Compositions and methods for determining the concentration of live cells in a population of cells comprising cell aggregates

Abstract

The present invention relates to methods for conducting cell assays for determining a concentration of live cells in a composition of cells that comprises cell aggregates, including pluripotent stem cells.

Description

Attorney Docket No. 132042-5008- WOCOMPOSITIONSAND METHODS FOR DETERMINING THE CONCENTRATION OF LIVE CELLS IN A POPULATION OF CELLS COMPRISING CELL AGGREGATESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to U.S. Patent Application No. 63 / 748,594 filed on January 23, 2025, entitled “COMPOSITIONS AND METHODS FOR DETERMINING THE CONCENTRATION OF LIVE CELLS IN A POPULATION OF CELLS COMPRISING CELL AGGREGATES,’’ which is incorporated herein by reference in its entirety.FIELD

[0002] The disclosure generally relates to methods for determining the concentration of live cells in a population of cells including, for example, a population of cells containing cell aggregates.BACKGROUND

[0003] During multiple stages of cell therapy product manufacturing, release, and formulation, it is required to know the concentration of viable cells within such product, intermediate, or starting material. Automated cell counting assays have been developed to determine the concentration of viable cells in cell suspensions. However, for compositions comprising cell aggregates, some assays provide more accurate total cell counts with inaccurate percentage viability measurements, whereas other assays provide more accurate percentage viability measurements with inaccurate total cell counts. Without accurate values for either measurement, a lower concentration of viable cells may be reported, which can result in inaccurate dosing during product, intermediate, or starting material preparations, thus impacting the yield and / or quality of a downstream product.

[0004] Thus, there is a need for improved methods for determining the concentration of viable cells in cell compositions containing cell aggregates, including pluripotent stem cells (PSCs).SUMMARY

[0005] The present disclosure addresses the above need by providing a method for determining the concentration of live cells in a cell composition that comprises cell aggregates.Attorney Docket No. 132042-5008- WO

[0006] In an aspect, described herein is a method of determining a concentration of live (e.g., viable) cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; calculating a first concentration of total cells in the first aliquot of cells; and calculating a percentage viability of cells in the second aliquot of cells, wherein the concentration of live cells in the cell composition is determined as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0007] In some embodiments, the first concentration of total cells in the first aliquot of cells is calculated by quantifying a total concentration of nuclei in the first aliquot of cells.

[0008] In some embodiments, the step of calculating the first concentration of total cells in the first aliquot of cells comprises contacting the first aliquot of cells with a composition comprising a cell lysis reagent.

[0009] In some embodiments, the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0010] In some embodiments, the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium dihydrate.

[0011] In some embodiments, the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0012] In some embodiments, the method further comprises staining nuclei of the lysed cells with a stain.

[0013] In some embodiments, the stain comprises 4',6-diamidino-2-phenylindole (DAPI).

[0014] In some embodiments, the first concentration of total cells in the first aliquot of cells is calculated by quantifying the total concentration of nuclei of the lysed cells.

[0015] In some embodiments, the step of calculating the percentage viability of cells in the second aliquot of cells comprises staining the second aliquot of cells with one or more stains.

[0016] In some embodiments, the one of more stains comprises DAPI to stain nuclei of dead cells and / or acridine orange (AO) to stain nuclei of dead cells and live cells.

[0017] In some embodiments, the percentage viability of cells in the second aliquot of cells is calculated by: (a) quantifying a concentration of dead cells and a second concentration of totalAttorney Docket No. 132042-5008- WOcells; (b) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (c) dividing the total number of live cells per volume by the second concentration of total cells.

[0018] In some embodiments, the step of calculating the first concentration of total cells in the first aliquot of cells comprises: (a) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (b) staining nuclei of the lysed cells with a stain; and (c) quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells.

[0019] In some embodiments, the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0020] In some embodiments, the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0021] In some embodiments, the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0022] In some embodiments, the stain comprises DAPI.

[0023] In some embodiments, the step of calculating the percentage viability of cells in the second aliquot of cells comprises: (a) contacting the second aliquot of cells with one or more stains; (b) quantifying a concentration of dead cells and a second concentration of total cells; (c) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (d) calculating the percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the second concentration of total cells.

[0024] In some embodiments, the one or more stains is DAPI and / or AO.

[0025] In some embodiments, the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0026] In some embodiments, the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0027] In some embodiments, an aggregate in the cell aggregates comprises about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.Attorney Docket No. 132042-5008- WO

[0028] In an aspect described herein is a method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (ii) staining nuclei of the lysed cells with a stain; and (iii) quantifying the total concentration of nuclei in the lysed cells; wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells; and (d) determining the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0029] In some embodiments, the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0030] In some embodiments, the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0031] In some embodiments, the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0032] In some embodiments, step of staining nuclei of the lysed cells with a stain comprises contacting the lysed cells with DAPI.

[0033] In some embodiments, the step of contacting the second aliquot of cells with one or more stains comprises contacting the second aliquot of cells with DAPI and / or AO.

[0034] In some embodiments, the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0035] In some embodiments, the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).Attorney Docket No. 132042-5008- WO

[0036] In some embodiments, an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0037] In an aspect, described herein is a method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate; (ii) staining nuclei of the lysed cells with a stain, wherein the stain comprises DAPI; and (iii) quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains, wherein the one or more stains comprise DAPI and / or AO; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells; and (d) determining the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0038] In an aspect, described herein is a method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) determining a total concentration of nuclei of cells in lysed cells in the first aliquot of cells, wherein the total concentration of nuclei in the lysed cells corresponds to a first concentration of total cells in the first aliquot; (c) determining a percentage viability of cells in the second aliquot of cells; and (d) calculating the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0039] In some embodiments, the step of determining the total concentration of nuclei in the first aliquot of cells comprises: (a) contacting the first aliquot of cells with a composition comprisingAttorney Docket No. 132042-5008- WOa cell lysis reagent to produce the lysed cells; (b) staining nuclei of the lysed cells with a stain; and (c) quantifying the total concentration of nuclei in the lysed cells.

[0040] In some embodiments, the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0041] In some embodiments, the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0042] In some embodiments, the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0043] In some embodiments, the stain comprises DAPI.

[0044] In some embodiments, the step of determining the percentage viability of cells in the second aliquot of cells comprises: (a) contacting the second aliquot of cells with one or more stains; (b) quantifying a concentration of dead cells and a second concentration of total cells; (c) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (d) calculating the percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the second concentration of total cells.

[0045] In some embodiments, the one or more stains comprises DAPI and / or AO.

[0046] In some embodiments, the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0047] In some embodiments, the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0048] In some embodiments, an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0049] In an aspect, described herein is a method for separately determining a first concentration of total cells and a percentage viability of cells within a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells, (ii) staining nuclei of the lysed cells with a stain; and (iii) quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysedAttorney Docket No. 132042-5008- WOcells corresponds to the first concentration of total cells in the first aliquot of cells; and (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells.

[0050] In some embodiments, the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0051] In some embodiments, the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0052] In some embodiments, the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0053] In some embodiments, the stain comprises DAPI.

[0054] In some embodiments, the one or more stains comprises DAPI and / or AO.

[0055] In some embodiments, the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0056] In some embodiments, the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cell (PRPs).

[0057] In some embodiments, an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0058] In an aspect, described herein is a system for determining a concentration of live cells of a cell composition comprising cell aggregates, the system comprising: (a) a means for obtaining a portion of the cell composition and splitting the portion into a first aliquot and a second aliquot of cells; (b) a means for measuring a total concentration of nuclei of total cells in the first aliquot of cells, wherein the total concentration of nuclei of total cells corresponds to a concentration of total cells in the first aliquot of cells; (c) a means for measuring a percentage viability of cells in the second aliquot of cells; and (d) a means for determining the concentration of live cells in the cell composition as the concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.Attorney Docket No. 132042-5008- WO

[0059] In as aspect, described herein is a method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (c) contacting the lysed cells with 4',6-diamidino-2-phenylindole (DAPI) to stain nuclei from the cells in the first aliquot of cells; (d) determining a concentration of stained nuclei in the first aliquot of cells; (e) contacting the second aliquot of cells with DAPI to stain nuclei in dead cells and acridine orange (AO) to stain live cells and dead cells; (f) determining a concentration of dead cells and a concentration of total cells; (g) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the concentration of total cells; and (h) calculating a percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the concentration of total cells, wherein the concentration of live cells in the cell composition is determined as the concentration of stained nuclei in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0060] In an aspect, described herein is a method of determining a percentage post-thaw attachment value for post-thaw cells comprising cell aggregates, the method comprising: (a) adding a cell dye to post-thaw cells; (b) determining a number of live cells in the post-thaw cells once per hour for twenty-four hours after addition of the cell dye; and (c) calculating the percentage post-thaw attachment value as a concentration of live cells at a time point greater than one hour divided by a concentration of live cells at a one hour time point.

[0061] In some embodiments, the post-thaw cells are proliferative if the percentage postthaw attachment value is greater than 70%.

[0062] In some embodiments, the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0063] In some embodiments, the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0064] In some embodiments, an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0065] In an aspect, described herein is a method of determining if post-thaw cells comprising cell aggregates reach a target cell confluence, the method comprising: (a) seedingAttorney Docket No. 132042-5008- WOthe post-thaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2; (b) adding a cell dye to the post-thaw cells; (c) incubating the post-thaw cells for twenty-four hours; (d) exchanging the medium with a fresh medium every twenty-four hours; (e)measuring a percentage of confluence after ninety-six hours; and (f) determining if the post-thaw cells reach the target cell confluence, wherein the target cell confluence is about 60% to about 70%.

[0066] In some embodiments, the post thaw cells reach the target cell confluence if the percentage confluence value is greater than 60% after ninety-six hours.

[0067] In some embodiments, the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0068] In some embodiments, the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0069] In some embodiments, an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0070] In an aspect, described herein is a method of determining quality of post-thaw cells comprising cell aggregates, the method comprising: (a) adding a cell dye for a first time to the post-thaw cells and seeding the post-thaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2; (b) determining a number of live cells in the post-thaw cells once per hour for twenty-four hours after adding the cell dye for the first time; (c) calculating a percentage post-thaw attachment value as a number of live cells six hours after adding the cell dye for the first time divided by a number of live cells one hour after adding the cell dye for the first time; (d) exchanging the medium with a fresh medium and adding the cell dye for a second time to the post-thaw cells twenty-four hours after adding the cell dye for the first time; (e) incubating the post-thaw cells for seventy-two hours after adding the cell dye for a second time while exchanging the medium with a fresh medium every twenty-four hours; and (f) determining a percentage confluence value ninety-six hours after adding the cell dye for the first time, wherein a percentage post-thaw attachment value greater than 70% in step (c) and a percentage confluence value greater than 60% in step (f) indicates a high quality cell composition.

