method comprises (a) providing an electrical resistance meter and an injection pipette having a tip.
  • the tip is designed for cell penetration and injection of a material.
  • the electrical resistance meter is connected directly or indirectly to the injection pipette.
  • the method comprises (b) measuring electrical resistance of the injection pipette tip outside the cell.
  • the method comprises (c) advancing the tip of the injection pipette towards the cell while measuring electrical resistance of the injection pipette repeatedly.
  • the method comprises (d) determining membrane piercing by the pipette tip when an increase in resistance is detected.
  • the method comprises (a) providing an electrical capacitance meter and an injection pipette having a tip.
  • the tip is designed for cell penetration and injection of a material.
  • the electrical capacitance meter is connected directly or indirectly to the injection pipette tip.
  • the method comprises (b) measuring electrical capacitance of the injection pipette tip outside the cell.
  • the method comprises (c) inserting the tip of the injection pipette into the cell and measuring electrical capacitance of the injection pipette tip inside the cell.
  • the method comprises (d) calculating capacitance of the cell by subtracting the capacitance outside the cell of step (b) from the capacitance inside the cell of step (c). In certain embodiments, the method comprises (e) determining cell membrane piercing of the cell by comparing the capacitance of the cell of step (d) with a control.
  • the invention further provides a method of performing intracytoplasmic sperm injection into an oocyte.
  • the method comprises determining viability of the oocyte by any method contemplated within the invention.
  • the method comprises determining membrane piercing of the oocyte by any method
  • the method comprises injecting a sperm cell into the viable oocyte through the injection pipette tip.
  • the invention further provides a method of injecting material into a cell.
  • the method comprises determining viability of the cell by any method contemplated within the invention.
  • the method comprises determining membrane piercing of the cell by any method contemplated within the invention.
  • the method comprises injecting material into the cell through the injection pipette tip.
  • FIG. 1 depicts a schematic diagram of a method according to an embodiment of the present disclosure.
  • FIG. 2 depicts a photograph of a non-limiting example of a multimeter suitable for measuring resistance.
  • FIG. 3 shows the experiment design for testing an exemplary method of measuring membrane resistance using ICSI pipettes according to the methods described herein.
  • FIG. 4 shows absolute resistances obtained using the exemplary method of ICSI pipette-based measurement.
  • FIGS. 5A-5B show ROC analysis of the change in resistance (AR) in the
  • FIG. 6 depicts an egg with visually intact membrane.
  • FIG. 7 depicts an egg with fragmented membrane.
  • FIG. 9 (comprising Panels A-P) shows screenshots from a video documenting zona pellucida penetration by the ICSI pipette tip but no plasmatic membrane penetration before or after positive pressure application through the pipette (negative control group).
  • FIG. 10 depicts screenshots from a video documenting zona pellucida and plasmatic membrane penetration by the ICSI pipette tip as well as membrane rupture following the application of positive pressure through the pipette (positive control group).
  • FIGS. 11A-11F depict characterization of electrical resistance when piercing versus not piercing the oolemma.
  • the resistance is about 9 MW before penetrating the oocyte (FIG. 11 A).
  • the resistance increases to 14 MW after penetrating the oocyte (FIG. 11B).
  • the application of positive pressure through the ICSI pipette tip leads to oolemma rupture and, in return, the resistance decreases back to about 9 MW (FIG. 11C).
  • FIGS. 11D-11E different oocyte
  • FIGS. 11D-11E demonstrates resistance measurements when the pipette tip has never penetrated the oocyte.
  • FIG. 12 depicts electrical resistance measurements in oocytes with intact oolemma vs. fragmented mouse oocytes. Fragmented oocytes are considered non-viable. In this experiment, the fragmented mouse oocytes showed a resistance increase of up to 2.2 MW. Therefore, intact mouse oocytes showing resistance increase equal or less than 2.2 MW are likely to be non-viable. Intact mouse oocytes showing resistance increase of more than 2.2 MW are considered viable.
  • FIG. 13 illustrates exemplary measurement of electrical resistance change in human oocytes using a commercial ICSI system.
  • leaning against the oolemma does not result in a significant resistance increase.
  • FIG. 14 depicts electrical resistance measurements in oocytes with intact oolemma vs. fragmented human oocytes. Fragmented oocytes are considered non-viable. In this experiment, the fragmented human oocytes showed a resistance increase of up to 0.3 MW. Therefore, intact human oocytes showing resistance increase equal or less than 0.3 MW are likely to be non-viable. Intact human oocytes showing resistance increase of more than 0.3 MW are considered viable. AUC, area under the curve. Cl, confidence interval.
  • FIG. 15A depicts an exemplary adaptor for holding a pipette as contemplated herein.
