Methods for determining the percentage viability of cells within cell aggregates

Abstract

The present disclosure relates to methods of determining percentage viability of a cell composition comprising cell aggregates. Such methods may include 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, measuring a total number of live cells in the first aliquot of cells; measuring a total number of cells in the second aliquot of cells, and determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number cells in the second aliquot of cells.

Description

METHODS FOR DETERMINING THE PERCENTAGE VIABILITY OF CELLS WITHIN CELL AGGREGATESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit and priority to United States Provisional Application No. 63 / 651 ,035, filed on May 23, 2024, which is incorporated herein by reference in its entirety.FIELD

[0002] The disclosure generally relates to methods for determining the percentage viability of 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 percentage of viable cells within such product. The percentage of cell viability is conventionally determined by first separately determining the counts of total cells (i.e., both live cells and dead cells) as one attribute and the counts of dead or live cells as another. Percentage viability is then determined by subtracting the number of dead cells from the number of total cells to determine the number of live cells, and subsequently dividing that value by the number total cells. Alternatively, percentage viability may be calculated by separately determining the live cells and the total cells and dividing the two. However, cell aggregates within certain drug products can occlude cells which have taken up viability dyes, leading to inaccurate cell counts. Consequently, such methods regularly employ dissociation reagents to break apart any cell aggregates, aiding the accessibility of all cells to be exposed to dyes and thus their visualization. Unfortunately, these reagents may lyse dead cells in the sample instead of simply dissociating them leading to an overestimation of the percentage of viable cells within the aggregates. Consequently, a risk of inaccurate dosing arises when a product includes cell aggregates due to an erroneous calculation of the percentage of viable cells within the aggregates.

[0004] Thus, there is a need for improved methods for calculating the percentage of cell viability of a population of cells containing cell aggregates.SUMMARY

[0005] The present disclosure addresses the above need by providing a method for determining the percentage viability of cell compositions that comprise cell aggregates.

[0006] In an aspect, described herein is a method of determining percentage viability of a cell composition comprising cell aggregates. Such methods may comprise: (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; (b) measuring a total number of live cells in the first aliquot of cells (a concentration of live cells); (c) measuring a total number of cells in the second aliquot of cells (a concentration of total cells); and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number cells in the second aliquot of cells.

[0007] In some embodiments, the total number of live cells in the first aliquot of cells is measured by quantifying the total number of nuclei of live cells.

[0008] In some embodiments, the step of measuring the total number of live cells in the first aliquot of cells comprises contacting the first aliquot of cells with a cell dissociation reagent.

[0009] In some embodiments, the cell dissociation reagent is TrypLE™.

[0010] In some embodiments, the method comprises triturating the cells in the dissociation reagent to produce a population of dissociated cells.

[0011] In some embodiments, the method comprises staining nuclei of live cells in the population of dissociated cells with a stain.

[0012] In some embodiments, the stain comprises acridine orange (AO).

[0013] In some embodiments, the method comprises counting a total number of stained nuclei in the second aliquot of cells.

[0014] In some embodiments, the stain comprises propidium iodide (PI).

[0015] In some embodiments, the step of measuring the total number of live cells in the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total numberof stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells.

[0016] In some embodiments, the cell dissociation reagent is TrypLE™.

[0017] In some embodiments, the total number of cells in the second aliquot of cells is determined by measuring the total number of nuclei of lysed cells in the second aliquot.

[0018] In some embodiments, the step of measuring the total number of cells in the second aliquot of cells comprises contacting the cells with a cell lysis buffer.

[0019] In some embodiments, the cell lysis buffer comprises one or more agents selected from the group consisting of: <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate. In some embodiments, the cell lysis buffer comprises or consists of <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

[0020] In some embodiments, the method comprises triturating the cells in the cell lysis buffer to produce a population of lysed cells.

[0021] In some embodiments, the method comprises staining nuclei in the population of lysed cells.

[0022] In some embodiments, the method comprises counting the total number of stained nuclei.

[0023] In some embodiments, the step of measuring the total number of cells in the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells.

[0024] In some embodiments, the cell lysis buffer comprises one or more agents selected from the group consisting of: <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate. In some embodiments, the cell lysis buffer comprises or consists of <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

[0025] In some embodiments, the cell composition comprises induced pluripotent stem cells (iPSCs).

[0026] In some embodiments, the cell composition comprises photoreceptor precursor cell (PRPs).

[0027] In some embodiments, the cell aggregates comprise about 1 to about 1,000 cells.

[0028] In some embodiments, the percentage viability of the cell composition is between about 10% and about 100%.

[0029] In some embodiments, the percentage viability of the cell composition is between about 30% and about 70%.

[0030] In an aspect, described herein is a method of determining percentage viability of a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells; (b) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells; wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0031] In some embodiments, staining the nuclei of the live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

[0032] In some embodiments, staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

[0033] In some embodiments, the cell composition comprises induced pluripotent stem cells (iPSCs).

[0034] In some embodiments, the cell composition comprises photoreceptor precursor cell (PRPs).

[0035] In some embodiments, the cell aggregates comprise about 1 to about 1,000 cells.

[0036] In some embodiments, the cell dissociation reagent is TrypLE™.

[0037] In some embodiments, the cell lysis buffer comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and / or <0.2% w / w trinsodium citrate dihydrate.

[0038] In some embodiments, the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0039] In some embodiments, the percentage viability of the cell composition is between about 10% and about 100%.

[0040] In some embodiments, the percentage viability of the cell composition is between about 30% and about 70%.

[0041] In an aspect, described herein is a method of determining percentage viability of a cell composition of photoreceptor precursor cells (PRPs) comprising cell aggregates in a drug substance or drug product, 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 a substantially similar concentration of cells; (b) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with TrypLE™; ii) triturating the cells in the TrypLE™ to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells, wherein staining the nuclei of the live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO); and iv) counting the total number of stained nuclei; wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate; ii) triturating the cells in the composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5; and iv) counting the total number of stained nuclei in the population of lysed cells; wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) determining thepercentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0042] In an aspect, described herein is a method for determining percentage viability of a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells; (b) measuring a total number of nuclei of live cells in dissociated cells from the first aliquot of cells, wherein the total number of nuclei of the live cells corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of nuclei of lysed cells from the second aliquot of cells, wherein the total number of nuclei of the lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0043] In some embodiments, the step of measuring the total number of nuclei of live cells in dissociated cells from the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting the total number of stained nuclei.

[0044] In some embodiments, staining nuclei of live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

[0045] In some embodiments, the step of measuring the total number of nuclei of lysed cells from the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells.

[0046] In some embodiments, staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

[0047] In some embodiments, the cell composition comprises induced pluripotent stem cells (iPSCs).

[0048] In some embodiments, the cell composition comprises photoreceptor precursor cell (PRPs).

[0049] In some embodiments, the cell aggregates comprise about 1 to about 1,000 cells.

[0050] In some embodiments, the cell dissociation reagent is TrypLE™.

[0051] In some embodiments, the cell lysis buffer comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and / or <0.2% w / w trinsodium citrate dihydrate.

[0052] In some embodiments, the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0053] In some embodiments, the percentage viability of the cell composition is between about 10% and about 100%.

[0054] In some embodiments, the percentage viability of the cell composition is between about 30% and about 70%.

[0055] In an aspect, described herein is a method for determining a number of 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; (b) measuring a total number of nuclei of live cells in dissociated cells from the first aliquot of cells, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of nuclei of lysed cells from the second aliquot of cells, wherein the total number of stained nuclei of the lysed cells corresponds to the total number of cells in the second aliquot of cells; (d) determining a percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells; and (e) determining the number of viable cells in the cell composition by multiplying the percentage viability by a total number of cells in the cell composition.

