Methods for cell expansion and differentiation

Abstract

The present disclosure concerns a method for the expansion and / or differentiation of stem cells in perfusion cell culture.

Description

[0001] METHODS FOR CELL EXPANSION AND DIFFERENTIATION

[0002] Field of the disclosure

[0003] The present disclosure concerns a method for the expansion and / or differentiation of stem cells in perfusion cell culture.

[0004] Background of the disclosure

[0005] Commercialization of cell-based technologies is a growing area of interest for multiple scientific and industrial fields. Stem cells in particular are an appealing cell type for biomedical and therapeutic applications. Researchers have made progress in providing methods for the large-scale manufacture of cells for use in these different fields, whilst keeping costs to a minimum. However, this is still a major commercial barrier.

[0006] Current technologies for large-scale manufacture of cells include the use of bioreactors. Bioreactors support the biological growth environment for cells, thus potentially enabling cell expansion and differentiation on a large-scale. Increasingly there is a focus on the culture of certain cell types, such as iPSCs, as 3D aggregates in bioreactors. This is partly related to cost benefits associated with avoiding use of microcarrier and microencapsulation technology. However, there are a number of technical challenges associated with culture of 3D cell aggregates, including controlling the size of the aggregates. Increased cell aggregate size is associated with negative physical and physiological properties, including inefficient nutrient transport, reduced cell quality and viability.

[0007] Another significant challenge is developing a culture system that is scalable. Perfusion systems have been developed for expanding hPSCs but using an in situ cell retention device (for example, see Manstein et al., (2021, Stem Cells Translational Medicine, Vol. 10, 7, July 2021, pages 1063-1080). The limitation of systems using in situ cell retention devices is that the waste / harvest lines become clogged with cells and have to be removed from the system to be cleaned or replaced. For this reason, this type of system cannot be used on a large scale.

[0008] Accordingly, there is an ongoing requirement for improved cell culture systems that can be used for expanding and / or differentiating 3D stem cell aggregates, in particular systems suitable for use on an industrial scale.

[0009] Thus, it is an object of the present disclosure to provide an improved method for cell expansion and / or differentiation, where large-scale production is achieved with improved yield, and maintained or improved cell quality and viability.

[0010] Summary of the disclosure

[0011] The present disclosure solves the above-mentioned technical problem.

[0012] Provided herein is a method for expanding and / or differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in matrix free 3D suspension conditions in a bioreactor; and expanding and / or differentiating the cells; wherein the cells and culture medium are circulated over a membrane filter in an alternating tangential flow and wherein the filter has a pore size suitable to retain cells in the bioreactor, while allowing removal of spent culture medium through an outlet in the bioreactor.

[0013] Further provided herein is a method for differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in a bioreactor under conditions that stimulate the stem cells to differentiate, wherein the bioreactor comprises an inlet for introduction of fresh culture medium, an outlet for extraction of spent culture medium and a cell retention device. Further provided herein is a method for differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in a bioreactor and a. extracting the cells from the bioreactor, b. removing spent culture medium from the bioreactor, c. supplying fresh culture medium to the bioreactor, d. removing non-viable (delaminated) single cells from the bioreactor; and e. reintroducing the extracted cells into the bioreactor, wherein the stem cells are cultured in conditions that direct the stem cells to differentiate.

[0014] In some embodiments, the filter is a hollow fiber filter.

[0015] In some embodiments, the filter has a pore size of approximately 0.1 pm to 10 pm, including about 2 pm.

[0016] In some embodiments, the alternating tangential flow is provided by a diaphragm pump.

[0017] In some embodiments, the alternating tangential flow rate is the alternating tangential flow rate is between 60 mL / min and 120 mL / min when expanding the stem cells and / or between 70 mL / min and 140 mL / min when differentiating the stem cells.

[0018] The alternating tangential flow rate can be surface-normalized with the membrane filter i.e. adjusted for the specific surface area of the membrane filter. In some embodiments, the ATF flow rate is 3800mL / min / m2to 4500mL / min / m2including 4000mL / min / m2to 4200mL / min / m2. In some embodiments, the ATF flow rate is 4050mL / min / m2to 4150mL / min / m2' including 4090mL / min / m2. Thus, if the hollow fiber membrane area is 0.022m2the ATF flow rate is 90mL / min, for example. If the hollow fiber membrane area is 0.13m2the ATF flow rate is 531mL / min.

[0019] In some embodiments, the bioreactor is a stirred tank bioreactor.

[0020] In some embodiments, the perfusion rate is between 0.2 vessel volumes per day (VVD) to 4 VVD.

[0021] In some embodiments, the dissolved oxygen concentration is maintained at 45% to 70% optionally wherein the oxygen concentration is about 65% during the expansion phase and / or about 50% during the differentiation phase.

[0022] In some embodiments, the stem cells are mammalian. In one embodiment, the stem cells are bovine, murine, avian or human stem cells.

[0023] In some embodiments, the stem cells are selected from any one of pluripotent stem cells (PSCs), muscle stem cells / satellite cells (MuSC / SC), adipose derived stem cells (ADSC), adipocytes, epithelial cells, mesenchymal stem / stromal cells (MSCs), fibro- adipogenic progenitors (FAPS), induced-pluripotent stem cells (iPSCs), breast milk stem cells (BMSCs), embryonic-like stem cells (ELC-Cs), chemically induced pluripotent stem cells (CiPSCs) and chemically induced totipotent stem cells (CiTotiSCs).

[0024] In some embodiments, the culture medium is serum free medium. In one embodiment, the culture medium is animal-component free media.

[0025] In some embodiments, the cells are differentiated into ectoderm cell lineage, endoderm cell lineage or mesoderm cell lineage. In some embodiments, the stem cells are iPSCs and the cells are differentiated into lactocytes.

