Methods for large-scale propagation of PSCs

Abstract

This disclosure relates to applications of cell culture, and more specifically to applications of cell culture for growing cells such as stem cells (e.g., PSCs). The method comprises seeding cells in a first container and optionally transferring the cells from the first container to a second container. The cells in the first container, and, if applicable, the cells in the second container, are brought into contact with at least one externally added extracellular matrix or extracellular matrix protein. The method of this disclosure yields a population of cells that are significantly and rapidly grown while maintaining cell quality. The method of this disclosure may also improve the cell seeding efficiency of cells that are significantly and rapidly cultured / grown in a monolayer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 460,751, filed on April 20, 2023, the entire contents of which are incorporated herein by reference.

[0002] Technical field This disclosure relates to the application of cell culture, more specifically to the application of cell culture using stem cells, and even more specifically to the application of cell culture related to stem cells such as pluripotent stem cells (PSCs). [Background technology]

[0003] Scaling up and / or accelerating cell culture and growth protocols is of interest in many applications, including, to name a few, cell therapy applications, protein production applications, and regenerative medicine applications. Such applications require large quantities of high-quality cells that can be obtained reproducibly in a cost-effective manner.

[0004] Pluripotent stem cells and other stem cells possess unique characteristics, including self-renewal ability and the ability to differentiate into downstream cell lineages, posing the potential to revolutionize cell therapy, drug discovery, disease modeling, and regenerative medicine. In particular, the use of cells in clinical trials requires a very large number of cells per patient dose.

[0005] Therefore, there remains an unmet need for efficient and scalable methods for mass-producing high-quality cells such as stem cells to meet growing commercial and clinical needs. [Overview of the project]

[0006] This disclosure relates to a method for scaling up cell proliferation, such as in a monolayer. The method may also apply to stem cells, such as pluripotent stem cells.

[0007] One aspect of the present disclosure provides a method for growing cells such as PSCs in a monolayer. The method of the present disclosure may include providing a first suspension of cells in a cell culture medium or other support medium, settling the cells from the first suspension into a first container, and culturing the settled cells as a monolayer in a cell culture medium.

[0008] The method of the present disclosure may further include contacting the precipitated cells with at least one externally added extracellular matrix or extracellular matrix protein (before culturing the precipitated cells). In one embodiment, the first container is not pre-coated with at least one externally added extracellular matrix or extracellular matrix protein.

[0009] The extracellular matrix or extracellular matrix protein of this disclosure is not particularly limited, but in some embodiments it may be Matrigel®, or isolated or purified extracellular matrix proteins such as fibronectin, vitronectin, or laminin isoforms, or fragments thereof.

[0010] The method of the present disclosure may further include separating a monolayer (of cells) and preparing a second suspension of cells in a cell culture medium or other support medium.

[0011] The method of the present disclosure may further include precipitating the cells of a second suspension in a second container and culturing the precipitated cells as a monolayer in a cell culture medium. The method of the present disclosure may further include contacting the precipitated cells with at least one externally added extracellular matrix or extracellular matrix protein (before culturing such precipitated cells). In one embodiment, the second container is not pre-coated with at least one externally added extracellular matrix or extracellular matrix protein.

[0012] In the method of the present disclosure, the precipitated cells may be contacted with at least one extracellular matrix or extracellular matrix protein added from the outside before, after, or simultaneously with precipitation in the first or second container.

[0013] In one aspect of the present disclosure, a method for growing cells such as PSCs in a monolayer (e.g., a method that is rapidly processed) is provided. The method of the present disclosure includes providing a first suspension of cells in a cell culture medium or other supporting liquid, precipitating the cells of the first suspension in a first container, culturing the precipitated cells as a monolayer in a cell culture medium, separating the monolayer to prepare a second suspension of cells, and precipitating and culturing the cells of the second suspension of cells to obtain a proliferation exceeding 1000-fold in two weeks or less, essentially as described above.

[0014] The method of this aspect of the present disclosure may further include contacting the precipitated cells with at least one extracellular matrix or extracellular matrix protein added from the outside (before culturing the precipitated cells). In one embodiment, the first and / or second container is not pre-coated with at least one extracellular matrix or extracellular matrix protein added from the outside.

[0015] In another aspect of the present disclosure, a method for improving the cell seeding efficiency of cells cultured in a monolayer is provided. The method of the present disclosure may include providing a first suspension of cells in a cell culture medium or other supporting liquid, precipitating the cells of the first suspension in a first container, and culturing the precipitated cells as a monolayer in a cell culture medium.

[0016] The method of the present disclosure may further include contacting the precipitated cells with an extracellular matrix or an extracellular matrix protein added from at least one external source (before culturing the precipitated cells). In one embodiment, the first container is not pre-coated with an extracellular matrix or an extracellular matrix protein added from at least one external source.

[0017] The method of this aspect of the present disclosure may further include separating a monolayer of cells and preparing a second suspension of cells in a cell culture medium or other supporting liquid.

[0018] The method of this aspect of the present disclosure may further include precipitating the cells of the second suspension in a second container and culturing the precipitated cells as a monolayer in a cell culture medium. The method of this aspect of the present disclosure may further include contacting the precipitated cells with an extracellular matrix or an extracellular matrix protein added from at least one external source (before culturing such precipitated cells). In one embodiment, the second container is not pre-coated with an extracellular matrix or an extracellular matrix protein added from at least one external source.

[0019] In another aspect of the present disclosure, a method for improving the cell seeding efficiency of cells cultured as a monolayer is provided. The method of this aspect of the present disclosure includes providing a first suspension of cells in a cell culture medium or other supporting liquid, precipitating the cells of the first suspension in a first container, culturing the precipitated cells as a monolayer in a cell culture medium, separating the monolayer, preparing a second suspension of cells, and precipitating and culturing the cells of the second suspension of cells, essentially as described above, to obtain, for example, more than 1000-fold growth in less than or equal to two weeks.

[0020] In any embodiment, the extracellular matrix or extracellular matrix proteins of the Disclosure are not particularly limited, but in some embodiments they may be isolated or purified extracellular matrix proteins such as Matrigel®, or fibronectin, vitronectin, or laminin isoforms, or fragments thereof. In any embodiment, at least one externally added extracellular matrix protein is recombinant, or the extracellular matrix is ​​a mixture of two or more extracellular matrix proteins.

[0021] In any embodiment, the total or combined culture surface area of ​​the second container may be greater than the total or combined culture surface area of ​​the first container. In one embodiment, the culture surface area of ​​the second container is at least 10 times greater than the culture surface area of ​​the first container.

[0022] In any embodiment, the method of the present disclosure may further include separating a monolayer from the second container to obtain a third suspension. The amount of cells in the second (or third) suspension is at least three orders of magnitude greater than the amount of cells in the first suspension, and the culture period may be two weeks or less, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days.

[0023] The method of this disclosure may further include contacting the precipitated cells with an adhesion aid (by supplementing the culture medium). In one embodiment, the adhesion aid comprises albumin and one or more fatty acids. Contact with the adhesion aid may increase the seeding efficiency of the precipitated cells.

[0024] Compared to precipitation without contact with at least one externally added extracellular matrix or extracellular matrix protein, when contact is made with an adhesion aid and / or at least one externally added extracellular matrix or extracellular matrix protein, a larger proportion of the precipitated cells may adhere to the walls of the first or second container via contact with the adhesion aid and / or at least one externally added extracellular matrix or extracellular matrix protein.

[0025] Compared to seeding in a culture medium lacking deposition additives and / or at least one externally added extracellular matrix or extracellular matrix protein, when seeding in the presence of a culture medium and an adhesion aid and / or at least one externally added extracellular matrix or extracellular matrix protein, a larger proportion of the precipitated cells in one or both of the first and second suspensions may adhere to the walls of the first and second containers, respectively, via contact with the adhesion aid and / or at least one externally added extracellular matrix or extracellular matrix protein.

[0026] Compared to culturing cells precipitated in contact with culture medium and in the absence of adhesion aids and / or at least one externally added extracellular matrix or extracellular matrix protein, culturing cells precipitated in contact with culture medium and in the presence of adhesion aids and / or at least one externally added extracellular matrix or extracellular matrix protein rapidly promotes proliferation of more than 1000-fold via contact with adhesion aids and / or at least one externally added extracellular matrix or extracellular matrix protein.