[0056] In some embodiments, the step of measuring the total number of nuclei of live cells in dissociated cells from the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting the total number of stained nuclei.

[0057] In some embodiments, staining nuclei of live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

[0058] In some embodiments, the step of measuring the total number of nuclei of lysed cells from the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii)staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells.

[0059] In some embodiments, staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, and / or DRAQ5.

[0060] In some embodiments, the cell composition comprises induced pluripotent stem cells (iPSCs).

[0061] In some embodiments, the cell composition comprises photoreceptor precursor cell (PRPs).

[0062] In some embodiments, the cell aggregates comprise about 1 to about 1,000 cells.

[0063] In some embodiments, the cell dissociation reagent is TrypLE™.

[0064] In some embodiments, the cell lysis buffer comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and / or <0.2% w / w trinsodium citrate dihydrate.

[0065] In some embodiments, the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0066] In some embodiments, the percentage viability of the cell composition is between about 10% and about 100%.

[0067] In some embodiments, the percentage viability of the cell composition is between about 30% and about 70%.

[0068] In an aspect, described herein is a method for separately counting viable cells and total 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) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; ill) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; and (c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stainednuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells.

[0069] In some embodiments, staining nuclei of live cells in the population of dissociated cells comprises contacting the population of dissociated cells with acridine orange (AO).

[0070] In some embodiments, staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, and / or DRAQ5.

[0071] In some embodiments, the cell composition comprises induced pluripotent stem cells (iPSCs).

[0072] In some embodiments, the cell composition comprises photoreceptor precursor cell (PRPs).

[0073] In some embodiments, the cell aggregates comprise about 1-1 ,000 cells.

[0074] In some embodiments, the cell dissociation reagent is TrypLE™.

[0075] In some embodiments, the cell lysis buffer comprises <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and / or <0.2% w / w trinsodium citrate dihydrate.

[0076] In some embodiments, the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0077] In an aspect, described herein is a system for determining percentage viability 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 of cells and a second aliquot of cells; (b) a means for measuring a total number of nuclei of live cells in the first aliquot of cells, wherein the total number of nuclei of live cells corresponds to the total number of live cells in the first aliquot of cells; (c) a means for measuring a total number of nuclei of lysed cells in the second aliquot of cells, wherein the total number of nuclei of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) a means for determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.BRIEF DESCRIPTION OF THE OF THE DRAWINGS

[0078] 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.

[0079] Figure 1 : Live cell concentration (left y-axis, darker bar) and percentage viability (right y-axis, lighter bar) of cell compositions comprising photoreceptor precursor cells containing cell aggregates over time at storage between 1-8 °C.

[0080] Figure 2: Normalized dead cell counts of single cells after treatment with different enzymatic and non-enzymatic dissociation reagents for 0 or 30 minutes.

[0081] Figure 3: Normalized live cell counts of single cells after treatment with different enzymatic and non-enzymatic dissociation reagents for 0 or 30 minutes.

[0082] Figure 4: Schematic of the hybrid two-count cell viability assay using total cell counts via cell lysis and nuclei staining (top) and live cell counts via dissociation and cell staining (bottom).

[0083] Figure 5A-5C: Panel A shows an untreated cell composition sample loaded on the Cellaca™ MX High Throughput Cell Counter having large aggregates with both live and dead cells. Panel B shows dissocated aggregates as live cells that are AO postive. Panel C shows PI positive total nuclei after isolation with a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octyl phenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.DETAILED DESCRIPTION

[0084] Provided herein are methods for determining the percentage viability of a cell composition comprising cell aggregates (e.g., iPSCs or PRPs). Such methods may comprise measuring a number of live cells and a number of total cells and determining the percentage viability of the cell composition as the ratio between the number of live cells and the total number of cells (e.g., dividing live cell counts by total cell counts). Advantageously, the methods disclosed herein provide for increased accuracy over conventional methods used for cell viability assessments of cell aggregates including, for example, those which involve counting dead cells. Definitions

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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 and / 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 trinsodium 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, or mixing.

[0090] The term “cell dissociation,” “dissociation,” “cell disaggregation,” or “disaggregation” refers to providing a cell composition containing cell aggregates or aggregated cells and separating the aggregated cells into singularized cells and / or reduced-size cell aggregates. Cell dissociation may be mechanical, physical, enzymatic, chemical, or any combination thereof. In an example, cell dissociation may be induced via incubation with TrypLE™ (Thermo Fisher Scientific, Waltham, MA, Catalog No.: A1217701). Cell dissociation may further involve agitation, vortexing, trituration, or mixing in order to break cell aggregates apart.Methods of Determining a Percentage Viability of Cells Within Cell Aggregates

[0091] Provided herein are methods of determining the percentage viability of cells within a cell composition (e.g., a drug substance or a drug product) comprising cell aggregates. The percentage viability may be determined as a ratio between the total number of live cells in a first aliquot of cells obtained from the cell composition and the total number of cells in a second aliquot of cells obtained from the cell composition.

[0092] 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; (b) measuring a total number of live cells in the first aliquot of cells (e.g., by contacting the first aliquot of cells with a cell dissociation reagent; triturating the cells in the dissociation reagent to produce a population of dissociated cells; staining nuclei of live cells in the population of dissociated cells; and counting a total number of live stained nuclei, wherein the total number of live stained nuclei corresponds to the total number of live cells in the first aliquot of cells); (c) measuring a total number of cells in the second aliquot of cells (e.g., by contacting the second aliquot of cells with a cell lysis buffer; triturating the cells in the cell lysis buffer to produce a population of lysed cells; staining the nuclei in the population of lysed cells; and counting the total number of stained nuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells); and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number cells in the second aliquot of cells.

[0093] In some embodiments, the cell compositions comprise a concentration of cells of about 100,000 cells / mL to about 10,000,000 cells / mL. In some embodiments, the cell compositions comprise a concentration of cells of about 100,000 cells / mL, about 110,000 cells / mL, about 120,000 cells / mL, about 130,000 cells / mL, about 140,000 cells / mL, about 150,000 cells / mL, about 160,000 cells / mL, about 170,000 cells / mL, about 180,000 cells / mL, about 190,000 cells / mL, about 200,000 cells / mL, about210,000 cells / mL, about 220,000 cells / mL, about 230,000 cells / mL, about 240,000 cells / mL, about 250,000 cells / mL, about 260,000 cells / mL, about 270,000 cells / mL, about 280,000 cells / mL, about 290,000 cells / mL, about 300,000 cells / mL, about 310,000 cells / mL, about 320,000 cells / mL, about 330,000 cells / mL, about 340,000 cells / mL, about 350,000 cells / mL, about 360,000 cells / mL, about 370,000 cells / mL, about 380,000 cells / mL, about 390,000 cells / mL, about 400,000 cells / mL, about 410,000 cells / mL, about 420,000 cells / mL, about 430,000 cells / mL, about 440,000 cells / mL, about 450,000 cells / mL, about 460,000 cells / mL, about 470,000cells / mL, about 480,000 cells / mL, about 490,000 cells / mL, about 500,000 cells / mL, about 510,000 cells / mL, about 520,000 cells / mL, about 530,000 cells / mL, about 540,000 cells / mL, about 550,000 cells / mL, about 560,000 cells / mL, about 570,000 cells / mL, about 580,000 cells / mL, about 590,000 cells / mL, about 600,000 cells / mL, about 610,000 cells / mL, about 620,000 cells / mL, about 630,000 cells / mL, about 640,000 cells / mL, about 650,000 cells / mL, about 660,000 cells / mL, about 670,000 cells / mL, about 680,000 cells / mL, about 690,000 cells / mL, about 700,000 cells / mL, about 710,000 cells / mL, about 720,000 cells / mL, about 730,000 cells / mL, about 740,000 cells / mL, about 750,000 cells / mL, about 760,000 cells / mL, about 770,000 cells / mL, about 780,000 cells / mL, about 790,000 cells / mL, about 800,000 cells / mL, about 810,000 cells / mL, about 820,000 cells / mL, about 830,000 cells / mL, about 840,000 cells / mL, about 850,000 cells / mL, about 860,000 cells / mL, about 870,000 cells / mL, about 880,000 cells / mL, about 890,000 cells / mL, about 900,000 cells / mL, about 910,000 cells / mL, about 920,000 cells / mL, about 930,000 cells / mL, about 940,000 cells / mL, about 950,000 cells / mL, about 960,000 cells / mL, about 970,000 cells / mL, about 980,000 cells / mL, about 990,000 cells / mL, about 1 ,000,000 cells / mL, about 1 ,100,000 cells / mL, about 1 ,200,000 cells / mL, about 1 ,300,000 cells / mL, about 1 ,400,000 cells / mL, about 1 ,500,000 cells / mL, about 1 ,600,000 cells / mL, about 1 ,700,000 cells / mL, about 1 ,800,000 cells / mL, about 1 ,900,000 cells / mL, about 2,000,000 cells / mL, about 3,000,000 cells / mL, about 4,000,000 cells / mL, about 5,000,000 cells / mL, about 6,000,000 cells / mL, about 7,000,000 cells / mL, about 8,000,000 cells / mL, about 9,000,000 cells / mL, or about 10,000,000 or more cells per milliliter.

