Method for cell expansion

Abstract

A method for expanding mammalian cells, and products produced by the method, the method comprising dynamically agitating cells in a bioreactor to produce expanded cell cultures.

Description

Technical Field

[0001] The present invention relates to a method for expanding mammalian cells, as well as products produced by said method and their use, the method comprising dynamically stirring cells in a bioreactor to produce an expanded cell culture. Background Art

[0002] The commercialization of cell-based technologies is a growing area of ​​interest across multiple scientific and industrial fields. Specifically, stem cells are an attractive cell type for biomedical and therapeutic applications. Researchers have made progress in providing methods for large-scale manufacturing of cells for use in these diverse fields while keeping costs to a minimum. However, this remains a major commercial hurdle.

[0003] The technology of large-scale production of cells currently includes the use of bioreactors. Bioreactors support the biological growth environment of cells, so large-scale cell expansion can be carried out. Specifically, stirred tank bioreactors are usually used because these bioreactors provide 3-dimensional (3D) controlled environments compatible with different culture medium replacement schemes, which ultimately lead to improved cell growth.

[0004] However, cell expansion methods, including those using stirred tank bioreactors, still have various disadvantages. Recent studies have shown that cell aggregates formed during expansion have large size variability. Increased cell aggregate size is associated with negative physical and physiological properties, including inefficient nutrient transport, reduced cell quality and viability.

[0005] The key to overcoming these problems is to control the size of cell aggregates in culture.

[0006] It is therefore an object of the present invention to provide an improved method for cell expansion, wherein large-scale production with improved cell quality and viability is achieved. Summary of the invention

[0007] The present invention solves the above technical problems.

[0008] Provided herein is a method for expanding mammalian cells in a stirred tank bioreactor, the bioreactor comprising cells and a cell culture medium, the method comprising dynamically agitating the cells in the bioreactor to produce an expanded cell culture.

[0009] In one embodiment, the dynamic agitation comprises agitating the cells at an agitation rate that varies during production of the expanded cell culture.

[0010] In one embodiment, the stirring speed is increased from a first stirring speed to a second stirring speed.

[0011] In one embodiment, the stirring speed ranges from 150 RPM to 800 RPM.

[0012] In one embodiment, the first agitation speed is increased to the second agitation speed at a rate of 6.25 RPM to 20 RPM per hour.

[0013] In one embodiment, the first agitation speed is from 150 RPM to 400 RPM, and optionally, the second agitation speed is from 450 RPM to 500 RPM.

[0014] In one embodiment, the first agitation speed is 250 RPM, and optionally, the second agitation speed is 400 RPM.

[0015] In one embodiment, the stirring power is 3 W / m 3 Up to 600W / m 3 within the range.

[0016] In one embodiment, the first stirring power is 2.35 W / m per hour. 3 Up to 23.53W / m 3 The rate is increased to the second stirring power.

[0017] In one embodiment, the first stirring speed is 3 W / m 3 Up to 75W / m 3 , and optionally, the second stirring speed is 105 W / m 3 Up to 150W / m 3 .

[0018] In one embodiment, the first stirring power is about 18 W / m 3 , including 18.19W / m 3 , and optionally, the second stirring power is about 75 W / m 3 , including 74.52W / m 3 .

[0019] In one embodiment, the dynamic agitation of the amplification method has a duration of 3 to 30 days, optionally 5 days.

[0020] In one embodiment, the agitation speed is changed from day 2 to day 3, wherein the amplification method is initiated on day 0.

[0021] In one embodiment, the cells used for expansion are human cells, and / or wherein the cells used for expansion are stem cells.

[0022] In one embodiment, the cells used for expansion are selected from any one of pluripotent stem cells (PSC), muscle stem cells / satellite cells (MuSC / SC), adipose-derived stem cells (ADSC), adipocytes, epithelial cells, mesenchymal stem cells / stromal cells (MSC), fibroadipogenic progenitor cells (FAPS), induced pluripotent stem cells (iPSC), breast milk stem cells (BMSC), embryonic-like stem cells (ELC-C), chemically induced pluripotent stem cells (CiPSC) and chemically induced omnipotent stem cells (CiTotiSC), optionally wherein the cells used for expansion are iPSCs.

[0023] Also provided herein is a method for expanding mammalian cells in a stirred tank bioreactor, the method comprising:

[0024] i) inoculating a bioreactor with a single cell sample,

[0025] ii) dynamically stirring cells in a bioreactor, and

[0026] iii) Isolating the expanded cell culture.

[0027] In some embodiments, the mammalian cell is a mammalian pluripotent stem cell (PSC).

[0028] In one embodiment, the method comprises:

[0029] i) inoculating a bioreactor with a single cell sample,

[0030] ii) dynamically stirring cells in a bioreactor, wherein:

[0031] a) agitating the cells from day 0 to day 2 at a first agitation speed of optionally 250 RPM,

[0032] b) increasing the first agitation speed, optionally at a rate of 6.25 RPM per hour, to a second agitation speed from day 2 to day 3, and

[0033] c) agitating the cells at a second agitation speed, optionally 400 RPM, from day 3 to day 5,

[0034] iii) After 5 days the expanded cell culture was isolated.

[0035] In one embodiment, the method comprises:

[0036] i) inoculating a bioreactor with a single cell sample of PSC, wherein the single cell sample is a sample of iPSC,

[0037] ii) dynamically stirring cells in a bioreactor, wherein:

[0038] a) agitating the cells from day 0 to day 2 at a first agitation speed of optionally 250 RPM,

[0039] b) increasing the first agitation speed, optionally at a rate of 6.25 RPM per hour, to a second agitation speed from day 2 to day 3, and

[0040] c) agitating the cells at a second agitation speed, optionally 400 RPM, from day 3 to day 5,

[0041] iii) After 5 days the expanded cell culture was isolated.

[0042] In one embodiment, the method comprises:

[0043] i) inoculating a bioreactor with a single cell sample of PSC, wherein the single cell sample is a sample of iPSC,

[0044] ii) dynamically stirring cells in a bioreactor, wherein:

[0045] a) agitating the cells from day 0 to day 2 at a first agitation speed, optionally from 150 RPM to 400 RPM,

[0046] b) increasing the first agitation speed, optionally at a rate of 6.25 RPM per hour, to a second agitation speed from day 2 to day 3, and

[0047] c) agitating the cells at a second agitation speed, optionally 400 RPM to 500 RPM, from day 3 to day 5, wherein the first agitation speed and the second agitation speed are different, iii) separating the expanded cell culture after 5 days.

[0048] In one embodiment, the method comprises:

[0049] i) inoculating a bioreactor with a single cell sample,

[0050] ii) dynamically stirring cells in a bioreactor, wherein:

[0051] a) from day 0 to day 2 at optionally about 18 W / m 3 (including 18.19W / m 3 ) of the first stirring power stirring cell,

[0052] b) from the 2nd day to the 3rd day, the first stirring power is optionally increased to 2.35 W / m per hour. 3 The rate is increased to a second stirring speed, and

[0053] c) from day 3 to day 5 at, optionally, about 75 W / m 3 (including 74.52W / m 3) of the second stirring power stirring cell,

[0054] iii) After 5 days the expanded cell culture was isolated.

[0055] In one embodiment, the method comprises:

[0056] i) inoculating a bioreactor with a single cell sample of PSC, wherein the single cell sample is a sample of iPSC,

[0057] ii) dynamically stirring cells in a bioreactor, wherein:

[0058] a) from day 0 to day 2 at optionally about 18 W / m 3 (including 18.19W / m 3 ) of the first stirring power stirring cell,

[0059] b) from day 2 to day 3, the first stirring speed is optionally increased to 2.35 W / m per hour. 3 The rate is increased to the second stirring power, and

[0060] c) from day 3 to day 5 at, optionally, about 75 W / m 3 (including 74.52W / m 3 ) of the second stirring power stirring cell,

[0061] iii) After 5 days the expanded cell culture was isolated.