[0026] In some embodiments, the expanded cells are cultured to a viable density of at least 10 million cells per mL and then isolated. In some embodiments, the differentiated cells are cultured to a viable density of at least 10 million cells per mL and then isolated.

[0027] Also provided herein is a method for producing a cell secreted product, comprising conducting any method as described herein and collecting product secreted by the differentiated stem cells via an outlet in the bioreactor. Further provided is a method for producing a cell product, comprising conducting any method as described herein and collecting the expanded and / or differentiated cell from the bioreactor.

[0028] Detailed description of the disclosure

[0029] Definitions

[0030] Within the context of the present disclosure, the term "cell expansion" (or similar) refers to the method by which cells are cultivated to produce a larger number of cells of the same cell type. The term "cell expansion" may be used interchangeably with "cell cultivation".

[0031] Within the context of the present disclosure, the term "cell differentiation" (or similar) refers to the method by which a stem cell changes into a differentiated cell.

[0032] Within the context of the present disclosure, the term "cell viability" refers to healthy, live cells. Viability may be calculated as the % number of cells in a sample that are healthy. Within the context of the present disclosure, the term "pluripotency" refers to the ability of a cell to develop into the three primary germ cell layers of the early embryo and therefore into all cells of the adult body.

[0033] Within the context of the present disclosure, the term "perfusion culture" includes a culture method that keeps cells in a bioreactor while continuously exchanging culture medium. "Perfusion culture" can also include continuous harvesting of a cellular product.

[0034] Within the context of the present disclosure, the term "alternating tangential flow" means an alternating flow, which flows generally tangentially to the membrane filter.

[0035] Within the context of the present disclosure, the term "3D suspension culture" means an artificially created environment in which cells are permitted to grow or interact with their surroundings in three dimensions. 3D suspension culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors. 3D suspension culture is matrix / microcarrier free.

[0036] Within the context of the present disclosure, the term "seed culture" refers to a small sample of viable single-cells.9

[0037] Within the context of the present disclosure, the term "culture medium" refers to any suitable medium that enables cell expansion of the cell type of interest and / or differentiation into a cell type of interest. The culture medium can comprise basal media (e.g. DMEM and / or F12) and growth factors, for example, fibroblast growth factor, insulin, transferrin and / or transforming growth factor beta. For example, in the case of an induced pluripotent stem cell expansion method, the culture medium may comprise mTeSR™. mTeSR™ is serum-free and comprises basal medium and recombinant human basic fibroblast growth factor and recombinant human transforming growth factor . An exemplary culture medium is described in Kuo et al. 2020, which is herein incorporated by reference (Kuo, H. H., Gao, X., DeKeyser, J. M., Fetterman, K. A., Pinheiro, E. A., Weddle, C. J. & Burridge, P. W. (2020). Negligible-cost and weekend-free chemically defined human iPSC culture. Stem Cell Reports, 14(2), 256-270). For example, in the case of an induced pluripotent stem cell differentiation method, the culture medium can be serum-free and comprise basal media comprising hydrocortisone, insulin, FGF10 and HGF.

[0038] Within the context of the present disclosure, the term "cell aggregate" (or similar) refers to a cluster of adhered cells of a same cell type.

[0039] Methods according to the present disclosure

[0040] The present disclosure relates to methods for cell expansion and / or differentiation, wherein cell yield is improved compared to methods described in the art and wherein the methods can be conducted on a large scale.

[0041] During cell expansion processes, the expanding cells form cell aggregates. If these aggregates become too large, this can disrupt nutrient transport to the cells and cause build-up of metabolic waste products from the cells, which negatively impacts cell quality and viability.

[0042] In the present disclosure, the cells are cultured in a perfusion system comprising an alternating tangential flow. It has been surprisingly shown in the present disclosure that cell aggregates cultured in matrix free 3D-suspension conditions in bioreactors using an alternating tangential flow system leads to an increased yield. In addition, this approach leads to smaller cell aggregates with a more homogenous size distribution, which is also beneficial.

[0043] Perfusion systems using ATF filtration are known for culture of recombinant CHO cells and human cell lines such as HEK-293, HeLa, Vero, and SF9, for example. However, it has never previously been considered that this type of culture system is likely to be successful for the expansion and differentiation of aggregating stem cells cultured in matrix free 3D-suspension conditions.

[0044] The present disclosure provides a method for expanding and / or differentiating stem cells in perfusion cell culture comprising: culturing the cells in culture medium in matrix free 3D-suspension conditions in a bioreactor; and expanding and / or differentiating the cells; wherein the cells and culture medium are circulated over a membrane filter in an alternating tangential flow and wherein the filter has a pore size suitable to retain cells in the bioreactor, while allowing removal of spent culture medium through an outlet in the bioreactor.

[0045] The method disclosed above is conducted in 3-dimensional (3D) suspension conditions, such as a hollow-fiber bioreactor. The method disclosed above is conducted in matrix free conditions i.e. cell-only aggregates. Thus, aggregated cells are retained in the bioreactor.

[0046] In some embodiments, the membrane filter is a hollow fiber filter.

[0047] The term "hollow fiber" refers to a tubular membrane. The internal diameter of the tube is preferably between 0.3 and 6.0 mm, more preferably between 0.5 and 3.0 mm, most preferably between 0.5 and 2.0 mm. Preferably, the pore size in the membrane is chosen such that the size of the pores in the mesh is close to the diameter of the cells, ensuring a high retention of cells while cell debris can pass the filter. Preferably, the mesh size is between 0.1 pm and 10 pm, for example about 0.2 pm to 3 pm.

[0048] The pore size of the membrane can also be suitable to allow cell secreted product to either be retained in the bioreactor with the retained cells or to allow the secreted product to be removed with the spent culture medium.

[0049] In some embodiments, the ATF is provided by a diaphragm pump. A diaphragm pump can comprise a diaphragm membrane which is moved up and down by pressurised air or vacuum, generating alternating flow.