  • FIG. 15B depicts a zoomed-in view of the adaptor highlighting a wire connected to the electrode wire on one end and connected to an alligator clip of a cable that can connect to one or more measuring devices.
  • FIGS. 16A-16B depict cross-sectional drawings of an exemplary adaptor as contemplated herein.
  • FIG. 17 illustrate a comparison of the concept of evaluating the viability a cell using electrical resistance versus using light microscopy.
  • the terms“treat” or“treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub- clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • the subject is a human.
  • the term“cell” refers to any eukaryotic cell, including mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells, whether located in vitro or in vivo.
  • the term“cell culture” refers to any in vitro population of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
  • ICSI intracytoplasmic sperm injection
  • oocyte refers to a female gametocyte or germ cell involved in reproduction. In other words, it is an immature or mature ovum, or egg cell. An oocyte is produced in the ovary during female gametogenesis.
  • oocytes for use in the invention are mammalian, including but not limited to human, livestock (including but not limited to bovine, porcine, and ovine) and companion animal (including but not limited to canine and feline).
  • sperm refers to a male gametocyte or germ cell involved in reproduction.
  • a sperm cell is produced in the testis during male gametogenesis.
  • sperm for use in the invention are mammalian, including but not limited to human, livestock (including but not limited to bovine, porcine, and ovine) and companion animal (including but not limited to canine and feline).
  • membrane potential “membrane potential”,“transmembrane potential”, and“membrane voltage” are used interchangeably herein to refer to the difference in electric potential between the interior and the exterior of a biological cell.
  • electrical conductor refers to an object or a type of material that allows the flow of an electrical charge (such as an electron, a proton, and/or an ion) in one or more directions.
  • electrical conductors include metal (e.g., copper, gold, silver), non-metal material (such as graphite), and fluid (e.g., a liquid or a gel) containing charged particles (e.g., electrolytes).
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • Electric potential across the cell membrane is a common feature of living cells. Its magnitude depends on the type as well as the physiologic status of the cell. Different cell types can have different membrane potentials. Cells of a certain type (e.g., oocytes) of different species also can have different membrane potentials. Further, certain cells can have different membrane potentials at different stages in development. For example, oocytes can have different membrane potentials before and after fertilization, as well as at first cleavage (see, e.g., US 6,927,049; Tyler et al, Biological Bulletin, 1965, 111(1): 153-177; Morrill et al., J. Cell Physiol., 1965, 67: 85-92).
  • Membrane potential is the difference in electric potential between the interior and the exterior of a biological cell. With respect to the exterior of the cell, typical values of membrane potential range from -10 mV to -80 mV. All animal cells are surrounded by a membrane composed of a lipid bilayer with proteins embedded in it. The membrane serves as both an insulator and a diffusion barrier to the movement of ions, including positively and negatively charged ions (e.g., K + , Na + ’ Ca 2+ , Cl ) via transmembrane proteins such as ion channels and ion transporters. Ion transporter or ion pump proteins actively move ions across the membrane to establish ion concentration gradients across the membrane, while ion channels allow ions to move across the membrane down those concentration gradients.
  • ions including positively and negatively charged ions (e.g., K + , Na + ’ Ca 2+ , Cl ) via transmembrane proteins such as ion channels and ion transporters.
  • Active ion pumps and passive ion channels can be thought of as equivalent to a set of batteries and resistors inserted in the membrane, and therefore create a voltage between the two sides of the membrane.
  • eukaryotic cells including cells from animals, plants, and fungi
  • maintain a non-zero transmembrane potential usually with a negative voltage in the cell interior as compared to the cell exterior.
  • the membrane potential can enable a cell to function as a battery, providing power to operate a variety of“molecular devices” embedded in the membrane.
  • the membrane potential can also enable transmitting signals between different parts of a cell.
  • Signals are generated by opening or closing of ion channels at one point in the membrane, producing a local change in the membrane potential.
  • the membrane potential is held at a relatively stable value, called the resting potential.
  • the difference in potential between any two points can be measured by inserting an electrode at each point, for example one inside and one outside the cell, and connecting both electrodes to the leads of a voltmeter, an electrical resistance meter, and/or an electrical capacitance meter.
  • an electrode at each point for example one inside and one outside the cell
  • the zero potential value is assigned to the outside of the cell and the sign of the potential difference between the outside and the inside is determined by the potential of the inside relative to the outside zero.
  • the membrane potential in a cell derives from at least two factors: electrical force and diffusion.
  • Electrical force arises from the mutual attraction between particles with opposite electrical charges (positive and negative) and the mutual repulsion between particles with the same type of charge (both positive or both negative).