[0062] In one embodiment, the method comprises:

[0063] i) inoculating a bioreactor with a single cell sample of PSC, wherein the single cell sample is a sample of iPSC,

[0064] ii) dynamically stirring cells in a bioreactor, wherein:

[0065] a) from day 0 to day 2 at, optionally, about 3 W / m 3 Up to 75W / m 3 The first stirring power stirring cell,

[0066] b) from day 2 to day 3, the first stirring speed is optionally increased to 2.35 W / m per hour. 3 The rate is increased to the second stirring power, and

[0067] d) From day 3 to day 5, optionally 75 W / m 3 Up to 150W / m 3 a second stirring power to stir the cell, wherein the first stirring power and the second stirring power are different,

[0068] iii) After 5 days the expanded cell culture was isolated.

[0069] In one embodiment, the method produces cell aggregates, wherein the method reduces the size of the cell aggregates compared to a method that does not include dynamic agitation.

[0070] In one embodiment, the method increases the circularity of cell aggregates compared to a method that does not include dynamic agitation.

[0071] In one embodiment, the method increases cell viability compared to a method that does not include dynamic agitation.

[0072] In one embodiment, the cells used for expansion are pluripotent cells, and the method increases the pluripotency of the cells compared to a method that does not include dynamic agitation.

[0073] In one embodiment, the expanded cell culture is suitable for use as a food product or a pharmaceutical.

[0074] Finally, provided herein is an expanded cell culture produced according to any of the methods of the invention described herein, optionally wherein the cells are PSCs. DETAILED DESCRIPTION

[0075] definition

[0076] In the context of the present invention, the term "cell expansion" (or similar terms) refers to a method by which cells are cultured to produce a large number of cells of the same cell type. The term "cell expansion" can be used interchangeably with "cell culture".

[0077] In the context of the present invention, the term "stirring speed" refers to the rotational speed. In some cases, the stirring speed is described as revolutions per minute (RPM). In some cases, the stirring speed can be expressed as stirring power in units of power / volume (P / V). Power can be measured in watts (W). Volume can be measured in m 3 The stirring speed can be converted to stirring power, and the formula is described in Rotondi, M. et al. 2021, which is incorporated herein by reference (Rotondi, M. et al. Design and development of new bioreactor vessel for improved cell and gene therapy application. Biotechnology Letter 43,1103-1116, doi:10.1007 / s10529-021-03076-3(2021)).

[0078] In the context of the present invention, the term "dynamic agitation" (or similar terms) refers to a process of varying the characteristics of agitation.

[0079] In the context of the present invention, the term "static stirring" (or similar terms) refers to a process of stirring at a single speed, wherein the speed does not vary.

[0080] In the context of the present invention, the term "cell viability" refers to healthy living cells. Viability can be calculated as the % number of healthy cells in a sample.

[0081] In the context of the present invention, the term "pluripotency" refers to the ability of a cell to develop into the three primary germ cell layers of the early embryo and hence into all cells of the adult.

[0082] In the context of the present invention, the term "seed culture" refers to a small sample of living single cells.

[0083] In the context of the present invention, the term "culture medium" refers to any suitable culture medium capable of achieving cell expansion of the cell type of interest. The culture medium may comprise a basal medium (e.g., DMEM and / or F12) and growth factors, such as fibroblast growth factor, insulin, transferrin, and / or transforming growth factor β. For example, in the case of an induced pluripotent stem cell expansion method, the culture medium may comprise mTeSR TM .mTeSR TM It is serum-free and contains a basal medium and recombinant human basic fibroblast growth factor and recombinant human transforming growth factor β. Exemplary culture media are described in Kuo et al. 2020, which is incorporated herein by reference (Kuo, HH, Gao, X., DeKeyser, JM, Fetterman, KA, Pinheiro, EA, Weddle, CJ & Burridge, PW (2020). Negligible-cost and weekend-free chemically defined human iPSC culture. Stem Cell Reports, 14 (2), 256-270).

[0084] In the context of the present invention, the term "cell circularity" refers to the morphological circularity of cell aggregates and can be quantified by any method known in the art, such as by using image processing algorithms, e.g., NIS Elements software. Cell circularity ranges from 0 to 1 (arbitrary units), where 1 is a perfect circle.

[0085] In the context of the present invention, the term "cell aggregate" (or similar terms) refers to a cluster of attached cells of the same cell type.

[0086] Method according to the invention

[0087] The present invention relates to methods for cell expansion, wherein cell quality and viability are improved compared to expansion methods described in the art, optionally wherein the cells are PSCs.

[0088] During the expansion process, the expanded cells form cell aggregates. If these aggregates become too large, this can disrupt the transport of nutrients to the cells and lead to the accumulation of metabolic waste products from the cells, which adversely affects cell quality and viability.

[0089] In the present invention, the cells are dynamically agitated within the bioreactor during the expansion method.Preferably, the agitation speed is increased during the expansion method, such as from a first speed to a second speed.

[0090] It has surprisingly been demonstrated in the present invention that controlling the aggregate size of expanded cells using a dynamic agitation method results in the formation of smaller cell aggregates with a more uniform size distribution and improved circularity.

[0091] It has also been surprisingly demonstrated in the present invention that utilizing a dynamic agitation method during cell expansion improves cell viability.

[0092] Furthermore, it has surprisingly been shown in the present invention that utilizing a dynamic agitation method during cell expansion of pluripotent cells improves the pluripotency of the cells.

[0093] Agitation speed is related to shear stress, and it is known that shear stress on cells during expansion methods can adversely affect cell properties (such as quality and viability). Therefore, it is surprising that changing the agitation speed during a cell expansion method (such as in the present invention) does not adversely affect the cells, and in addition, actually improves cell quality and viability.

[0094] Thus, the present invention provides a method for expanding mammalian cells in a stirred tank bioreactor, the bioreactor comprising cells and a cell culture medium, the method comprising dynamically agitating the cells in the bioreactor to produce an expanded cell culture.

[0095] In some embodiments, the method comprises:

[0096] i) inoculating a bioreactor with a single cell sample,

[0097] ii) dynamically stirring the contents of the bioreactor, and

[0098] iii) Isolating the expanded cell culture.

[0099] In some embodiments, the method comprises:

[0100] i) inoculating a bioreactor with a single cell sample of PSC,

[0101] ii) dynamically stirring the contents of the bioreactor, and

[0102] iii) Isolating the expanded cell culture.

[0103] In some embodiments, the method includes first culturing the cell of interest to produce a seed cell culture of a single cell. Optionally, the cell is a PSC. In some embodiments, the culture is carried out in a 2-dimensional (2D) planar culture, such as a 2D T-flask. In some embodiments, the method includes inoculating a bioreactor with a seed cell culture. In some embodiments, the bioreactor is inoculated with 50,000 to 5,000,000 cells per milliliter of bioreactor container. In some embodiments, 50,000 to 5,000,000 cells per milliliter of bioreactor container; 150,000 to 1,000,000 cells per milliliter of bioreactor container; 200,000 to 500,000 cells per milliliter of bioreactor container; or 250,000 to 500,000 cells per milliliter of bioreactor container. In some embodiments, the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor container.

[0104] The method includes expanding cells in a bioreactor for a specific period of time, as described anywhere herein. In some embodiments, the method includes separating the expanded cells. The expanded cells are in the form of cell aggregates. In some embodiments, the cells have been expanded at least 10 times. In some embodiments, the cells have been expanded at least 10 times in 4 days. In some embodiments, the expanded cells are separated by centrifugation. In some embodiments, the expanded cells are separated by gravimetric analysis. 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 a differentiation medium.

[0105] Certain features of the method of the present invention will now be described in more detail.

[0106] Dynamic stirring

[0107] The methods of the present invention require dynamic agitation of the cells in the bioreactor.