[0094] In some embodiments, the cell aggregates comprise about 1 cell to about 50 cells. In some embodiments, the cell aggregates comprise about 1 cell, about 2 cells, about 3 cells, about 4 cells, about 5 cells, about 6 cells, about 7 cells, about 8 cells, about 9 cells, about 10 cells, about 11 cells, about 12 cells, about 13 cells, about 14 cells, about 15 cells, about 16 cells, about 17 cells, about 18 cells, about 19 cells, about 20 cells, about 21 cells, about 22 cells, about 23 cells, about 24 cells, about 25 cells, about 26 cells, about 27 cells, about 28 cells, about 29 cells, about 30 cells, about 31 cells, about 32 cells, about 33 cells, about 34 cells, about 35 cells, about 36 cells, about 37 cells, about 38 cells, about 39 cells, about 40 cells, about 41 cells, about 42 cells, about 43 cells, about 44 cells, about 45 cells, about 46 cells, about 47 cells, about 48 cells, about 49 cells, about 50 or more cells. In some embodiments, the cell aggregates comprise about 10 cells to about 1 ,000 cells. In other embodiments, the cell aggregates may comprise about 10 cells, about 20 cells, about 30 cells, about 40 cells, about 50 cells, about 60 cells, about 70 cells, about 80 cells, about 90 cells, about 100 cells, about 110 cells, about 120 cells, about 130 cells, about 140 cells, about 150 cells, about 160 cells, about 170 cells, about 180 cells, about 190 cells, about 200 cells, about 210 cells, about 220 cells, about 230 cells, about 240 cells, about 250 cells, about260 cells, about 270 cells, about 280 cells, about 290 cells, about 300 cells, about 310 cells, about320 cells, about 330 cells, about 340 cells, about 350 cells, about 360 cells, about 370 cells, about380 cells, about 390 cells, about 400 cells, about 410 cells, about 420 cells, about 430 cells, about440 cells, about 450 cells, about 460 cells, about 470 cells, about 480 cells, about 490 cells, about500 cells, about 510 cells, about 520 cells, about 530 cells, about 540 cells, about 550 cells, about560 cells, about 570 cells, about 580 cells, about 590 cells, about 600 cells, about 610 cells, about620 cells, about 630 cells, about 640 cells, about 650 cells, about 660 cells, about 670 cells, about680 cells, about 690 cells, about 700 cells, about 710 cells, about 720 cells, about 730 cells, about740 cells, about 750 cells, about 760 cells, about 770 cells, about 780 cells, about 790 cells, about800 cells, about 810 cells, about 820 cells, about 830 cells, about 840 cells, about 850 cells, about860 cells, about 870 cells, about 880 cells, about 890 cells, about 900 cells, about 910 cells, about920 cells, about 930 cells, about 940 cells, about 950 cells, about 960 cells, about 970 cells, about980 cells, about 990 cells, or about 1 ,000 or more cells.

[0095] In some embodiments, a cell aggregate is about 10 pm to about 1 ,000 pm in diameter. In some embodiments, the cell aggregates are about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, or about 100 pm in diameter. In some embodiments, the cell aggregates may be about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, or about 1 ,000 pm in diameter. In some embodiments, a cell aggregate is about 40 pm in diameter.

[0096] The cell aggregates may comprise any cell type. In some embodiments, the cell aggregates 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 comprise photoreceptor progenitor cells. In some embodiments, the cell aggregates comprise induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In other embodiments, the cell aggregates comprise cells derived or differentiated from iPSCs or ESCs. In some embodiments, the cell aggregates comprise glial and / or amacrine cells. In some embodiments, the cell aggregates comprise microglia. The microglia may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates comprise cardiomyocytes. The cardiomyocytes may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, thecell aggregates comprise dopaminergic neurons. The dopaminergic neurons may be induced, derived, or differentiated from pluripotent stem cells. In some embodiments, the cell aggregates comprise mixtures of two or more different cell types. In some embodiments, the cell aggregates comprise mixtures of two or more different cell types. In some embodiments, the cell aggregates comprise photoreceptor progenitor cells and ESCs, iPSCs and / or PSCs. In some embodiments, the cell aggregates comprise microglia and ESCs, iPSCs and / or PSCs. In some embodiments, the cell aggregates comprise cardiomyocytes and ESCs, iPSCs and / or PSCs. In some embodiments, the cell aggregates comprise dopaminergic neurons and ESCs, iPSCs and / or PSCs.

[0097] 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.

[0098] In some embodiments, a singularized cell is about 1 pm to about 30 pm in diameter. In some embodiments, the singularized live may be about 1 pm, about 2 pm, about 3 pm, about4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, or about 30 pm in diameter. In some embodiments, a singularized live cell is about 7 pm in diameter.

[0099] In some embodiments, a singularized dead cell is about 1 pm to about 13 pm in diameter. In some embodiments, the singularized dead cell may be about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, or about 13 pm in diameter. In some embodiments, a singularized dead cell is about 9 pm in diameter.

[0100] 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%, 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%, about91%, 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.

[0101] 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.

[0102] The cell composition comprising cell aggregates may be treated with a dissociation reagent to dissociate the cell aggregates (e.g., dissociate the cells within the cell aggregates into singularized cells and / or smaller cell aggregates). In some embodiments, the dissociation of the cell composition comprising cell aggregates may break the cell aggregates apart into singularized cells. In other embodiments, the dissociation of the cell composition comprising cell aggregates may break the cell aggregates apart into singularized cells and smaller aggregates. In some embodiments, the dissociated cell composition may contain live and dead cells. In other embodiments, the dissociated cell composition may contain only live cells.

[0103] 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.

[0104] The cell aggregates may be dissociated. In some embodiments, the dissociation of cell aggregates may break apart cell aggregates into singularized cells. In other embodiments, the dissociation of cell aggregates may break apart the aggregates into singularized cells and smaller aggregates. In some embodiments, the dissociation of cell aggregates may result in substantially all singularized cells. In other embodiments, the dissociation of cell aggregates may result in singularized cells and cell aggregates. In some embodiments, the dissociated cell aggregates may contain live and dead cells. In other embodiments, the dissociated cell aggregates may contain only live cells.