[0050] In some embodiments, the ATF flow rate is between 50 mL / min and 150 mL / min. In one embodiment, the ATF flow rate is between 60 mL / min and 110 mL / min, for example between 85 mL / min and 94 mL / min, including about 90 mL / min, when expanding the stem cells. In one embodiment, the ATF flow rate is between 70 mL / min and 120 mL / min, for example between 95 mL / min and 105 mL / min, including about 100 mL / min, when differentiating the stem cells.

[0051] The alternating tangential flow rate can be surface-normalized with the membrane filter i.e. adjusted for the specific surface area of the membrane filter. In some embodiments, the ATF flow rate is 3800mL / min / m2to 4500mL / min / m2including 4000mL / min / m2to 4200mL / min / m2. In some embodiments, the ATF flow rate is 4050mL / min / m2to 4150mL / min / m2' including 4090mL / min / m2.

[0052] Thus, if the hollow fiber membrane area is 0.022m2the ATF flow rate is 90mL / min, for example. If the hollow fiber membrane area is 0.13m2the ATF flow rate is 531mL / min. In some embodiments, perfusion starts 24 hours to 56 hours after inoculation of the bioreactor, including at about 40 to 50 hours after inoculation.

[0053] In some embodiments, the perfusion rate is between 0.2 vessel volumes per day (VVD) to 4 VVD.

[0054] In some embodiments, perfusion starts 24 hours to 56 hours after inoculation of the bioreactor at a rate of between 0.3 VVD to 0.7 VVD, including about 0.5 VVD. In some embodiments, the perfusion rate is increased to between 1 VVD and 2 VVD at between 65 and 75 hours after inoculation.

[0055] In some embodiments, the perfusion rate is between 1 VVD and 2 VVD during the cell expansion phase.

[0056] In some embodiments, perfusion starts 24 hours to 56 hours after inoculation of the bioreactor at a rate of between 0.3 VVD to 0.7 VVD, including about 0.5 VVD. In some embodiments, the perfusion rate is increased to between 1 VVD and 2 VVD at between 65 and 75 hours after inoculation.

[0057] In some embodiments, the perfusion rate is between 0.5 VVD and 2.5VV from 5 or 6 days after inoculation.

[0058] In some embodiments, the perfusion rate is between 0.5 VVD and 2.5VV during the cell differentiation phase.

[0059] In some embodiments, the outlet for removing spent media is located at a section of the membrane filter proximal to the bioreactor. In some embodiments, the bioreactor comprises a second or further outlet located at a section of the membrane filter end proximal to the diaphragm pump. This second outlet can be used for removal of spent media and / or cell secreted product.

[0060] In some embodiments, the bioreactor additionally comprises a bleed line. The bleed line can be used for removing spent culture medium.

[0061] In some embodiments, cell expansion is carried out in a stirred tank bioreactor, wherein the cells are stirred at a first stirring speed of between 120 RPM to 180 RPM, including about 150 RPM and at a second stirring speed of between 220 RPM and 280 RPM, including about 250 RPM.

[0062] In some embodiments, cell expansion is carried out in a stirred tank bioreactor, wherein the cells are stirred at a first stirring speed of between 3.32 W / m3to 11.2 W / m3, including about 6.5 W / m3and at a second stirring speed of between 20.5 W / m3and 42.2 W / m3, including about 30 W / m3.

[0063] In some embodiments, cell expansion is carried out in a stirred tank bioreactor, wherein the cells are stirred at a first stirring speed of between 120 RPM to 180 RPM for the first 1 to 2 days following inoculation, including about 150 RPM, and the speed is increased to a second stirring speed of between 220 RPM and 280 RPM, including about 250 RPM, over a period of 16 to 48 hours. In some embodiments, the culture continues to be stirred at a speed of between 220 RPM and 280 RPM, including about 250 RPM, until completion of expansion. In some embodiments, the culture continues to be stirred at a speed of between 220 RPM and 280 RPM, including about 250 RPM, in the cell differentiation phase.

[0064] In some embodiments, cell expansion is carried out in a stirred tank bioreactor, wherein the cells are stirred at a first stirring speed of between 3.32 W / m3to 11.2 W / m3for the first 1 to 2 days following inoculation, including about 6.5 W / m3, and the speed is increased to a second stirring speed of between 20.5 W / m3 and 42.2 W / m3, including about 30 W / m3, over a period of 16 to 48 hours. In some embodiments, the culture continues to be stirred at a speed of between 20.5 W / m3and 42.2 W / m3, including about 30 W / m3, until completion of expansion. In some embodiments, the culture continues to be stirred at a speed of between 20.5 W / m3and 42.2 W / m3, including about 30 W / m3, in the cell differentiation phase.

[0065] In some embodiments, the method comprises inoculating the bioreactor with the seed cell culture. In some embodiments, the bioreactor is inoculated with 50,000 to 6,000,000 cells per ml of the bioreactor vessel. In some embodiments, the bioreactor is inoculated with 50,000 to 6,000,000 cells per ml of the bioreactor vessel; 150,000 to 1,000,000 cells per ml of the bioreactor vessel; 200,000 to 500,000 cells per ml of the bioreactor vessel; or 250,000 to 500,000 cells per ml of the bioreactor vessel. In some embodiments, the bioreactor is inoculated with 3,000,000 to 6,000,000 cells per ml of the bioreactor vessel.

[0066] In one embodiment, the method comprises expanding cells in the bioreactor for a specific time period as described anywhere herein. For example, the cells can be expanded for up to 5 days following inoculation. In some embodiments, the method comprises isolating the expanded cells. The expanded cells are in the form of cell aggregates. In some embodiments, the cells have expanded by at least 10 fold. In some embodiments, the cells have expanded by at least 10 fold in 4 days. In some embodiments, the expanded cells are isolated by centrifugation. In some embodiments, the expanded cells are isolated by gravimetry. In some embodiments, the expanded cells are dissociated into single-cells for further expansion. In some embodiments, the expanded cells are dissociated into single-cells for storage in a biobank. In some embodiments, the expanded cells are exposed to differentiation media. Culture conditions for expanding and differentiating stem cells are described in WO2021 / 219634, WO2021 / 219635, W02023 / 073107, W02023 / 073106,

[0067] WO2023 / 073121, WO2023 / 073119 and W02023 / 073108, for example, the contents of which are hereby incorporated by reference.