[0027] Compared to culturing cells precipitated in a first suspension in contact with culture medium but without contact with adhesion aids and / or at least one externally added extracellular matrix or extracellular matrix protein, culturing cells precipitated in a first suspension in contact with culture medium and adhesion aids and / or at least one externally added extracellular matrix or extracellular matrix protein rapidly promotes proliferation of more than 1000-fold via contact with adhesion aids and / or at least one externally added extracellular matrix or extracellular matrix protein.

[0028] In any embodiment of the present disclosure, the precipitated cells may be brought into contact with at least one externally added extracellular matrix or extracellular matrix protein before, after, or simultaneously with, the precipitation in a first or second container.

[0029] In any embodiment of the present disclosure, the precipitated cells may be brought into contact with an adhesion aid before, after, or simultaneously with, the precipitation in the first or second container.

[0030] The method of the present disclosure may further include combining at least one externally added extracellular matrix or extracellular matrix protein with a culture medium before contacting the precipitated cells with at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aid.

[0031] The method of the present disclosure may further include adding or supplementing a cell culture medium with at least one extracellular matrix or extracellular matrix protein and / or adhesion aid, the medium of which may be pre-distributed into a first or second container.

[0032] In any aspect of this disclosure, the method may be automated.

[0033] Other features and advantages of the present invention will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art through this detailed description, so it should be understood that this detailed description and specific examples, while illustrating preferred embodiments of the present invention, are provided for illustrative purposes only.

[0034] To better understand the various embodiments described herein and to clearly illustrate how these various embodiments may be carried out, we now refer to and describe, by example, the accompanying drawings showing at least one exemplary embodiment. The drawings are not intended to limit the scope of the teachings described herein. [Brief explanation of the drawing]

[0035] [Figure 1] This shows the proliferation of STi0003-A PSCs in a monolayer. Representative microscopic images at 20x and 100x magnification show the morphology and growth level of cells cultured in contact with either pre-coated or uncoated ("spike-in") extracellular matrix on the container. Under both conditions, the extracellular matrix was present at either 4 μg / cm² or 9 μg / cm² (A). The bar graphs show the proliferation (B) and viable cell count (C) of cells at either pre-coated or uncoated (spike-in) Matrigel® at 4, 5, 6, 7, 8, and 9 μg / cm². For (B), the data is normalized against 9 μg / cm² of Matrigel®. The scale bars represent 100 μm and 500 μm at 100× and 20× magnifications, respectively. [Figure 2](A) A bar graph characterizing the viability of proliferated cells on day 5 cultured according to the uncoated method of this disclosure. (B) A bar graph characterizing the cell yield per mL of culture medium of proliferated cells on day 5 cultured according to the uncoated method of this disclosure. (C) A bar graph characterizing the growth rate of proliferated cells on day 5 cultured according to the uncoated method of this disclosure. The bars represent the mean ± SD of seven biological replicate tests. [Figure 3] (A) Shows the evaluation of cell quality between proliferating cells. Bar graph quantifies the expression of undifferentiated markers OCT4 and TRA-1-60 among proliferating PSCs (SCTi003-A, WLS-1C, and H9) cultured on day 5 according to the uncoated method of this disclosure. Data are shown as mean ± SD of two technical replicate tests. (B) Bar graph of the recovery rate (recovered viable cells / seeded viable cells × 100) of cells grown according to the uncoated method of this disclosure in a scale-out container compared to a corresponding small control plate of cells precipitated in a 96-well plate (12 wells per condition) pre-coated with Matrigel®. Data are shown as mean ± standard deviation of two technical replicate tests. [Figure 4] (A) A bar graph summarizing the % survival rate of cells after thawing on day 0 is shown. (B) A bar graph summarizing the % survival rate of cells after thawing on day 5 is shown. (C) A bar graph summarizing the proliferation rate of cells after thawing on day 5 is shown. The thawed cells were cultured according to the uncoated method of this disclosure. Data are shown as mean ± SD. [Figure 5] This graph quantifies the number of cell clusters of H9 cells cultured in direct comparative experiments with or without pre-coating (spike-in) different extracellular matrices or extracellular matrix proteins. Data are presented as the mean ± SD of four technically replicated tests. [Figure 6]This shows the maintenance of SCTi003-A, WLS-1C, H9, and STiPS-R038 cells in mTeSR®Plus or TeSR®-AOF, where Matrigel® is pre-coated on the culture vessel or cultured according to the uncoated method of this disclosure. Representative microscopic images at 2x magnification show the morphology of colonies after 10-20 passages. Scale bars represent 500 μm. Flow cytometry analysis of positive expression % of undifferentiated state markers (OCT4 and TRA160) after 10-20 passages of cells cultured in mTeSR®Plus or TeSR®-AOF under pre-coated conditions, under uncoated conditions only, or in combination with polymer surfactants. Bars represent the mean ± SD of two biological replicates for WLS-1C and STiPS-R038 cells, and four biological replicates for H9 and SCTi003-A cells. [Figure 7] The characteristics of cells cultured according to the uncoated method of this disclosure are quantified. After culturing the cells on pre-coated Matrigel® plates (PC), under the uncoated conditions of this disclosure (CF), or under the uncoated conditions with polymer surfactants (CF+S), the cells were cultured in mTeSR® Plus medium for 10-20 passages using enzymatic separation (A) or 1-3 passages using ReLeSR®-based scrape separation (B). Seeding efficiency of cells cultured as described in (A) and (B) of either mTeSR® Plus (C) or TeSR®-AOF (D). Seeding efficiency was calculated by dividing the total number of seeded cell aggregates by the total number of colonies at the end of passage. [Figure 8]The graphs show the potential for trilinear differentiation of cells cultured in mTeSR® plus medium (A, C, and E) under pre-coated Matrigel® conditions (PC) and uncoated conditions with and without surfactants. The graphs also show the potential for trilinear differentiation of cells cultured in TeSR®-AOF medium under the same conditions as (A), (C), and (E) (B, D, F). The bar graphs quantify the percentage of cells positive for ectoderm markers Nestin and PAX6 (8A and 8B), mesoderm markers NCAM and Brachury (8C and 8D), and endoderm markers SOX17 and CXCR4 (8E and 8F). Data are shown as mean ± SD deviation. Each bar represents two technical replicates. [Figure 9] This report shows the morphology and proliferation of porcine PSCs (pPSCs) cultured with spike-in LN511 compared to a control pre-coated with LN511. Representative microscopic images at 4x magnification show the morphology of pPSCs at the end of Phase 3 on day 5. 4000 cells were seeded under each condition. LN511 was added to the culture at 50%, 75%, and 100% of the LN511 concentration (0.375 mg / mL) of the pre-coated plate. [Figure 10] The morphology and proliferation of pPSCs cultured with spike-in LN521 are shown compared to a control pre-coated with LN511. Representative microscopic images show the morphology of pPSCs captured at the end of phase 0. LN511 was pre-coated at a certain concentration (0.375 mg / mL), while LN521 was added to the culture at concentrations of 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, and 10 μg / mL. [Modes for carrying out the invention]

[0036] This disclosure relates to culture medium compositions, adjuvants, and methods for growing monolayer-like cells. More specifically, this disclosure relates to growing monolayer-like cells in the presence of at least one externally added extracellular matrix or extracellular matrix protein, which may be added to or in contact with a cell suspension.

[0037] As used in this disclosure, the term “stem cell” refers to a cell capable of self-renewal and differentiation into at least one downstream lineage. As used specifically in this disclosure, the term “pluripotent stem cell” or “PSC,” or its plural variation, refers to a cell capable of self-renewal and differentiation into all three germ layers and beyond. The conditions and media requirements for culturing stem cells, and in the case of PSCs, the conditions and media requirements for maintaining them in an undifferentiated state, are widely known, but improvements to these conditions are the subject of ongoing research. PSCs include embryonic stem cells (“ESCs”) and induced pluripotent stem cells (“iPSCs”), etc. Stem cells like PSCs are important models in the study of differentiation mechanisms and disease modeling, and offer great potential in the medical field. Stem cells may be obtained from, derived from, or induced from any source species, but in this disclosure, stem cells are preferably mammalian.