[0071] In some embodiments, the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0072] In some embodiments, in the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).Attorney Docket No. 132042-5008- WO

[0073] In some embodiments, an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.BRIEF DESCRIPTION OF THE OF THE DRAWINGS

[0074] The foregoing summary, as well as the following detailed description, will be better understood when read in conjunction with the appended figures. For the purpose of illustration, shown in the figures are embodiments. It should be understood, however, that the summary, detailed description, and figures are not limited to the precise arrangements, examples, and instrumentalities shown.

[0075] Figure 1A-1C: A) Percentage post-thaw attachment over 96 hours for sequentially degraded Freeze-Hold (FH) samples initially seeded at 20,000 cells / cm2. B) Percentage confluence over 96 hours for sequentially degraded FH samples initially seeded at 20,000 cells / cm2. C) Percentage post-thaw attachment at the 6 hour time point for sequentially degraded FH samples initially seeded at 20,000 cells / cm2. UT = untreated, 1xFH = 1 freeze-hold cycle, and 2xFH = 2 freeze-hold cycles. Error bars indicate the standard error of the mean (SEM). Statistical significance levels are denoted as: **p<0.01, ***p<0.001.

[0076] Figure 2A-2D: Percentage post-thaw attachment as measured by live cells / mm2after six hours for three seeding densities (20,000 cells / cm2, 40,000 cells / cm2, and 60,000 cells / cm2) for A) Lot #2, B) Lot #2, C) Lot #3, and D) Lot #4. R2values for each curve are indicated on the corresponding plot.DETAILED DESCRIPTION

[0077] Provided herein are methods for determining the concentration of live cells in a cell composition comprising cell aggregates (e.g., PSCs, ESCs, or iPSCs). Such methods may comprise measuring a concentration of total cells and a percentage viability and determining the concentration of live cells in the cell composition by multiplying the concentration of total cells and the percentage viability. Advantageously, the methods disclosed herein provide for increased accuracy and efficiency over conventional methods used for cell concentration assessments of compositions containing cell aggregates.Definitions

[0078] Definitions of certain terms to be used herein are provided. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood.Attorney Docket No. 132042-5008- WO

[0079] The terms “approximately,” “substantially,” and “about” may be used to mean within ±20% of a target value in some embodiments, within ±10% of a target value in some embodiments, within ±5% of a target value in some embodiments, within ±2% of a target value in some embodiments. The terms “approximately,” “substantially,” and “about” may include the target value.

[0080] The term “cells in aggregates,” “aggregated cells,” “aggregates,” “clumps,” “clusters,” “cell clusters,” or “cell aggregates” refers to cells associated with one another by means of intercellular adhesion or other mechanisms of association.

[0081] The term “aliquot,” “sample,” or “portion” refers to a subset of a larger composition that may be taken for the purposes of executing a method. Any subsets of a larger composition may be taken or separated from the larger composition at the same time or at about the same time to ensure that each subset of the larger sample is representative of the entire composition. The composition may be in the form of a solution or a suspension. A composition may be split or separated into multiple subsets to create aliquots, samples, or portions.

[0082] The term “cell lysis” refers to the breaking down and / or permeabilization of the plasma membrane enclosing the contents of a cell. In some examples, lysis may be performed by physical or chemical means. In some examples, lysis may be induced via incubation with a composition comprising about <2.5% w / w citric acid monohydrate (CAS-no. 5949-29-1), about <1% w / w octylphenol ethoxylate (Triton X-100) (CAS-no. 9036-19-5), and about <0.2% w / w trisodium citrate dihydrate (Cas no. 6132-04-03) including, for example, Solution 10™ (Chemometec, Allerod, Denmark, Product No.: 910-3010). Cell lysis may involve incubation, agitation, vortexing, trituration, and / or mixing.

[0083] The term “post-thaw cell” or “post-thawed cells” refers to a composition of cells that have been frozen and then thawed to be used for a downstream process or product (e.g., research, cell therapy, drug product, or drug substance). The term may refer to compositions that were stored in liquid nitrogen or in a freezer at a temperature between 0 °C and -80 °C. Post-thaw cells may have been thawed by a variety of methods, including but not limited to water baths, bead baths, or other thawing methods known in the art. The post-thaw cells may have been frozen with a cryopreservant (e.g., CryoStorlO™ and / or DMSO).

[0084] The term “cell bank,” “master cell bank,” or “working cell bank” refers to a collection of cells that are preserved or maintained for future use (e.g., research, drug development, or cell therapy). The collection of cells may be preserved by freezing or storage in liquid nitrogen.Attorney Docket No. 132042-5008- WOCryopreservation may be used to keep the composition of cells intact. In this method, cells are frozen using liquid nitrogen to maintain their viability over extended periods of time. The collection of cells may be derived from a single source, such as a cell line, primary cell culture, or production lot.

[0085] The term “medium,” “media,” “growth media,” “growth medium,” “cell culture media,” or “cell culture medium,” used interchangeably herein, refers to a liquid or gel that provides nutrients and other components to support cellular growth and function. The media may contain amino acids, vitamins, carbohydrates, inorganic salts, trace elements, growth factors, hormones, buffers, or any combination thereof.

[0086] The term “confluence” or “confluency” refers to a degree to which an area is covered by adherent cells in a culture dish. Confluence may be measured by a percentage of surface area in the culture dish covered by the adherent cells. Complete confluence may be when a culture dish is approximately 100% covered by adherent cells. Subconfluence may refer to a culture dish that is incompletely covered by adherent cells. As used herein, a population of cells at a target confluence percentage may refer to a population of cells with at least 60% confluence. A composition of cells may be considered to have reached proliferation to target confluence if the composition of cells reaches a percentage confluence of 60% or more after four days of growth when seeded at a density of 20,000 cells / cm2.

[0087] The term “percentage recovery” or “percent recovery” refers to a proportion of a measured number of cells with respect to an expected number of cells. Percentage recovery may be calculated in relation to total cells per ml_, live cells per ml_, or percentage viability. A calculation of percentage recovery may enable assessment of the accuracy of a method by comparing an expected value to an observed value. Specifically, percentage recovery may be calculated by dividing an observed value by an expected value and multiplying the quotient by 100.

[0088] The term “percentage post-thaw attachment” or “percent post-thaw attachment” refers to a proportion of live cells that have adhered to a culture dish or plate with respect to the number of live cells initially seeded onto the culture dish or plate. Percentage post-thaw attachment may be calculated at any time point after initial seeding of the plate. A percentage post-thaw attachment greater than 50% six hours after initial seeding of the plate may indicate a healthy cell composition.

[0089] The term “seed,” “seeding,” “plate,” or “plating” refers to a process of spreading cells (e.g., pluripotent stem cells) in or on a culture vessel for cell culture. The culture vessel may beAttorney Docket No. 132042-5008- WOmade of any suitable material known in the art. Cells can be seeded at any density, including but not limited to 10,000 cells / cm2up to 100,000 cells / cm2.Methods of Determining a Live Cell Concentration Within in a Cell Composition Comprising Cell Aggregates

[0090] Provided herein are methods of determining the concentration of live cells within a cell composition (e.g., a drug substance or a drug product) comprising cell aggregates. The concentration of live cells may be determined by multiplying the concentration of total cells in a first aliquot of cells obtained from the cell composition and the percentage viability of a second aliquot of cells obtained from the cell composition.

[0091] Exemplary methods may comprise one or more of the following steps: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) calculating a first concentration of total cells in the first aliquot of cells; and (c) calculating a percentage viability of cells in the second aliquot of cells, wherein the concentration of live cells in the cell composition is determined as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0092] In some embodiments, the cell compositions may comprise concentrations of about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270, 000, about 280,000, about 290,000, about 300,000, about 310,000, about 320,000, about 330,000, about 340,000, about 350,000, about 360,000, about 370,000, about 380,000, about 390,000, about 400,000, about 410,000, about 420,000, about 430,000, about 440,000, about 450,000, about 460,000, about 470,000, about 480,000, about 490,000, about 500,000, about 510,000, about 520,000, about 530,000, about 540,000, about 550,000, about 560,000, about 570,000, about 580,000, about 590,000, about 600,000, about 610,000, about 620,000, about 630,000, about 640,000, about 650,000, about 660,000, about 670,000, about 680,000, about 690,000, about 700,000, about 710,000, about 720,000, about 730,000, about 740,000, about 750,000, about 760,000, about 770,000, about 780,000, about 790,000, about 800,000, about 810,000, about 820,000, about 830,000, about 840,000, about 850,000, about 860,000, about 870,000, about 880,000, about 890,000, about 900,000, about 910,000, about 920,000, about 930,000, about 940,000, about 950,000, about 960,000, about 970,000, about 980,000, about 990,000, about 1,000,000, about 1,100,000, about 1,200,000, about 1,300,000, about 1,400,000, aboutAttorney Docket No. 132042-5008- WO1,500,000, about 1,600,000, about 1,700,000, about 1,800,000, about 1,900,000, about 2,000,000, about 3,000,000, about 4,000,000, about 5,000,000, about 6,000,000, about 7,000,000, about 8,000,000, about 9,000,000, or about 10,000,000 or more cells per milliliter.

[0093] In some embodiments, the cell aggregates may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 or more cells. In other embodiments, the cell aggregates may comprise about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, about 910, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990, or about 1 ,000 or more cells.

[0094] In some embodiments, a cell aggregate is about 40 pm in diameter. In some embodiments, the cell aggregates may be about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 pm in diameter. In some embodiments, the cell aggregates may be about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1 ,000 pm in diameter.

[0095] The cell aggregates may comprise any cell type. In some embodiments, the cell aggregates may comprise retinal cells (e.g., photoreceptor cells, retinal epithelial cells, retinal ganglion cells, neuroretinal cells, photoreceptor progenitor cells, progenitors thereof, or any combination of photoreceptor cells). In some embodiments, the cell aggregates may compriseAttorney Docket No. 132042-5008- WOphotoreceptor progenitor cells. In some embodiments, the cell aggregates may comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs). In other embodiments, the cell aggregates may comprise cells derived or differentiated from iPSCs or ESCs. In some embodiments, the cell aggregates may comprise glial and / or amacrine cells. In some embodiments, the cell aggregates may comprise microglia. In some embodiments, the microglia may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates may comprise cardiomyocytes. In some embodiments, the cardiomyocytes may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates may comprise dopaminergic neurons. In some embodiments, the dopaminergic neurons may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates may comprise mixtures of two or more different cell types. In some embodiments, the cell aggregates may comprise photoreceptor progenitor cells and ESCs, iPSCs and / or PSCs. In some embodiments, the cell aggregates may comprise microglia and ESCs, iPSCs and / or PSCs. In some embodiments, the cell aggregates may comprise cardiomyocytes and ESCs, iPSCs and / or PSCs. In some embodiments, the cell aggregates may comprise dopaminergic neurons and ESCs, iPSCs and / or PSCs.