[0108] In one embodiment, dynamic stirring includes changing the stirring speed during the amplification method. In one embodiment, the stirring speed changes more than once, such as twice, three times or four times during the amplification method. In one embodiment, the stirring speed is changed from a first stirring speed to a second stirring speed. In one embodiment, the stirring speed is gradually changed from a first stirring speed to a second stirring speed.

[0109] In one embodiment, the stirring speed is increased during the amplification method. In one embodiment, the stirring speed is increased more than once, such as twice, three times or four times during the amplification method. In one embodiment, the stirring speed is increased from a first stirring speed to a second stirring speed. In one embodiment, the stirring speed is gradually increased from a first stirring speed to a second stirring speed.

[0110] In some embodiments, the stirring speed is 150RPM to 800RPM. In some embodiments, the stirring speed changes in the range of 150RPM to 800RPM. In some embodiments, each stirring speed is selected from the range of 150RPM to 800RPM.

[0111] In some embodiments, the stirring speed is 200RPM to 800RPM. In some embodiments, the stirring speed changes in the range of 200RPM to 800RPM. In some embodiments, each stirring speed is selected from the range of 200RPM to 800RPM.

[0112] In some embodiments, the first stirring speed is 150RPM to 400RPM. In some embodiments, the first stirring speed is 150RPM to 300RPM. In some embodiments, the first stirring speed is 200RPM to 400RPM. In some embodiments, the first stirring speed is 200RPM to 300RPM. In some embodiments, the first stirring speed is 225RPM to 275RPM. In some embodiments, the first stirring speed is selected from 150RPM, 200RPM, 250RPM, 300RPM, 350RPM and 400RPM. In a preferred embodiment, the first stirring speed is 250RPM.

[0113] In some embodiments, the second stirring speed is 300RPM to 800RPM. In some embodiments, the second stirring speed is 300RPM to 600RPM. In some embodiments, the second stirring speed is 350RPM to 450RPM. In some embodiments, the second stirring speed is selected from 300RPM, 350RPM, 400RPM, 450RPM, 500RPM, 550RPM, 600RPM, 650RPM, 700RPM, 750RPM and 800RPM. Preferably, the second stirring speed is selected from 350RPM, 400RPM and 450RPM. In preferred embodiments, the second stirring speed is 400RPM to 500RPM, including 400RPM.

[0114] In some embodiments, a first stirring speed is between 150 RPM and 400 RPM, which is increased to a second stirring speed between 300 RPM and 800 RPM during the amplification method, wherein the first stirring speed and the second stirring speed are different.

[0115] In some embodiments, a first agitation speed is between 150 RPM and 300 RPM, which is increased to a second agitation speed between 300 RPM and 800 RPM during the amplification method, wherein the first agitation speed and the second agitation speed are different.

[0116] In some embodiments, a first stirring speed is between 150 RPM and 400 RPM, which is increased to a second stirring speed between 300 RPM and 800 RPM during the amplification method, wherein the first stirring speed and the second stirring speed are different.

[0117] In some embodiments, a first stirring speed is between 150 RPM and 400 RPM, which is increased to a second stirring speed between 300 RPM and 600 RPM during the amplification method, wherein the first stirring speed and the second stirring speed are different.

[0118] In some embodiments, a first stirring speed is between 200 RPM and 300 RPM, which is increased to a second stirring speed between 300 RPM and 500 RPM during the amplification method, wherein the first stirring speed and the second stirring speed are different.

[0119] In some embodiments, the first agitation speed is less than 300 RPM and the second agitation speed is greater than 300 RPM.

[0120] In some embodiments, the first agitation speed is selected from 150RPM, 200RPM, 250RPM, 300RPM, and 400RPM; and the second agitation speed is selected from 350RPM, 400RPM, 450RPM, 500RPM, 550RPM, and 600RPM.

[0121] In some embodiments, the first agitation speed is 250 RPM and the second agitation speed is 400 RPM.

[0122] In some embodiments, the first stirring speed increases to the second stirring speed at a rate of 6.25RPM to 20RPM per hour. In a preferred embodiment, the first stirring speed increases to the second stirring speed at a rate of 6.25RPM per hour.

[0123] In some embodiments, the first agitation speed is 150 RPM to 400 RPM, which increases at a rate of 6.25 RPM to 20 RPM per hour to a second agitation speed of 300 RPM to 800 RPM.

[0124] In some embodiments, the first agitation speed is 150 RPM to 300 RPM, which increases at a rate of 6.25 RPM to 20 RPM per hour to a second agitation speed of 300 RPM to 800 RPM.

[0125] In some embodiments, the first agitation speed is 150 RPM to 400 RPM, which increases to a second agitation speed of 300 RPM to 600 RPM at a rate of 6.25 RPM to 20 RPM per hour.

[0126] In some embodiments, the first agitation speed is 200 RPM to 300 RPM, which increases to a second agitation speed of 300 RPM to 500 RPM at a rate of 6.25 RPM to 20 RPM per hour.

[0127] In a preferred embodiment, the first stirring speed is 250 RPM, which increases at a rate of 6.25 RPM per hour to a second stirring speed of 400 RPM.

[0128] In some embodiments, the stirring power is 3 W / m 3 Up to 600W / m 3 In some embodiments, the stirring power is 3 W / m 3 Up to 600W / m 3 In some embodiments, each stirring power is selected from 3W / m 3 Up to 600W / m 3 range.

[0129] In some embodiments, the stirring power is 3.93 W / m 3 Up to 596.16W / m 3 In some embodiments, the stirring power is 3.93 W / m 3 Up to 596.16W / m 3 In some embodiments, each stirring power is selected from 3.93W / m 3 Up to 596.16W / m 3 range.

[0130] In some embodiments, the stirring power is 9 W / m 3 Up to 600W / m 3 In some embodiments, the stirring power is 9 W / m 3 Up to 600W / m 3 In some embodiments, each stirring power is selected from 9W / m 3 Up to 600W / m 3 range.

[0131] In some embodiments, the stirring power is 9.32 W / m 3 To 596.16W / m 3 In some embodiments, the stirring power is 9.32 W / m 3 Up to 596.16W / m 3 In some embodiments, each stirring power is selected from 9.32W / m 3 Up to 596.16W / m 3 range.

[0132] In some embodiments, the first stirring power is 3 W / m 3 Up to 75W / m 3 In some embodiments, the first stirring power is 3 W / m 3 Up to 32W / m 3 In some embodiments, the first stirring power is 9 W / m 3 Up to 75W / m 3 In some embodiments, the first stirring power is 9 W / m 3 Up to 32W / m 3 In some embodiments, the first stirring power is 13 W / m 3 Up to 24W / m 3 In some embodiments, the first stirring power is selected from 3 W / m 3 , 9W / m 3 、18W / m 3 、31W / m 3 and 74W / m3 In a preferred embodiment, the first stirring power is about 18 W / m 3 , including 18.19W / m 3 .

[0133] In some embodiments, the first stirring power is 3.93 W / m 3 Up to 74.52W / m 3 In some embodiments, the first stirring power is 3.93 W / m 3 Up to 31.44W / m 3 In some embodiments, the first stirring power is 9.32 W / m 3 Up to 74.52W / m 3 In some embodiments, the first stirring power is 9.32 W / m 3 Up to 31.44W / m 3 In some embodiments, the first stirring power is 13.26 W / m 3 Up to 24.22W / m 3 In some embodiments, the first stirring power is selected from 3.93 W / m 3 , 9.32W / m 3 、18.19W / m 3 、31.44W / m 3 and 74.52W / m 3 .

[0134] In some embodiments, the second stirring power is 30 W / m 3 Up to 600W / m 3 In some embodiments, the second stirring power is 30 W / m 3 Up to 255W / m 3 In some embodiments, the second stirring power is 48 W / m 3 Up to 108W / m 3 In some embodiments, the second stirring power is selected from 31 W / m 3 、50W / m 3 、74W / m 3 、106W / m 3 、145W / m 3 、193W / m 3 、251W / m 3 、319W / m 3 、399W / m 3 、491W / m 3 and 596W / m 3 Preferably, the second stirring power is selected from 49W / m 3 、74W / m 3 and 106W / m3 In a preferred embodiment, the second stirring power is 74 W / m 3 Up to 145W / m 3 , including 74W / m 3 .