[0038] As used in this disclosure, the terms “extracellular matrix,” “extracellular matrix protein,” or “ECM” refer to one or more molecules produced or secreted by cells that provide structural and biochemical support to cells, for example, by promoting cell adhesion, intercellular communication, and / or differentiation of a given tissue or cell culture. The extracellular matrix may include an interconnected mesh of fibrous proteins and glycosaminoglycans. In the context of this disclosure, when mammalian cells (e.g., stem cells or PSCs) are cultured in a monolayer, whether seeded as single cells or as clusters, it may be desirable or necessary to do so in the presence of a suitable matrix or matrix protein, which may be present in the cell culture medium or cell suspension (cell culture medium or other supporting medium, e.g., buffer). The matrix or matrix protein may be any that supports the proliferation of one or more mammalian stem cells, and both natural and synthetic extracellular matrices or proteins are intended within the scope of this disclosure. The extracellular matrix may include one or more extracellular matrix proteins. In one embodiment, the extracellular matrix is ​​a mixture of two or more extracellular matrix proteins. The components of the extracellular matrix, and by extension the extracellular matrix within the scope of this disclosure, may include one or more of the following proteins: collagen, fibronectin, laminin, and vitronectin. The extracellular matrix used in or with the culture medium described herein may be a mixture of gelatinous proteins secreted by cells such as fibroblasts, chondrocytes, Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, for example, Matrigel®. One or more extracellular matrix proteins may be recombinant proteins and / or fusion proteins. In one embodiment, the extracellular matrix proteins(s) of this disclosure are human and / or mouse proteins, or fragments thereof. In one embodiment, one or more externally added extracellular matrix proteins may be of natural origin.In one embodiment, one or more extracellular matrix proteins may be genetically engineered. In one embodiment, one or more extracellular matrix proteins may be whole proteins or fragments thereof, such as peptide fragments.

[0039] As used in this disclosure, the term “cell culture medium” refers to a liquid, semi-liquid, or gelatinous substance containing nutrients on which cells or tissues can be cultured (e.g., proliferate and / or maintained). Cell culture media typically supply the components that cells need to meet their nutritional needs as they proliferate, differentiate, or are maintained in vitro. The cell culture media of this disclosure may be serum-free and / or heterogeneous and / or animal component-free. The cell culture media of this disclosure may be used under conditions or environments without feeder cells. The cell culture media of this disclosure may include a basic medium. The basic medium may require the addition of one or more additives to form a complete medium. In one embodiment, PSCs may be maintained or proliferated in a commercially available cell culture medium, e.g., a product of the TeSR® brand (e.g., mTeSR® 1, TeSR®-AOF, TeSR®-E8, or mTeSR® Plus). Other commercially available media for supporting stem cell growth / proliferation are known. In one embodiment, porcine PSCs may be maintained and / or grown in a porcine PSC medium containing a WNT pathway inhibitor, FGF, and a member of the transforming growth factor beta (TGF-β) family of proteins. In one embodiment, the porcine PSC medium may contain activin A, FGF, and XAV939.

[0040] As used in this disclosure, the term “adhesion aid” refers to a cell culture aid that facilitates the initial and / or continued adhesion of cells to the surface of a cell culture vessel (e.g., cell culture flasks and other cell culture vessels). Adhesion aids may include, in non-limiting examples, carriers such as albumin, micelles, liposomes, extracellular vesicles, exosomes, cyclodextrins, and nanostructured lipid carriers. In one embodiment, the carrier is or comprises lipid-containing albumin or lipid-enriched albumin. In embodiments where the carrier is albumin, the albumin may be derived from any source (e.g., BSA, HSA, recombinant, or others), and certain albumins may be more suitable for stem cell culture. Adhesion aids comprising lipid-containing carriers or lipid-enriched carriers may contain one or more lipids / fatty acids, or more lipids / fatty acids. As a non-limiting example, one or more lipids / fatty acids, or more lipids / fatty acids, may be one or more of mead acid, arachidic acid, palmitoleic acid, oleic acid, myristic acid, palmitic acid, myristoleic acid, linoleic acid, stearic acid, α-linolenic acid, arachidonic acid, cholesterol, DL-α-tocopheryl, and Kolliphor P188.

[0041] As used in this disclosure, the terms “spike-in” or “added” are interchangeable with the terms “externally added” or “uncoated” and refer to extracellular matrix or one or more extracellular matrix proteins added directly to a cell suspension or cell culture medium. In one embodiment, a desired amount or concentration of matrix or one or more proteins may be added to a cell culture medium containing a population of cells or a suspension. In one embodiment, a desired amount or concentration of matrix or one or more proteins may be added to a cell culture medium that will later come into contact with a population of cells or a suspension. In one embodiment, a desired amount or concentration of matrix or one or more proteins may be added to a culture vessel containing a cell culture medium or a cell suspension or both.

[0042] method One aspect of the present disclosure provides a method for growing cells, such as stem cells (e.g., PSCs), in an optional monolayer. The cells of the present disclosure may be grown in a large-scale cell culture vessel (e.g., a “scale-out vessel”) such as Cell Factory® or CELLSTACK®.

[0043] The method of the present disclosure may include providing a first suspension of cells. The first suspension of cells may be provided as a pellet in a cell culture medium or in another support medium, for example, in a buffer.

[0044] The first cell suspension is not particularly limited and may contain any cell type, such as stem cells, that can proliferate during culture. Stem cells such as MSCs, epithelial stem cells, neural stem cells, HSPCs, and PSCs can self-replicate under appropriate culture conditions.

[0045] Certain embodiments of this disclosure relate to PSCs, and therefore the first suspension of stem cells may be a suspension of PSCs such as ESCs or iPSCs. Stem cells such as PSCs may be derived from any animal or mammalian species, including humans, primates, rodents, or livestock sources. In one embodiment, the PSCs are human PSCs (hPSCs), such as human ESCs (hESCs) or human iPSCs (hiPSCs). In one embodiment, the PSCs are livestock-derived PSCs, such as pig PSCs (pPSCs), bovine PSCs, goat PSCs, or sheep PSCs.

[0046] The first suspension of stem cells may be obtained from a suitable cell culture vessel, for example, one or more wells of a 96-well, 48-well, 24-well, 12-well, or 6-well plate, or one or more dishes, for example, 35 mm or 10 cm dishes. Typically, a single well of a 6-well plate with a concentration density of 75-95% contains approximately 1 × 10⁶ cells. 6 ~4×10 6 Cells between (e.g., stem cells), or about 2 × 10 6 ~3×10 6 It contains such cells between them.

[0047] The method of the present disclosure may further include seeding or precipitating the cells of the first suspension in the first container. The cells of the first suspension precipitated or seeded in the first container may be seeded as single cells or as cell aggregates composed of about 3, about 5, about 8, about 10, about 15, about 20, about 50, about 100, or even more cells.

[0048] (The cells of the first suspension) may be precipitated or seeded in the first container at an appropriate density considering the culture surface area of the first container and the amount of cells in the first suspension. The seeding density may be between about 5000 - 15000 cells / cm 2 , between about 7000 - 13000 cells / cm 2 , or between about 8500 - 11500 cells / cm 2 . More specifically, the seeding density may be in the range of about 9000 cells / cm 2 ± 1000 cells, or in the range of 10000 cells / cm 2 ± 1000 cells, or more.

[0049] The nature of the first container is not particularly limited. In some cases, it may be important that the culture surface area of the first container is larger than the culture surface area of the cell culture container from which the first suspension was obtained. For example, if the cell culture container from which the first suspension was obtained is a well such as a 6-well dish, the first container may be a T-150 or T-175 flask. Alternatively, as a non-limiting example, if the cell culture container from which the first suspension was obtained is each well such as a 6-well dish, the first container has a culture surface area larger than the combined surface area of the parent cultures, and potentially has a culture surface area 5 times, 10, 15 times, 20 times or more larger. The foregoing description is not limited to the 6-well plate format and includes other plate or flask formats. Exemplary first (or second) containers may include cell stacks or cell hotels as commercially available from Nunc™ or Corning™.