[0096] In some embodiments, the cell composition may comprise cell aggregates and / or singularized (e.g., non-aggregated) cells. In some embodiments, the cell composition may comprise only cell aggregates. In other embodiments, the cell composition may comprise only singularized cells. In some embodiments, the cell composition may comprise living cells and dead cells. In another embodiment, the cell composition may comprise only living cells. In another embodiment, the cell composition may comprise only dead cells.

[0097] In some embodiments, a singularized live cell is about 7 pm in diameter. In some embodiments, the singularized live may be about 1, about 2, about 3, about 4, about 5, about 6, about?, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 pm in diameter.

[0098] In some embodiments, a singularized dead cell is about 9 pm in diameter. In some embodiments, the singularized dead cell may be about 1, about 2, about 3, about 4, about 5, about6, about ?, about 8, about 9, about 10, about 11, about 12, or about 13 pm in diameter.

[0099] In some embodiments, cell aggregates comprise about 50% to about 100% of the cell composition’s biomass. In some embodiments, cell aggregates comprise about 50%, aboutAttorney Docket No. 132042-5008- WO55%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the cell composition’s biomass. In some embodiments, cell aggregates comprise about 70% or at least about 70% of the cell composition’s biomass.

[0100] In some embodiments, singularized cells comprise about 0% to about 60% of the cell composition’s biomass. In some embodiments, singularized cells comprise about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 45%, about 50%, about 55%, or about 60% the cell composition’s biomass. In some embodiments, singularized cells comprise about 30% of the cell composition’s biomass.

[0101] In some embodiments, the cell aggregates may comprise living cells and / or dead cells. In another embodiment, the cell aggregates may comprise only living cells. In another embodiment, the cell aggregates may comprise only dead cells.

[0102] The cell compositions may be provided as a first or a second aliquot. In some embodiments, the first and second aliquots may be provided from the same vial, lot, or production run. In some embodiments, the first and second aliquots may have the same concentration of cells, equal concentration of cells, about the same concentration of cells, substantially similar concentration of cells, or different concentrations of cells. In some embodiments, the first and second aliquots may be obtained from a homogenously distributed solution or suspension. In some embodiments, the first and second aliquots may be provided from a cell composition comprising cell aggregates that has been mixed or homogenized. In some embodiments, the first and / or second aliquots may be measured to quantify the cell concentration. In other embodiments, the cell concentration may be determined using a cell counter. In some embodiments, the concentration of cells may be measured as cells / mL. In some embodiments, the first and second aliquot may contain the same number of cells, equal numbers of cells, about the same number of cells, or substantially similar numbers of cells.Attorney Docket No. 132042-5008- WO

[0103] Additionally, the cell composition comprising cell aggregates or aliquots thereof may be contacted with a cell lysis reagent. Exemplary cell lysis reagents may include a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate. In some embodiments, the cell lysis reagent is Solution 10™ (Chemometec, Allerod, Denmark, Product No.: 910-3010). In some embodiments, the cell lysis reagent is a composition comprising citric acid monohydrate, octylphenol ethoxylate, and trinsodium citrate dihydrate. In some embodiments, the cell lysis reagent comprises octylphenol ethoxylate. In some embodiments, the cell lysis reagent is a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate. In some embodiments, the composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate may be at about 50% full strength. In further embodiments, the composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate may be at about 94% full strength solution. In some embodiments, the composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate may be at about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% full strength. In some embodiments, the composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate may be at about 100% full strength.

[0104] In some embodiments, the cell lysis reagent may be incubated with the cell composition for about 0 seconds to about 90 seconds. In some embodiments, the cell lysis reagent may be incubated with the cell composition for about 0 seconds, about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 11 seconds, about 12 seconds, about 13 seconds, about 14 seconds, about 15 seconds, about 16 seconds, about 17 seconds, about 18 seconds, about 19 seconds, about 20 seconds, about 21 seconds, about 22 seconds, about 23 seconds, about 24 seconds, about 25 seconds, about 26 seconds, about 27 seconds, about 28 seconds, about 29 seconds, about 30 seconds, about 31 seconds, about 32 seconds, about 33 seconds, about 34 seconds, about 35 seconds, about 36 seconds, about 37 seconds, about 38 seconds, about 39 seconds, about 40 seconds, about 41 seconds, about 42 seconds, about 43 seconds, about 44 seconds, about 45 seconds, about 46 seconds, about 47 seconds, about 48 seconds, about 49 seconds, about 50 seconds, about 51 seconds, about 52 seconds, about 53 seconds, about 54 seconds, about 55 seconds, about 56 seconds, about 57Attorney Docket No. 132042-5008- WOseconds, about 58 seconds, about 59 seconds, about 60 seconds, about 61 seconds, about 62 seconds, about 63 seconds, about 64 seconds, about 65 seconds, about 66 seconds, about 67 seconds, about 68 seconds, about 69 seconds, about 70 seconds, about 71 seconds, about 72 seconds, about 73 seconds, about 74 seconds, about 75 seconds, about 76 seconds, about 77 seconds, about 78 seconds, about 79 seconds, about 80 seconds, about 81 seconds, about 82 seconds, about 83 seconds, about 84 seconds, about 85 seconds, about 86 seconds, about 87 seconds, about 88 seconds, about 89 seconds, or about 90 seconds. In some embodiments, the cell lysis reagent may be incubated with the cell composition for about 30 seconds to about 60 seconds. In some embodiments, the cell lysis reagent may be incubated with the cell composition for about 45 seconds.

[0105] In some embodiments, the cell composition or aliquots thereof may be mixed by pipetting. In some embodiments, the mixing by pipetting may be performed on cell compositions containing singularized cells. In other embodiments, the mixing by pipetting may be performed on cell compositions containing cell aggregates. In other embodiments, the mixing by pipetting may be performed on cell compositions containing singularized cells and cell aggregates. In some embodiments, the mixing by pipetting may be performed on cell compositions that have been lysed. In other embodiments, the mixing by pipetting may be performed on cell compositions that have been contacted with a cell lysis reagent.

[0106] In some embodiments, mixing by pipetting may be performed with a P1000 ora P200 pipette. In some embodiments, mixing by pipetting may be performed with a serological pipet. In some embodiments, the serological pipet may be a 10 mL pipet. In some embodiments, the mixing volume may be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, aboutAttorney Docket No. 132042-5008- WO96%, about 97%, about 98%, about 99%, about 100% or more of the total solution or suspension volume of the cell composition.

[0107] In some embodiments, the mixing by pipetting may be performed about 1 time to about 100 times. In some embodiments, the mixing by pipetting may be performed about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 , about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 times. In some embodiments, the mixing by pipetting may be performed about 3 times. In some embodiments, the mixing by pipetting may be performed about 4 times. In some embodiments, the mixing by pipetting may be performed about 5 times.

[0108] In some embodiments, mixing may be performed by vortexing, agitating, mixing, or shaking the cell composition or aliquots thereof. In other embodiments, mixing may be performed with gentle, intermediate, or heavy force. In some embodiments, mixing may be performed with gentle force.

[0109] The cell composition containing cell aggregates or aliquots thereof may be stained with a cell staining reagent. In some embodiments, the cells and the stain are mixed at a 1:1 volume ratio (e.g., 50 pl of cells and 50 pL of cell staining reagent). In some embodiments, the cell staining reagent is DAPI. In some embodiments, the cell staining reagent is Acridine Orange (AO). In other embodiments, the cell staining reagent is Propidium Iodide (PI). In other embodiments, the cells may be stained with any suitable reagent for counting, including Acridine Orange (AO), Propidium Iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green, SYTOX red, DAPI, NucLight Rapid Red, and / or DRAQ5. In some embodiments, the cells may be stained with Acridine Orange and Propidium Iodide at the same time (e.g., AOPI). In some embodiments, the cell staining reagent stains the nuclei of cells.

[0110] In some embodiments, the stained cells may be counted with a cell counter. In some embodiments, the stained cells may be counted with a NucleoCounter® NC-200™ (Chemometec;Attorney Docket No. 132042-5008- WOAllerod, Denmark, Cat# 900-0201) according to the manufacturer’s instructions. In some embodiments, cells may be counted after contact with a cell lysis reagent, a cell staining reagent, and / or a nuclei staining reagent. In some embodiments, cells may be counted after contact with a cell lysis reagent and a nuclei staining reagent. In some embodiments, cells may be counted after contact with a nuclei staining reagent.

[0111] The percentage viability of cells within a composition (e.g., a composition comprising cell aggregates) may be calculated by determining a ratio between the number of live cells and the number of total cells within the composition including, for example an aliquot obtained from the composition. For example, the percentage viability may be calculated by dividing the number of live cells by the number of total cells. In further embodiments, the number of total cells in a composition may be calculated by dividing the number of live cells by the percentage viability.

[0112] The methods herein may be used for determining a concentration of live cells in a cell composition comprising cell aggregates. Such methods may comprise one or more of the following steps: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (ii) staining nuclei of the lysed cells with a stain; and (iii) quantifying the total concentration of nuclei in the lysed cells; wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells; and (d) determining the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0113] The methods herein may be used for separately determining a first concentration of total cells and a percentage viability of cells within a cell composition comprising cell aggregates. Such methods may comprise one or more of the following steps: (a) providing a first aliquot of cells and a second aliquot of cells; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising aAttorney Docket No. 132042-5008- WOcell lysis reagent to produce lysed cells, (ii) staining nuclei of the lysed cells with a stain; and (iii) quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; and (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells.

[0114] The disclosure provides systems for determining a concentration of live cells of a cell composition comprising cell aggregates. Such systems may comprise one or more of the following components: (a) a means for obtaining a portion of the cell composition and splitting the portion into a first aliquot and a second aliquot of cells; (b) a means for measuring a total concentration of nuclei of total cells in the first aliquot of cells, wherein the total concentration of nuclei of total cells corresponds to a concentration of total cells in the first aliquot of cells; (c) a means for measuring a percentage viability of cells in the second aliquot of cells; and (d) a means for determining the concentration of live cells in the cell composition as the concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0115] The methods herein may be used for determining a concentration of live cells in a cell composition comprising cell aggregates. Such methods may comprise one or more of the following steps: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (c) contacting the lysed cells with 4',6-diamidino-2-phenylindole (DAPI) to stain nuclei from the cells in the first aliquot of cells; (d) determining a concentration of stained nuclei in the first aliquot of cells; (e) contacting the second aliquot of cells with DAPI to stain nuclei in dead cells and acridine orange (AO) to stain live cells and dead cells; (f) determining a concentration of dead cells and a concentration of total cells; (g) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the concentration of total cells; and (h) calculating a percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the concentration of total cells, wherein the concentration of live cells in the cell composition is determined as theAttorney Docket No. 132042-5008- WOconcentration of stained nuclei in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.Methods of Determining Health and / or Quality of Post-Thaw Pluripotent Stem Cells

[0116] Provided herein are methods of determining percentage post-thaw attachment value and / or confluence for a post-thaw cell composition (e.g., a drug substance or a drug product) comprising cell aggregates. The percentage post-thaw attachment may be determined by dividing a concentration of live cells at a time point greater than one hour by the number of cells at the one hour time point. The confluence of the cell population may be determined by seeding cells and determining if cells reach a target confluence (e.g., percentage confluence greater than 60 or 70%) after 96 hours.