[0135] In some embodiments, the second stirring power is 31.44 W / m 3 To 596.16W / m 3 In some embodiments, the second stirring power is 31.44 W / m 3 Up to 251.51W / m 3 In some embodiments, the second stirring power is 49.92 W / m 3 Up to 106.1W / m 3 In some embodiments, the second stirring power is selected from 31.44 W / m 3 、49.92W / m 3 、74.52W / m 3 、106.1W / m 3 、145.55W / m 3 、193.72W / m 3 、251.51W / m 3 、319.77W / m 3 、399.38W / m 3 、491.22W / m 3 and 596.16W / m 3 Preferably, the second stirring power is selected from 49.92 W / m 3 、74.52W / m 3 and 106.1W / m 3 In a preferred embodiment, the second stirring power is 74.52 W / m 3 Up to 145.55W / m 3 , including 74.52W / m 3 .

[0136] In some embodiments, the first stirring power is 3 W / m 3 Up to 75W / m 3 The first stirring power is increased to 30 W / m3 to 600 W / m during the amplification method. 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0137] In some embodiments, the first stirring power is 3.93 W / m 3 Up to 74.52W / m 3 , the first stirring power increases to 31.44 W / m during the amplification method 3To 596.16W / m 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0138] In some embodiments, the first stirring power is 18 W / m 3 Up to 75W / m 3 , the first stirring power is increased to 30 W / m during the amplification method 3 Up to 600W / m 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0139] In some embodiments, the first stirring power is 18.19 W / m 3 Up to 74.52W / m 3 , the first stirring power increases to 31.44 W / m during the amplification method 3 To 596.16W / m 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0140] In some embodiments, the first stirring power is 3 W / m 3 Up to 32W / m 3 , the first stirring power is increased to 32 W / m during the amplification method 3 Up to 600W / m 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0141] In some embodiments, the first stirring power is 3.93 W / m 3 Up to 31.44W / m 3 , the first stirring power increases to 31.44 W / m during the amplification method 3 To 596.16W / m 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0142] In some embodiments, the first stirring power is 9 W / m 3 Up to 32W / m 3 , the first stirring power is increased to 32 W / m during the amplification method 3 Up to 150W / m 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0143] In some embodiments, the first stirring power is 9.32 W / m 3 Up to 31.44W / m 3 , the first stirring power increases to 31.44 W / m during the amplification method3 Up to 145.55W / m 3 A second stirring power, wherein the first stirring power and the second stirring power are different.

[0144] In some embodiments, the first stirring power is less than 30 W / m 3 And the second stirring power is greater than 30W / m 3 .

[0145] In some embodiments, the first stirring power is less than 31.44 W / m 3 And the second stirring power is greater than 31.44W / m 3 .

[0146] In some embodiments, the first stirring power is selected from about 3 W / m 3 , 9W / m 3 、18W / m 3 、31W / m 3 and 74W / m 3 ; and the second stirring power is selected from about 50W / m 3 、74W / m 3 、106W / m 3 、145W / m 3 、193W / m 3 and 251W / m 3 .

[0147] In some embodiments, the first stirring power is selected from 3.93 W / m 3 、9.32W / m 3 、18.19W / m 3 、31.44W / m 3 and 74.52W / m 3 ; and the second stirring power is selected from 49.92W / m 3 、74.52W / m 3 、106.1W / m 3 、145.55W / m 3 、193.72W / m 3 and 251.51W / m 3 .

[0148] In some embodiments, the first stirring power is about 18 W / m 3 , including 18.19W / m 3 , and the second stirring power is about 75W / m 3 , including 74.52W / m 3 .

[0149] In some embodiments, the first stirring power is 2.35 W / m per hour.3 Up to 23.53W / m 3 In a preferred embodiment, the first stirring power is increased to a second stirring power at a rate of 2.35 W / m per hour. 3 The rate is increased to the second stirring power.

[0150] In some embodiments, the first stirring power is 3 W / m 3 Up to 32W / m 3 The first stirring power is 2.35W / m per hour. 3 Up to 23.53W / m 3 The rate increased to 32W / m 3 Up to 600W / m 3 The second stirring power.

[0151] In some embodiments, the first stirring power is 3.93 W / m 3 Up to 31.44W / m 3 The first stirring power is 2.35W / m per hour. 3 Up to 23.53W / m 3 The rate increased to 31.44W / m 3 To 596.16W / m 3 The second stirring power.

[0152] In some embodiments, the first stirring power is 9 W / m 3 Up to 32W / m 3 The first stirring power is 2.35W / m per hour. 3 Up to 23.53W / m 3 The rate increased to 32W / m 3 Up to 150W / m 3 The second stirring power.

[0153] In some embodiments, the first stirring power is 9.32 W / m 3 Up to 31.44W / m 3 The first stirring power is 2.35W / m per hour. 3 Up to 23.53W / m 3 The rate increased to 31.44W / m 3 Up to 145.55W / m 3 The second stirring power.

[0154] In a preferred embodiment, the first stirring power is about 18 W / m 3 , including 18.19W / m 3 The first stirring power is 2.35W / m per hour. 3 The rate increases to about 75W / m 3(including 74.52W / m 3 )'s second stirring power.

[0155] The first stirring speed / power and the second stirring speed / power are different. The first stirring speed and the second stirring speed may differ by at least 50RPM, 100RPM, 150RPM or 200RPM. The first stirring power and the second stirring power may differ by at least 45W / m 3 、50W / m 3 、60W / m 3 、70W / m 3 or 80W / m 3 .

[0156] As described anywhere herein, the dynamic agitation step of the expansion method is performed over a certain number of days. As described anywhere herein, the dynamic agitation step includes a duration for which the cells are agitated.

[0157] On day 0 the dynamic stirring step was started.

[0158] In one embodiment, the duration of the dynamic stirring step is 3 to 30 days, such as 3 days, 4 days, 5 days, 7 days, 10 days, 15 days, 25 days or 30 days. Preferably, the duration is 5 days.

[0159] In one embodiment, the cells are stirred at the first stirring speed for at least 2 days. In one embodiment, the cells are stirred at the first stirring speed for 2 days. In one embodiment, the cells are stirred at the first stirring speed from day 0 to day 2 (i.e., for 48 hours).

[0160] In one embodiment, the stirring speed is changed at least on day 2, such as on day 2, day 3, day 4 and / or day 5, preferably on day 3.

[0161] In one embodiment, the stirring speed is gradually changed for at least 1 day, such as 1 day, 2 days or 3 days, preferably 1 day (ie, 24 hours). In this way, the stirring speed is gradually changed from the first speed to the second speed.

[0162] In one embodiment, the stirring speed is changed from day 2 to day 3. In one embodiment, the stirring speed is changed for 24 hours from day 2 to day 3.

[0163] In one embodiment, the cells are stirred at the second stirring speed for at least 2 days. In one embodiment, the cells are stirred at the second stirring speed for 2 days. In one embodiment, the cells are stirred at the second stirring speed from the 3rd day to the 5th day.

[0164] Preferably, in one embodiment, the duration of dynamic stirring is 5 days in total, and the stirring speed is increased from the first stirring speed to the second stirring speed for 24 hours from the 2nd day to the 3rd day.

[0165] In one embodiment, the amplification method is an iterative method, ie, the method is repeated.

[0166] It will be appreciated that any characteristics of the stirring speed described anywhere herein may be combined with any characteristics of the duration described anywhere herein.