[0105] 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.

[0106] The cell composition comprising cell aggregates or aliquots thereof may be contacted with a dissociation reagent (e.g., the dissociation reagent may be added to the cell composition, mixed with the cell composition, and / or incubated with the cell composition). Exemplary dissociation reagents may include TrypLE™, Accutase™, dispase, NeuroPapain™, and NSDK. In some embodiments, the dissociation reagent is TrypLE™. In further embodiments, the TrypLE™ may be at an about 1X concentration. In other embodiments, the TrypLE™ may be at about 10X concentration. In some embodiments, the TrypLE™ is at about 1X to about 15X concentration. In some embodiments, the TrypLE™ may be at about 1X, about 2X, about 3X, about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 11X, about 12X, about 13X, about 14X, or about 15X concentration. In some embodiments, the dispase may be at an about 0.6 U / mL concentration. In other embodiments, the dispase may be at an about 2.4 U / mL concentration. In some embodiments, the dissociation reagent is incubated with the cell compositions for about 5 minutes to about 60 minutes at about 30 °C to about 45 °C. In some embodiments, the dissociation agent may be incubated with the cell composition for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes at about 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, or 45 °C. In some embodiments, the dissociation reagent is incubated with the cell compositions for about 35 minutes at about 37 °C. In some embodiments, the dissociation reagentis incubated with the cell compositions for about 0 minutes at about 37 °C. In some embodiments, the dissociation reagent is incubated with the cell compositions for about 30 minutes at about 37 °C. In some embodiments, contacting may mean vortexing or agitating the cells with a reagent.

[0107] Additionally, the cell composition comprising cell aggregates or aliquots thereof may be contacted with a cell lysis reagent. Exemplary cell lysis reagents may include Triton-X 100, Saponin, Pluronic® F-68, NP-40, and / or a composition comprising one or more agents selected from the group consisting of: <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate. In some embodiments, the cell lysis reagent is a composition comprising or consisting of <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 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 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% full strength.

[0108] In some embodiments, the cell lysis reagent is incubated with the cell composition for about 1 minute to about 10 minutes. In some embodiments, the cell lysis reagent is incubated with the cell composition for about, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes. In some embodiments, the cell lysis reagent is incubated with the cell composition for about 5 minutes.

[0109] In some embodiments, the Triton-X 100 may be at an about 0.2% concentration. In some embodiments, the Saponin may be at an about 0.2 mg / mL, about 0.4 mg / mL, about 0.6 mg / mL, about 0.8 mg / mL, about 1.0 mg / mL, about 1.2 mg / mL, about 1.4 mg / mL, about 1.6 mg / mL, about 1.8 mg / mL, or about 2.0 mg / mL concentration. In some embodiments, the NP-40 may be at an about 0.5%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35% or about 0.4% concentration. In some embodiments, the Pluronic® F-68 may be at an about 0.5%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35% orabout 0.4% concentration. In some embodiments, the cell lysis reagent may be incubated with the cell composition for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes at about room temperature.

[0110] In some embodiments, the cell composition or aliquots thereof may be triturated. In some embodiments, the trituration may be performed on cell compositions containing singularized cells. In other embodiments, the trituration may be performed on cell compositions containing cell aggregates. In other embodiments, the trituration may be performed on cell compositions containing singularized cells and cell aggregates. In some embodiments, the trituration may be performed on cell compositions that have been dissociated. In other embodiments, the trituration may be performed on cell compositions that have been lysed. In other embodiments, the trituration may be performed on cell compositions that have been contacted with a dissociation reagent or a cell lysis reagent. In other embodiments, the trituration may be performed on cell compositions that have been contacted with a dissociation reagent and a cell lysis reagent.

[0111] In some embodiments, trituration of the cell composition or aliquots thereof may be performed with a pipette. In some embodiments, the pipette may be a P1000 or a P200. In some embodiments, trituration of the cell composition or aliquots thereof may be performed with a serological pipet. In some embodiments, the serological pipet may be a 10 mL pipet. In some embodiments, the trituration volume is about 1% to about 100% or more of the total solution or suspension volume of the cell composition. In some embodiments, the trituration 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%, about 96%, about 97%,about 98%, about 99%, about 100% or more of the total solution or suspension volume of the cell composition.

[0112] Trituration of the cell composition or aliquots thereof may be performed about 1 time to about 100 times. In some embodiments, the trituration is performed about 1 time, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 21 times, about 22 times, about 23 times, about 24 times, about 25 times, about 26 times, about 27 times, about 28 times, about 29 times, about 30 times, about 31 times, about 32 times, about 33 times, about 34 times, about 35 times, about 36 times, about 37 times, about 38 times, about 39 times, about 40 times, about 41 times, about 42 times, about 43 times, about 44 times, about 45 times, about 46 times, about 47 times, about 48 times, about 49 times, about 50 times, about 51 times, about 52 times, about 53 times, about 54 times, about 55 times, about 56 times, about 57 times, about 58 times, about 59 times, about 60 times, about 61 times, about 62 times, about 63 times, about 64 times, about 65 times, about 66 times, about 67 times, about 68 times, about 69 times, about 70 times, about 71 times, about 72 times, about 73 times, about 74 times, about 75 times, about 76 times, about 77 times, about 78 times, about 79 times, about 80 times, about 81 times, about 82 times, about 83 times, about 84 times, about 85 times, about 86 times, about 87 times, about 88 times, about 89 times, about 90 times, about 91 times, about 92 times, about 93 times, about 94 times, about 95 times, about 96 times, about 97 times, about 98 times, about 99 times, or about 100 times. In some embodiments, trituration of the cell composition or aliquots thereof is performed about 30 times. In some embodiments, trituration of the cell composition or aliquots thereof is performed about 20 times. In some embodiments, trituration of the cell composition or aliquots thereof is performed about 25 times.

[0113] In some embodiments, trituration of the cell composition or aliquots thereof is performed by vortexing, agitating, mixing, or shaking the cell composition or aliquots thereof. In other embodiments, trituration is performed with gentle, intermediate, or heavy force. In some embodiments, trituration of the cell composition or aliquots thereof is performed with gentle force. In some embodiments, trituration of the cell composition or aliquots thereof is performed with heavy force.

[0114] The cell composition or aliquots thereof may be mixed by pipetting. In some embodiments, mixing the cell composition or aliquots thereof by pipetting is performed on cell compositions containing singularized cells. In other embodiments, mixing the cell composition oraliquots thereof by pipetting is performed on cell compositions containing cell aggregates. In other embodiments, mixing the cell composition or aliquots thereof by pipetting is performed on cell compositions containing singularized cells and cell aggregates. In some embodiments, mixing the cell composition or aliquots thereof by pipetting is performed on cell compositions that have been lysed. In other embodiments, mixing the cell composition of aliquots thereof by pipetting is performed on cell compositions that have been contacted with a cell lysis reagent.

[0115] Mixing the cell composition or aliquots thereof by pipetting may be performed with a P1000 or a P200 pipette. The mixing of the cell composition or aliquots thereof may be a volume of about 1% to about 100% or more of the total solution or suspension volume of the cell composition. In some embodiments, the mixing of the cell composition or aliquots thereof may be a volume of 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%, about24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31 %, about32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71 %, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, about 100% or more of the total solution or suspension volume of the cell composition.