[0117] Exemplary methods for determining a percentage post-thaw attachment value for post-thaw cells comprising cell aggregates (e.g., post-thaw cell composition) may comprise one or more of the following steps: (a) adding a cell dye to post-thaw cells; (b) determining a number of live cells in the post-thaw cells once per hour for twenty-four hours after addition of the cell dye; and (c) calculating the percentage post-thaw attachment value as a concentration of live cells at a time point greater than one hour divided by a concentration of live cells at a one hour time point.

[0118] Exemplary methods for determining if post-thaw cells comprising cell aggregates reach a target cell confluence may comprise one or more of the following steps: seeding the postthaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2; (b) adding a cell dye to the post-thaw cells; (c) incubating the post-thaw cells for twenty-four hours; (d) exchanging the medium with a fresh medium every twenty-four hours; (e) measuring a percentage of confluence after ninety-six hours; and (f) determining if the post-thaw cells reach the target cell confluence, wherein the target cell confluence is about 60% to about 70%.

[0119] In some embodiments, the post-thaw cell composition may comprise concentrations of about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270, 000, about 280,000, about 290,000, about 300,000, about 310,000, about 320,000, about 330,000, about 340,000, about 350,000, about 360,000, about 370,000, about 380,000, about 390,000, about 400,000, about 410,000, about 420,000, about 430,000, about 440,000, about 450,000, about 460,000, about 470,000, about 480,000, about 490,000, about 500,000, about 510,000, about 520,000, about 530,000, about 540,000, about 550,000, about 560,000, about 570,000, about 580,000, about 590,000, about 600,000, about 610,000, about 620,000, about 630,000,Attorney Docket No. 132042-5008- WOabout 640,000, about 650,000, about 660,000, about 670,000, about 680,000, about 690,000, about 700,000, about 710,000, about 720,000, about 730,000, about 740,000, about 750,000, about 760,000, about 770,000, about 780,000, about 790,000, about 800,000, about 810,000, about 820,000, about 830,000, about 840,000, about 850,000, about 860,000, about 870,000, about 880,000, about 890,000, about 900,000, about 910,000, about 920,000, about 930,000, about 940,000, about 950,000, about 960,000, about 970,000, about 980,000, about 990,000, about 1,000,000, about 1,100,000, about 1,200,000, about 1,300,000, about 1,400,000, about 1,500,000, about 1,600,000, about 1,700,000, about 1,800,000, about 1,900,000, about 2,000,000, about 3,000,000, about 4,000,000, about 5,000,000, about 6,000,000, about 7,000,000, about 8,000,000, about 9,000,000, or about 10,000,000 or more cells per milliliter.

[0120] In some embodiments, the cell aggregates in the post-thaw cell composition may comprise about 1 to about 1,000 or more cells. In some embodiments, the cell aggregates in the post-thaw cell composition may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 or more cells. In other embodiments, the cell aggregates may comprise about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, about 910, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990, or about 1,000 or more cells.

[0121] In some embodiments, a cell aggregate in the post-thaw cell composition is about 10 pm to about 1,000 pm in diameter. In some embodiments, the cell aggregates may be about 10,Attorney Docket No. 132042-5008- WOabout 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 m in diameter. In some embodiments, the cell aggregates may be about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1,000 pm in diameter. In some embodiments, a cell aggregate in the post-thaw cell composition is about 40 pm in diameter

[0122] The cell aggregates in the post-thaw cell composition may comprise any cell type. In some embodiments, the cell aggregates may comprise retinal cells (e.g., photoreceptor cells, retinal epithelial cells, retinal ganglion cells, neuroretinal cells, photoreceptor progenitor cells, progenitors thereof, or any combination of photoreceptor cells). In some embodiments, the cell aggregates may comprise photoreceptor progenitor cells. In some embodiments, the cell aggregates may comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs). In other embodiments, the cell aggregates may comprise cells derived or differentiated from iPSCs. In some embodiments, the cell aggregates may comprise glial and / or amacrine cells. In some embodiments, the cell aggregates may comprise microglia. In some embodiments, the microglia may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates may comprise cardiomyocytes. In some embodiments, the cardiomyocytes may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates may comprise dopaminergic neurons. In some embodiments, the dopaminergic neurons may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates may comprise mixtures of two or more different cell types. In some embodiments, the cell aggregates may comprise photoreceptor progenitor cells and iPSCs and / or PSCs. In some embodiments, the cell aggregates may comprise microglia and iPSCs and / or PSCs. In some embodiments, the cell aggregates may comprise cardiomyocytes and iPSCs and / or PSCs. In some embodiments, the cell aggregates may comprise dopaminergic neurons and iPSCs and / or PSCs.

[0123] In some embodiments, the post-thaw cells may comprise cell aggregates and / or singularized (e.g., non-aggregated) cells. In some embodiments, the post-thaw cells may comprise only cell aggregates. In other embodiments, the post-thaw cells may comprise only singularized cells. In some embodiments, the post-thaw cells may comprise living cells and deadAttorney Docket No. 132042-5008- WOcells. In another embodiment, the post-thaw cells may comprise only living cells. In another embodiment, the post-thaw cells may comprise only dead cells.

[0124] In some embodiments, cell aggregates in the post-thaw cell composition comprise about 50% to about 100% of the cell composition’s biomass. In some embodiments, cell aggregates comprise about 50%, about 55%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the cell composition’s biomass. In some embodiments, cell aggregates in the post-thaw cell composition comprise about 70% or at least about 70% of the cell composition’s biomass.

[0125] In some embodiments, singularized cells comprise about 0% to about 60% of the post-thaw cell composition’s biomass. In some embodiments, singularized cells comprise about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 45%, about 50%, about 55%, or about 60% the post-thaw cell composition’s biomass. In some embodiments, singularized cells comprise about 30% of the post-thaw cell composition’s biomass.

[0126] In some embodiments, the cell aggregates in the post-thaw cell composition may comprise living cells and / or dead cells. In another embodiment, the cell aggregates in the postthaw cell composition may comprise only living cells. In another embodiment, the cell aggregates in the post-thaw cell composition may comprise only dead cells.

[0127] In some embodiments, the post-thaw cells containing cell aggregates or aliquots thereof may be stained with a cell staining reagent. In some embodiments, the post-thaw cells and the stain are mixed ata 1:1 volume ratio (e.g., 50 pl of cells and 50 pL of cell staining reagent). In some embodiments, the cell staining reagent is NucLight Rapid Red (Sartorius; Cat. No. 4717). In other embodiments, the post-thaw cells may be stained with any suitable reagent for counting, including Acridine Orange (AO), Propidium Iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green, SYTOX red, DAPI, NucLight Rapid Red, and / or DRAQ5. In some embodiments, the postAttorney Docket No. 132042-5008- WOthaw cells may be stained with NucLight Rapid Red dye prior to plating. In some embodiments, the staining reagent stains the nuclei of cells of the post-thaw cell composition.

[0128] In some embodiments, the post-thaw cells containing cell aggregates or aliquots thereof may be contacted with a rho kinase inhibitor (ROCKi). Exemplary rho kinase inhibitors include Fasudil, Y27632, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE-104, Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, H-1152P, ROKa inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012, and quinazoline. For example, rho kinase inhibitors are described in Liao et. al., J Cardiovasc Pharmacol. 2007 Jul; 50(1): 17— 24, which is herein incorporated by reference in its entirety. In some embodiments, the ROCKi is Y27632. In some embodiments, the post-thaw cells are contacted with the ROCKi before plating and / or seeding the post-thaw cells. In some embodiments, the post-thaw cells are cryopreserved in medium containing ROCKi. In some embodiments, the ROCKi is removed from the composition of post-thaw cells.

[0129] In some embodiments, the ROCKi is removed from the composition of post-thaw cells about 12 hours to about 36 hours after plating. In some embodiments, the ROCKi is removed from the composition of post-thaw cells about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours after plating. In some embodiments, a ROCKi is removed from the composition of post-thaw cells about 24 hours after plating.

[0130] In some embodiments, the post-thaw cells in a medium are seeded and / or plated at a density of about 5,000 cells / cm2to about 100,000 cells / cm2. In some embodiments, the postthaw cells in a medium are seeded and / or plated at a density of about 5,000 cells / cm2, about 10,000 cells / cm2, about 15,000 cells / cm2, about 20,000 cells / cm2, about 25,000 cells / cm2, about 30,000 cells / cm2, about 35,000 cells / cm2, about 40,000 cells / cm2, about 45,000 cells / cm2, about 50,000 cells / cm2, about 55,000 cells / cm2, about 60,000 cells / cm2, about 65,000 cells / cm2, about 70,000 cells / cm2, about 75,000 cells / cm2, about 80,000 cells / cm2, about 85,000 cells / cm2, about 90,000 cells / cm2, about 95,000 cells / cm2, or about 100,000 cells / cm2. In some embodiments, the post-thaw cells in a medium are seeded and / or plated at a density of about 20,000 cells / cm2, about 40,000 cells / cm2, or about 60,000 cells / cm2. In some embodiments, the post-thaw cells in a medium are seeded and / or plated at a density of about 20,000 cells / cm2. In some embodiments,Attorney Docket No. 132042-5008- WOthe post-thaw cells in a medium are seeded and / or plated at a density of about 40,000 cells / cm2. In some embodiments, the post-thaw cells in a medium are seeded and / or plated at a density of about 60,000 cells / cm2.

[0131] In some embodiments, the post-thaw cells may be counted with a cell counter. In some embodiments, the post-thaw cells may be counted with a NucleoCounter® NC-200™ (Chemometec; Allerod, Denmark, Cat# 900-0201) according to the manufacturer’s instructions. In some embodiments, the post-thaw cells may be counted after contact with a cell lysis reagent, a cell staining reagent, and / or a nuclei staining reagent. In some embodiments, the post-thaw cells may be counted after contact with a cell lysis reagent and a nuclei staining reagent. In some embodiments, the post-thaw cells may be counted after contact with a nuclei staining reagent (e.g., stained cells). In some embodiments, the NC-200™ may be used to determine a percentage viability of a composition of post-thaw cells.