[0050] The method of the present disclosure may further include contacting the precipitated cells (of the first suspension) with at least one externally added extracellular matrix or extracellular matrix protein. Preferably, the first container is not pre-coated with at least one externally added extracellular matrix or extracellular matrix protein. However, in certain cases (e.g., certain steps of the no-coating method disclosed herein), the first container may be pre-coated with at least one or more externally added extracellular matrix or extracellular matrix proteins.

[0051] The extracellular matrix or extracellular matrix protein(s) of this disclosure are not particularly limited, whether they come into contact with, or are made into contact with, the precipitated cells before, after, or simultaneously with the precipitated cells in the (first) container. Non-limiting examples of at least one externally added extracellular matrix protein include vitronectin, laminin isoforms, and / or vitronectin, or the aforementioned fragments. Non-limiting examples of at least one externally added extracellular matrix include Matrigel®, which may be produced by a population of cells (e.g., Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells). The at least one externally added extracellular matrix or extracellular matrix protein(s) may be a single matrix or protein. Alternatively, the at least one externally added extracellular matrix or extracellular matrix protein(s) may be a mixture of one or more matrices or proteins.

[0052] The concentration of extracellular matrix or extracellular matrix proteins is not particularly limited, provided that it is non-toxic, does not otherwise adversely affect cell proliferation / growth, and / or causes gelation of the cell culture medium. Depending on the properties of the matrix or proteins, it may be more appropriate to express the concentration in terms of ratio (dilution) or density. For example, the concentration of extracellular matrix or extracellular matrix proteins may be relative to the volume of the cell culture medium (such as the first suspension of stem cells), and may be, for example, between about 0 and 20% (v / v), or between about 0.05 and 10%, or between about 0.05 and 5%, or between about 0.1 and 1%. Alternatively, the concentration of extracellular matrix or extracellular matrix proteins may be expressed as a dilution, such as in Matrigel®, where this dilution is about 1:50, 1:100, 1:150, 1:200, 1:250, 1:300, or higher. Alternatively, the concentration of the extracellular matrix or extracellular matrix protein may be in the range of approximately 0.1 μg / mL to 1 mg / mL, approximately 1 μg / mL to 500 μg / mL, approximately 3 μg / mL to 200 μg / mL, approximately 5 μg / mL to 150 μg / mL, or approximately 10 μg / mL to 100 μg / mL. Alternatively, the concentration of the extracellular matrix or extracellular matrix protein may be in the range of approximately 1 μg / mL to 5 μg / mL.

[0053] In some cases, at least one externally added extracellular matrix or extracellular matrix protein is added to the first cell suspension before the cells in the first suspension settle into the first container. Alternatively, at least one externally added extracellular matrix or extracellular matrix protein is added to the cell culture medium pre-distributed in the first cell culture vessel before the cells in the first cell suspension come into contact with it. Alternatively, the cells settled in the first cell culture vessel may then be brought into contact with at least one externally added extracellular matrix or extracellular matrix protein by adding or supplementing the matrix or protein.

[0054] The method of the present disclosure may further include culturing cells in contact with at least one externally added extracellular matrix or extracellular matrix protein as a monolayer in a cell culture medium.

[0055] The cell culture media described herein are not particularly limited in that they support cell proliferation (e.g., self-renewal of stem cells). A variety of culture medium formulations are commercially available and tailored to specific cell types. However, a medium optimized for a particular cell type (e.g., XVIVO medium) may not be suitable for other cell types (e.g., PSCs). Exemplary PSC cell culture media include those well known in the art and marketed by STEMCELL Technologies under the TeSR® brand name.

[0056] Precipitated cells, which may be brought into contact with at least one externally added extracellular matrix or extracellular matrix protein, may be cultured for a length of time appropriate to yield a concentration density between approximately 60% and 100%, or between approximately 70% and 95%. However, an appropriate seeding density (e.g., approximately 7500 to 12500 cells / cm³) is required for the cells and the surface area of ​​the first container. 2 Provided that ) is used, the desired collection density may be reached in less than one week, about six days or less, about five days or less, about four days or less, or about three days or less.

[0057] The method of the present disclosure may further include separating a monolayer of cells (e.g., stem cells) from a first container and preparing a second suspension of cells.

[0058] A monolayer of adherent cells, such as stem cells, may be separated using conventional reagents and methodologies, for example, by separation using enzymes or chemical reagents. Non-limiting examples of enzyme separation reagents include trypsin-containing solutions, such as Accutase® or TrypLE®. Non-limiting examples of chemical separation reagents may include chelating agents such as EDTA, or commercially available reagents such as ReLeSR®.

[0059] In any embodiment of the methods of this disclosure, the extracellular matrix or extracellular matrix protein added from an external source may further include a surfactant. While not theoretically limited, separation may be improved by contacting the monolayer of this disclosure (in a first container, a second container, or otherwise) with the extracellular matrix or extracellular matrix protein added from an external source containing a surfactant. The surfactants are known and, in particular, may be polymers, copolymers, or block copolymers. As a non-limiting example, the surfactants of this disclosure may be ionic or nonionic polymers, copolymers, or block copolymers containing Pluronic F-68, Pluronic F-127, Pluronic F-108, or polyethylene glycol (PEG). The surfactants of this disclosure may be used at concentrations ranging from 0.001% to 10%, or from 0.01% to 1%, or from about 0.05% ± 0.03%.

[0060] The adhered monolayer may be contacted with a proteolytic reagent or chemical separation reagent, and then the cells may be incubated in the reagent to separate the cell-adhered monolayer. Gentle tapping and / or gentle tritulation may help separate the cell-adhered monolayer and obtain a second cell suspension. Alternatively, scraping with a cell scraper or micropipette / pipette tip may help separate and collect cells in the well or container to obtain a second cell suspension.

[0061] The second suspension of cells prepared as described above may be resuspended in a suitable liquid or cell culture medium, such as a buffer as described herein. The second suspension may contain a population of grown cells, for example, if the first container is a scale-out container.

[0062] However, the second suspension does not have to be the final proliferated cell population if further proliferation is desired. In such a case, the method of the present disclosure may further include settling or seeding the cells of the second suspension into a second container. The cells of the second suspension may be settled (as described above) as individual cells or as aggregates of cells, or seeded.

[0063] Furthermore, as described above, the cells of the second suspension are settled or seeded into the second container at an appropriate density, taking into account the cell type, the number of cells in the second suspension, and the properties of the second container. For example, the cells of the second suspension may be approximately 5,000 to 15,000 cells / cm³. 2 During that period, approximately 7,000 to 13,000 cells / cm² 2 Between, or approximately 8500-11500 cells / cm² 2 The seeds may be sown in a second container at a density between these two densities. In one embodiment, the seeding density is approximately 9000 cells / cm³. 2 ±1000 cells, or 10000 cells / cm² 2 The range is ±1000 cells or more.

[0064] The second container of this disclosure is not particularly limited, provided that it has a larger culture surface area than the first container. For example, the culture surface area of ​​the second container may be at least 5, 10, 15, 20, 50, 100, or 200 times larger than the culture surface area of ​​the first container. More specifically, the culture surface area of ​​the second container may be about 10 times larger than the culture surface area of ​​the first container. If the first container is each well of a 6-well plate, a T-150, or a T-175 flask, the second container may be a further scale-out container, such as a commercially available one (e.g., a Nunc® Cell Factory® or Corning® CELLSTACK®). The foregoing discussion is not limited to the 6-well plate format and cell stacks or cell hotels, but merely illustrates scale-up containers.

[0065] Essentially as described above, with respect to the cells of the first suspension precipitated in the first container, the cells of the second suspension precipitated in the second container may be brought into contact with at least one externally added extracellular matrix or extracellular matrix protein. The description of the at least one externally added extracellular matrix or extracellular matrix protein that comes into contact with the cells of the second suspension is as described herein in terms of type, concentration, nature of contact, coating or addition, etc.

[0066] In some cases, at least one externally added extracellular matrix or extracellular matrix protein is added to the second cell suspension before the cells in the first suspension settle into the second container. Alternatively, at least one externally added extracellular matrix or extracellular matrix protein is added to the cell culture medium pre-distributed in the second cell culture vessel before the cells in the second cell suspension come into contact with it. Alternatively, the cells settled in the second cell culture vessel may then be brought into contact with at least one externally added extracellular matrix or extracellular matrix protein by adding or supplementing the matrix or protein.