[0068] Cells expanded according to the present disclosure express pluripotency markers. Pluripotency may be measured according to specific pluripotency markers such as TRA 1-81, SSEA-4, SSEA-1, OCT3 / 4, SOX2, NANOG and / or any other known markers in the art. High levels of expression of these markers indicate a high level of pluripotency and ability to differentiate into different lineages. A suitable assay is gene expression profiling using Nanostring technology. Another suitable assay for testing pluripotency markers is germ layer analysis performed by differentiating cell aggregates into different lineages using STEMdiff™ Trilineage Differentiation Assay Kit (StemCell Technologies).

[0069] In some embodiments, pluripotency markers are selected from one or more of TRA-1- 81, SSEA-4, SSEA-1, OCT3 / 4, NANOG and SOX2.

[0070] In some embodiments, the stem cells are differentiated into ectoderm cell lineage, endoderm cell lineage or mesoderm cell lineage.

[0071] In some embodiments, the stem cells are iPSCs and are differentiated into lactocytes. Differentiation into lactocytes can be confirmed by detection of GATA3. In some embodiments, differentiation into lactocytes can be confirmed by detection of one or more, or all of luminal markers EpCAM, GATA3, CD49f, KRT18 and / or KRT8.

[0072] In some embodiments, the cells are mammary epithelial cells or breast milk stem cells and are differentiated into lactocytes. The disclosure further provides a method for differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in a bioreactor under conditions that stimulate the stem cells to differentiate, wherein the bioreactor comprises an inlet for introduction of fresh culture medium, an outlet for extraction of spent culture medium and a cell retention device.

[0073] All embodiments described above are also envisaged for use in this method for differentiating stem cells. Thus, this method can be conducted in matrix free 3D- suspension conditions. This method can also be conducted in a bioreactor comprising a membrane filter, which the cells and culture medium are circulated over in an alternating tangential flow.

[0074] A cell retention device retains cells inside the bioreactor while components such as cell debris, spent media and cell products can be removed.

[0075] In some embodiments, the cell retention device is ex situ. This means it is located on a waste line and / or harvest line outside of the bioreactor vessel.

[0076] In some embodiments, the cell retention device is not an acoustic filter. In some embodiments, the cell retention device is a physical device. In some embodiments, the cell retention device is a mesh or a membrane.

[0077] In some embodiments, the cell retention device is a hollow fiber filter. Preferably, the mesh size is between 0.1 and 10 pm, for example about 0.2 pm.

[0078] Further, the disclosure provides a method for differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in a bioreactor and a. extracting the cells from the bioreactor, b. removing spent culture medium from the bioreactor, c. supplying fresh culture medium to the bioreactor, d. removing non-viable single cells from the bioreactor; and e. reintroducing the extracted cells into the bioreactor, wherein the stem cells are cultured in conditions that direct the stem cells to differentiate.

[0079] Further provided herein is a method for differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in a bioreactor and continuously a. extracting the cells from the bioreactor, b. retaining aggregated cells using a hollow-fiber membrane, c. removing spent culture medium from the hollow-fiber filter, d. supplying fresh culture medium to the bioreactor, e. removing non-viable (delaminated) single cells from the bioreactor; and f. reintroducing the retained cells into the bioreactor, wherein the stem cells are cultured in conditions that direct the stem cells to differentiate.

[0080] This method is a 'semi-perfusion' method, which mimics perfusion.

[0081] A non-viable cell can be considered to be a delaminated cell.

[0082] In one embodiment, the method comprises differentiating cells in the bioreactor for a specific time period as described anywhere herein. For example, the cells can be differentiated from 5 days following inoculation. In some embodiments, the method comprises isolating the differentiated cells. In some embodiments, the methods disclosed herein are conducted in 3-dimensional (3D) suspension conditions, such as a hollow-fiber bioreactor. In some embodiments, the methods disclosed above are conducted in matrix free conditions i.e. cell-only aggregates.

[0083] Cell types

[0084] The method of the present disclosure relates to expanding and / or differentiating bovine, murine, avian or human cells.

[0085] In one embodiment, the present disclosure relates to expanding and / or differentiating mammalian cells.

[0086] Cells for use in the present disclosure may include any mammalian cell type that is required to be expanded and / or differentiated.

[0087] In one embodiment, the cells are human cells. In one embodiment, the cells are bovine cells. In one embodiment, the cells are stem cells. In one embodiment, the cells are human stem cells. In one embodiment, the cells are bovine stem cells. In one embodiment, the cells are adult human stem cells.

[0088] In one embodiment, the cells may be selected from any one of pluripotent stem cells (PSCs), muscle stem cells / satellite cells (MuSC / SC), adipose derived stem cells (ADSC), adipocytes, epithelial cells, mesenchymal stem / stromal cells (MSCs), fibro-adipogenic progenitors (FAPS), induced-pluripotent stem cells (iPSCs), breast milk stem cells (BMSCs), embryonic-like stem cells (ELC-Cs), chemically induced pluripotent stem cells (CiPSCs) and chemically induced totipotent stem cells (CiTotiSCs).

[0089] In a preferred embodiment, the cells are iPSCs. In another preferred embodiment, the cells are BMSCs. In a further preferred embodiment, the cells are epithelial cells. In some embodiments, the cells are not neural stem cells.

[0090] It will be understood that the features of the cell type as described herein may be combined with any of the methods of the disclosure, such as methods relating to expanding cells and methods relating to differentiating cells.