  • Diffusion arises from the tendency of particles to redistribute from regions where they are highly concentrated to regions where the concentration is low. Both of these factors influence the movement of ions across the cell membrane, which leads to the generation of electrical signals.
  • the plasma membrane intrinsically has a high electrical resistivity, in other words a low intrinsic permeability to ions. This low permeability is countered by the presence of transmembrane proteins that either actively transport ions from one side of the membrane to the other or provide channels through which they can move or diffuse.
  • the plasma membrane functions as a combined resistor and capacitor (see, e.g., Rettinger I, Schwarz S., Schwarz W. (2016) Basics: Theory. In:
  • the membrane impedes the movement of ions across it, it can be considered a resistor.
  • the thinness of the lipid bilayer (about 7-8 nanometers) enables an accumulation of charged particles on one side of the membrane, which gives rise to an electrical force that pulls oppositely charged particles toward the other side, and thus provides the capacitance.
  • the capacitance of the membrane is relatively unaffected by the molecules that are embedded in it, so it has a more or less invariant value.
  • the conductance of a pure lipid bilayer is generally very low, so that it is generally dominated by the conductance of alternative pathways provided by the transmembrane proteins. Thus, the capacitance of the membrane is more or less fixed, but the resistance is highly variable.
  • a cell’s resistance is a measure of how easily ions can move through the membrane. Generally, the fewer channels there are for ions to flow through, the higher the resistance of the cell will be. In other words, the resistance of a lipid bilayer membrane to the passage of ions across it is very high, but transmembrane proteins can greatly enhance ion movement, either actively or passively, via facilitated transport and facilitated diffusion, respectively. Ion channels provide passageways through which ions can passively move (i.e., facilitated diffusion). Generally, an ion channel is permeable only to specific types of ions (e.g., sodium and potassium but not chloride or calcium), and the permeability can vary depending on the ion’s concentration gradient.
  • ions e.g., sodium and potassium but not chloride or calcium
  • Ion channel proteins have different configurations that open and close the channel (also called a pore) and can change conformation based on voltage changes across the membrane, binding of a ligand to the channel proteins, and in response to various stimuli (e.g., heat, light).
  • Ion pumps also known as ion transporters or carrier proteins, actively transport specific types of ions from one side of the membrane to the other, sometimes using cellular energy (e.g., ATP) to do so, and thus can move ions against their concentration gradient.
  • Common ion pumps include sodium-potassium pumps and sodium- calcium exchangers.
  • a cell capacitance determines how quickly the membrane potential can respond to a change in current.
  • a capacitor is made up of two conducting materials separated by an insulator.
  • the extracellular and intracellular fluids are the conductors, and the lipid bilayer membrane is the insulator.
  • the capacitance When there is a voltage difference (such as the resting membrane potential) across an insulator, charge will build up at the interface because current cannot flow directly across the insulator.
  • the constant that describes the relationship between the voltage and the charge that builds up is called the capacitance.
  • an induced (capacitive) current is produced, which can change the membrane voltage.
  • the ion pumps and channels can open to allow charge to move across the membrane.
  • the capacitance of the cell membrane is high because it is only two molecules (phospholipids) thick, meaning that not much voltage is needed to separate charges across the membrane.
  • the specific capacitance of biological membranes is very close to what is obtained simply from the dielectric constant of lipids and the thickness of the and, unlike the conductance, the capacitance of a cell membrane is generally constant. Further, the membrane capacitance can be measured in terms of the area of the membrane, such that the larger the area, the larger the capacitance.
  • cell membranes can be measured by, e.g., electrodes (including arrays of electrodes), and pipettes (see, e.g., Rettinger I, Schwarz S., Schwarz W. (2016) Basics: Theory. In: Electrophysiology, Basics, Modem Approaches and Applications. Springer International Publishing; Narahashi T, Principles of electrophysiology: an overview. Curr Protoc Toxicol., Nov. 2003, Chapter 11).
  • pipettes suitable for use in measuring membrane potential must be of a small diameter, in order to avoid significantly disrupting the cell membrane and intracellular environment and thus damaging the cell.
  • These pipettes can have a relatively small bore diameter that is about 1 micrometer to about 2.5 micrometers.
  • the cell may not be able to fully correct the massive disruption of the intercellular ionic concentrations, and therefore the resting membrane potential may be forced to depolarize and even cancel completely.
  • the majority of the eggs recover and regain their negative physiological resting membrane potential.
  • small bore pipettes can inhibit the rapid mixture of solutions, they generally are not suitable for sperm injection due to their small tip diameter.
  • the electrolyte solution used in small bore regular electrophysiological pipettes generally contains ionic concentrations similar to those of the intracellular environment rather than the extracellular environment, and therefore can lead to less disruption of the intracellular environment.