[0116] The mixing of the cell composition or aliquots thereof by pipetting may be performed about 1 time to about 100 times. For example, mixing of the cell composition or aliquots thereof by pipetting may be performed about 1 time, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 21 times, about 22 times, about 23 times, about 24 times, about 25 times, about 26 times, about 27 times, about 28 times, about 29 times, about 30 times, about 31 times, about 32 times, about 33 times, about 34 times, about 35 times, about 36 times, about 37 times, about 38 times, about 39 times, about 40 times, about 41times, about 42 times, about 43 times, about 44 times, about 45 times, about 46 times, about 47 times, about 48 times, about 49 times, about 50 times, about 51 times, about 52 times, about 53 times, about 54 times, about 55 times, about 56 times, about 57 times, about 58 times, about 59 times, about 60 times, about 61 times, about 62 times, about 63 times, about 64 times, about 65 times, about 66 times, about 67 times, about 68 times, about 69 times, about 70 times, about 71 times, about 72 times, about 73 times, about 74 times, about 75 times, about 76 times, about 77 times, about 78 times, about 79 times, about 80 times, about 81 times, about 82 times, about 83 times, about 84 times, about 85 times, about 86 times, about 87 times, about 88 times, about 89 times, about 90 times, about 91 times, about 92 times, about 93 times, about 94 times, about 95 times, about 96 times, about 97 times, about 98 times, about 99 times, or about 100 times. In some embodiments, the mixing of the cell composition or aliquots thereof by pipetting may be performed about 30 times. In some embodiments, the mixing of the cell composition or aliquots thereof by pipetting is performed about 20 times. In some embodiments, the mixing of the cell composition or aliquots thereof by pipetting is performed about 25 times.

[0117] In some embodiments, mixing of the cell composition or aliquots thereof is performed by vortexing, agitating, mixing, and / or shaking the cell composition or aliquots thereof. In other embodiments, mixing is performed with gentle, intermediate, and / or heavy force. In some embodiments, mixing of the cell composition or aliquots thereof is performed with gentle force. In some embodiments, mixing of the cell composition or aliquots thereof is performed with heavy force.

[0118] 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 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, and / or DRAQ5. In some embodiments, the cells may be stained with acridine orange and propidium iodide at the same time (i.e., AOPI). In some embodiments, the cell staining reagent stains the nuclei of cells.

[0119] The stained cells may be counted with a cell counter. In some embodiments, the stained cells may be counted with a Cellaca™ MX High Throughput Cell Counter (Revvity, Waltham, MA, Part No.: MX-AOPI) according to the manufacturer’s instructions. In some embodiments, cells may be counted after contact with a dissociation reagent, a cell lysis reagent,a cell staining reagent, and / or a nuclei staining reagent In some embodiments, cells are counted after contact with a cell lysis reagent and a nuclei staining reagent. In some embodiments, cells are counted after contact with a dissociation reagent and a cell staining reagent. In some embodiments, cells are counted after contact with a nuclei staining reagent. In some embodiments, cells are counted after contact with a cell staining reagent.

[0120] 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.

[0121] Also provided herein are methods for determining a number of viable 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; (b) measuring a total number of nuclei of live cells in dissociated cells from the first aliquot of cells, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of nuclei of lysed cells from the second aliquot of cells, wherein the total number of stained nuclei of the lysed cells corresponds to the total number of cells in the second aliquot of cells; (d) determining a percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells; and (e) determining the number of viable cells in the cell composition by multiplying the percentage viability by a total number of cells in the cell composition.

[0122] The disclosure also provides methods of determining percentage viability of a cell composition of photoreceptor precursor cells (PRPs) comprising cell aggregates in a drug substance or drug product. 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 a substantially similar concentration of cells; (b) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with TrypLE™; ii) triturating the cells in the TrypLE™ to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells, wherein staining the nuclei of the live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO); and iv) counting the total number of stained nuclei; wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c)measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate; ii) triturating the cells in the composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells, wherein staining the nuclei in the population of lysed cells may comprise contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5; and iv) counting the total number of stained nuclei in the population of lysed cells; wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0123] The methods herein may be used for separately counting viable cells and total 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) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; and (c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells.

[0124] The disclosure provides systems for determining percentage viability 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 of cells and a second aliquot of cells; (b) a means for measuring a total number of nuclei of live cells in the first aliquot of cells, wherein the total number of nuclei of live cells corresponds to the total number of live cells in the first aliquot of cells; (c) a means for measuring a total number of nuclei of lysed cells in the second aliquot of cells, wherein the total number ofnuclei of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) a means for determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.Illustration of Subject Technology as Clauses

[0125] 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.

[0126] Clause 1 : A method of determining percentage viability of 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; (b) measuring a total number of live cells in the first aliquot of cells; (c) measuring a total number of cells in the second aliquot of cells; and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number cells in the second aliquot of cells.

[0127] Clause 2: The method of clause 1 , wherein the total number of live cells in the first aliquot of cells is measured by quantifying the total number of nuclei of live cells.

[0128] Clause 3: The method of clause 1 or 2, wherein the step of measuring the total number of live cells in the first aliquot of cells comprises contacting the first aliquot of cells with a cell dissociation reagent.

[0129] Clause 4: The method of clause 3, wherein the cell dissociation reagent is TrypLE™.

[0130] Clause 5: The method of clause 3 or 4, further comprising triturating the cells in the dissociation reagent to produce a population of dissociated cells.

[0131] Clause 6: The method of clause 5, further comprising staining nuclei of live cells in the population of dissociated cells with a stain.

[0132] Clause 7: The method of clause 6, wherein the stain comprises acridine orange (AO).

[0133] Clause 8: The method of any one of clauses 1 to 6, further comprising counting a total number of stained nuclei in the second aliquot of cells.

[0134] Clause 9: The method of clause 8, wherein the stain comprises propidium iodide (PI).

[0135] Clause 10: The method of clause 1, wherein the step of measuring the total number of live cells in the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells.

[0136] Clause 11 : The method of clause 10, wherein the cell dissociation reagent is TrypLE™

[0137] Clause 12: The method of any one of the preceding clauses, wherein the total number of cells in the second aliquot of cells is determined by measuring the total number of nuclei of lysed cells in the second aliquot.

[0138] Clause 13: The method of any one of the preceding clauses, wherein the step of measuring the total number of cells in the second aliquot of cells comprises contacting the cells with a cell lysis buffer.

[0139] Clause 14: The method of clause 13, wherein the cell lysis buffer is a composition comprising about <2.5% w / w citric acid monohydrate, about <1% w / w octylphenol ethoxylate, and about <0.2% w / w trinsodium citrate dihydrate .

[0140] Clause 15: The method of clause 13 or 14, further comprising triturating the cells in the cell lysis buffer to produce a population of lysed cells.

[0141] Clause 16: The method of clause 15, further comprising staining nuclei in the population of lysed cells.

[0142] Clause 17: The method of clause 16, further comprising counting the total number of stained nuclei.

[0143] Clause 18: The method of clause 1, wherein the step of measuring the total number of cells in the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells.

[0144] Clause 19: The method of clause 18, wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate .

[0145] Clause 20: The method of any one of the preceding clauses, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

[0146] Clause 21 : The method of any one of the preceding clauses, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

[0147] Clause 22: The method of any one of the preceding clauses, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.

[0148] Clause 23: The method of any one of the preceding clauses, wherein the percentage viability of the cell composition is between about 10% and about 100%.

[0149] Clause 24: The method of any one of the preceding clauses, wherein the percentage viability of the cell composition is between about 30% and about 70%.

[0150] Clause 25: A method of determining percentage viability of a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells; (b) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells; wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0151] Clause 26: The method of clause 25, wherein staining the nuclei of the live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

[0152] Clause 27: The method of clause 25 or 26, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

[0153] Clause 28: The method of clause 25, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

[0154] Clause 29: The method of any one of clauses 25 to 28, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

[0155] Clause 30: The method of any one of clauses 25 to 29, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.