[0091] Cell culture vessel and conditions

[0092] Methods of the present disclosure relate to cell expansion and / or differentiation in a perfusion bioreactor. In one embodiment, the bioreactor is a stirred tank bioreactor.

[0093] To achieve stirring of the cell culture, the bioreactor may comprise one or more impellers. Preferably, the impeller is a single 'elephant ear' impeller. A single 'elephant ear' impeller having a 30 mm diameter and 45° pitched-blade angle is described in by Rotondi, M et al. 2021 (incorporated herein by reference) and may be used in any embodiments of the present disclosure described herein. Hence, the power number (Np) to convert the stirring speed to PPV is 2.07.

[0094] In one embodiment, the bioreactor comprises one or more bioreactor chambers. In some embodiments, the reactor chamber has an internal volume of 0.1-100,000 L. In some embodiments, the working volume is up to 50,000L, including 100 mL to 50,000L and up to 10,000 L, including 100 mL to 10,000 L. The working volume can also be 100 to 500 mL, 150 to 400 mL, 200 to 300 mL, or 200 to 250 mL.

[0095] In some embodiments, the fluid density is 800 to 1,200 kg / m3of aqueous solution. Preferably, the fluid density is 1,000 kg / m3of aqueous solution.

[0096] The stirred tank bioreactor comprises a cell culture medium. The cell culture medium facilitates cell expansion and / or differentiation by providing necessary nutrients to the cells. Any suitable cell culture medium known in the art may be used, according to the cell type that is being expanded or differentiated.

[0097] In one embodiment, the cell culture medium is a serum-free culture medium. In one embodiment, the cell culture medium is an animal-component free culture medium. In one embodiment, the cell culture medium is any suitable stem cell maintenance medium, such as mTeSR™ or E8 medium. Culture medium supplementation can include mTeSR plus and B8 media with Nutrient Mixture F12 (DMEM F12) as basal medium. An exemplary culture medium is described in Kuo et al. 2020, which is herein incorporated by reference (Kuo, H. H., Gao, X., DeKeyser, J. M., Fetterman, K. A., Pinheiro, E. A., Weddle, C. J. & Burridge, P. W. (2020). Negligible-cost and weekend- free chemically defined human iPSC culture. Stem Cell Reports, 14(2), 256-270).

[0098] The conditions within the cell culture vessel may also be controlled to facilitate cell expansion or differentiation, for example pH, nutrient supply, toxic by-product disposal and dissolved oxygen (DO) levels may be controlled. Any suitable conditions known in the art may be used, according to the cell type that is being expanded or differentiated.

[0099] In one embodiment, the nutrient supply comprises glucose, optionally within the range of 5 to 30nM. In one embodiment, the nutrient supply comprises glutamine (or similar compounds such as GLUTAMAX™), optionally within the range of 1 to lOnM.

[0100] In one embodiment, the toxic by-products comprise lactate, optionally wherein the level of lactate in the cell culture vessel is removed from the bioreactor such that the concentration of lactate does not exceed 60mM. In one embodiment, the concentration of lactate does not exceed 3g / L. In one embodiment, the toxic byproducts comprise ammonia, optionally wherein the level of ammonia in the cell culture vessel is removed from the bioreactor such that the concentration of ammonia does not exceed 5mM. In one embodiment, the pH of the cell culture is above pH 6. In another embodiment the pH of the cell culture is pH 6 to 7.5. In a preferred embodiment, the pH of the cell culture is 7 to 7.5, more preferably 7.1 to 7.2. In one embodiment, the nutrient supply comprises glucose and / or amino acids. In one embodiment, the spent culture medium comprises lactate and / or ammonia.

[0101] In one embodiment, the dissolved oxygen (DO) is in the range of 30 to 70%. In one embodiment, the dissolved oxygen (DO) is in the range of 45 to 70%.

[0102] In some embodiments, the dissolved oxygen (DO) is 60% to 70% when the cells are being expanded and / or 45% to 55% when the cells are being differentiated. In some embodiments, the dissolved oxygen (DO) is about 65% when the cells are being expanded and / or about 50% when the cells are being differentiated.

[0103] In some embodiments, the cells are cultivated with pH controlled at 7 to 7.5 and dissolved oxygen (DO) controlled at 60% to 70% when the cells are being expanded, optionally wherein the temperature is 35°C to 40°C, preferably 37°C.

[0104] In some embodiments, the cells are cultivated with pH controlled at 7 to 7.5 and dissolved oxygen (DO) controlled at 45% to 55% when the cells are being differentiated, optionally wherein the temperature is 35°C to 40°C, preferably 37°C.

[0105] In some embodiments, the cells are cultivated with pH controlled at 7.2 and dissolved oxygen (DO) controlled at about 65% when the cells are being expanded and / or about 50% when the cells are being differentiated, optionally wherein the temperature is 35°C to 40°C, preferably 37°C. Gas is supplemented to the headspace. In one embodiment, headspace gassing is carried out at 150 to 250 mL / min with a mixture of N2, O2, and CO2. Dissolved oxygen is controlled with O2gas overlay (0 - 100%). Percentage of CO2overlay is used to control pH, ranging from 0% to 20%.

[0106] In some embodiments, cell expansion is carried out in a stirred tank bioreactor, wherein the cells are stirred at a first stirring speed of between 3.32 W / m3to 11.2 W / m3, including about 6.5 W / m3and at a second stirring speed of between 20.5 W / m3and 42.2 W / m3, including about 30 W / m3.

[0107] In some embodiments, cell expansion is carried out in a stirred tank bioreactor, wherein the cells are stirred at a first stirring speed of between 3.32 W / m3to 11.2 W / m3for the first 1 to 2 days following inoculation, including about 6.5 W / m3, and the speed is increased to a second stirring speed of between 20.5 W / m3 and 42.2 W / m3, including about 30 W / m3, over a period of 16 to 48 hours. In some embodiments, the culture continues to be stirred at a speed of between 20.5 W / m3and 42.2 W / m3, including about 30 W / m3, until completion of expansion. In some embodiments, the culture continues to be stirred at a speed of between 20.5 W / m3and 42.2 W / m3, including about 30 W / m3, in the cell differentiation phase.