  • the present invention provides methods for measuring cell resistance using one or more large bore pipettes such as ICSI pipettes.
  • the methods of the present invention produce a clear, reproducible, and stable increase in measured resistance upon piercing of a cell membrane using these methods.
  • These pipettes can have bore diameters that are about 4 micrometers to about 10 micrometers, or about 4 micrometers to about 7 micrometers, or about 4 micrometers to about 6 micrometers, or about 4 micrometers, or about 5 micrometers, or about 6 micrometers.
  • the pipettes can be made of any suitable material as understood in the art, including for example, one or more
  • the pipette may include one or more pipettes having an electrical wire in the pipette lumen and/or one or more pipettes having one or more conductive material adhered to the inner pipette wall (e.g., glass pipette-carbon fiber).
  • the large bore pipettes also enable real-time measurement of the membrane resistance and/or capacitance to enable real-time determinations of viability and membrane piercing.
  • Intracytoplasmic sperm injection has been found to be an effective method of achieving fertilization and treating male factor infertility.
  • the progress of the ICSI technique has been dependent primarily on trial and error strategies, with success rates heavily dependent on the experience of the practitioner performing the procedure (Polcz, T.E. et al, Fertility and Sterility, 1997, 68, 4, 735-738; Neri et al, Cell Calcium, 2014 Jan; 55(1), 24-37).
  • penetration of the cell membrane of the oocyte presents the greatest difficulty.
  • One technique to facilitate and confirm penetration is to aspirate ooplasm into the pipette before injecting the sperm cell into the egg. This method may be disadvantageous in that it may disrupt cell structures such as the cytoskeleton, spindles, or genetic material.
  • One method of confirming oocyte membrane penetration involves measuring the electrical potential of the membrane with an electrode through a small bore pipette.
  • incorporating an electrophysiological pipette-based confirmation of membrane penetration adds another step, as the pipette for confirming penetration is of too small a diameter to inject a sperm cell or spermatozoon.
  • the general knowledge in the field suggests that the diameter of pipettes used to determine membrane potential must be small in order to allow an accurate measurement, and also to prevent intracellular contents from leaking out.
  • Non-limiting examples of such materials include a peptide, a protein, a fatty acid, a carbohydrate, a metal, a basic element, a nucleic acid (e.g., DNA, RNA), a vector (e.g., a viral vector), a microparticle (e.g., a virus, nanoparticle, liposome), a cell (e.g., a sperm cell), or a molecule (e.g., a small molecule, a dye, a fluorescent molecule, a reporter, a pharmaceutical).
  • a nucleic acid e.g., DNA, RNA
  • a vector e.g., a viral vector
  • a microparticle e.g., a virus, nanoparticle, liposome
  • a cell e.g., a sperm cell
  • a molecule e.g., a small molecule, a dye, a fluorescent molecule, a reporter, a pharmaceutical
  • the present invention provides methods for determining oocyte viability.
  • the methods include first penetrating the membrane of a cell using one or more ICSI pipettes as described herein.
  • the methods further include measuring the resistance of the cell.
  • the resistance can be measured using standard techniques as understood in the art, including using a standard multi-meter capable of measuring resistance.
  • Embodiments of the methods further include analyzing the measured resistance by comparing the measured resistance value to a threshold control resistance.
  • the threshold control is about 0.5 MW to about 1 MW, about 1 MW to about 1.5 MW, about 1.5 MW to about 2 MW, abot 2 MW to about 2.5 MW, about 2.5 MW to about 3 MW. About 3 MW to about 3.5 MW. About 3.5 MW to about 4 MW and so on.
  • the threshold resistance is about 0.9 MW. In certain embodiments, the threshold resistance is about 2.2 MW.
  • Embodiments of the methods further include determining cell viability of the measured cell.
  • the cell is determined to be viable if the measured resistance is greater than the threshold resistance.
  • the cell is determined to the viable if the measured change in resistance ( ⁇ R) exceeds the threshold resistance by a non-zero value (i.e. has a difference in resistance ⁇ R) of between about 0.001 MW and about 0.01 MW, about 0.1 MW and about 0.5 MW, about 0.5 MW and about 1 MW , about 1 MW and about 1.5 MW , about 1.5 MW and about 2.0 MW , about 2.0 MW and about
  • an intact, viable cell may have a change in resistance value that is low (e.g., below a threshold value) and appear visually intake. This may indicate that the cell has not been sufficiently punctured or penetrated, that the cell is in fact non- viable, and/or that there is a large leak current.