[0167] In one embodiment, the duration of dynamic stirring is 5 days, and the stirring speed is increased during the amplification method. In another embodiment, the duration of dynamic stirring is 5 days, and the stirring speed is increased from the first stirring speed to the second stirring speed. In another embodiment, the stirring speed is increased from the first stirring speed to the second stirring speed from the 2nd day to the 3rd day. In another embodiment, the duration of dynamic stirring is 5 days, wherein the stirring speed is increased from the first stirring speed of 250RPM to the second stirring speed of 400RPM at a rate of 6.25RPM per hour from the 2nd day to the 3rd day. In another embodiment, the duration of dynamic stirring is 5 days, wherein the stirring speed is increased from the first stirring speed of 250RPM to the second stirring speed of 400RPM at a rate of 2.35W / m per hour from the 2nd day to the 3rd day. 3 The stirring power is increased from about 18W / m 3 (including 18.19W / m 3 ) is increased to about 75 W / m 3 (including 74.52W / m 3 )'s second stirring speed.

[0168] Cell Type

[0169] The methods of the present invention involve expanding mammalian cells.

[0170] Cells used in the present invention may include any mammalian cell type that requires expansion.

[0171] In one embodiment, the cell is a human cell. In one embodiment, the cell is a bovine cell. In one embodiment, the cell is a stem cell. In one embodiment, the cell is a human stem cell. In one embodiment, the cell is a bovine stem cell. In one embodiment, the cell is an adult stem cell.

[0172] In one embodiment, the cells can be selected from any one of pluripotent stem cells (PSC), muscle stem cells / satellite cells (MuSC / SC), adipose-derived stem cells (ADSC), adipocytes, epithelial cells, mesenchymal stem cells / stromal cells (MSC), fibroadipogenic progenitor cells (FAPS), induced pluripotent stem cells (iPSC), breast milk stem cells (BMSC), embryonic-like stem cells (ELC-C), chemically induced pluripotent stem cells (CiPSC) and chemically induced omnipotent stem cells (CiTotiSC).

[0173] In a preferred embodiment, the cell is an iPSC. In another preferred embodiment, the cell is a BMSC. In another preferred embodiment, the cell is an epithelial cell.

[0174] It will be appreciated that the features of the cell types as described herein may be combined with any other features of the methods of the invention, such as the dynamic agitation features described anywhere herein.

[0175] Cell culture vessels and conditions

[0176] The method of the present invention relates to cell expansion in a cell culture vessel, in particular a stirred tank bioreactor.

[0177] To achieve agitation of the cell culture, the bioreactor may include one or more impellers. Preferably, the impeller is a single 'elephant ear' impeller. A single 'elephant ear' impeller with a 30 mm diameter and a 45° pitch angle is described in Rotondi, M et al. 2021 (incorporated herein by reference) and can be used in any embodiment of the invention described herein. Therefore, the power index (Np) for converting the agitation speed to PPV is 2.07.

[0178] In one embodiment, the bioreactor includes one or more bioreactor chambers. In some embodiments, the reactor chamber has an internal volume of 0.1L to 100,000L. In some embodiments, the working volume is up to 50,000L, including 100mL to 50,000L, and up to 10,000L, including 100mL to 10,000L. The working volume can also be 100mL to 500mL, 150mL to 400mL, 200mL to 300mL, or 200mL to 250mL.

[0179] In some embodiments, the fluid density is 800 kg / m 3 Up to 1,200kg / m 3 Aqueous solution. Preferably, the fluid density is 1,000 kg / m 3 Aqueous solution.

[0180] In some embodiments, the bioreactor is inoculated with a seed cell culture. In some embodiments, the bioreactor is inoculated with 50,000 to 5,000,000 cells per milliliter of the bioreactor container. In some embodiments, the bioreactor is inoculated with 50,000 to 5,000,000 cells per milliliter of the bioreactor container; 150,000 to 1,000,000 cells per milliliter of the bioreactor container; 200,000 to 500,000 cells per milliliter of the bioreactor container; or 250,000 to 500,000 cells per milliliter of the bioreactor container. In some embodiments, the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of the bioreactor container.

[0181] The stirred tank bioreactor contains a cell culture medium. The cell culture medium promotes cell expansion by providing essential nutrients to the cells. Depending on the type of cells being expanded, any suitable cell culture medium known in the art can be used.

[0182] In one embodiment, the bioreactor is a batch bioreactor. In one embodiment, the bioreactor is a fed-batch bioreactor. In one embodiment, the bioreactor is a continuous (perfusion) bioreactor.

[0183] In one embodiment, the cell culture medium is a serum-free mammalian culture medium. In one embodiment, the cell culture medium is any suitable stem cell maintenance medium, such as mTeSR TM Or E8 culture medium. Culture medium supplementation may include mTeSR plus and B8 culture medium, with Nutrient Mixture F12 (DMEM F12) as basal culture medium. Exemplary culture medium is described in Kuo et al. 2020, which is incorporated herein by reference (Kuo, HH, Gao, X., DeKeyser, JM, Fetterman, KA, Pinheiro, EA, Weddle, CJ & Burridge, PW (2020). Negligible-cost and weekend-free chemically defined human iPSC culture. Stem Cell Reports, 14 (2), 256-270).

[0184] Conditions within the cell culture vessel may also be controlled to promote cell expansion, for example pH, nutrient supply, toxic byproduct disposal, and dissolved oxygen (DO) levels may be controlled. Depending on the cell type being expanded, any suitable conditions known in the art may be used.

[0185] In one embodiment, the nutrient supply comprises glucose, optionally in the range of 5 nM to 30 nM. In one embodiment, the nutrient supply comprises glutamine (or a similar compound such as GLUTAMAX ) optionally in the range of 1 nM to 10 nM. TM ).

[0186] In one embodiment, the toxic byproduct comprises lactate, optionally wherein the level of lactate in the cell culture vessel is controlled so that the concentration of lactate does not exceed 60 mM. In one embodiment, the toxic byproduct comprises ammonia, optionally wherein the level of ammonia in the cell culture vessel is controlled so that the concentration of ammonia does not exceed 5 mM.

[0187] 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 7.2. In another preferred embodiment, the pH of the cell culture is 7.35. In one embodiment, the nutrient supply comprises glucose and / or amino acids. In one embodiment, the toxic byproducts include lactate and / or ammonia. In one embodiment, the DO is 5% to 80%, including 5% to 60%.

[0188] In one embodiment, the dissolved oxygen (DO) is in the range of 30% to 70%. In some embodiments, the dissolved oxygen (DO) is in the range of 40% to 60%. In a preferred embodiment, the dissolved oxygen (DO) is 50%.

[0189] In some embodiments, the cells are cultured at a controlled pH of 7 to 7.5 and a controlled dissolved oxygen (DO) of 40% to 60%, optionally wherein the temperature is 35°C to 40°C, preferably 37°C.

[0190] In some embodiments, the cells are cultured at a controlled pH of 7.2 and a controlled dissolved oxygen (DO) of 50%, optionally wherein the temperature is between 35°C and 40°C, preferably 37°C.

[0191] In some embodiments, the cells are cultured at a controlled pH of 7.35 and a controlled dissolved oxygen (DO) of 50%, optionally wherein the temperature is between 35°C and 40°C, preferably 37°C.

[0192] Air and CO 2 In one embodiment, the air mixture is 15 mL / min to 25 mL / min and 4% to 8% CO 2 (including 5% CO 2 ) is performed under headspace aeration. In the embodiment, the headspace is aerated at 19 mL / min of air mixture and 5% CO2 Inflate the headspace.

[0193] In some embodiments, cell expansion is performed in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, optionally wherein the bioreactor uses a single 'elephant ear' impeller.

[0194] In some embodiments, cell expansion is performed in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are agitated at a first agitation speed of 250 RPM and a second agitation speed of 400 RPM, optionally wherein the bioreactor uses a single 'elephant ear' impeller.

[0195] In some embodiments, cell expansion is performed in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are agitated at a first agitation speed of 250 RPM, which first agitation speed is optionally increased at a rate of 6.25 RPM to 20 RPM per hour to a second agitation speed of 400 RPM, and optionally wherein the bioreactor uses a single 'elephant ear' impeller.