[0132] In some embodiments, the post-thaw cells may be tracked, monitored, or imaged with a live-cell imaging and tissue culture incubator. In some embodiments, the post-thaw cells may be imaged (e.g., monitored or tracked) with a IncuCyte® Live-Cell Analysis System (Sartorius; Gottingen, Germany, Cat. No. SX5) according to the manufacturer’s instructions. In some embodiments, the post-thaw cells may be tracked, monitored, or imaged after contact with a cell lysis reagent, a cell staining reagent, and / or a nuclei staining reagent. In some embodiments, the cell staining reagent and / or nuclei staining reagent is NucLight Rapid Red (Sartorius; Cat. No.4717). In some embodiments, the post-thaw cells may be imaged (e.g., monitored or tracked) after contact with a nuclei staining reagent. In some embodiments, the post-thaw cells may be imaged every two hours for four days after contact with a nuclei staining reagent.

[0133] In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red Dye and then imaged about every 5 minutes to about every 150 minutes with the IncuCyte® Live-Cell Analysis System. In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye and then imaged about every 5 minutes, about every 10 minutes, about every 15 minutes, about every 20 minutes, about every 25 minutes, about every 30 minutes, about every 35 minutes, about every 40 minutes, about every 45 minutes, about every 50 minutes, about every 55 minutes, about every 60 minutes, about every 65 minutes, about every 70 minutes, about every 75 minutes, about every 80 minutes, about every 85 minutes, about every 90 minutes, about every 95 minutes, about every 100 minutes, about every 105 minutes, about every 110 minutes, about every 115 minutes, about every 120 minutes, about every 125 minutes, about every 130 minutes, about every 135 minutes, about every 140 minutes, about every 145 minutes,Attorney Docket No. 132042-5008- WOor about every 150 minutes with the IncuCyte® Live-Cell Analysis System. In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye and then imaged about every 15 minutes with the IncuCyte® Live-Cell Analysis System. In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye and then imaged about every 120 minutes with the IncuCyte® Live-Cell Analysis System. In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye, imaged about every 15 minutes for about the first 60 minutes, then imaged about every 120 minutes for the following 23 hours, and then stained again with NucLight Rapid Red Dye, and imaged every 120 minutes thereafter with the IncuCyte® Live-Cell Analysis System.

[0134] In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye and then imaged (e.g., monitored or tracked) for about 1 hour to about 120 hours with the IncuCyte® Live-Cell Analysis System. In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye and then imaged for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about 105 hours, about 106 hours, about 107 hours, about 108 hours, about 109 hours, about 110 hours, about 111 hours, about 112 hours, about 113 hours, about 114 hours, about 115 hours, about 116 hours, about 117 hours, about 118 hours, about 119 hours, or about 120 hours with the IncuCyte® Live-Cell Analysis System. In some embodiments, the post-thaw cells may be stainedAttorney Docket No. 132042-5008- WOwith NucLight Rapid Red dye and then imaged for about 96 hours with the IncuCyte® Live-Cell Analysis System. In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye and then imaged for about 24 hours with the IncuCyte® Live-Cell Analysis System, stained again with NucLight Rapid Red dye and then imaged for an additional 72 hours with the IncuCyte® Live-Cell Analysis System .

[0135] In some embodiments, the NucLight Rapid Red dye may be added to the post-thaw cells about 1 time to about 5 times. In some embodiments, the NucLight Rapid Red dye may be added to the post-thaw cells about 1 time, about 2 times, about 3 times, about 4 times, or about 5 times within 96 hours after plating the post-thaw cells. In some embodiments, the post-thaw cells may be stained with NucLight Rapid Red dye and then imaged for about 24 hours with the IncuCyte® Live-Cell Analysis System, and then more NucLight Rapid Red dye may be added and then imaged for an additional 72 hours.

[0136] In some embodiments, the NucLight Rapid Red dye is mixed with the post-thaw cells at a 1:4,000 ratio to a 1:500 ratio. In some embodiments, the NucLight Rapid Red dye is mixed with the post-thaw cells at a 1:2,000 ratio during initial cell seeding. In some embodiments, the NucLight Rapid Red dye is mixed with the post-thaw cells ata 1:1,000 ratio during media change.

[0137] In some embodiments, a medium of the post-thaw cells may be changed about every 12 hours to about every 36 hours. In some embodiments, the medium of the post-thaw cells may be changed about every 12 hours, about every 13 hours, about every 14 hours, about every 15 hours, about every 16 hours, about every 17 hours, about every 18 hours, about every 19 hours, about every 20 hours, about every 21 hours, about every 22 hours, about every 23 hours, about every 24 hours, about every 25 hours, about every 26 hours, about every 27 hours, about every 28 hours, about every 29 hours, about every 30 hours, about every 31 hours, about every 32 hours, about every 33 hours, about every 34 hours, about every 35 hours, or about every 36 hours. In some embodiments, a medium of the post-thaw cells may be changed about every 24 hours.

[0138] The methods herein may be used for determining quality of post-thaw cells comprising cell aggregates. Such methods may comprise one or more of the following steps: (a) adding a cell dye for a first time to the post-thaw cells and seeding the post-thaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2; (b) determining a number of live cells in the post-thaw cells once per hour for twenty-four hours after adding the cell dye for the first time; (c) calculating a percentage post-thaw attachment value as a number of live cells six hours after adding the cell dye for the first time divided by a number of live cells one hour after adding theAttorney Docket No. 132042-5008- WOcell dye for the first time; (d) exchanging the medium with a fresh medium and adding the cell dye for a second time to the post-thaw cells twenty-four hours after adding the cell dye for the first time; (e) incubating the post-thaw cells for seventy-two hours after adding the cell dye for a second time while exchanging the medium with a fresh medium every twenty-four hours; and (f) determining a percentage confluence value ninety-six hours after adding the cell dye for the first time, wherein a percentage post-thaw attachment value greater than 70% in step (c) and a percentage confluence value greater than 60% in step (f) indicates a high quality cell composition.Kits

[0139] The invention also provides kits. The kits include cells, cell lysis solution (e.g., a cell lysis solution comprising octylphenol ethoxylate, and optionally further comprising citric acid monohydrate, and / or trisodium citrate dihydrate), and / or cell dyes (e.g., DAPI, acridine orange, and / or propidium iodide), either alone or in combination in suitable packaging, and written material that can include instructions for use. Such kits may also include information, such as scientific literature references, package insert materials, and / or summaries of these and the like, which indicate or establish the activities and / or advantages of the composition, and / or which describe methods of their use. Such information may be based on the results of various studies, for example, studies using PSCs (e.g., ESCs or iPSCs), cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells. The kit may further contain another reagent . Suitable packaging and additional articles for use (e.g., cassettes, pipette tips, and / or microcentrifuge tubes) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and / or promoted to clinics, researchers, pharmaceutical companies, and the like. In an embodiment, the invention provides a kit comprising cells, a cell lysis reagent, and / or cell dye(s) for use in determining a concentration of live cells in a cell composition comprising cell aggregates, or any of the other methods described herein.

[0140] The individual components of the kits can be packaged in a variety of containers, e.g., vials, tubes, microtiter well plates, bottles, and the like. Other reagents can be included in separate containers and provided with the kit; e.g., positive control samples, negative control samples, buffers, cell culture media, etc. Preferably, the kits will also include instructions for use.Illustration of Subject Technology as Clauses

[0141] Various examples of aspects are described as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples, and do not limit the subject technology. Identifications of the figures and reference numbers are provided below merely as examples and for illustrative purposes, and the clauses are not limited by those identifications.Attorney Docket No. 132042-5008- WO

[0142] Clause 1: A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) calculating a first concentration of total cells in the first aliquot of cells; and (c) calculating a percentage viability of cells in the second aliquot of cells, wherein the concentration of live cells in the cell composition is determined as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0143] Clause 2: The method of clause 1, wherein the first concentration of total cells in the first aliquot of cells is calculated by quantifying a total concentration of nuclei in the first aliquot of cells.

[0144] Clause 3: The method of clause 1, wherein the step of calculating the first concentration of total cells in the first aliquot of cells comprises contacting the first aliquot of cells with a composition comprising a cell lysis reagent.

[0145] Clause 4: The method of clause 3, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0146] Clause 5: The method of clause 4, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium dihydrate.

[0147] Clause 6: The method of clause 5, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0148] Clause 7: The method of clause 6, further comprising staining nuclei of the lysed cells with a stain.

[0149] Clause 8: The method of clause 7, wherein the stain comprises 4',6-diamidino-2-phenylindole (DAPI).

[0150] Clause 9: The method of clause 8, wherein the first concentration of total cells in the first aliquot of cells is calculated by quantifying the total concentration of nuclei of the lysed cells.

[0151] Clause 10: The method of clause 1, wherein the step of calculating the percentage viability of cells in the second aliquot of cells comprises staining the second aliquot of cells with one or more stains.Attorney Docket No. 132042-5008- WO

[0152] Clause 11 : The method of clause 10, wherein the one of more stains comprises DAPI to stain nuclei of dead cells and / or acridine orange (AO) to stain nuclei of dead cells and live cells.

[0153] Clause 12: The method of clause 11, wherein the percentage viability of cells in the second aliquot of cells is calculated by: (a) quantifying a concentration of dead cells and a second concentration of total cells; (b) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (c) dividing the total number of live cells per volume by the second concentration of total cells.

[0154] Clause 13: The method of clause 1, wherein the step of calculating the first concentration of total cells in the first aliquot of cells comprises: (a) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (b) staining nuclei of the lysed cells with a stain; and (c) quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells.

[0155] Clause 14: The method of clause 13, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0156] Clause 15: The method of clause 14, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0157] Clause 16: The method of clause 15, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0158] Clause 17: The method of clause 16, wherein the stain comprises DAPI.

[0159] Clause 18: The method of clause 1, wherein the step of calculating the percentage viability of cells in the second aliquot of cells comprises: (a) contacting the second aliquot of cells with one or more stains; (b) quantifying a concentration of dead cells and a second concentration of total cells; (c) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (d) calculating the percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the second concentration of total cells.

[0160] Clause 19: The method of clause 18, wherein the one or more stains is DAPI and / or AO.Attorney Docket No. 132042-5008- WO

[0161] Clause 20: The method of clause 1, wherein the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0162] Clause 21: The method of clause 1, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0163] Clause 22: The method of clause 1, wherein an aggregate in the cell aggregates comprises about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0164] Clause 23: A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (ii) staining nuclei of the lysed cells with a stain; and (iii) quantifying the total concentration of nuclei in the lysed cells; wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells; and (d) determining the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0165] Clause 24: The method of clause 23, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0166] Clause 25: The method of clause 24, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0167] Clause 26: The method of clause 25, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0168] Clause 27: The method of clause 23, wherein the step of staining nuclei of the lysed cells with a stain comprises contacting the lysed cells with DAPI.Attorney Docket No. 132042-5008- WO

[0169] Clause 28: The method of clause 23, wherein the step of contacting the second aliquot of cells with one or more stains comprises contacting the second aliquot of cells with DAPI and / or AO.