[0067] Furthermore, the second container does not need to be pre-coated with at least one externally added extracellular matrix or extracellular matrix protein. Therefore, in some embodiments, neither the first nor the second container is pre-coated with at least one externally added extracellular matrix or extracellular matrix protein.

[0068] The method of the present disclosure may further include culturing the cells of a second suspension (in contact with at least one externally added extracellular matrix or extracellular matrix protein) as a monolayer in a cell culture medium as described above.

[0069] The cells in the second suspension may be cultured for an appropriate length of time to obtain a concentration density between approximately 60% and 100%, or between approximately 70% and 95%, as described above. An appropriate seeding density (e.g., approximately 7500–12500 cells / cm³) should be applied to the cells. 2 Provided that the above-mentioned density is used, the density may be reached in less than one week, less than approximately six days, less than approximately five days, less than approximately four days, or less than approximately three days.

[0070] The method of the present disclosure may further include separating the cell monolayer from a second container (e.g., a cell hotel or cell stack) as described above with respect to the cell monolayer in the first container. Thus, a third suspension of cells or a suspension of grown cells (e.g., stem cells) may be obtained after separating the cell monolayer from the second container. In fact, a larger quantity of cells may be present in the third suspension of cells compared to the initial quantity of cells seeded in the first container and the quantity of grown cells in the second container. The quantity of cells in the third suspension may be at least one, two, three, four, or five orders of magnitude greater than the quantity of cells in the first suspension.

[0071] A proliferation method in any aspect of this disclosure, beginning with a first suspension of cells to a second and / or third suspension of cells, is optimized for speed while maintaining high overall stem cell quality. As described above, the amount of cells in the third suspension may be, for example, one, two, three, four, or five orders of magnitude greater than the amount of cells in the first suspension, and such cells may exhibit qualities characteristic of the starting cells with respect to, for example, proliferation rate, (un)differentiated state, differentiation potential, karyotype, etc.

[0072] The method of the present disclosure may further include contacting the precipitated cells (of the first suspension and / or the second suspension) with an adhesion aid. The adhesion aid may promote precipitation and / or substrate adhesion of the cells of the applicable suspension, whether added before, during, or after exposure. As described above, the cell suspension (in contact with the cell culture medium and the adhesion aid) may contain single cells, clusters / cell aggregates, or a mixture of the two.

[0073] As described herein, the adhesion aid is not particularly limited and may include one or more carriers such as albumin. The adhesion aid may further include one or more lipids (or at least one fatty acid) and / or lipid-like substances as described herein. In one embodiment, the adhesion aid includes albumin and one or more fatty acids. In one embodiment, the adhesion aid may be a reagent branded Clone® (STEMCELL Technologies).

[0074] In any aspect of this disclosure, the concentration of the adhesion aid depends on the volume of cells precipitated in the first and / or second culture vessel. The adhesion aid may be used at 5%, 10%, 20%, or more of the volume of precipitated or seeded cells in the first and / or second cell suspension. In one embodiment, the adhesion aid is at least 10% (v / v) of the volume of the culture medium or seeding medium. Exemplary adhesion aids may include commercially available albumins, such as AlbuMax® brand products, regardless of whether they are recombinant or not, and regardless of the species from which the albumin is derived or isolated (e.g., human, bovine, etc.). Other exemplary adhesion aids may include Clone® brand reagents (STEMCELL Technologies).

[0075] When at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aid comes into contact with the cells in the cell suspension, a larger proportion of the precipitated cells adhere to the walls of the first or second container, respectively, when in contact with the at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aid than when precipitated without contact with the at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aid.

[0076] In another embodiment, the method of the present disclosure may also relate to the rapid processing of arbitrary monolayer proliferation of cells such as stem cells (e.g., PSCs). The cells of the present disclosure may be grown in a large-scale cell culture vessel (e.g., a “scale-out vessel”) such as Cell Factory® or CELLSTACK®.

[0077] As described herein, the cells of this disclosure are not particularly limited, but are preferably stem cells (e.g., PSCs) or progenitor cells.

[0078] A method in this embodiment may include (a) providing a first cell suspension (as described herein), (b) settling the cells of the first suspension into a first container (as described herein), (c) contacting the settled cells with at least one externally added extracellular matrix or extracellular matrix protein (as described herein), (d) culturing the contacted cells as a monolayer in a cell culture medium (as described herein), (e) separating the monolayer and preparing a second cell suspension (as described herein), (f) repeating at least steps (b) to (d) in a second container with the second cell suspension (as described herein), and (g) obtaining a proliferation of more than 1000 times (as described herein) in two weeks or less.

[0079] In such embodiments, the properties of the first and second containers are as described herein, including differences in culture surface area, and whether or not such containers are pre-coated with at least one externally added extracellular matrix or extracellular matrix protein.

[0080] In such embodiments, the properties of at least one externally added extracellular matrix or extracellular matrix protein, including its type, concentration, and when and how the cells in the suspension come into contact with it, are as described herein. For example, the precipitated cells may be brought into contact with at least one externally added extracellular matrix or extracellular matrix protein before, after, or simultaneously with, the precipitation in the first or second container.

[0081] The method in this embodiment may further include separating a monolayer from a second container to obtain a proliferated suspension. More specifically, in practice of the method of the present disclosure (in any embodiment disclosed herein), cell growth of up to 100-fold, 200-fold, 500-fold, 1000-fold, or up to 5000-fold or more may be obtained. Thus, growth rates of 1000-fold or more may be obtained, for example, in two weeks or less, 12 days or less, or about 10 days ± 1 day.

[0082] This method or any embodiment thereof may include contacting the precipitated cells with an adhesion aid (such as those described herein). The cells may be contacted with the adhesion aid before, during, or after precipitation in a cell culture vessel. As described herein, cells in contact with an adhesion aid may precipitate and / or adhere to the substrate more rapidly and more firmly than in the absence of the adhesion aid, and such cells may begin to proliferate more rapidly, so that their proliferation may be rapidly handled.

[0083] Accordingly, another aspect of the present disclosure provides a method for improving the cell seeding efficiency of cells cultured in a monolayer. The method of this aspect of the present disclosure includes providing a first suspension of cells in a cell culture medium; settling the cells of the first suspension into a first container; culturing the settled cells as a monolayer in a cell culture medium; separating the monolayer; preparing a second suspension of cells; and, essentially as described above, settling and culturing the cells of the second suspension of cells to obtain, for example, a more than 1000-fold proliferation in two weeks or less.

[0084] As described above, in any aspect of the present disclosure, a larger proportion of the precipitated cells adhere to the walls of the first or second container, respectively, when the cells are brought into contact with at least one externally added extracellular matrix or extracellular matrix protein, compared to when the cells are precipitated without contact with at least one externally added extracellular matrix or extracellular matrix protein.

[0085] As described above, in any aspect of the present disclosure, a greater proportion of the precipitated cells adhere to the walls of the first or second container when the cells are brought into contact with at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aid compared to when the cells are precipitated without contact with at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aid.

[0086] As described above, in any aspect of this disclosure, growth of more than 1000-fold is rapidly processed when cells are cultured in contact with culture medium and at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aids compared to culturing cells precipitated in contact with culture medium and in the absence of at least one externally added extracellular matrix or extracellular matrix protein and / or adhesion aids.

[0087] A method in any aspect of the present disclosure may include combining at least one externally added extracellular matrix or extracellular matrix protein with a culture medium before contacting the precipitated cells with at least one externally added extracellular matrix or extracellular matrix protein.

[0088] A method according to any aspect of the present disclosure may include adding at least one extracellular matrix or extracellular matrix protein to a cell culture medium that has been pre-distributed in a first or second container.

[0089] In one embodiment, when seeded in the presence of the culture medium and adhesion aid of the present disclosure, a larger proportion of cells in the first suspension may adhere to the wall of the first container than when seeded in a culture medium lacking the adhesion aid. In one embodiment, when seeded in the presence of the culture medium and adhesion aid of the present disclosure, a larger proportion of cells in the second suspension will adhere to the wall of the second container than when seeded in a culture medium lacking the adhesion aid.

[0090] Any method of any aspect of this disclosure may be suitable for automated culture or may be performed by automation. In particular, avoiding the need to pre-coat cell culture vessels may significantly reduce processing / preparation time and may be suitable for processes using liquid processing robots. Thus, the methods disclosed herein improve cell scale-up processes, such as by producing large quantities of high-quality cells in a simplified workflow.