[0196] In some embodiments, cell expansion is performed in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are agitated at a first agitation speed of 200 RPM to 300 RPM, which is increased to a second agitation speed of 300 RPM to 500 RPM during the expansion method, wherein the first agitation speed and the second agitation speed are different.

[0197] In some embodiments, cell expansion is performed in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are agitated at a first agitation speed of 200 to 300 RPM, which is optionally increased at a rate of 6.25 to 20 RPM per hour to a second agitation speed of 300 to 500 RPM.

[0198] In some embodiments, the cells are expanded in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are grown at 3 W / m 3 With 75W / m 3 The first stirring power between 75W / m 3and 255W / m 3 The first stirring power and the second stirring power differ by at least 45 W / m 3 or 50W / m 3 , and optionally wherein the bioreactor uses a single 'elephant ear' impeller.

[0199] In some embodiments, the cells are expanded in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are heated at 3.93 W / m 3 and 74.52W / m 3 The first stirring power between 74.52W / m 3 and 251.51W / m 3 The bioreactor is stirred at a second stirring power of 1000 m / s, wherein the first stirring power and the second stirring power are different, and optionally wherein the bioreactor uses a single 'elephant ear' impeller.

[0200] In some embodiments, the cells are expanded in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are heated at about 18 W / m 3 (including 18.19W / m 3 ) and about 75W / m 3 (including 74.52W / m 3 ), optionally wherein the bioreactor uses a single 'elephant ear' impeller.

[0201] In some embodiments, the cells are expanded in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are heated at about 18 W / m 3 (including 18.19W / m 3 ) is stirred at a first stirring power of 2.35 W / m per hour. 3 The rate increases to about 75W / m 3 (including 74.52W / m 3 ) and further optionally wherein the bioreactor uses a single 'elephant ear' impeller.

[0202] In some embodiments, the cell expansion is performed in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are heated at 9 W / m 3 Up to 32W / m 3(including 9.32W / m 3 Up to 31.44W / m 3 ) and the first stirring power was increased to 32 W / m during the amplification method. 3 Up to 150W / m 3 (including 31.44W / m 3 Up to 145.55W / m 3 ), wherein the first stirring power and the second stirring power are different, optionally differing by at least 50 W / m 3 .

[0203] In some embodiments, the cell expansion is performed in a stirred tank bioreactor, wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, and wherein the cells are heated at 9 W / m 3 Up to 32W / m 3 (including 9.32W / m 3 Up to 31.44W / m 3 ) is stirred at a first stirring power of 2.35 W / m per hour. 3 Up to 23.53W / m 3 The rate increased to 32W / m 3 Up to 150W / m 3 (including 31.44W / m 3 Up to 145.55W / m 3 )'s second stirring power.

[0204] In some embodiments, cell expansion is performed in a stirred tank bioreactor using a single 'elephant ear' impeller with a working volume of 200 mL to 250 mL and a fluid density of 800 kg / m 3 Up to 1,200kg / m 3 aqueous solution, and wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, optionally wherein the bioreactor is cultured in an air mixture of 15 mL / min to 25 mL / min and 5% CO 2 Inflate the headspace.

[0205] In some embodiments, cell expansion is performed in a stirred tank bioreactor using a single 'elephant ear' impeller with a working volume of 200 mL to 250 mL and a fluid density of 1,000 kg / m 3 aqueous solution, and wherein the bioreactor is inoculated with 200,000 to 400,000 cells per milliliter of bioreactor vessel, optionally wherein the bioreactor is cultured in a 19 mL / min air mixture and 5% CO 2 Inflate the headspace.

[0206] Cell characteristics

[0207] The methods of the present invention produce cell aggregates, such as PSC aggregates, having surprising properties. Specifically, the cell aggregates produced by the methods of the present invention have reduced aggregate size compared to cell aggregates produced by expansion methods that do not include a dynamic agitation method (i.e., a method that does not include agitation) or include a static agitation method in which the agitation speed is not changed.

[0208] In one embodiment, the size of the cell aggregates produced by the method of the present invention is 190 μm to 210 μm. In one embodiment, compared with the cell aggregates produced by the amplification method that does not include the dynamic stirring method, these cell aggregates are at least 5 μm smaller. In one embodiment, compared with the cell aggregates produced by the amplification method that does not include the dynamic stirring method, the size of these cell aggregates is at least 10%, such as 10%, 11%, 12%, 13%, 14% or 15% smaller. In one embodiment, the size of these cell aggregates is roughly the same, because they have a size distribution less than 15 μm. In one embodiment, the cell aggregates are uniform in size.

[0209] In another embodiment, the cell aggregates produced by the method of the present invention have an increased circularity compared to the cell aggregates produced by the amplification method that does not include the dynamic agitation method. In one embodiment, the cell aggregates have a circularity of 0.85 to 0.90. In one embodiment, the cell aggregates are at least 0.015 more rounded than the cell aggregates produced by the amplification method that does not include the dynamic agitation method. In one embodiment, the cell aggregates have at least 4%, such as 4%, 5%, 6%, 7%, 8%, 9% or 10% circularity increase compared to the cell aggregates produced by the amplification method that does not include the dynamic agitation method.

[0210] In another embodiment, the cells produced by the method of the present invention have increased vigor compared to cells produced by an amplification method that does not include a dynamic agitation method. In some embodiments, after the amplification method is completed, at least 90% of the cells are alive. Preferably, after the amplification method is completed, at least 95% of the cells are alive. In some embodiments, after the amplification method is completed, at least 90% of the cells are alive, and the duration of dynamic agitation is a total of 5 days. In some embodiments, after the 2nd day of the amplification method, 95% of the cells are alive, and the duration of dynamic agitation is a total of 5 days. In some embodiments, after the 2nd day of the amplification method, 95% of the cells are alive, and during the amplification method, the viability of the cells changes by no more than 5%. In one embodiment, compared to the cell aggregates produced by the amplification method that does not include a dynamic agitation method, the method produces at least 10% more viable cells.

[0211] When the method of the invention includes expansion of pluripotent cells, the method produces cells, such as PSCs, having additional surprising properties. Specifically, the cells produced by the method of the invention have improved pluripotency compared to cells produced by an expansion method that does not include a dynamic agitation method (i.e., a method that does not include agitation) or includes a static agitation method in which the agitation speed is not changed.

[0212] Pluripotency can be measured according to specific pluripotency markers such as TRA-1-81, TRA-1-60 and / or any other marker known in the art. Pluripotency markers include TRA-1-81, TRA-1-60 and SSEA-4. Additional germ layer differentiation assays are performed, including analysis of various markers such as Sox2, Pax6, Sox17 and CXCR4. High expression levels of these markers indicate high levels of pluripotency and the ability to differentiate into different lineages. A suitable assay for testing pluripotency markers is by using STEMdiff TM Tri-lineage Differentiation Assay Kit (StemCell Technologies) Germ layer analysis by differentiating cell aggregates into different lineages.

[0213] In the present invention, in one embodiment, the cells produced by the method of the present invention have improved pluripotency compared to the cells produced by the amplification method that does not include the dynamic agitation method. In one embodiment, these cells have one or more pluripotency markers of increased level compared to the cells produced by the amplification method that does not include the dynamic agitation method. In one embodiment, the pluripotency marker is selected from TRA-1-81 and TRA-1-60. In one embodiment, the pluripotency marker is selected from TRA-1-81, TRA-1-60 and SSEA-4.

[0214] In one embodiment, at least 85% of the cells produced by the expansion method of the present invention are TRA-1-81 + , such as at least 85%, 86%, 87%, 88% or more. In one embodiment, at least 86% of the cells produced by the amplification method of the present invention are TRA-1-60 + , such as at least 86%, 87%, 88% or more. In one embodiment, at least 85% of the cells produced by the amplification method of the present invention are TRA-1-81 + , such as at least 85%, 86%, 87%, 88% or more, and at least 86% of the cells produced by the expansion method of the present invention are TRA-1-60 + , such as at least 86%, 87%, 88% or more.