[0156] Clause 31 : The method of any one of clauses 25 to 30, wherein the cell dissociation reagent is TrypLE™.

[0157] Clause 32: The method of any one of clauses 25 to 31, wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

[0158] Clause 33: The method of any one of clauses 25 to 32, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0159] Clause 34: The method of any one of clauses 25 to 33, wherein the percentage viability of the cell composition is between about 10% and about 100%.

[0160] Clause 35: The method of any one of clauses 25 to 34, wherein the percentage viability of the cell composition is between about 30% and about 70%.

[0161] Clause 36: A method of determining percentage viability of a cell composition of photoreceptor precursor cells (PRPs) comprising cell aggregates in a drug substance or drug product, 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 a substantially similar concentration of cells; (b) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with TrypLE™; ii) triturating the cells in the TrypLE™ to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells, wherein staining the nuclei of the live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO); and iv) counting the total number of stained nuclei; wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a totalnumber of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate; ii) triturating the cells in the composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5; and iv) counting the total number of stained nuclei in the population of lysed cells; wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0162] Clause 37: A method for determining percentage viability of a cell composition comprising cell aggregates, the method comprising: (a) providing a first aliquot of cells and a second aliquot of cells; (b) measuring a total number of nuclei of live cells in dissociated cells from the first aliquot of cells, wherein the total number of nuclei of the live cells corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of nuclei of lysed cells from the second aliquot of cells, wherein the total number of nuclei of the lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0163] Clause 38: The method of clause 37, wherein the step of measuring the total number of nuclei of live cells in dissociated cells from the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting the total number of stained nuclei.

[0164] Clause 39: The method of clause 38, wherein staining nuclei of live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

[0165] Clause 40: The method of any one of clauses 37 to 39, wherein the step of measuring the total number of nuclei of lysed cells from the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer toproduce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells.

[0166] Clause 41: The method of clause 40, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

[0167] Clause 42: The method of any one of clauses 37 to 41 , wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

[0168] Clause 43: The method of any one of clauses 37 to 42, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

[0169] Clause 44: The method of any one of clauses 37 to 43, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.

[0170] Clause 45: The method of clause 38 or 39, wherein the cell dissociation reagent is TrypLE™.

[0171] Clause 46: The method of clause 40 or 41 , wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

[0172] Clause 47: The method of any one of clauses 37 to 46, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0173] Clause 48: The method of any one of clauses 37 to 47, wherein the percentage viability of the cell composition is between about 10% and about 100%.

[0174] Clause 49: The method of any one of clauses 37 to 48, wherein the percentage viability of the cell composition is between about 30% and about 70%.

[0175] Clause 50: A method for determining a number of 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; (b) measuring a total number of nuclei of live cells in dissociated cells from the first aliquot of cells, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; (c) measuring a total number of nuclei of lysed cells from the second aliquot of cells, wherein the total number of stained nuclei of the lysed cells corresponds to the total number of cells in the second aliquot of cells; (d) determining a percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells; and (e) determiningthe number of viable cells in the cell composition by multiplying the percentage viability by a total number of cells in the cell composition.

[0176] Clause 51 : The method of clause 50, wherein the step of measuring the total number of nuclei of live cells in dissociated cells from the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting the total number of stained nuclei.

[0177] Clause 52: The method of clause 51, wherein staining nuclei of live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

[0178] Clause 53: The method of any one of clauses 50 to 52, wherein the step of measuring the total number of nuclei of lysed cells from the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells.

[0179] Clause 54: The method of clause 53, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

[0180] Clause 55: The method of any one of clauses 50 to 54, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

[0181] Clause 56: The method of any one of clauses 50 to 55, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

[0182] Clause 57: The method of any one of clauses 50 to 56, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.

[0183] Clause 58: The method of clause 51 or 52, wherein the cell dissociation reagent is TrypLE™.

[0184] Clause 59: The method of clause 53 or 54, wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

[0185] Clause 60: The method of any one of clauses 50 to 59, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0186] Clause 61 : The method of any one of clauses 50 to 60, wherein the percentage viability of the cell composition is between about 10% and about 100%.

[0187] Clause 62: The method of any one of clauses 50 to 61 , wherein the percentage viability of the cell composition is between about 30% and about 70%.

[0188] Clause 63: A method for separately counting viable cells and total 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) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; and (c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells.

[0189] Clause 64: The method of clause 63, wherein staining nuclei of live cells in the population of dissociated cells comprises contacting the population of dissociated cells with acridine orange (AO).

[0190] Clause 65: The method of clause 63 or 64, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

[0191] Clause 66: The method of any one of clauses 63 to 65, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

[0192] Clause 67: The method of any one of clauses 63 to 66, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

[0193] Clause 68: The method of any one of clauses 63 to 67, wherein the cell aggregates comprise 1-1 ,000 cells.

[0194] Clause 69: The method of any one of clauses 63 to 68, wherein the cell dissociation reagent is TrypLE™.

[0195] Clause 70: The method of any one of clauses 63 to 69, wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

[0196] Clause 71 : The method of any one of clauses 63 to 70, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

[0197] Clause 72: A system for determining percentage viability 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 of cells and a second aliquot of cells; (b) a means for measuring a total number of nuclei of live cells in the first aliquot of cells, wherein the total number of nuclei of live cells corresponds to the total number of live cells in the first aliquot of cells; (c) a means for measuring a total number of nuclei of lysed cells in the second aliquot of cells, wherein the total number of nuclei of lysed cells corresponds to the total number of cells in the second aliquot of cells; and (d) a means for determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

[0198] 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.

[0199] 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 : Conventional Cell Counting Methods Undercount Dead Cells Leading to Inaccurate Measurements of Percentage Viability Which Results in a Lack of Reproducibility

[0200] Cell compositions comprising aggregates of photoreceptor precursor cells were assessed for cell recovery and viability. Briefly, aggregated photoreceptor precursor cells were dissociated with 10X TrypLE™ at 37 °C for 35±5 minutes. Subsequently, a quench reagent(DMEM / F12 + B27 + DNase) was added at 2X volume. Samples were then triturated with a pipette and stored at 1-8 °C for up to four days.

[0201] The cells were then stained at a 1 :1 ratio with AOPI (a stain comprising acridine orange (AO) and propidium iodide (PI)) and were counted on a Cellaca™ MX High Throughput Cell Counter. AO is cell permeable and can pass through intact cell membranes thereby staining living cells. In contrast, PI is cell impermeable and cannot enter through the plasma membrane. However, PI can selectively stain nucleic acids within nuclei from lysed or dead cells. Cell counts were measured using a Cellaca™ MX High Throughput Cell Counter cell counter in brightfield and fluorescence collection modes across two sub-channels. Cell diameter for Channel 1 (FL1) was set between 3.0 to 25.0 pm and cell diameter for Channel 2 (FL2) was set between 3.0 and 20.0 pm. Total cells were calculated by adding the number of live cells (AO positive) to the number of dead cells (PI positive). Percent viability of the cells was then calculated by determining the ratio of live cells (AO positive) to the calculated value of total cells. Only a small sample was taken to determine the counts, so back-calculations were used to obtain the concentration of living cells in the original sample.