[0170] Clause 29: The method of clause 23, wherein the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0171] Clause 30: The method of clause 23, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0172] Clause 31: The method of clause 23, wherein an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0173] Clause 32: A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate; (ii) staining nuclei of the lysed cells with a stain, wherein the stain comprises DAPI; and (iii) quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains, wherein the one or more stains comprise DAPI and / or AO; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells; and (d) determining the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0174] Clause 33: A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have aboutAttorney Docket No. 132042-5008- WOthe same concentration of cells of the cell composition; (b) determining a total concentration of nuclei of cells in lysed cells in the first aliquot of cells, wherein the total concentration of nuclei in the lysed cells corresponds to a first concentration of total cells in the first aliquot; (c) determining a percentage viability of cells in the second aliquot of cells; and (d) calculating the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0175] Clause 34: The method of clause 33, wherein the step of determining the total concentration of nuclei in the first aliquot of cells comprises: (a) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce the lysed cells; (b) staining nuclei of the lysed cells with a stain; and (c) quantifying the total concentration of nuclei in the lysed cells.

[0176] Clause 35: The method of clause 34, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0177] Clause 36: The method of clause 35, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0178] Clause 37: The method of clause 36, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0179] Clause 38: The method of clause 37, wherein the stain comprises DAPI.

[0180] Clause 39: The method of clause 33, wherein the step of determining the percentage viability of cells in the second aliquot of cells comprises: (a) contacting the second aliquot of cells with one or more stains; (b) quantifying a concentration of dead cells and a second concentration of total cells; (c) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (d) calculating the percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the second concentration of total cells.

[0181] Clause 40: The method of clause 39, wherein the one or more stains comprises DAPI and / or AO.

[0182] Clause 41: The method of clause 39, wherein the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).Attorney Docket No. 132042-5008- WO

[0183] Clause 42: The method of clause 39, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0184] Clause 43: The method of clause 39, wherein an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0185] Clause 44: A method for separately determining a first concentration of total cells and a percentage viability of cells within a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells; (b) calculating a first concentration of total cells in the first aliquot of cells, comprising: (i) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells, (ii) staining nuclei of the lysed cells with a stain; and (iii) quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; and (c) calculating a percentage viability of cells in the second aliquot of cells, comprising: (i) contacting the second aliquot of cells with one or more stains; (ii) quantifying a concentration of dead cells and a second concentration of total cells; (iii) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; and (iv) dividing the total number of live cells per volume by the second concentration of total cells.

[0186] Clause 45: The method of clause 44, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

[0187] Clause 46: The method of clause 45, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

[0188] Clause 47: The method of clause 46, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

[0189] Clause 48: The method of clause 44, wherein the stain comprises DAPI.

[0190] Clause 49: The method of clause 44, wherein the one or more stains comprises DAPI and / or AO.

[0191] Clause 50: The method of clause 44, wherein the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).Attorney Docket No. 132042-5008- WO

[0192] Clause 51: The method of clause 44, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cell (PRPs).

[0193] Clause 52: The method of clause 44 wherein an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0194] Clause 53: A system for determining a concentration of live cells of a cell composition comprising cell aggregates, the system comprising: (a) a means for obtaining a portion of the cell composition and splitting the portion into a first aliquot and a second aliquot of cells; (b) a means for measuring a total concentration of nuclei of total cells in the first aliquot of cells, wherein the total concentration of nuclei of total cells corresponds to a concentration of total cells in the first aliquot of cells; (c) a means for measuring a percentage viability of cells in the second aliquot of cells; and (d) a means for determining the concentration of live cells in the cell composition as the concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0195] Clause 54: A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition; (b) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells; (c) contacting the lysed cells with 4',6-diamidino-2-phenylindole (DAPI) to stain nuclei from the cells in the first aliquot of cells; (d) determining a concentration of stained nuclei in the first aliquot of cells; (e) contacting the second aliquot of cells with DAPI to stain nuclei in dead cells and acridine orange (AO) to stain live cells and dead cells; (f) determining a concentration of dead cells and a concentration of total cells; (g) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the concentration of total cells; and (h) calculating a percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the concentration of total cells, wherein the concentration of live cells in the cell composition is determined as the concentration of stained nuclei in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

[0196] Clause 55: A method of determining a percentage post-thaw attachment value for post-thaw cells comprising cell aggregates, the method comprising: (a) adding a cell dye to postthaw cells; (b) determining a number of live cells in the post-thaw cells once per hour for twenty-four hours after addition of the cell dye; and (c) calculating the percentage post-thaw attachmentAttorney Docket No. 132042-5008- WOvalue as a concentration of live cells at a time point greater than one hour divided by a concentration of live cells at a one hour time point.

[0197] Clause 56: The method of clause 55 wherein, the post-thaw cells are proliferative if the percentage post-thaw attachment value is greater than 70%.

[0198] Clause 57: The method of clause 55, wherein the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0199] Clause 58: The method of clause 55, wherein the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0200] Clause 59: The method of clause 55, wherein an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0201] Clause 60: A method of determining if post-thaw cells comprising cell aggregates reach a target cell confluence, the method comprising: (a) seeding the post-thaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2; (b) adding a cell dye to the post-thaw cells; (c) incubating the post-thaw cells for twenty-four hours; (d) exchanging the medium with a fresh medium every twenty-four hours; (e) measuring a percentage of confluence after ninety-six hours; and (f) determining if the post-thaw cells reach the target cell confluence, wherein the target cell confluence is about 60% to about 70%.

[0202] Clause 61 : The method of clause 60, wherein the post thaw cells reach the target cell confluence if the percentage confluence value is greater than 60% after ninety-six hours.

[0203] Clause 62: The method of clause 60, wherein the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0204] Clause 63: The method of clause 60, wherein the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0205] Clause 64: The method of clause 60, wherein an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0206] Clause 65: A method of determining quality of post-thaw cells comprising cell aggregates, the method comprising: (a) adding a cell dye for a first time to the post-thaw cells and seeding the post-thaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2; (b) determining a number of live cells in the post-thaw cells once per hour for twenty-four hoursAttorney Docket No. 132042-5008- WOafter adding the cell dye for the first time; (c) calculating a percentage post-thaw attachment value as a number of live cells six hours after adding the cell dye for the first time divided by a number of live cells one hour after adding the cell dye for the first time; (d) exchanging the medium with a fresh medium and adding the cell dye for a second time to the post-thaw cells twenty-four hours after adding the cell dye for the first time; (e) incubating the post-thaw cells for seventy-two hours after adding the cell dye for a second time while exchanging the medium with a fresh medium every twenty-four hours; and (f) determining a percentage confluence value ninety-six hours after adding the cell dye for the first time, wherein a percentage post-thaw attachment value greater than 70% in step (c) and a percentage confluence value greater than 60% in step (f) indicates a high quality cell composition.

[0207] Clause 66: The method of clause 65, wherein the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

[0208] Clause 67: The method of clause 65, wherein the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

[0209] Clause 68: The method of clause 65, wherein an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

[0210] The present disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0211] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.EXAMPLESExample 1: Percentage Recovery Using Two Methods on NC-200™Attorney Docket No. 132042-5008- WO

[0212] Pluripotent stem cell compositions comprising cell aggregates were assessed for percentage recovery using the NucleoCounter® NC-200™ (Chemometec; Cat# 900-0201). A twofold dilution series was then made ranging from 6.25x104cells to 4*106cells for a total of seven samples. Cells were prepared for counting by transferring 100 pL of a cell composition comprising cell aggregates to a microcentrifuge tube. The suspension of cells was gently mixed by pipetting. Each dilution was mixed with 100 L of lysis buffer (<2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate) for a 1:1 ratio of cell sample to lysis buffer. The mixtures were then incubated for approximately 45 seconds, and then gently mixed by pipetting 3 to 5 times. Each dilution was then sampled by a Via-1 Cassette™ on the NC-200™ using the “Viability and Cell Count - Aggregated Cells Assay”. Briefly, the cell sample was loaded by inserting the tip of the Via 1 -Cassette™ into the cell suspension and the piston was pressed. The Vial -Cassette™ was then immediately placed into the NucleoCounter® NC-200™. The “Viability and Cell Count - Aggregated Cells Assay” protocol was then run.

[0213] Prepared cell suspensions not mixed with the lysis buffer were also counted using the “Viability and Cell Count Assay” protocol. Briefly, the cell sample was mixed and subsequently loaded by inserting the tip of the Via 1 -Cassette™ into the cell suspension and the piston was pressed. The Vial -Cassette™ was then immediately placed into the NucleoCounter® NC-200™. The “Viability and Cell Count Assay” protocol was then run.

[0214] The assay was performed with multiple technical replicates. As shown in Table 1 , the “Viability and Cell Count - Aggregated Cells Assay” produces reproducible and high percentage recovery values using a dilution series, whereas the “Viability and Cell Count Assay” produces largely reproducible yet substantially lower percentage recovery values.Table 1. Calculated Percentage Recovery using “Viability and Cell Count - Aggregated Cells Assay” or “Viability and Cell Count Assay” Protocol on the NC-200™.Attorney Docket No. 132042-5008- WOExample 2: Percentage Viability ofO% Viable Cells Using Two Protocols on NC-200™

[0215] The NC-200™ was also used to assess percentage viability for cells exposed to 70% ethanol to produce a population of dead cells. Briefly, for seven replicates, cell suspensions comprising cell aggregates were spun down at 200 x g for 5 min. The supernatants were removed, and 270 pL of medium and 730 pL of 100% ethanol were added to each tube. The mixtures were incubated for 2 minutes on ice and then spun down at 1,000 x g for 5 minutes. Next, two washes were performed with 1 mL of media and 1 ,000 x g for 5-minute spins. A control sample comprising non-aggregated cells was also assessed using the same method described. The cell compositions were confirmed to be dead by Trypan Blue staining. Each biological replicate was then sampled by a Via-1™ cassette on the NC-200™ using the “Viability and Cell Count -Aggregated Cells Assay” and the “Viability and Cell Count Assay” protocol. The results shown in Table 2 demonstrate that for samples confirmed by cell staining to be dead, the “Viability and Cell Count - Aggregated Cells Assay” inaccurately reported values much higher than 0% for calculated percentage viability for each biological replicate, whereas the ““Viability and Cell Count Assay” reported values much closer to 0%.Table 2. Calculated Percentage Viability for 0% Viable Samples using “Viability and Cell Count Aggregated Cells Assay” Protocol on the NC-200™.Example 3: Two-Protocol Method to Determine Accurate Cell Concentrations and Percentage Viability on the NC-200™

[0216] As demonstrated in Examples 1 and 2, the “Viability and Cell Count - Aggregated Cells Assay” reports higher values for percentage recovery of total cells / mL and total cell concentrations with higher accuracy, but inaccurate percentage viability values (e.g., live cells / mLAttorney Docket No. 132042-5008- WOdivided by total cells / mL). Conversely, the “Viability and Cell Count Assay” reports lower values for percentage recovery of total cells / mL yet more accurate percentage viability values. Using either method alone may produce an inaccurate value for cell concentration. As such, a hybrid, two-protocol method may be used to obtain accurate values for both total cells / mL and percentage viability in order to correctly calculate a concentration of live cells in a cell composition comprising cell aggregates. Therefore, a new method was developed, which involved acquiring total cells / mL using the Cell Count of Aggregated Cells Assay and percentage viability using the Viability and Cell Count Assay. To obtain the live cells / mL result, the total cells / mL result from the Count of Aggregate Cells Assay would be multiplied by the percentage viability from the Viability and Cell Count Assay. A custom NC200 protocol, “Viability and Cell Count - Aggregated (1: Total & 2: Viability)”, was created to combine the two default NC200 protocols and automatically calculate live cells / mL using this new approach.