[0091] The following non-limiting embodiments illustrate the present disclosure. [Examples]

[0092] Example 1: Maintenance and recovery of PSCs Human pluripotent stem cells (PSCs) were maintained in mTeSR® brands such as mTeSR® Plus or AOF (STEMCELL Technologies) on Corning® Matrigel®. PSCs were subculturified as described by the manufacturers; generally, cultures were ready for subculturing when they had a concentration of approximately 60–80% and the majority of colonies were large, dense, and had high-density multilayer centers. Various iPSC cell lines (SCTi003-A, WLS-1C, H9, and R038) were used in downstream proliferation experiments. PSCs may be seeded as single cells or cell aggregates.

[0093] A collection density of over 60% (approximately 2 x 10) from one well of a 6-well plate coated with Matrigel®. 6 PSCs (from living cells) were seeded in a T-175 flask. When harvested at an even lower density, 2 × 10⁶ 6 In some cases, cells from multiple wells may be required to obtain viable cells.

[0094] To isolate the cells, they were rinsed with PBS and contacted with approximately 1 mL of a trypsin-containing solution such as TrypLE® Express (ThermoFisher). The enzyme was inactivated with either 1 mL of mTeSR® Plus or ACF enzyme inhibitor solution (STEMCELL Technologies), and the cells were extracted by pipetting and dispensing 1-2 times. The single cell suspension was transferred to a Falcon® tube, pelletized, and then resuspended in 1-2 mL of seeding medium (mTeSR® Plus + 10% Clone® 2 (3.7 mL of Clone® 2 + 33.3 mL of mTeSR® Plus to prepare 37 mL)), and the viable cells were counted.

[0095] Example 2: Seeding and harvesting of PSCs as single cells in a T-175 flask PSCs were maintained in mTeSR® Plus on 6-well plates pre-coated with hESC-compliant Matrigel® and separated, essentially as described in Example 1. For seeding into T-175 flasks, Matrigel was thawed and added to the seeding medium to achieve a final concentration of 0.3–0.6% (e.g., T-175 seeding volume = 20 mL, 0.3% × 20 mL = 0.06 mL of Matrigel).

[0096] On the day of sowing, 9000 cells / cm² were planted in 35 mL (0.3-0.6% Matrigel) seeding medium. 2The cells were added to a T-175 flask that had not been pre-coated with extracellular matrix. The flask was shaken back and forth to evenly distribute the cells, and the cells were cultured at 37°C for 4-5 days. Complete medium changes with mTeSR®Plus medium (-Matrigel) may be performed on days 1, 3, and 4 after seeding, or daily.

[0097] On day 5 of growth, or when the culture reached a concentration of 90-95%, the cells were rinsed with PBS and contacted with 12 mL of a trypsin-based solution such as TryPLE® Express. An equal volume of mTeSR® Plus or ACF enzyme inhibitor solution was added to inactivate the enzyme, and the cells were gently extracted by approximately three pipette aspirations and dispensings. The single-cell suspension was transferred to a tube, pelletized, and then resuspended in a small amount (approximately 20 mL) of seeding medium, and the number of single viable cells was counted. 9000 cells / cm² 2 The cells were seeded in a 10-layer scale-out container using the cell count calculated from the seeding density. The seeded PSCs cultured for 5 days as described above were approximately 7-10 × 10⁶ cells. 7 It produced cells.

[0098] Example 3: Seeding of PSCs into a 10-layer scale-out container and replacement of the culture medium. For scaling out the growth of STi003-A cells, the cell suspension obtained in Example 2 was seeded into a 10-layer flask such as the Nunc® Cell Factory System or Corning® CELLSTACK®. An appropriate number of STi003-A cells were added in 2 L bottles / containers to approximately 1300 mL of seeding medium (10% Clone® 2 in mTeSR® Plus) (e.g., 9000 cells / cm³). 2 ×6360cm 2 The surface area of ​​the culture = 5.724 × 10 7 cell).

[0099] Since the 10-layer scale-out container cannot be observed under a standard bright-field microscope, a "small control flask" such as a T-75 flask is used to monitor the cell health and density, and the same cell density (cm³) as the scale-out container is used. 2 Seeds are sown at a rate of approximately 3 cm³, and mTeSR(trademark)Plus is used in a proportional volume (cm³) at a similar frequency. 2 The culture medium was changed (for the period).

[0100] The amount of Matrigel added to the cell suspension before seeding into the scale-out container was calculated according to the formula in Example 2. Approximately 1300 mL of the cell suspension was poured into the open port to seed into the scale-out container, ensuring that the culture medium was equilibrated in all layers. The container was then carefully placed in the incubator to prevent spillage between layers.

[0101] The culture medium was changed on days 1, 3, and 4 after seeding for cells in a 10-layer scale-out container and a small control T-75 flask. Optionally, the culture medium may be changed daily for cells in both containers.

[0102] Example 4: Recovery of PSCs from a 10-layer scale-out container The cell density in the small control T-75 flask of Example 3 was monitored, and when the cell density reached 90-95% (5-6 days after seeding), the cells in both containers were collected.

[0103] Cells in the scale-out container were rinsed with 200 mL of PBS, incubated with 200 mL of a trypsin-containing solution such as TrypLE Express or 0.25% trypsin-EDTA, inactivated with 200 mL of ACF enzyme inhibitory solution or mTeSR® Plus, and the isolated cells were transferred to sterile 1 L bottles / containers. The remaining cells in the scale-out container were collected in PBS and transferred to the same sterile collection bottles. Using a 50 mL serum pipette, the collected cell suspension was tritulated to ensure that aggregates were separated into single cells, divided equally into conical centrifuge tubes, and centrifuged at 300 × g for 5 minutes to remove any residual separation reagents. The cells were resuspended in mTeSR® Plus with optionally 10% CloneR® 2, and then cell counting and differentiation or freezing protocols were performed.

[0104] Cells in a small control T-75 flask may be harvested in parallel.

[0105] Example 5: Comparable morphology and proliferation rate of PSCs in a method without pre-coating The viable cell proliferation rate and morphology of PSCs grown as described in Examples 3 and 4, or by conventional pre-coating methods, were compared.

[0106] 4 μg / cm³ 2 or 9 μg / cm³ 2 Similar morphology and concentration were observed under both conditions, examined using either Matrigel (Figure 1A). On day 5, the concentration was 9 μg / cm³. 2 Control conditions with pre-coated Matrigel and 9 μg / cm³ 2 The average growth rates of PSCs under conditions where Matrigel was added were found to be 8.36 and 8.61, respectively (data not shown).

[0107] Furthermore, comparable viable cell proliferation rates on day 5 were observed for PSCs under multiple Matrigel conditions for both pre-coating and additive protocols (Figure 1B). Additionally, the day 5 proliferation rates among PSCs were similar across standard Matrigel concentrations (8.9 μg / cm³). 2 ) or 4 μg / cm³ 2 The results were equivalent under either of the following conditions (Figure 1B). The total number of viable cells was also equivalent under both the pre-coating and addition conditions (Figure 1C).

[0108] Overall, this data suggests that the time-consuming process of pre-coating culture vessels can be avoided, and that under such conditions, cells are unlikely to be adversely affected.

[0109] Example 6: Growth of PSCs in an uncoated scale-out container The proliferation capacity of PSCs (iPSCs: STi003-A and WLS-1C, ESC: H9) according to Examples 3 and 4 was 8,000 to 11,500 cells / cm³. 2 The evaluation was performed starting from the optimized seeding density.

[0110] All three PSC strains showed cell viability of over 95% when grown in a 10-layer scale-out container (Figure 2A), and averaged 6.83 × 10⁶ cells per 5 days from the 10-layer scale-out container. 5 Live cells / mL (Figure 2B), average of over 3 billion cells (Figure 2B) (3.5 × 10⁻¹⁰) 9 The PSCs showed viability, which corresponds to a proliferation rate of approximately 40 to 60 times within 5 days (Figure 2C).

[0111] Example 7: Quality and adhesion of PSCs grown in a scale-out container The quality of PSCs (iPSCs: SCTi003-A and WLS-1C, and ESCs: H9) was quantified by evaluating the percentage of cells expressing the undifferentiated markers OCT4 and TRA-1-60.