[0202] The results show that live cell concentration (calculated from living AO-positive cells) declines as length of storage at 1-8 °C increases (FIG. 1, left y-axis). A commensurate decline in living cell concentration and percentage viability was expected. However, calculated cell percentage viability remains relatively constant (FIG. 1, right y-axis). The lack of consistency between the measurements suggests that the viability assessment may be undercounting dead cells and therefore leading to a false inflation of the percentage viability.Example 2: Dissociation Reagents Negatively Affect Dead Cell Counts But Not Live Cell Counts

[0203] A potential cause of a decreased dead cell count may be due to removal of dead cells with the use of dissociation reagents such as TrypLE™. Consequently, singularized photoreceptor precursor cells were used to quantify the effects of various dissociation agents on dead cell count and live cell count after incubation for 0 or 30 minutes.

[0204] Dead cell counts for each dissociation reagent and timepoint were normalized to a negative control sample incubated with Balanced Salt Solution with Human Serum Albumin (BSS+HSA). Neither of the other non-enzymatic treatments (DMEM / F12 + B27 + DNase or Versene / EDTA) significantly decreased dead cell count after a 0- or 30-minute incubation (FIG. 2). However, all tested enzymatic dissociation reagents decreased dead cell counts after 0 or 30 minutes (with the exception of Accutase at 0 minutes), suggesting that any enzymatic dissociationreagent removes dead cells from the count and likely leads to an artificial increase in calculated percentage viability of cells.

[0205] Next, the effects of various dissociation reagents on live cell counts were quantified after incubation for 0 or 30 minutes. Live cell counts for each dissociation reagent and timepoint were normalized to a negative control sample incubated with BSS+HSA. All non-enzymatic conditions (BSS+HSA, DMEM / F12 + B27 + DNase, and Versene / EDTA) showed similar live cell counts after a 0- and 30-minute incubation (FIG. 3). In contrast to dead cell counts in FIG. 2, the tested dissociation reagents showed little effect on live cell counts after a 0-minute incubation. Two of the tested enzymes (10X TrypLE™ and NSDK (Neurosphere Dissociation Kit, Miltenyi Biotec)) showed increased cell counts after incubation at 30 minutes, suggesting that the tested cells may not be entirely singularized.

[0206] The results suggest that obtaining accurate cell counts when using dissociation reagents depends on whether the cells are dead or alive. The use of enzymatic dissociation reagents leads to decreases in dead cell counts but does not negatively affect living cell counts. The greatest number of cells were observed in samples dissociated with 10X TrypLE, suggesting this enzyme performed the best at singularizing cells.Example 3: Measurement of Percentage Viability of Cells Within a Cell Aggregate Using a Hybrid Counting Method

[0207] A hybrid counting method was tested using total and live cell counts to determine percentage cell viability in cell compositions containing aggregates of photoreceptor precursor cells. This hybrid counting approach does not rely on counting dead cells but instead directly measures the number of live cells and the number of total cells (FIG. 4). In the hybrid method, live cells are measured similarly to the protocol in Example 1 , but total cells are measured independently of both live and dead cells using a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate as a lysis reagent. The composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate permeabilizes the cell membrane allowing for PI to label cell nuclei.

[0208] Total cell counts were determined by combining 30 pL of aggregated cells and 470 pL of a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate. The solution was mixed with a pipette, and samples were then incubated for up to 5 minutes at room temperature. Subsequently,samples were triturated up to 30±2 times using a P1000 pipette set to 400 pL with force to isolate the nuclei. Samples were then transferred to a plate (3 replicates, 50 pL samples), and 50 pL of AOPI was mixed with the cells. Total cell counts (PI positive cells) were measured within two hours on a Cellaca™ MX High Throughput Cell Counter. Brightfield and fluorescence collection modes were used to count total cells across two sub-channels, with cell diameter for Channel 1 set between 3.0 to 25.0 pm and cell diameter for Channel 2 set between 3.0 and 20.0 pm.

[0209] Live cell counts were also determined from the same starting cell composition. Briefly, 30 pL of aggregated cells were combined with 400 pL 10X TrypLE™. Next, the cells were incubated for 35±5 minutes at 37 °C. Subsequently, 770 pL of quench reagent (DMEM / F12 + B27 + DNase) was added, and each sample was triturated up to 20±2 times with a P1000 pipette set to 770 pL. Samples were then transferred to a plate (3 replicates, 50 pL samples), and 50 pL of AOPI was mixed with the cells. Live cell counts (AO positive cells) were measured within two hours on a Cellaca™ MX High Throughput Cell Counter. Brightfield and fluorescence collection modes were used to count live cells across two sub-channels, with cell diameter for Channel 1 set between 3.0 to 25.0 pm and cell diameter for Channel 2 set between 3.0 and 20.0 pm.

[0210] Cells were visualized before and after nuclei isolation or enzymatic dissociation on the Cellaca™ MX High Throughput Cell Counter. Untreated cells were loaded on the Cellaca™ MX High Throughput Cell Counter and showed large aggregates with both live and dead cells (FIG. 5A). After dissociation, live cells represented by AO positive cells were also visualized (FIG. 5B). Total nuclei were visualized for PI positive cells ( / .e., total cells) after treatment with a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate (FIG. 5C).

[0211] The hybrid method was tested using seven different production lots containing aggregated cells. The total and live cell counts were used to calculate percentage viability by taking the ratio between live cell counts and the total cell counts. As shown in Table 1 , percentage viability varies across each production lot, but within each lot, the calculated viability remains relatively consistent (e.g., is reproducible) as indicated by a low standard deviation. Importantly, without the use of dead cells in the calculation of percentage viability, the observed value is more accurate.Table 1 : Percentage Viability of Cells Within Cell Aggregates by Lot.

[0212] 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.

[0213] 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.

[0214] 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 thespecification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained.

[0215] 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 the specification should be construed as indicating any non-claimed element essential to the practice of the description.

[0216] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and.”

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.”

[0223] 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, 19 20, 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.

[0224] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as described herein.

Claims

WHAT IS CLAIMED IS:

1. A method of determining percentage viability of 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;(b) measuring a total number of live cells in the first aliquot of cells;(c) measuring a total number of cells in the second aliquot of cells; and(d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number cells in the second aliquot of cells.

2. The method of claim 1 , wherein the total number of live cells in the first aliquot of cells is measured by quantifying the total number of nuclei of live cells.

3. The method of claim 1 or 2, wherein the step of measuring the total number of live cells in the first aliquot of cells comprises contacting the first aliquot of cells with a cell dissociation reagent.

4. The method of claim 3, wherein the cell dissociation reagent is TrypLE™.

5. The method of claim 3 or 4, further comprising triturating the cells in the dissociation reagent to produce a population of dissociated cells.

6. The method of claim 5, further comprising staining nuclei of live cells in the population of dissociated cells with a stain.

7. The method of claim 6, wherein the stain comprises acridine orange (AO).

8. The method of any one of claims 1 to 6, further comprising counting a total number of stained nuclei in the second aliquot of cells.

9. The method of claim 8, wherein the stain comprises propidium iodide (PI).

10. The method of claim 1 , wherein the step of measuring the total number of live cells in the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells.

11. The method of claim 10, wherein the cell dissociation reagent is TrypLE™.

12. The method of any one of the preceding claims, wherein the total number of cells in the second aliquot of cells is determined by measuring the total number of nuclei of lysed cells in the second aliquot.

13. The method of any one of the preceding claims, wherein the step of measuring the total number of cells in the second aliquot of cells comprises contacting the cells with a cell lysis buffer.

14. The method of claim 13, wherein the cell lysis buffer is a composition comprising about <2.5% w / w citric acid monohydrate, about <1% w / w octylphenol ethoxylate, and about <0.2% w / w trinsodium citrate dihydrate.

15. The method of claim 13 or 14, further comprising triturating the cells in the cell lysis buffer to produce a population of lysed cells.