[0108] It will be understood that the features of the cell culture as described herein may be combined with any methods of the disclosure, such as methods relating to expanding cells and methods relating to differentiating cells.

[0109] Uses of the present disclosure

[0110] The present disclosure provides novel methods for the large-scale manufacture of cells, e.g. PSCs. The cell produced by the methods of the disclosure may be useful as a cell product, or products secreted by the cells may be useful, in a variety of industries. Thus, the disclosure provides a method of producing a cell secreted product, comprising conducting any of the methods described herein for differentiating stem cells and collecting product secreted by the differentiated stem cells via an outlet in the bioreactor. In some embodiments, the outlet for collecting secreted product is the same as the outlet for removing spent culture medium.

[0111] The disclosure further provides a method of producing a cell product, comprising conducting any method as described herein and collecting expanded and / or differentiated cells via an outlet in the bioreactor.

[0112] In one embodiment, the expanded and / or differentiated cell culture is suitable for use as a food product, for example in the cultivated meat industry. In one embodiment, the expanded and / or differentiated cell culture is suitable for use in the manufacture of a food product.

[0113] In some embodiments, the secreted product is a milk like product, optionally a human milk like product.

[0114] In one embodiment, the expanded and / or differentiated cell culture is suitable for use as a medicine. In one embodiment, the expanded and / or differentiated cell culture is suitable for use in the development of a medicine.

[0115] The disclosure also provides an expanded and / or differentiated cell culture, e.g. PSC cell culture, produced according to the method as described anywhere herein. Figures

[0116] Fig. 1: Semi-Perfusion matrix-free iPSC 3D culture system.

[0117] Fig. 2: ATF-bioreactor set up. A indicates the feed line, used for providing culture medium to the bioreactor. B indicates an optional bleed line. C indicates the preferred outlet for removing spent media and / or product (i.e. in a section of the membrane filter proximal to the bioreactor). In operation, the culture medium containing cells is pumped out of the bioreactor and circulated across the membrane filter (shown here as a hollow fiber filter). The cell-free supernatant (permeate) is removed through outlet C and the concentrated cells are returned to the bioreactor.

[0118] Fig. 3: iPSC 3D expansion (aggregate morphology).

[0119] Fig. 4: illustrates a X4 improvement in cell daily fold expansion when culturing iPSC aggregate with ex-situ ATF perfusion set-up.

[0120] Fig. 5: cells cultured in ex-situ hollow-fiber ATF perfusion method demonstrated high levels of expression of pluripotency markers.

[0121] Fig. 6: semi-perfusion method increased the cell yield (VCD - Viable Cell Density) throughout the process.

[0122] Fig. 7: illustrates organoid morphology - 3D Directed differentiation under perfusion (with ATF) and semi-perfusion conditions compared to the repeated batch method (control). Fig. 8: flow cytometry marker demonstrates comparable differentiation efficiency in the perfusion (with ATF) method and the semi-perfusion method compared to the repeated batch method (control).

[0123] Fig 9: shows expression of mammary gland progenitor marker GATA3 using NanoString technology for gene expression profiling.

[0124] Fig 10: shows expression of Oct4 and NaNog as a marker for pluripotency, determined using NanoString technology for gene expression profiling.

[0125] Fig 11: three germ layer analysis - differentiation capacity of cells cultured in the perfusion method compared to the repeated batch method (control).

[0126] Experimental section

[0127] Here we describe exemplary methods of the disclosure. The examples should not be construed as limiting in anyway.

[0128] Example 1

[0129] Methods

[0130] Pluripotent Stem Cell Expansion in semi-perfusion system

[0131] Semi-perfusion bioreactor operation set-up is shown in Figure 1. This set-up is representative of a perfusion system (mimicking a perfusion process).

[0132] Mimicking perfusion (semi-perfusion) process is performed in a robotic automated micro-bioreactor system (e.g. AMBR®250). The process was performed using an unbaffled 'elephant-ear' single-impeller vessel type, suitable for stem cell culture. Cells were seeded at 0.5 million cells per mL using B8 media for iPSC culture. Feeding is initiated 24 hours after the cell seeding.

[0133] Semi-perfusion cell culture utilizes automated robotic liquid-handler to perform spent- media removal and feeding. Semi-perfusion is a semi-continuous cell cultivation method that breaks down feeding regimen of fed-batch process into smaller- increment with more frequency. One small semi-perfusion batch will replace 50 mL of the culture volume out of 250 mL total volume (20%), with one semi-perfusion cycle will replace lOmL of the volume (one batch contains 5 cycles). The step-by-step of one semi-perfusion feeding cycle is as follows:

[0134] 1. Aspiration of cells and spent media from bioreactor (10 mL volume)

[0135] 2. Settling of cells inside the liquid-handler tip (varying from 20 sec to 3 min depending on the size of the aggregated cells)

[0136] 3. Cell retention by returning the cells back into the bioreactor (10% of aspirated volume)

[0137] 4. Removal / collection of the spent media Gassing with 19 mL / min, headspace, N2 gas, 02 overlay from 0-100% to control the DO. CO2used to control the pH (bring the pH down if needed), with overlay percentage from 0 - 20%.

[0138] Pluripotent Stem Cells Expansion in ATF perfusion system

[0139] Bioreactor description

[0140] Bioreactor operation with Alternating Tangential Flow (ATF) perfusion set-up is shown in Figure 2. The set up consist of Benchtop Biostat-B bioreactor (Sartorius Stedim Biotech, Germany) and XCell ATF perfusion set up (Repligen, USA). ATF line is connected to one of the bioreactor dip-tube with an approximately 35 cm silicone tubing.