[0217] The “Viability and Cell Count - Aggregated (1 : Total & 2: Viability)” method was then compared to aforementioned methods. Briefly, four samples of varying expected approximate cell viability (0%, 50%, 70%, and stock) were assessed by each protocol (“Viability and Cell Count -Aggregated Cells Assay,” “Viability and Cell Count Assay,” and “Viability and Cell Count -Aggregated (1: Total & 2: Viability)”) for percentage viability. Each of the cell compositions assessed were comprised of cell aggregates. For each expected viability sample, 200 pL of cells were transferred to a microcentrifuge tube and gently mixed by pipetting 3 to 5 times. Then, 100 pL of cells were transferred to a Tube 1 and 100 pL of cells were transferred to a Tube 2. The Tube 2 samples were placed on ice during Tube 1 processing.

[0218] The cells in Tube 1 were mixed with 100 pL of lysis buffer (Solution 10™) for a 1:1 ratio of cell sample to lysis buffer. The Tube 1 mixture was incubated for approximately 45 seconds, and then gently mixed by pipetting 3 to 5 times. The Tube 1 mixture was then sampled by a first Via-1™ cassette on the NC-200™ for the Viability and Cell Count - Aggregated Cells Assay and Viability and Cell Count- Aggregated (1: Total & 2: Viability) to obtain a value for total cells / mL. Next, Tube 2 was mixed by pipetting 3 to 5 times and sampled with a second Via-1™ cassette on the NC-200™ using each assay. The calculated percentage viability for each assay is shown in Table 3, which demonstrates that the hybrid two-count method, “Viability and Cell Count - Aggregated (1 : Total & 2: Viability),” produces percentage viability results that align with the Viability and Cell Count assay result, which was previously shown to give a more accurate readout of percentage viability.Attorney Docket No. 132042-5008- WOTable 3. Calculated Percentage Viability for Varying Viability Samples using Three Protocols on the NC-200™.&Example 4: Replating Assay for Post-Thaw Cells

[0219] Post-thaw percentage viability of pluripotent stem cells (PSCs), as measured by automated cell counting, has been found to be highly variable both between and among specific cell banks, which limits the ability of the cell banks to support downstream manufacturing. The replating assay presented herein accounts for an accurate initial count of live cells, post-plating latent cell death, percentage post-thaw attachment, culture stabilization, proliferation initiation, and active proliferation of post-thaw cells.

[0220] In the replating assay, cells were first thawed in a water bath. A percentage viability was determined using a NC-200™. NucLight Rapid Red dye (Sartorius; Cat. No. 4717) and rho kinase inhibitors (ROCKi) were added to a media diluent. The thawed cell suspension was then added to the media-dye diluent at three cell densities (20,000 cells / cm2, 40,000 cells / cm2, and 60,000 cells / cm2). The samples were plated and the plate was then placed into the IncuCyte® Live-Cell Analysis System (Sartorius; Gottingen, Germany, Cat. No. SX5). Images of the samples were taken every 15 minutes for the first 45 minutes, and then every two hours for up to 24 hours. Twenty-four hours after plating, the ROCKi were removed, old media was exchanged for fresh media, and fresh NucLight Rapid Red dye was added. The cells were returned to the IncuCyte® and imaged every two hours for the next seventy-two hours, with media changes every twenty-four hours, for a total of ninety-six hours of analysis post-plating. Percentage post-thaw attachment was determined as the concentration of live cells at a time point greater than one hour divided by the concentration of live cells at the one-hour time point. The addition of NucLight Rapid Red dye prior to plating allowed for accurate labeling and segmentation of individual cells in aggregates. Furthermore, consistent imaging of the stained cells (e.g., every four hours) in addition to one hour post-plating allowed thorough tracking of the cell culture to capture theAttorney Docket No. 132042-5008- WOmoment of cell attachment, post-plating latent cell death, culture stabilization, proliferation initiation, and active proliferation.

[0221] To evaluate the specificity of the replating assay, three samples of PSCs were tested with different levels of degradation (Untreated - UT, 1 Freeze-Hold Cycle - 1xFH, and 2 Freeze-Hold Cycles -2xFH) across multiple technical replicates. A Freeze-Hold Cycle was performed by removing cells from liquid nitrogen storage and placing at -30 °C for three hours. The percentage viability of the post-thaw cells was calculated prior to the addition of media and NucLight Rapid Red dye. The results presented in Table 4 demonstrate that the percentage viability correlates with the degradation state of the post-thaw cells.Table 4. Percentage Viability of Post-Thaw Cells using the NC-200™.

[0222] As shown in Figure 1A, percentage post-thaw attachment increased over time and was correlated with degradation state. For example, the 2xFH samples displayed the lowest percentage post-thaw attachment over time, consistent with dead and / or degraded cells, whereas the untreated samples showed percentage post-thaw attachment greater than 100% after 48 hours, consistent with highly proliferative, living cells. At the six hour time point, the percentage post-thaw attachment again correlates with the degradation state of the PSCs (Figure 1C).

[0223] Percentage confluence was also measured for 96 hours post-plating using the replating assay. Figure 1B shows that the percentage confluence at the 96 hour time point is greatest for the untreated samples, and much lower for the 1xFH and 2xFH degraded samples. Furthermore, the low variance among technical replicates at each time point for each degradation state remained low, further demonstrating the specificity of the assay.

[0224] To evaluate the linearity of the replating assay, three different seeding densities for four different lots of PSCs were tested for percentage post-thaw attachment. PSCs from each lot were seeded at 20,000 cells / cm2, 40,000 cells / cm2, or 60,000 cells / cm2and assessed for percentage post-thaw attachment after 6 hours (Figures 2A-D). The results showed that the assay was reproducible across varying seeding densities for all four lots.

[0225] The replating assay described herein is specific for high (e.g., untreated) and low (e.g., freeze-hold degraded) viability samples, with little variance among replicates as measuredAttorney Docket No. 132042-5008- WOby both percentage post-thaw attachment and percentage confluence. Moreover, the assay is robust for cells plated at densities between 20,000 cells / cm2to 60,000 cells / cm2. Thus, the replating assay can be used to accurately measure the health and / or quality of populations of PSCs, including those comprising cell aggregates.

[0226] Specific embodiments provided herein can be further limited in the claims using “consisting of” or “consisting essentially of’ language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments so claimed are inherently or expressly described and enabled herein.

[0227] In cases where numerical values are indicated herein, the skilled person will understand that the technical effect of the feature in question is ensured within an interval of accuracy, which typically encompasses a deviation of the numerical value given of ± 10% or of ± 5%. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed considering the number of reported significant digits and by applying ordinary rounding techniques.

[0228] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight and median size, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained.

[0229] The terms “a,” “an,” “the” and similar referents used in the context of the description herein (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the specification and does not pose a limitation on the scope of the claims. No language in theAttorney Docket No. 132042-5008- WOspecification should be construed as indicating any non-claimed element essential to the practice of the description.

[0230] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and.”

[0231] Groupings of alternative elements or embodiments provided herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.

[0232] Certain embodiments are described herein, including the best mode known for carrying out methods provided herein. Of course, variations on these described embodiments will become apparent upon reading the foregoing description. One can be expected to employ such variations as appropriate and can be practiced other than as specifically described herein. Accordingly, this description includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the description unless otherwise indicated herein or otherwise clearly contradicted by context.

[0233] It is to be understood that the embodiments provided herein are illustrative of the principles of the description herein. Other modifications that can be employed are within the scope of the description. Thus, by way of example, but not of limitation, alternative configurations can be utilized in accordance with the teachings herein. Accordingly, the presented information is not limited to that precisely as shown and described.

[0234] While the present description has been described and illustrated herein by references to various specific materials, procedures, and examples, it is understood that the description is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the specification being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.Attorney Docket No. 132042-5008- WO

[0235] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood. Although other probes, compositions, methods, and kits similar, or equivalent, to those described herein can be used in the practice described herein, the materials and methods are described herein. It is to be understood that the terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting.

[0236] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance, for example within 2 standard deviations of the mean. About is understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0237] A stated range is understood to be any value between and at the limits of the stated range. As examples, a range between 1 and 5 includes 1, 2, 3, 4, and 5; a range between 1 and 10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and a range between 1 and 100 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100.

[0238] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as described herein.

Claims

Attorney Docket No. 132042-5008- WOWHAT IS CLAIMED IS:

1. A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising:a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition;b) calculating a first concentration of total cells in the first aliquot of cells; and c) calculating a percentage viability of cells in the second aliquot of cells, wherein the concentration of live cells in the cell composition is determined as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

2. The method of claim 1, wherein the first concentration of total cells in the first aliquot of cells is calculated by quantifying a total concentration of nuclei in the first aliquot of cells.

3. The method of claim 1 , wherein the step of calculating the first concentration of total cells in the first aliquot of cells comprises contacting the first aliquot of cells with a composition comprising a cell lysis reagent.

4. The method of claim 3, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

5. The method of claim 4, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium dihydrate.

6. The method of claim 5, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

7. The method of claim 6, further comprising staining nuclei of the lysed cells with a stain.

8. The method of claim 7, wherein the stain comprises 4',6-diamidino-2-phenylindole (DAPI).Attorney Docket No. 132042-5008- WO9. The method of claim 8, wherein the first concentration of total cells in the first aliquot of cells is calculated by quantifying the total concentration of nuclei of the lysed cells.

10. The method of claim 1, wherein the step of calculating the percentage viability of cells in the second aliquot of cells comprises staining the second aliquot of cells with one or more stains.