  • the method may further include injecting the cell if the cell is determined to be viable, and not injecting the cell if the cell is determined to be not viable. If the cell is determined to be viable, the cell may be injected with one or more materials including a peptide, a protein, a fatty acid, a carbohydrate, a metal, a basic element, a nucleic acid (e.g., DNA, RNA), a vector (e.g., a viral vector), a microparticle (e.g., a virus, nanoparticle, liposome), a cell (e.g., a sperm cell), or a molecule (e.g., a small molecule, a dye, a fluorescent molecule, a reporter, a pharmaceutical), as contemplated herein.
  • a nucleic acid e.g., DNA, RNA
  • a vector e.g., a viral vector
  • microparticle e.g., a virus, nanoparticle, lipo
  • Embodiments of the methods may further include measuring resistance as contemplated herein in order to confirm that an embryo has been successfully deployed from a transfer catheter.
  • Embodiments of the methods may include measuring resistance as contemplated herein in order to confirm successful lumber puncture (LP) and intravenous (IV) access.
  • LP lumber puncture
  • IV intravenous
  • Embodiments of the methods may include measuring resistance of Xenopus laevis eggs using the methods as contemplated herein. Measurement of a first resistance increase may be used to indicate piercing of the plasmatic membrane of Xenopus laevis eggs.
  • Measurement of a second resistance increase may be used to indicate piercing the nuclear envelope.
  • the methods of the invention can be performed by a human or can be automated such that a robotic system can perform them.
  • Non-limiting exemplary automated systems are described in WO 2008/034249; WO 2012/037642; WO 2013/158658; Huang et al, IEEE Transactions on Robotics, 2009, 25(3), 727-737; Lu et al, IEEE
  • mice Female mice were injected with Follicular Stimulating Hormone (FSH) and human Chorionic Gonadotropin (hCG) in order to mature their eggs. The mice were sacrificed and their ovaries and fallopian tubes were harvested. The eggs were separated from the ovaries and tubes under the microscope. The eggs were transferred into commercial Mil medium (water based solution) in order to keep them viable.
  • FSH Follicular Stimulating Hormone
  • hCG human Chorionic Gonadotropin
  • a droplet of commercial Mil medium was placed on a petri dish. Mineral oil was added until it fully covered the droplet to prevent droplet evaporation.
  • a single egg was placed in the droplet using a commercial transfer pipette.
  • the petri dish with the egg was placed under the microscope of the electrophysiological setup.
  • Two robotic micromanipulators were used:
  • a commercial egg/cell glass holding pipette with a tip curved at 30 degrees was attached to the left micromanipulator.
  • a commercial egg/cell glass injection pipette with a tip curved at 30 degrees was attached to the right manipulator.
  • the injection pipette was prefilled with Mil medium and was attached to an electrophysiological headstage (available, e.g., from Axon instruments).
  • a silver wire originating from the headstage was in contact with the Mil medium in the injection pipette.
  • the headstage was connected to a standard amplifier (available, e.g., from Axon instruments), which was fully controlled by a computer software.
  • the egg was atached to the holding pipete by applying vacuum.
  • An electrical ground wire (electrode) was placed into the petri dish liquid (Mil medium droplet surrounded by oil). Additional Mil medium is added to the petri dish in order to create a continuous aqueous extracellular solution between the egg and the ground electrode.
  • FIG. 1 An exemplary cell setup is depicted in FIG. 1.
  • a cell (1) is placed in a Petri dish (7) with extracellular solution.
  • the cell (1) is held in place with a holding micropipete (3) which is coupled to an electrical ground connection (8).
  • Material can be injected into the cell (1) via an injection micropipette (2) that is coupled to an
  • Each of the holding micropipete and the injection micropipette are also coupled to air or oil microinjectors that apply a controlled vacuum or pressure.
  • the injection pipete may be integrated with one or more adaptors (FIGS. 15A and 15B) for ease of handling.
  • a short test pulse of 10 mV was generated by the headstage and amplifier.
  • the generated electrical current is measured through the tip of the glass injection pipete (by the amplifier).
  • the injection pipete resistance was initially measured in the Mil solution outside the egg. Then, the injection pipete was advanced into the egg. The pipete resistance was measured while its tip was in the egg. Then, the pipete was withdrawn and the resistance was again measured when its tip was outside the egg (this was done in order to demonstrate effect reversal).
  • the egg’s resistance is calculated according to the following:
  • Egg resistance (pipete resistance when its tip is inside the egg) - (pipete resistance when its tip is outside the egg).
  • FIG. 1 A first figure.
  • the ICSI pipette-based method of determining cell membrane piercing via measurement of membrane electrophysiology described herein was tested in comparison with a known visual method of determining cell membrane piercing via light microscopy.