[0141] Cell culture

[0142] Human CDI-iPSC-603 was cultured in the bioreactor with chemically defined media ( serum-free media comprising basal medium and a proliferation supplement cocktail)7heparin and hydrocortisone. Cells were thawed from a cell bank vial and were expanded in T-flask or CellStack, approximately 6 passages prior to the bioreactor seeding. Cells were cultured in an humidified incubator with temperature of 37°C, 8% CO2.

[0143] Bioreactor and ATF preconditioning

[0144] Prior to the bioreactor culture, Single-Use (SU) UniVessel (Sartorius Stedim Biotech., Germany) was prepared and preconditioned. SU UniVessel are connected aseptically with media feed line, ATF line, and effluent line. Headspace gas line was connected with 4 different gas types: CO2, O2, N2, and air. Bioreactor was filled with 1.5L DMEM / F12 basal media (Life Technologies, USA) for preconditioning for at least 2 hours. Bioreactor preconditioning was performed with the following conditions:

[0145] • Temperature : 37°C

[0146] • Headspace gas flow rate : 150 ccm, 100% air

[0147] • Stirring speed : 100 RPM Afterwards, ATF line and permeate line was primed to wet the hollow-fiber filter. Two hours after the preconditioning, a sample was withdrawn from the bioreactor for bioanalysis with Nova Flex2. The obtained pH and O2saturation values were used to calibrate the bioreactor online measured values. Prior to the seeding, 3.75 mL of 400X Hi-Def B8 supplement (Defined Biosciences, USA), and 850 pL of 200 nM Rho-kinase inhibitor (Y-27632, ABCAM) was supplemented to the bioreactor, resulting in a complete B8 media with 10 pM Y-27632.

[0148] Bioreactor seeding

[0149] Prior to the seeding, iPSC was detached from T-flasks or CellStack using Accutase as dissociation reagent. Viable cell concentrations and culture viability were assessed using NC-202 (Chemometek, Denmark). About 850 million viable cells were separated from the preculture, centrifuged, and resuspended in a final seed volume of around 200 mL. The inoculum (seed) culture should have approximately 4.25 million cells per mL. Cells were seeded to the preconditioned bioreactor, resulting in approximately 0.5 million cells per mL of viable cell concentration.

[0150] Bioreactor operation parameters

[0151] Feeding and ATF flow

[0152] Note: perfusion rate was adjusted based on lactate level. Lactate was maintained below 40 mM.

[0153] Differentiation supplement cocktail (equivalent for full bioreactor volume) was added to the bioreactor prior to the start of the differentiation. Afterwards, the feeding line was replaced with differentiation media prior to resuming the ATF process.

[0154] Stirring speed

[0155] Other operating conditions pH : 7.2

[0156] DO : 65% oxygen saturation, controlled with %O2headspace gassing variable

[0157] (100 ccm gas flow)

[0158] Three-germ layer analysis of 3D iPSC aggregate cultured in perfusion process iPSC 3D Aggregate Sampling from Bioreactor Induced pluripotent stem cell (iPSC) aggregates were sampled from the ATF perfusion bioreactor under sterile conditions using the designated sampling port. The collected aggregates were transferred into 50 mL conical tubes and allowed to settle by gravity or centrifuged at 100xg for 3 minutes. The supernatant was carefully removed, and aggregates were resuspended in pre-warmed complete B8 media. iPSC 3D Dissociation into Single Cells using Accutase

[0159] For dissociation into single cells, aggregates were washed once with calcium- and magnesium-free PBS and incubated with pre-warmed Accutase for 10-15 minutes at 37°C, with gentle pipetting every 5 minutes to facilitate dissociation. The reaction was quenched by adding an equal volume of complete B8 media supplemented with 10 pM Y-27632 (ROCK inhibitor), followed by centrifugation at 200 x g for 5 minutes. The resulting cell pellet was resuspended in fresh medium containing ROCK inhibitor, and cell counts were determined prior to seeding. iPSC Single Cell Seeding into 2D Monolayer Culture

[0160] Single-cell suspensions were seeded onto Vitronectin-coated plates at a density of 1.5-2.5 x 104cells / cm2in complete B8 media supplemented with ROCK inhibitor. Cultures were maintained at 37°C and 5% CO2, and after 24 hours, the medium was replaced with complete B8 media without ROCK inhibitor. Daily medium changes were performed until cells reached the desired confluency for three-germ-layer analysis.

[0161] Three Germ-Layer Analysis Using StemCell Technologies Kit To assess the differentiation potential of iPSCs into the three germ layers, the STEMdiff™ Trilineage Differentiation Kit (StemCell Technologies) was employed following the manufacturer's instructions.

[0162] Briefly, iPSCs at 70-80% confluency were washed with PBS and seeded into separate Vitronectin-coated wells for ectoderm, mesoderm, and endoderm induction. Lineagespecific induction media were applied, and cultures were maintained for 5-7 days with daily medium changes. At the end of the differentiation period, cells were washed with PBS and fixed using the kit's recommended fixation and permeabilization buffer (perm buffer) for the appropriate duration at room temperature. Following fixation, cells were incubated with primary antibodies targeting germ-layer markers: PAX6 and SOX2 for ectoderm; Brachyury (T) & CD140b for mesoderm; and SOX17 and CD184 for endoderm. Flow cytometry analysis was performed to assess the respective protein expression in each lineages.

[0163] Repeated batch culture (control)

[0164] The perfusion methods were compared to a repeated batch culture approach, which increased iPSC cell mass through repeated re-inoculation of part or all of the cells from one batch of cultured cells into the next batch. In a repeated-batch process, feeding was performed by the following steps:

[0165] 1. Stopping the agitation

[0166] 2. Removing the spent media from the bioreactor

[0167] 3. Feeding fresh media

[0168] 4. Starting the agitation

[0169] Results Figure 3 illustrates that smaller and more regular aggregate sizes were achieved with both the semi-perfusion and perfusion-ATF approaches, compared to the repeated batch approach.