[0112] After staining with PE anti-human TRA-1-60-R and Alexa Fluor® 488 anti-Oct4 (Oct3) antibody (BioLegend) (Figure 3A), and then determining by flow cytometry (GuavaEasyCyte), it was observed that more than 90% of cells grown according to the novel methods outlined in Examples 3 and 4 expressed OCT4 and TRA-1-60, confirming that differentiation was limited or absent during PSC proliferation by the methods of this disclosure.

[0113] The effect of high-density cell culture on adhesion during subsequent subculturing was investigated for three PSC strains (SCTi003-A, WLS-1C, and H9). Briefly, single cells recovered from scale-out containers and corresponding small control T-75 flasks were seeded in seeding medium (mTeSR® Plus with CloneR® 2) into different wells of a Matrigel-coated 96-well plate (12 wells per condition). After 24 hours of culture, cell recovery was examined for each well, and approximately 100% viable cell recovery was observed for all three PSC strains examined (Figure 3B).

[0114] Overall, these results confirm the potential for rapid production of approximately 3 billion PSCs, the fact that more than 90% of the proliferated cells remain in an undifferentiated state, and that virtually all cells are recovered.

[0115] Example 8: Survival rate, recovery rate, and proliferation rate of PSC strains frozen at high cell density Since it is sometimes desirable to store PSCs grown in scale-out culture for subsequent use, we investigated the viability, recovery rate, and growth rate of PSCs after thawing. Suspensions of PSCs (SCTi003-A, WLS-1C, and H9) recovered from scale-out containers were placed in FreSR-S (STEMCELL Technologies) on ice for up to 2 hours (T=0, T=1, and T=2 hours) to simulate the long vial filling process required for large recovery volumes.

[0116] The recovered PSCs were 1 x 10 7 The cells were packed into vials, frozen at -80°C using Mr. Frosty's on ice at T=0, T=1, and T=2 hours, and then transferred to LN2 after 24 hours.

[0117] After more than one week in LN2, vials from each time point were thawed, seeding was performed, and the survival rate on day 0 was evaluated. Cells cultured for 5 days were then harvested, and the survival rate on day 5 was determined. The survival rate after thawing on day 0 was over 90%, and the survival rate after thawing on day 5 was observed to be over 80% for all three hPSC strains examined (Figures 4A and 4B).

[0118] After more than one week in LN2, vials from each time point were thawed, and repeated seeding was performed 5 days after thawing to examine the growth rate. All three cell lines were determined to have grown more than 30 times in 5 days (Figure 4C).

[0119] Overall, cells grown using the scale-out protocols described herein were cryopreserved and showed significant post-thaw growth rates.

[0120] Example 9: Proliferation ratio of H9 viable cells in different spike-in ECM proteins A cell aggregate suspension of hES cell line H9 was prepared from a culture maintained with mTeSR Plus, and the number of viable cells in the suspension was counted: 3 × 10⁶ 4 Live H9 cells (corresponding to a cell cluster of 1345 cells) were seeded into four repeat wells of a 6-well plate.

[0121] The matrices examined were Matrigel (Corning), Vitronectin-XF (STEMCELL Technologies), Laminin 511-E8 fragment (Matrixome), and Laminin-521 (BioLamina). For each matrix, pre-coating control and spike-in conditions were established. The coating concentration was the concentration recommended by the manufacturer.

[0122] Regarding the coating conditions, please refer to the recommended concentration (μg / cm³) beforehand. 2 The matrix was added to each repeating well and incubated overnight at 4°C. The matrix solution was aspirated before the cell suspension was allowed to settle in the pre-coated wells. For the spike-in condition, the matrix was added directly to the cell suspension and then allowed to settle in the uncoated wells to a concentration equivalent to that of the pre-coated control.

[0123] 24 hours after seeding, 6-well plates were imaged, and the resulting images were analyzed using ImageJ software to quantify the number of hPSC aggregates that successfully adhered to various matrices. Conditions with Matrigel and laminin 511 fragments performed at an equal or slightly better level than the respective pre-coating conditions (Figure 5). Furthermore, the condition with laminin 521 performed slightly worse than the corresponding pre-coating conditions.

[0124] Overall, these results demonstrate the potential of individual extracellular matrix proteins in uncoated cell proliferation workflows. Example 9.1: Maintenance of hPSCs using uncoated laminin

[0125] Four human PSCs (iPS cells: SCTi003-A, WLS-1C, STiPS-R038; embryonic stem cells: H9) maintained in mTeSR®Plus (STEMCELL Technologies) or TeSR™ AOF (STEMCELL Technologies) medium were investigated for their suitability in an uncoated proliferation workflow over at least 10 passages. Cells were contacted with recombinant laminin fragments ("CF") at a concentration of approximately 1.25 μg / mL, or with the fragments plus approximately 0.02% surfactant ("CF+S"). The uncoated condition was compared to a Matrigel control pre-coated at the manufacturer's recommended concentration. The addition of surfactant may improve cell separation.

[0126] The matrix solution was aspirated before seeding the cell suspension into the pre-coated wells. For the spike-in condition, recombinant laminin-511 fragments were added directly to the cell suspension before the cells settled in the uncoated wells.

[0127] Cell separation was performed essentially as described in Example 1, or using ReLeSR® PSC Selection and Passaging Reagent (STEMCELL Technologies). Briefly, 1 mL / well of ReLeSR® was added and aspirated within 1 minute to expose the colonies to a thin film of liquid. Cells were incubated at 37°C according to the manufacturer's instructions. Typically, separation under uncoated conditions was 2-3 minutes longer than under Matrigel® control conditions, but less than 10 minutes. Scrape separation using ReLeSR® was also investigated, in which the well surface was scraped off instead of tapping the plate to dislodge the cells. 8 × 10⁶ cells per well were used in a 6-well plate containing 2 mL of cell culture medium. 3 ~1 × 10 4 Cells were seeded with live cells. The culture medium was changed throughout the week using a restricted medium change schedule, as outlined in the manufacturer's protocols for mTeSR®Plus and TeSR®-AOF.

[0128] Comparable morphology was observed between pre-coated Matrigel controls and uncoated conditions using either mTeSR® Plus or TeSR® AOF medium (Figures 6A and 6B). Round, densely layered colonies with distinct margins were observed under all conditions, with the only difference being that the colonies under the uncoated condition were slightly smaller (however, the overall colony density was comparable as there were more colonies under the uncoated condition). Therefore, the uncoated condition can support the maintenance of PSCs for at least 10 passages.

[0129] The quality of the proliferated PSC lines was assessed by quantifying the percentage of cells expressing OCT4 and TRA-1-60, and the quality remained high under all uncoated conditions for all cell lines examined in both culture media (Figure 6C).

[0130] Next, the growth rates of four cell lines maintained in mTeSR®Plus and TeSR™AOF medium were calculated by either enzymatic separation of cells (Figure 7A) or chemical separation (Figure 7B). Enzymatic separation showed comparable growth rates between pre-coated and uncoated conditions (Figure 7A). When cells were scraped and subcultured together with chemical separation, comparable or superior growth rates were observed in the uncoated condition compared to the pre-coated control (Figure 7B). The ability to recover all cells using the scrape subculture method indicated that all matrices had growth comparable to the pre-coated Matrigel® control. Further analysis of the seeding efficiency of the four cell lines cultured in mTeSR®Plus (Figure 7C) and TeSR™ AOF (Figure 7D) medium showed even higher seeding efficiency in the uncoated condition compared to the pre-coated Matrigel® control.

[0131] The ability of PSCs grown using an uncoated workflow (CF or CF+S) to differentiate into ectoderm, endoderm, and mesoderm lineages (tristrains) was further evaluated. Tristrain differentiation of WLS-1C and STiPS-R038 cells (maintained with mTeSR Plus®) and H9 and SCTi003-A (maintained with mTeSR Plus® or TeSR® AOF) was performed on Matrigel using the Stemdiff® Tristrain Differentiation Kit (STEMCELL Technologies) according to the manufacturer's instructions. Positive cell percentages for ectoderm (Nestin, PAX6), endoderm (SOX17, CXCR4), and mesoderm (NCAM, Brachury) related markers were evaluated by flow cytometry. The results in Figure 8 show that all uncoated PSCs showed tristrain differentiation, often equivalent to or better than the pre-coated Matrigel® control.