16. The method of claim 15, further comprising staining nuclei in the population of lysed cells.

17. The method of claim 16, further comprising counting the total number of stained nuclei.

18. The method of claim 1 , wherein the step of measuring the total number of cells in the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells.

19. The method of claim 18, wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

20. The method of any one of the preceding claims, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

21. The method of any one of the preceding claims, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

22. The method of any one of the preceding claims, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.

23. The method of any one of the preceding claims, wherein the percentage viability of the cell composition is between about 10% and about 100%.

24. The method of any one of the preceding claims, wherein the percentage viability of the cell composition is between about 30% and about 70%.

25. A method of determining percentage viability of a cell composition comprising cell aggregates, the method comprising:(a) providing a first aliquot of cells and a second aliquot of cells;(b) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with a cell dissociation reagent;ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells;(c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells; wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells; and(d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

26. The method of claim 25, wherein staining the nuclei of the live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

27. The method of claim 25 or 26, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

28. The method of any one of claims 25 to 27, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

29. The method of any one of claims 25 to 28, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

30. The method of any one of claims 25 to 29, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.31 . The method of any one of claims 25 to 30, wherein the cell dissociation reagent isTrypLE™.

32. The method of any one of claims 25 to 31 , wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

33. The method of any one of claims 25 to 32, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

34. The method of any one of claims 25 to 33, wherein the percentage viability of the cell composition is between about 10% and about 100%.

35. The method of any one of claims 25 to 34, wherein the percentage viability of the cell composition is between about 30% and about 70%.

36. A method of determining percentage viability of a cell composition of photoreceptor precursor cells (PRPs) comprising cell aggregates in a drug substance or drug product, 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 a substantially similar concentration of cells;(b) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with TrypLE™; ii) triturating the cells in the TrypLE™ to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells, wherein staining the nuclei of the live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO); and iv) counting the total number of stained nuclei; wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells;(c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate di hydrate;ii) triturating the cells in the composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5; and iv) counting the total number of stained nuclei in the population of lysed cells; wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells; and(d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

37. A method for determining percentage viability of a cell composition comprising cell aggregates, the method comprising:(a) providing a first aliquot of cells and a second aliquot of cells;(b) measuring a total number of nuclei of live cells in dissociated cells from the first aliquot of cells, wherein the total number of nuclei of the live cells corresponds to the total number of live cells in the first aliquot of cells;(c) measuring a total number of nuclei of lysed cells from the second aliquot of cells, wherein the total number of nuclei of the lysed cells corresponds to the total number of cells in the second aliquot of cells; and(d) determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

38. The method of claim 37, wherein the step of measuring the total number of nuclei of live cells in dissociated cells from the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; andiv) counting the total number of stained nuclei.

39. The method of claim 38, wherein staining nuclei of live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

40. The method of any one of claims 37 to 39, wherein the step of measuring the total number of nuclei of lysed cells from the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells.

41. The method of claim 40, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

42. The method of any one of claims 37 to 41 , wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

43. The method of any one of claims 37 to 42, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

44. The method of any one of claims 37 to 43, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.

45. The method of claim 38 or 39, wherein the cell dissociation reagent is TrypLE™.

46. The method of claim 40 or 41 , wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

47. The method of any one of claims 37 to 46, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

48. The method of any one of claims 37 to 47, wherein the percentage viability of the cell composition is between about 10% and about 100%.

49. The method of any one of claims 37 to 48, wherein the percentage viability of the cell composition is between about 30% and about 70%.

50. A method for determining a number of 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;(b) measuring a total number of nuclei of live cells in dissociated cells from the first aliquot of cells, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells;(c) measuring a total number of nuclei of lysed cells from the second aliquot of cells, wherein the total number of stained nuclei of the lysed cells corresponds to the total number of cells in the second aliquot of cells;(d) determining a percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells; and(e) determining the number of viable cells in the cell composition by multiplying the percentage viability by a total number of cells in the cell composition.51 . The method of claim 50, wherein the step of measuring the total number of nuclei of live cells in dissociated cells from the first aliquot of cells comprises: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting the total number of stained nuclei.

52. The method of claim 51 , wherein staining nuclei of live cells in the population of dissociated cells comprises contacting the dissociated cells with acridine orange (AO).

53. The method of any one of claims 50 to 52, wherein the step of measuring the total number of nuclei of lysed cells from the second aliquot of cells comprises: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells.

54. The method of claim 53, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

55. The method of any one of claims 50 to 54, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

56. The method of any one of claims 50 to 55, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

57. The method of any one of claims 50 to 56, wherein the cell aggregates comprise about 1 to about 1 ,000 cells.

58. The method of claim 51 or 52, wherein the cell dissociation reagent is TrypLE™.

59. The method of claim 53 of 54, wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1% w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.

60. The method of any one of claims 50 to 59, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.61 . The method of any one of claims 50 to 60, wherein the percentage viability of the cell composition is between about 10% and about 100%.

62. The method of any one of claims 50 to 61 , wherein the percentage viability of the cell composition is between about 30% and about 70%.

63. A method for separately counting viable cells and total 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) measuring a total number of live cells in the first aliquot of cells, comprising: i) contacting the first aliquot of cells with a cell dissociation reagent; ii) triturating the cells in the dissociation reagent to produce a population of dissociated cells; iii) staining nuclei of live cells in the population of dissociated cells; and iv) counting a total number of stained nuclei, wherein the total number of stained nuclei corresponds to the total number of live cells in the first aliquot of cells; and(c) measuring a total number of cells in the second aliquot of cells, comprising: i) contacting the second aliquot of cells with a cell lysis buffer; ii) triturating the cells in the cell lysis buffer to produce a population of lysed cells; iii) staining the nuclei in the population of lysed cells; and iv) counting the total number of stained nuclei in the population of lysed cells, wherein the total number of stained nuclei in the population of lysed cells corresponds to the total number of cells in the second aliquot of cells.

64. The method of claim 63, wherein staining nuclei of live cells in the population of dissociated cells comprises contacting the population of dissociated cells with acridine orange (AO).

65. The method of claim 63 or 64, wherein staining the nuclei in the population of lysed cells comprises contacting the population of lysed cells with propidium iodide (PI), ethidium bromide (EB), 7AAD, SYTOX green / red, DAPI, or DRAQ5.

66. The method of any one of claims 63 to 65, wherein the cell composition comprises induced pluripotent stem cells (iPSCs).

67. The method of any one of claims 63 to 66, wherein the cell composition comprises photoreceptor precursor cell (PRPs).

68. The method of any one of claims 63 to 67, wherein the cell aggregates comprise 1-1 ,000 cells.

69. The method of any one of claims 63 to 68, wherein the cell dissociation reagent is TrypLE™.

70. The method of any one of claims 63 to 69, wherein the cell lysis buffer is a composition comprising <2.5% w / w citric acid monohydrate, <1 % w / w octylphenol ethoxylate, and <0.2% w / w trinsodium citrate dihydrate.71 . The method of any one of claims 63 to 70, wherein the first aliquot of cells and the second aliquot of cells have about the same concentration of cells.

72. A system for determining percentage viability 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 of cells and a second aliquot of cells;(b) a means for measuring a total number of nuclei of live cells in the first aliquot of cells, wherein the total number of nuclei of live cells corresponds to the total number of live cells in the first aliquot of cells;(c) a means for measuring a total number of nuclei of lysed cells in the second aliquot of cells, wherein the total number of nuclei of lysed cells corresponds to the total number of cells in the second aliquot of cells; and(d) a means for determining the percentage viability of the cell composition as a ratio between the total number of live cells in the first aliquot of cells and the total number of cells in the second aliquot of cells.

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