  • the ICSI pipette-based electrophysiology testing was carried out according to the schematic shown in FIG. 3.
  • FIG. 5A raw data
  • FIG. 5B ROC curve
  • the ROC curve showed that the visual method is not sufficient for a diagnostic test (e.g., membrane integrity and cell viability). More specifically, the ROC curve showed the lower bound of the confidence interval to approach the diagonal linear line, which suggests that visual inspection alone is not sufficient to provide reliable cell viability data or confirm membrane piercing.
  • Measuring membrane resistance with a large bore pipette is safe and does not lead to cell destruction.
  • the eggs remained alive and kept their fertilization potential after the resistance measurement methods according to the invention.
  • membrane potential measurement is not a reliable indicator for cell penetration, membrane integrity, or cell viability when using large bore pipettes.
  • the leak current does not interfere with detecting increase in electrical resistance when the egg is penetrated.
  • An alternative to an electrical wire in the pipette lumen can be a conductive material adhered to the inner pipette wall (e.g., glass pipette-carbon fiber).
  • baseline resistance increase can detect dirty or clogged pipette.
  • baseline resistance decrease can detect and/or indicate pipette breakage.
  • resistance and conductance and impedance can be used interchangeably to evaluate and report cell viability since conductance and impedance are resistance-sensitive computed values.
  • resistance can be calculated by a direct measurement of generated current to specific voltage pulse or by direct measurement of generated voltage to specific current pulse.
  • Embodiment 1 provides a method of determining viability of a cell, the method comprising: (a) providing an electrical resistance meter and an injection pipette having a tip, wherein the tip is configured to penetrate a membrane of the cell, and wherein the electrical resistance meter is connected directly or indirectly to the injection pipette tip; (b) measuring electrical resistance of the injection pipette tip outside the cell; (c) inserting the tip of the injection pipette into the cell and measuring electrical resistance of the injection pipette tip inside the cell; (d) calculating resistance of the cell by subtracting the electrical resistance outside the cell of step (b) from the electrical resistance inside the cell of step (c), and determining viability of the cell by comparing the resistance of the cell of step (d) with a control.
  • Embodiment 2 provides a method of determining viability of a cell, the method comprising: (a) providing an electrical capacitance meter and an injection pipette having a tip, wherein the tip is designed for cell penetration and injection of a material, and wherein the electrical capacitance meter is connected directly or indirectly to the injection pipette tip; (b) measuring electrical capacitance of the injection pipette tip outside the cell; (c) inserting the tip of the injection pipette into the cell and measuring electrical capacitance of the injection pipette tip inside the cell; (d) calculating capacitance of the cell by subtracting the capacitance outside the cell of step (b) from the capacitance inside the cell of step (c); and (e) determining viability of the cell by comparing the capacitance of the cell of step d) with a control.
  • Embodiment 3 provides a method of determining piercing of a cell membrane, the method comprising: (a) providing an electrical resistance meter and an injection pipette having a tip, wherein the tip is designed for cell penetration and injection of a material, and wherein the electrical resistance meter is connected directly or indirectly to the injection pipette; (b) measuring electrical resistance of the injection pipette tip outside the cell; (c) advancing the tip of the injection pipette towards the cell while measuring electrical resistance of the injection pipette repeatedly; and (d) determining membrane piercing by the pipette tip when an increase in resistance is detected.
  • Embodiment 4 provides a method of determining piercing of a cell membrane, the method comprising: (a) providing an electrical capacitance meter and an injection pipette having a tip, wherein the tip is designed for cell penetration and injection of a material, and wherein the electrical capacitance meter is connected directly or indirectly to the injection pipette tip; (b) measuring electrical capacitance of the injection pipette tip outside the cell;
  • step (c) inserting the tip of the injection pipette into the cell and measuring electrical capacitance of the injection pipette tip inside the cell; (d) calculating capacitance of the cell by subtracting the capacitance outside the cell of step (b) from the capacitance inside the cell of step (c); and (e) determining cell membrane piercing of the cell by comparing the capacitance of the cell of step (d) with a control.
  • Embodiment 5 provides the method of any of Embodiments 1 and 3, further wherein an electrical ground is connected directly or indirectly to an extracellular side of the cell and to the electrical resistance meter, wherein the electrical ground stabilizes the measurement of the electrical resistance in steps (b) and (c).
  • Embodiment 6 provides the method of any of Embodiments 2 and 4, further wherein an electrical ground is connected directly or indirectly to an extracellular side of the cell and to the electrical capacitance meter, wherein the electrical ground stabilizes the measurement of the electrical capacitance resistance in steps (b) and (c).