[0132] Furthermore, microarray-based karyotype analysis showed no or limited reportable cytogenetic changes between uncoated and pre-coated conditions for all four cell cultures (not shown). Example 9.2: Maintenance of porcine PSCs (pPSCs) using uncoated laminin.

[0133] pPSCs derived from pig embryos were cultured in porcine PSC medium containing Activin A, FGF, and XAV939 in 6-well plates. Cells were isolated using enzymatic methods such as AccuMAX®, Accutase®, or TrypLE® according to the manufacturer's recommendations, then mechanically separated by tritulation and diluted in PSC medium. Cells were centrifuged and resuspended in PSC medium to create single-cell suspensions. These cell suspensions were mixed with 10 μM of RHO / Lock pathway inhibitor and seeded into 6-well plates in contact with pre-coated laminin 511 (0.375 mg / mL concentration), or laminin 511 (50%, 75%, or 100% concentration) or 521 (1 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, and 10 μg / mL) added to the PSC medium.

[0134] The added (e.g., uncoated) LN511 showed morphology and growth consistent with the pre-coated LN511 control (Figure 9). LN511 added at 50% of the pre-coated control concentration (0.375 mg / mL) showed morphology and growth comparable to the pre-coated LN511 control, while the 75% and 100% spike-in conditions resulted in even lower cell counts and slightly different morphology. The added LN521 showed morphology and growth consistent with the pre-coated LN511 control (Figure 10). LN521 added at even lower concentrations (e.g., 1 and 2.5 μg / mL) showed comparable growth to the pre-coated control at the end of passage 0. At even higher concentrations (e.g., above 5 μg / mL), LN521 was observed to produce more loosely packed cells with clearly defined colony boundaries (Figure 10). In subsequent subculturing, LN521 added at concentrations of 2.5 μg / mL and 5 μg / mL resulted in morphology and growth comparable to the pre-coated LN511 control (data not shown).

[0135] The exemplary embodiments described herein are illustrative only and not limiting. The embodiments described are susceptible to numerous modifications of composition, detail, and sequence of operations. The present invention is rather intended to encompass all such modifications within the scope of the claims, as defined by the claims, and this should be given a broad interpretation consistent with the entire description.

Claims

1. A method for growing cells in a single layer, To provide a first suspension of cells; To settle the cells of the first suspension into the first container; The precipitated cells are brought into contact with at least one externally added extracellular matrix or extracellular matrix protein, wherein the first container is not pre-coated with the at least one externally added extracellular matrix or extracellular matrix protein; The method comprising culturing the contacted cells as a monolayer in a cell culture medium.

2. The method according to claim 1, further comprising separating the monolayer and preparing a second suspension of cells.

3. moreover, The process involves settling the cells of the second suspension into the second container; The method involves bringing the precipitated cells into contact with at least one externally added extracellular matrix or extracellular matrix protein, wherein the second container is not pre-coated with the at least one externally added extracellular matrix or extracellular matrix protein; The method according to claim 2, further comprising culturing the contacted cells as a monolayer in the cell culture medium.

4. The method according to any one of claims 1 to 3, wherein the precipitated cells are brought into contact with the extracellular matrix or extracellular matrix protein added from the outside, before, after, or simultaneously with, the precipitated cells in the first or second container.

5. The method according to claim 3 or 4, wherein the surface area of ​​the second container is larger than the surface area of ​​the first container.

6. The method according to claim 5, wherein the surface area of ​​the second container is at least 10 times larger than the surface area of ​​the first container.

7. The method according to any one of claims 3 to 6, further comprising separating the single layer from the second container to obtain a third suspension.

8. The method according to claim 7, wherein the amount of cells in the second or third suspension is at least three orders of magnitude greater than the amount of cells in the first suspension.

9. The method according to claim 8, wherein the amount of cells at least three orders of magnitude greater than the above is the result of culturing for two weeks or less.

10. The method according to any one of claims 1 to 9, wherein the cells are stem cells.

11. The method according to any one of claims 1 to 10, wherein the at least one extracellular matrix protein added from an external source is recombinant, or the extracellular matrix is ​​a mixture of two or more extracellular matrix proteins.

12. The method according to any one of claims 1 to 11, further comprising contacting the precipitated cells with an adhesion aid containing albumin and one or more fatty acids.

13. The method according to any one of claims 1 to 12, wherein when the precipitate is brought into contact with the extracellular matrix or extracellular matrix protein added from an external source, a larger proportion of the precipitated cells adhere to the wall of the first container or the second container, respectively, compared to when the precipitate is brought into contact with the extracellular matrix or extracellular matrix protein added from an external source.

14. The method according to any one of claims 1 to 13, comprising combining the at least one externally added extracellular matrix or extracellular matrix protein with the culture medium before bringing the precipitated cells into contact with the at least one externally added extracellular matrix or extracellular matrix protein.

15. The method according to any one of claims 1 to 14, further comprising adding the at least one extracellular matrix or extracellular matrix protein to the cell culture medium pre-distributed in the first or second container.

16. A rapid processing method for growing cells in a single layer, a) To provide a first suspension of cells; b) Allowing the cells of the first suspension to settle in the first container; c) bringing the precipitated cells into contact with at least one externally added extracellular matrix or extracellular matrix protein, wherein the first container is not pre-coated with at least one externally added extracellular matrix or extracellular matrix protein; d) Culturing the contacted cells as a monolayer in a cell culture medium; e) Separating the monolayer and preparing a second suspension of cells; f) Repeating at least steps b) to d) in a second container with the second suspension of cells; g) The method comprising obtaining a more than 1,000-fold increase in two weeks or less.

17. The method according to claim 16, wherein the surface area of ​​the second container is larger than the surface area of ​​the first container.

18. The method according to claim 19, wherein the surface area of ​​the second container is at least 10 times larger than the surface area of ​​the first container.

19. The method according to any one of claims 16 to 18, wherein the precipitated cells are brought into contact with the extracellular matrix or extracellular matrix proteins added from the external source before, after, or simultaneously with, the first or second container.

20. The method according to any one of claims 16 to 19, further comprising separating the monolayer from the second container to obtain a cultivated suspension.

21. The method according to any one of claims 16 to 20, wherein the cells are stem cells.

22. The method according to any one of claims 16 to 21, wherein the at least one extracellular matrix protein added from an external source is recombinant, or the extracellular matrix is ​​a mixture of two or more extracellular matrix proteins.

23. The method according to any one of claims 16 to 22, further comprising contacting the precipitated cells with an adhesion aid containing albumin and one or more fatty acids.

24. The method according to any one of claims 16 to 23, wherein a larger proportion of the precipitated cells adhere to the walls of the first container or the second container, respectively, when the precipitate is brought into contact with the extracellular matrix or extracellular matrix protein added from an external source, compared to when the precipitate is brought into contact with the extracellular matrix or extracellular matrix protein added from an external source.

25. The method according to claim 23 or 24, wherein, when the cells are brought into contact with the extracellular matrix or extracellular matrix protein and / or the adhesion aid added from an external source, a larger proportion of the precipitated cells adhere to the wall of the first container or the second container, respectively, compared to when the cells are precipitated without contact with the extracellular matrix or extracellular matrix protein and / or the adhesion aid added from an external source.

26. The method according to claim 25, wherein when cells precipitated by contact with the culture medium and the extracellular matrix or extracellular matrix protein and / or the adhesion aid added from an external source are cultured, proliferation is processed more rapidly than 1,000 times compared to culturing cells precipitated in contact with the culture medium and in the absence of the extracellular matrix or extracellular matrix protein and / or the adhesion aid added from an external source.

27. The method according to any one of claims 1 to 26, comprising combining the at least one externally added extracellular matrix or extracellular matrix protein with the culture medium before bringing the at least one externally added extracellular matrix or extracellular matrix protein into contact with the precipitated cells.

28. The method according to any one of claims 1 to 26, further comprising adding the at least one extracellular matrix or extracellular matrix protein to the cell culture medium pre-distributed in the first or second container.

29. The method according to any one of claims 1 to 28, wherein the above method is automated.

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