11. The method of claim 10, wherein the one of more stains comprises DAPI to stain nuclei of dead cells and / or acridine orange (AO) to stain nuclei of dead cells and live cells.

12. The method of claim 11, wherein the percentage viability of cells in the second aliquot of cells is calculated by:a) quantifying a concentration of dead cells and a second concentration of total cells; b) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; andc) dividing the total number of live cells per volume by the second concentration of total cells.

13. The method of claim 1 , wherein the step of calculating the first concentration of total cells in the first aliquot of cells comprises:a) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells;b) staining nuclei of the lysed cells with a stain; andc) quantifying the total concentration of nuclei in the lysed cells,wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells.

14. The method of claim 13, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

15. The method of claim 14, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.Attorney Docket No. 132042-5008- WO16. The method of claim 15, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

17. The method of claim 16, wherein the stain comprises DAPI.

18. The method of claim 1, wherein the step of calculating the percentage viability of cells in the second aliquot of cells comprises:a) contacting the second aliquot of cells with one or more stains;b) quantifying a concentration of dead cells and a second concentration of total cells; c) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; andd) calculating the percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the second concentration of total cells.

19. The method of claim 18, wherein the one or more stains is DAPI and / or AO.

20. The method of claim 1, wherein the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

21. The method of claim 1, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

22. The method of claim 1, wherein an aggregate in the cell aggregates comprises about 3- 25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

23. A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising:a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition;b) calculating a first concentration of total cells in the first aliquot of cells, comprising:Attorney Docket No. 132042-5008- WOi. contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells;ii. staining nuclei of the lysed cells with a stain; andiii. quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells;c) calculating a percentage viability of cells in the second aliquot of cells, comprising:i. contacting the second aliquot of cells with one or more stains;ii. quantifying a concentration of dead cells and a second concentration of total cells;iii. determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; andiv. dividing the total number of live cells per volume by the second concentration of total cells; andd) determining the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

24. The method of claim 23, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

25. The method of claim 24, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

26. The method of claim 25, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

27. The method of claim 23, wherein the step of staining nuclei of the lysed cells with a stain comprises contacting the lysed cells with DAPI.

28. The method of claim 23, wherein the step of contacting the second aliquot of cells with one or more stains comprises contacting the second aliquot of cells with DAPI and / or AO.Attorney Docket No. 132042-5008- WO29. The method of claim 23, wherein the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

30. The method of claim 23, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

31. The method of claim 23, wherein an aggregate in the cell aggregates comprise about 3- 25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

32. A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising:a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition;b) calculating a first concentration of total cells in the first aliquot of cells, comprising:i. contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate;ii. staining nuclei of the lysed cells with a stain, wherein the stain comprises DAPI; andiii. quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells;c) calculating a percentage viability of cells in the second aliquot of cells, comprising:i. contacting the second aliquot of cells with one or more stains, wherein the one or more stains comprise DAPI and / or AO;ii. quantifying a concentration of dead cells and a second concentration of total cells;iii. determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; andAttorney Docket No. 132042-5008- WOiv. dividing the total number of live cells per volume by the second concentration of total cells; andd) determining the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

33. A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising:a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition;b) determining a total concentration of nuclei of cells in lysed cells in the first aliquot of cells, wherein the total concentration of nuclei in the lysed cells corresponds to a first concentration of total cells in the first aliquot;c) determining a percentage viability of cells in the second aliquot of cells; and d) calculating the concentration of live cells in the cell composition as the first concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

34. The method of claim 33, wherein the step of determining the total concentration of nuclei in the first aliquot of cells comprises:a) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce the lysed cells;b) staining nuclei of the lysed cells with a stain; andc) quantifying the total concentration of nuclei in the lysed cells.

35. The method of claim 34, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

36. The method of claim 35, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.Attorney Docket No. 132042-5008- WO37. The method of claim 36, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

38. The method of claim 37, wherein the stain comprises DAPI.

39. The method of claim 33, wherein the step of determining the percentage viability of cells in the second aliquot of cells comprises:a) contacting the second aliquot of cells with one or more stains;b) quantifying a concentration of dead cells and a second concentration of total cells; c) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; andd) calculating the percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the second concentration of total cells.

40. The method of claim 39, wherein the one or more stains comprises DAPI and / or AO.

41. The method of claim 39, wherein the cell composition comprises pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

42. The method of claim 39, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

43. The method of claim 39, wherein an aggregate in the cell aggregates comprise about 3- 25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

44. A method for separately determining a first concentration of total cells and a percentage viability of cells within a cell composition comprising cell aggregates, the method comprising:a) providing a first aliquot of cells and a second aliquot of cells;b) calculating a first concentration of total cells in the first aliquot of cells, comprising:Attorney Docket No. 132042-5008- WOi. contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells,ii. staining nuclei of the lysed cells with a stain; andiii. quantifying the total concentration of nuclei in the lysed cells, wherein the total concentration of nuclei in the lysed cells corresponds to the first concentration of total cells in the first aliquot of cells; and c) calculating a percentage viability of cells in the second aliquot of cells, comprising:i. contacting the second aliquot of cells with one or more stains; ii. quantifying a concentration of dead cells and a second concentration of total cells;iii. determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the second concentration of total cells; andiv. dividing the total number of live cells per volume by the second concentration of total cells.

45. The method of claim 44, wherein the composition comprising the cell lysis reagent comprises octylphenol ethoxylate.

46. The method of claim 45, wherein the composition comprising the cell lysis reagent further comprises citric acid monohydrate and trisodium citrate dihydrate.

47. The method of claim 46, wherein the composition comprising the cell lysis reagent comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trisodium citrate dihydrate.

48. The method of claim 44, wherein the stain comprises DAPI.

49. The method of claim 44, wherein the one or more stains comprises DAPI and / or AO.

50. The method of claim 44, wherein the cell composition comprises pluripotent stem cells (PSCs) , optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).Attorney Docket No. 132042-5008- WO51. The method of claim 44, wherein the cell composition comprises cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cell (PRPs).

52. The method of claim 44 wherein an aggregate in the cell aggregates comprise about 3-25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

53. A system for determining a concentration of live cells of a cell composition comprising cell aggregates, the system comprising:a) a means for obtaining a portion of the cell composition and splitting the portion into a first aliquot and a second aliquot of cells;b) a means for measuring a total concentration of nuclei of total cells in the first aliquot of cells, wherein the total concentration of nuclei of total cells corresponds to a concentration of total cells in the first aliquot of cells;c) a means for measuring a percentage viability of cells in the second aliquot of cells;andd) a means for determining the concentration of live cells in the cell composition as the concentration of total cells in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

54. A method of determining a concentration of live cells in a cell composition comprising cell aggregates, the method comprising:a) providing a first aliquot of cells and a second aliquot of cells, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells of the cell composition;b) contacting the first aliquot of cells with a composition comprising a cell lysis reagent to produce lysed cells;c) contacting the lysed cells with 4',6-diamidino-2-phenylindole (DAPI) to stain nuclei from the cells in the first aliquot of cells;d) determining a concentration of stained nuclei in the first aliquot of cells;e) contacting the second aliquot of cells with DAPI to stain nuclei in dead cells and acridine orange (AO) to stain live cells and dead cells;f) determining a concentration of dead cells and a concentration of total cells;Attorney Docket No. 132042-5008- WOg) determining a total number of live cells per volume in the second aliquot of cells by subtracting the concentration of dead cells from the concentration of total cells; andh) calculating a percentage viability of cells in the second aliquot of cells by dividing the total number of live cells per volume by the concentration of total cells, wherein the concentration of live cells in the cell composition is determined as the concentration of stained nuclei in the first aliquot of cells multiplied by the percentage viability of cells in the second aliquot of cells.

55. A method of determining a percentage post-thaw attachment value for post-thaw cells comprising cell aggregates, the method comprising:a) adding a cell dye to post-thaw cells;b) determining a number of live cells in the post-thaw cells once per hour for twenty- four hours after addition of the cell dye; andc) calculating the percentage post-thaw attachment value as a concentration of live cells at a time point greater than one hour divided by a concentration of live cells at a one hour time point.

56. The method of claim 55 wherein, the post-thaw cells are proliferative if the percentage post-thaw attachment value is greater than 70%.

57. The method of claim 55, wherein the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

58. The method of claim 55, wherein the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

59. The method of claim 55, wherein an aggregate in the cell aggregates comprise about 3- 25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

60. A method of determining if post-thaw cells comprising cell aggregates reach a target cell confluence, the method comprising:Attorney Docket No. 132042-5008- WOa) seeding the post-thaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2;b) adding a cell dye to the post-thaw cells;c) incubating the post-thaw cells for twenty-four hours;d) exchanging the medium with a fresh medium every twenty-four hours;e) measuring a percentage of confluence after ninety-six hours; andf) determining if the post-thaw cells reach the target cell confluence,wherein the target cell confluence is about 60% to about 70%.

61. The method of claim 60, wherein the post thaw cells reach the target cell confluence if the percentage confluence value is greater than 60% after ninety-six hours.

62. The method of claim 60, wherein the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

63. The method of claim 60, wherein the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

64. The method of claim 60, wherein an aggregate in the cell aggregates comprise about 3- 25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

65. A method of determining quality of post-thaw cells comprising cell aggregates, the method comprising:a) adding a cell dye for a first time to the post-thaw cells and seeding the post-thaw cells in a medium at a density of about 20,000 to about 60,000 cells / cm2; b) determining a number of live cells in the post-thaw cells once per hour for twenty- four hours after adding the cell dye for the first time;c) calculating a percentage post-thaw attachment value as a number of live cells six hours after adding the cell dye for the first time divided by a number of live cells one hour after adding the cell dye for the first time;d) exchanging the medium with a fresh medium and adding the cell dye for a second time to the post-thaw cells twenty-four hours after adding the cell dye for the first time;Attorney Docket No. 132042-5008- WOe) incubating the post-thaw cells for seventy-two hours after adding the cell dye for a second time while exchanging the medium with a fresh medium every twenty-four hours; andf) determining a percentage confluence value ninety-six hours after adding the cell dye for the first time,wherein a percentage post-thaw attachment value greater than 70% in step c) and a percentage confluence value greater than 60% in step f) indicates a high quality cell composition.

66. The method of claim 65, wherein the post-thaw cells comprise pluripotent stem cells (PSCs), optionally wherein the PSCs are induced PSCs (iPSCs) or embryonic stem cells (ESCs).

67. The method of claim 65, wherein the post-thaw cells comprise cardiomyocytes, dopaminergic neurons, microglia, or photoreceptor precursor cells (PRPs).

68. The method of claim 65, wherein an aggregate in the cell aggregates comprise about 3- 25 cells, about 3-50 cells, about 3-75 cells, or about 3-100 cells.

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