  • Embodiment 7 provides the method of any of Embodiments 1 -6, wherein the electrical resistance meter or electrical capacitance meter is connected directly or indirectly to the injection pipette tip by an electrical conductor.
  • Embodiment 8 provides the method of Embodiment 7, wherein the electrical conductor is selected from a wire and a solution.
  • Embodiment 9 provides the method of any of Embodiments 7-8, wherein the electrical conductor is a metal wire selected from a silver wire and a copper wire.
  • Embodiment 10 provides the method of any of Embodiments 7-8, wherein the electrical conductor is an electrolyte solution or an extracellular solution.
  • Embodiment 11 provides the method of any of Embodiments 1-10, wherein the cell is a prokaryotic cell.
  • Embodiment 12 provides the method of any of Embodiments 1-10, wherein the cell is a eukaryotic cell.
  • Embodiment 13 provides the method of any of Embodiments 1-10, wherein the cell is a mammalian cell, a yeast cell, a plant cell, a parasite cell, or a human cell.
  • Embodiment 14 provides the method of any of Embodiments 1-10, wherein the cell is an oocyte.
  • Embodiment 15 provides the method of any of Embodiments 1-14, wherein the cell is attached to a holding pipette.
  • Embodiment 16 provides the method of any of Embodiments 1-15, wherein the injection pipette tip has a bore diameter of about 1 micrometer to about 10 micrometers.
  • Embodiment 17 provides the method of Embodiment 16, wherein the injection pipette tip has a bore diameter of about 1 micrometer to about 3 micrometers.
  • Embodiment 18 provides the method of Embodiment 16, wherein the injection pipette tip has a bore diameter of about 4 micrometers to about 10 micrometers.
  • Embodiment 19 provides the method of Embodiment 16, wherein the injection pipette tip has a bore diameter of about 4 micrometers to about 7 micrometers.
  • Embodiment 20 provides the method of any of Embodiments 1-19, wherein the injection pipette comprises glass.
  • Embodiment 21 provides the method of any of Embodiments 1-20, further comprising freezing the cell for storage.
  • Embodiment 22 provides the method of any of Embodiments 1-21, wherein the control is a predetermined value.
  • Embodiment 23 provides the method of any of Embodiments 1-21, wherein the control is the electrical resistance or capacitance measured using a control viable cell under the same conditions.
  • Embodiment 24 provides a method of performing intracytoplasmic sperm injection into an oocyte, the method comprising: (a) determining viability of the oocyte by the method of any of Embodiments 1-2, and (b) injecting a sperm cell into the viable oocyte through the injection pipette tip.
  • Embodiment 25 provides a method of performing intracytoplasmic sperm injection into an oocyte, the method comprising: (a) determining membrane piercing of the oocyte by the method of any of Embodiments 3-4, and (b) injecting a sperm cell into the viable oocyte through the injection pipette tip.
  • Embodiment 26 provides the method of any of Embodiments 24-25, wherein the injection pipette is preloaded with a sperm cell.
  • Embodiment 27 provides the method of any of Embodiments 24-26, wherein the sperm cell is injected into the viable oocyte within one minute after the viability of the oocyte is determined.
  • Embodiment 28 provides the method of any of Embodiments 24-27, wherein the sperm cell is injected into the viable oocyte within thirty seconds after the viability of the oocyte is determined.
  • Embodiment 29 provides the method of any of Embodiments 24-28, wherein the cell is attached to a holding pipette.
  • Embodiment 30 provides the method of any of Embodiments 24-29, wherein the injection pipette tip has a bore diameter of about 1 micrometer to about 10 micrometers.
  • Embodiment 31 provides the method of Embodiment 30, wherein the injection pipette tip has a bore diameter of about 4 micrometers to about 10 micrometers.
  • Embodiment 32 provides the method of Embodiment 30, wherein the injection pipette tip has a bore diameter of about 4 micrometers to about 7 micrometers.
  • Embodiment 33 provides the method of any of Embodiments 24-32, wherein the control is a predetermined value.
  • Embodiment 34 provides the method of any of Embodiments 24-33, wherein the control is the electrical resistance or electrical capacitance measured using a control viable cell under the same conditions.
  • Embodiment 35 provides a method of injecting material into a cell, the method comprising: determining viability of the cell by the method of any of Embodiments 1-2, and injecting material into the cell through the injection pipette tip.
  • Embodiment 36 provides a method of injecting material into a cell, the method comprising: determining membrane piercing of the cell by the method of any of
  • Embodiments 3-4 and injecting material into the cell through the injection pipette tip.
  • Embodiment 37 provides the method of any of Embodiments 35-36, wherein the material is selected from the group consisting of a peptide, a protein, a fatty acid, a carbohydrate, a metal, a basic element, a nucleic acid, a microparticle, a vector, a small molecule, a dye, a fluorescent molecule, a reporter, a pharmaceutical, and a cell.