[0170] Figure 4 illustrates an improvement in cell expansion when culturing iPSC aggregates with the semi-perfusion or ex-situ ATF perfusion set-up. There was a four fold improvement when using to ATF perfusion set-up compared to the control repeated batch approach.

[0171] Cells cultured using the ex-situ hollow-fiber ATF perfusion method demonstrated high expression of pluripotency surface protein markers - SOX2, OCT3 / 4, TRA1-81, SSEA- and SSEA-1 - see Figure 5. These markers were quantified with flow cytometry technology.

[0172] The semi-perfusion method increased the cell yield (VCD - Viable Cell Density) throughout the differentiation process, as demonstrated in Figure 6.

[0173] Cell cultured in the perfusion and semi-perfusion conditions demonstrated 3D directed differentiation (see Figure 7). In addition, flow cytometry marker analysis demonstrates comparable differentiation efficiency by cells cultured in the perfusion semi-perfusion methods compared to the repeated batch method. The analysis shows EpCAM and CD49F double positive cells can be detected at an early progenitor stage.

[0174] Using NanoString technology for gene expression profiling, expression of differentiation markers GATA3, Oct4 and NaNog was assessed. Figure 9 shows gene expression of luminal marker (GATA3), evidencing differentiation into a luminal-like phenotype. Figure 10 shows expression of Oct4 and Nanog as markers for pluripotency decreasing as cells pass towards maturation and differentiation. The gene expression profile was quantified with Nanostring technology.

[0175] The differentiation capacity of cells cultured in the perfusion conditions was assessed using three germ layer analysis. Figure 11 shows an increased differentiation potential of iPSCs into the mesodermal and ectodermal lineages when using the perfusion approach, compared to the repeated batch approach.

Claims

Claims1. A method for expanding and / or differentiating stem cells in perfusion cell culture comprising: culturing the cells in culture medium in matrix free 3D-suspension conditions in a bioreactor; and expanding and / or differentiating the cells; wherein the cells and culture medium are circulated over a membrane filter in an alternating tangential flow and wherein the filter has a pore size suitable to retain cells in the bioreactor, while allowing removal of spent culture medium through an outlet in the bioreactor.

2. The method of claim 1, wherein the filter is a hollow fiber filter.

3. The method of claim 1 or claim 2, wherein the filter has a pore size of between 0.1 pm to 10 pm.

4. The method of any one of claims 1 to 3, wherein the alternating tangential flow is provided by a diaphragm pump.

5. The method of any one of claims 1 to 4, wherein the alternating tangential flow rate is between 60 mL / min and 120 mL / min when expanding the stem cells and / or between 70 mL / min and 140 mL / min when differentiating the stem cells.

6. A method for differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in a bioreactor under conditions that stimulate the stem cells to differentiate, wherein the bioreactor comprises an32inlet for introduction of fresh culture medium, an outlet for extraction of spent culture medium and a cell retention device.

7. The method of any one of claims 1 to 6, wherein the bioreactor is a stirred tank bioreactor.

8. The method of any one of claims 1 to 7 , wherein the perfusion rate is between 0.4 and 0.9 volumes per day (VVD), 1 VVD, between 1 and 2 VVD, or more than 2 VVD.

9. A method for differentiating stem cells in perfusion cell culture comprising culturing the cells in culture medium in a bioreactor and f. extracting the cells from the bioreactor, g. removing spent culture medium from the bioreactor, h. supplying fresh culture medium to the bioreactor, i. removing non-viable (delaminated) single cells from the bioreactor; and j. reintroducing the extracted cells into the bioreactor, wherein the stem cells are cultured in conditions that direct the stem cells to differentiate.

10. The method of any one of claims 1 to 9, wherein the dissolved oxygen concentration is maintained at 45% to 70% optionally wherein the oxygen concentration is about 65% during the expansion phase and / or about 50% during the differentiation phase.

11. The method of any one of claims 1 to 10, wherein the stem cells are mammalian.3312. The method of any one of claims 1 to 10, wherein the stem cells are bovine, murine, avian or human cells.

13. The method of any one of claims 1 to 12, wherein the stem cells are selected from any one of pluripotent stem cells (PSCs), muscle stem cells / satellite cells (MuSC / SC), adipose derived stem cells (ADSC), adipocytes, epithelial cells, mesenchymal stem / stromal cells (MSCs), fibro-adipogenic progenitors (FAPS), induced-pluripotent stem cells (iPSCs), breast milk stem cells (BMSCs), embryonic-like stem cells (ELC-Cs), chemically induced pluripotent stem cells (CiPSCs) and chemically induced totipotent stem cells (CiTotiSCs).

14. The method of any one of claims 1 to 13, wherein the culture medium is serum free medium.

15. The method of any one of claims 1 to 14, wherein the culture medium is animal-component free media.

16. The method of any one of claims 1 to 15, wherein the cells are differentiated into ectoderm cell lineage, endoderm cell lineage or mesoderm cell lineage.

17. The method of any one of claims 1 to 16, wherein the stem cells are iPSCs and the cells are differentiated into lactocytes.

18. The method of any one of claims 1 to 17, wherein the expanded cells are cultured to a viable density of at least 10 million cells per mL and then isolated.

19. The method of any one of claims 1 to 18, wherein the differentiated cells are cultured to a viable density of at least 10 million cells per mL and then isolated.

20. A method of producing a cell secreted product, comprising conducting the method of any one of claims 1 to 19 and collecting product secreted by the differentiated stem cells via an outlet in the bioreactor.

21. The method of claim 20, wherein the secreted product is a milk like product, optionally a human milk like product.

22. A method of producing a cell product, comprising conducting the method of any one of claims 1 to 19 and collecting expanded and / or differentiated cells from the bioreactor.

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