[0215] In one embodiment, at least 90% of the cells produced by the expansion method of the present invention are SSEA-4 + , such as at least 90%, 91%, 92%, 93% or more. In one embodiment, at least 85% of the cells produced by the amplification method of the present invention are TRA-1-81 + , such as at least 85%, 86%, 87%, 88% or more, and at least 90% of the cells produced by the expansion methods of the present invention are SSEA-4 + , such as at least 90%, 91%, 92%, 93% or more. In one embodiment, at least 90% of the cells produced by the expansion method of the present invention are SSEA-4 + , such as at least 90%, 91%, 92%, 93% or more, and at least 86% of the cells produced by the expansion methods of the invention are TRA-1-60 + , such as at least 86%, 87%, 88% or more. In one embodiment, at least 85% of the cells produced by the amplification method of the present invention are TRA-1-81 + , such as at least 85%, 86%, 87%, 88% or more, at least 90% of the cells produced by the expansion methods of the present invention are SSEA-4 + , such as at least 90%, 91%, 92%, 93% or more, and at least 86% of the cells produced by the expansion methods of the invention are TRA-1-60 + , such as at least 86%, 87%, 88% or more.

[0216] In one embodiment, at least 70% of the differentiated cells obtained by the expansion method of the invention express Sox2, such as at least 70%, 71%, 72%, 73% or more.

[0217] In one embodiment, at least 45% of the differentiated cells obtained by the expansion method of the invention express Pax6, such as at least 48%, 49%, 50%, 51%, 52%, 53% or more.

[0218] In one embodiment, at least 80% of the differentiated cells obtained by the expansion method of the invention express Sox17, such as at least 81%, 82%, 83%, 84%, 85%, 86% or more.

[0219] In one embodiment, at least 80% of the differentiated cells obtained by the expansion method of the invention express CXCR4, such as at least 81%, 82%, 83%, 84%, 85%, 86% or more.

[0220] In one embodiment, at least 85% of the differentiated cells obtained by the expansion method of the present invention express TRA-1-81, such as at least 85%, 86%, 87%, 88% or more, at least 90% of the differentiated cells obtained by the expansion method of the present invention express SSEA-4, such as at least 90%, 91%, 92%, 93% or more, at least 86% of the differentiated cells obtained by the expansion method of the present invention express TRA-1-60, such as at least 86%, 87%, 88% or more, and at least 70% of the differentiated cells obtained by the expansion method of the present invention express Sox2, such as at least 85%, 86%, 87%, 88% or more. At least 70%, 71%, 72%, 73% or more, at least 45% of the differentiated cells obtained by the expansion method of the present invention express Pax6, such as at least 48%, 49%, 50%, 51%, 52%, 53% or more, at least 80% of the differentiated cells obtained by the expansion method of the present invention express Sox17, such as at least 81%, 82%, 83%, 84%, 85%, 86% or more, and at least 80% of the differentiated cells obtained by the expansion method of the present invention express CXCR4, such as at least 81%, 82%, 83%, 84%, 85%, 86% or more.

[0221] Pluripotency can also be measured based on specific gene markers (transcriptome profiles) such as KLF4, MYC, NANOG, SOX2 and POU5F1 and / or any other gene markers known in the art. High levels of these markers indicate high levels of pluripotency. Suitable assays are gene expression profiles performed using RNA-Seq (RNA sequencing) as described in Example 3.

[0222] In one embodiment, the pluripotency gene profile expression marker is selected from KLF4, MYC, NANOG, SOX2 and POU5F1. Exemplary relative expression levels are provided in Figure 5 middle.

[0223] It should be understood that references to RPM in relation to agitation speed as disclosed herein encompass equivalent RPM values ​​to achieve the same agitation speed, depending on the bioreactor used.

[0224] In some embodiments, the stirring speed is 0.48 W / m 3 Up to 72W / m 3 In some embodiments, the stirring speed is 0.48 W / m 3 Up to 72W / m 3 In some embodiments, each stirring speed is selected from 0.48W / m 3 Up to 72W / m 3 range.

[0225] In some embodiments, the first stirring speed is 0.48 W / m 3 Up to 3.8W / m 3 In a preferred embodiment, the first stirring speed is 2.20 W / m 3 .

[0226] In some embodiments, the second stirring speed is 3.8 W / m 3 Up to 72W / m 3 In a preferred embodiment, the second stirring speed is 9 W / m 3 .

[0227] In some embodiments, the first stirring speed is 0.48 W / m 3 Up to 3.8W / m 3 The first stirring speed is increased to 3.8 W / m during the amplification process. 3 Up to 72W / m 3 A second stirring speed, wherein the first stirring speed and the second stirring speed are different.

[0228] In some embodiments, the first stirring speed is less than 3.8 W / m 3 And the second stirring speed is greater than 3.8W / m 3 .

[0229] In some embodiments, the first stirring speed is 2.20 W / m 3 And the second stirring speed is 9W / m 3 .

[0230] In some embodiments, the first stirring speed is 0.3 W / m per hour. 3 Up to 3W / m 3 The first stirring speed is increased to the second stirring speed at a rate of 0.3 W / m per hour. 3The rate is increased to the second stirring speed.

[0231] In some embodiments, the first stirring speed is 0.48 W / m 3 Up to 3.8W / m 3 The first stirring speed is 0.3W / m 3 Up to 3W / m 3 The rate increased to 3.8W / m 3 Up to 72W / m 3 The second stirring speed.

[0232] In a preferred embodiment, the first stirring speed is 2.20 W / m 3 The first stirring speed is 0.3W / m 3 The rate increased to 9W / m 3 The second stirring speed.

[0233] In one embodiment, the method comprises:

[0234] i) inoculating a bioreactor with a single cell sample,

[0235] ii) dynamically stirring cells in a bioreactor, wherein:

[0236] a) From day 0 to day 2, optionally 2.20 W / m 3 The first stirring speed stirs the cell,

[0237] b) from the second day to the third day, the first stirring speed is optionally increased to 0.3 W / m per hour. 3 The rate is increased to a second stirring speed, and

[0238] c) from day 3 to day 5, optionally 9 W / m 3 and iii) separating the expanded cell culture after 5 days.

[0239] Uses of the present invention

[0240] The present invention provides a novel method for large-scale production of cells (eg, PSCs) with improved quality and viability. The cell products of the methods of the present invention can be used in a variety of industries.

[0241] In one embodiment, the expanded cell culture is suitable for use as a food product. In one embodiment, the expanded cell culture is suitable for use in the manufacture of a food product.

[0242] In one embodiment, the expanded cell culture is suitable for use as a medicament. In one embodiment, the expanded cell culture is suitable for use in the development of a medicament.

[0243] The invention also provides an expanded cell culture, such as a PSC cell culture, produced according to a method as described anywhere herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0244] Figure 1 : Schematic diagram of the dynamic stirring speed protocol for 3D PSC aggregate culture. TM A single PSC inoculated stirred tank bioreactor of a 2D culture in plus medium. The bioreactor culture was started with a stirring speed of 250 RPM. The static culture was maintained with 250 RPM culture until the end of the culture. At the same time, the stirring speed in the dynamic culture was ramped up from day 2 to 400 RPM on day 3.

[0245] Figure 2 : Dynamic stirring speed improves 3D culture conditions of hPSC. A. PSC aggregate size in 3D culture maintained at a constant speed (static) or following a gradient speed increase (dynamic). B. PSC aggregate circularity in 3D culture. Aggregate circularity was assessed by microscopic analysis, with aggregates with circularity scores between 0.8 and 1 as the target, where 1 is a perfect circle. C. PSC aggregation viability. D. The ratio of live PSC cells positive for the pluripotency marker TRA-1-60. E. The ratio of live PSC cells positive for the pluripotency marker TRA-1-81. F. The percentage of cell populations positive for the SSEA-4 marker in static or dynamic bioreactor expansion. Data are shown as the mean ± SEM of three replicate measurements and represent two independent experiments. Statistical analysis was performed using a two-tailed unpaired t-test, with *p<0.05.