  • Embodiment 38 provides the method of any of Embodiments 35-37, wherein the biological material is a cell.
  • Embodiment 39 provides the method of any of Embodiments 35-38, wherein the cell is attached to a holding pipette.
  • Embodiment 40 provides the method of any of Embodiments 35-39, wherein the injection pipette tip has a bore diameter of about 1 micrometer to about 10 micrometers.
  • Embodiment 41 provides the method of Embodiment 40, wherein the injection pipette tip has a bore diameter of about 1 micrometer to about 3 micrometers.
  • Embodiment 42 provides the method of Embodiment 40, wherein the inj ection pipette tip has a bore diameter of about 4 micrometers to about 10 micrometers.
  • Embodiment 43 provides the method of Embodiment 40, wherein the inj ection pipette tip has a bore diameter of about 4 micrometers to about 7 micrometers.
  • Embodiment 44 provides the method of any of Embodiments 35-43, wherein the control is a predetermined value.
  • Embodiment 45 provides the method of any of Embodiments 35-44, wherein the control is the electrical resistance or electrical capacitance measured using a control viable cell under the same conditions.
  • Embodiment 46 provides the method of any of Embodiments 1-45, wherein the control is a known reference value.

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Abstract

La présente invention concerne des procédés d'évaluation d'informations, par exemple concernant la viabilité et la pénétration membranaire de cellules. Selon certains aspects, les procédés selon l'invention se rapportent à la détermination de la viabilité cellulaire et la perforation de membrane de cellules par mesure de la résistance électrique à travers les membranes cellulaires. Dans des modes de réalisation particuliers, les procédés selon la présente invention peuvent être utilisés conjointement avec une procédure de distribution de matériau biologique dans un espace intracellulaire. Dans un mode de réalisation, les procédés selon la présente invention peuvent être utilisés conjointement avec la fertilisation d'un ovocyte, par exemple au cours d'une procédure ICSI.
PCT/US2019/055906 2018-10-11 2019-10-11 Procédés d'évaluation de la viabilité cellulaire et de perforation de membrane cellulaire WO2020077244A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022112460A1 (fr) * 2020-11-26 2022-06-02 Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen Système et dispositif pour caractériser des changements dans le processus de maturation d'un ovocyte, et leur utilisation
GB2609944A (en) * 2021-08-18 2023-02-22 Agm Medical System for assessing cell viability, cell membrane piercing and selecting a cell for microinjection

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120156763A1 (en) * 2001-08-06 2012-06-21 Vanderbilt University Apparatus and Methods for Using Biological Material to Discriminate an Agent
US20160278366A1 (en) * 2008-11-11 2016-09-29 H. Randall Craig Microfluidic embryo and gamete culture systems

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120156763A1 (en) * 2001-08-06 2012-06-21 Vanderbilt University Apparatus and Methods for Using Biological Material to Discriminate an Agent
US20160278366A1 (en) * 2008-11-11 2016-09-29 H. Randall Craig Microfluidic embryo and gamete culture systems

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANDERSON ET AL.: "Electrical Detection of Cellular Penetration during Microinjection with Carbon Nanopipettes", NANOTECHNOLOGY, vol. 25, no. 24, 20 June 2014 (2014-06-20), XP020266137 *
BAU ET AL.: "Carbon Nanoelectrodes for Single- Cell Probing", NANOTECHNOLOGY, vol. 26, no. 18, 16 April 2015 (2015-04-16), pages 185101, XP020283959, DOI: 10.1088/0957-4484/26/18/185101 *
TAN ET AL.: "Robust computer-controlled system for intracytoplasmic sperm injection and subsequent cell electro-activation", INT J MED ROBOTICS COMPUT ASSIST SURG, vol. 5, 23 January 2009 (2009-01-23), pages 85 - 98, XP055704198 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022112460A1 (fr) * 2020-11-26 2022-06-02 Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen Système et dispositif pour caractériser des changements dans le processus de maturation d'un ovocyte, et leur utilisation
GB2609944A (en) * 2021-08-18 2023-02-22 Agm Medical System for assessing cell viability, cell membrane piercing and selecting a cell for microinjection
WO2023021447A1 (fr) * 2021-08-18 2023-02-23 Agm Medical Système d'évaluation de la viabilité cellulaire, perforation de membrane cellulaire et sélection d'une cellule pour micro-injection
GB2609944B (en) * 2021-08-18 2024-06-05 Agm Medical System for assessing cell viability, cell membrane piercing and selecting a cell for microinjection

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