[0246] Figure 3 : Microscopic images of cell aggregates under different expansion conditions (static / dynamic) and different culture days (D1: day 1; D7: day 7).

[0247] Figure 4 : Markers obtained in analysis of germ layers expanded in static or dynamic bioreactors.

[0248] Figure 5 : Gene expression profiles obtained by RNA sequencing (3D dynamic vs. 2D monolayer 'control'). For each gene, left bar = 2D 'control', right bar = 3D dynamic.

[0249] Figure 6 : For AMBR250 single "elephant ear" impeller, stirring speed (RPM) to stirring power (W / m 3 ) conversion.

[0250] Experimental Section

[0251] Example 1

[0252] Expansion of Pluripotent Stem Cells in Stirred Tank Bioreactors

[0253] method

[0254] Cells were harvested from 2D T-flasks to inoculate the bioreactor. Previously, 2D cultured cells in T-flasks were dissociated with Accutase (ThermoFisher; 00-4555-56). A stirred tank bioreactor (with elephant ear impeller) was used. 250 modular and mammalian culture vessels, Sartorius Stedim), were seeded with single cell suspensions at a starting viable cell density of 200,000 or 400,000 cells per ml in mTeSRTM plus (StemCell Technologies; 100-0276) + 10 μM Y-27632 ROCK inhibitor (Abcam; 120129). The cells were cultured at 37°C with pH and dissolved oxygen (DO) controlled at 7.35 and 50%. The culture was started with a static stirring speed of 250 RPM for the first 48 hours. After 48 hours, the dynamic stirring speed was started according to Table 1. From 72 hours on, the stirring speed was maintained at 400 RPM until the end of the culture ( Figure 1 ). Two medium changes were performed daily, approximately 8 to 12 hours apart. Approximately 80% of the medium was renewed in each medium change.

[0255]

[0256] Table 1. 250 Dynamic stirring speed in stirred tank bioreactors. Stirring speed is only applicable to those with elephant ear impellers 250 system (mammalian culture vessel). Agitation speed can be converted to a universal variable (power per volume; P / V) for any type of stirred tank bioreactor. In the conversion, the Newton number (Ne) of 0.64 specific for elephant ear vessels was used. P / V was calculated based on the formula reported by Rotondi, M et al. 2021 (incorporated herein by reference). Table 2 is a conversion table. Figure 6 It displays from RPM to W / m 3 The conversion curve diagram.

[0257] Table 2

[0258]

[0259] result

[0260] This paper describes a novel strategy to improve the pluripotency of hiPSC aggregates cultured in 3D stirred tank bioreactors ( Figure 1 ). The strategy included a range of bioreactor seed densities, initial agitation speeds (i.e., agitation power input / volume) and a gradient increase in agitation speed starting on day 2. The proposed approach reduced aggregate size ( Figure 2 A), using the aggregate circularity index to maintain a more uniform aggregate structure ( Figure 2 B), and improved cell viability ( Figure 2 C) Cell aggregates under different expansion conditions (static / dynamic) and different culture days (day 1; day 7) are shown in Figure 3 Therefore, the enhanced physical properties of the aggregates were confirmed by the pluripotency markers TRA1-60 and TRA1-81 ( Figure 2 D to 2E) and the pluripotency marker SSEA-4 ( Figure 2 F) resulted in improved cellular pluripotency.

[0261] Example 2

[0262] 3D bioreactor culture of iPSCs and analysis of the three germ layers

[0263] method

[0264] Human induced pluripotent stem cells (h-iPSCs) were cultured in 250 mL of B8 medium. The iPSCs were cultured in an HTP bioreactor system (specifically a single impeller 'elephant ear' container type). The bioreactor was inoculated with single-cell iPSCs in a suspension obtained from a 2D monolayer culture (e.g., a T-flask), with an initial stirring speed of 175 RPM. iPSC cell aggregates were formed approximately 12 to 24 hours after the start of the bioreactor culture. Dynamic stirring conditions were initiated 48 hours after the start of the culture, reaching a final stirring speed of 400 RPM within 24 hours. The cell aggregates were cultured in the bioreactor for 5 to 7 days. Afterwards, the complete cell aggregates were transferred to a well plate with a 5 mL working volume for germ layer analysis. Germ layer analysis was performed by differentiating the cell aggregates into different lineages. By using STEMdiff TM The analysis was performed using the Tri-lineage Differentiation Assay Kit (Stem Cell Technologies).

[0265] result

[0266] Intact aggregates obtained from static / dynamic expansion protocols in bioreactors were transferred to well plates for germ layer assays. These aggregates were exposed to lineage-specific differentiation media. Various markers were observed in germ layer analysis, including Sox2, Pax6, Sox17, and CXCR4 ( Figure 4 ). The results showed that intact aggregates expanded using the dynamic protocol could be more efficiently differentiated into various lineages.

[0267] Example 3

[0268] Gene Expression Profiling Using RNA-Seq

[0269] method

[0270] Total RNA was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter). Tissue was disrupted and lysed using Fastprep 2x 1' at speed 6. 400 μL of lysate was extracted and eluted in 50 μL. RNA was quantified using the Quant It Ribogreen assay (Life Technologies) and its quality was checked on a fragment analyzer. Reagents provided in the Stranded mRNA Library Preparation workflow, the Stranded mRNA Library Preparation protocol converts mRNA in total RNA samples into libraries of template molecules of known strand origin. 300ng of total RNA was used for each RNA sample. Bead-based mRNA capture was performed, followed by fragmentation, first- and second-strand synthesis, end repair and 3' adenylation, and indexed adapter ligation. Adapter-ligated cDNA library fragments were then enriched by PCR using 13 cycles of amplification. The indexed libraries were sequenced as paired-end (PE) with 59 bases on each end, read on 3 and 1 single P3100 NextSeq2000 flow cells.

[0271] Raw counts were obtained from sequences by mapping to the human reference genome using STAR v.2.5.3 (Dobin A, 2012) and counting using htseq-count v.0.6.1 (Anders S, 2014).

[0272] Filter features to remove low expressed genes. Genes with very low counts are unlikely to be differentially expressed between groups. Low expressed genes were filtered by selecting only genes with at least 30 reads in at least 3 samples.

[0273] A filtering step was performed on the CPM values ​​that took into account the library size. Here, it corresponded to a threshold of 1.012 on the CPM values.

[0274] Genes without annotations were also discarded. Using these filtering criteria, 13,600 features were retained. The trimmed mean (TMM) normalization of the M value (Robinson, MD & Oshlack, A 2010, A scaling normalization method for differential expression analysis of RNA-seq data. Genome biology, 11 (3), 1-9) is used to illustrate the compositional bias between libraries. Differential expression analysis between different groups was performed using edgeR v3.40.2 (Robinson & Oshlack, 2010), which fits a negative binomial log-linear model to the normalized counts of each feature. The empirical Bayesian method is used to mitigate the degree of overdispersion. The negative binomial model is further extended with the quasi-likelihood method to illustrate gene-specific variability. Differential expression was tested by the quasi-likelihood F-test, and the obtained p value was adjusted for multiple testing using the Benjamini and Hochberg method (BH).

[0275] result

[0276] The gene expression profile of cells cultured under 3D dynamic conditions was not significantly different from that of cells cultured under 2D monolayer conditions (control). Figure 5 Similar gene expression profiles of pluripotency genes (KLF4, POU5F1, NANOG and SOX2), glucose metabolism genes (HK1, GLUT1), G6PD, TCA Cyce genes (PDHA1 and PDHB) and lipid metabolism genes (ACLY, ACSS2) between 3D dynamic culture conditions and 2D monolayer cell culture conditions were shown.

Claims

1. A method for expanding mammalian cells in a stirred tank bioreactor, the bioreactor comprising mammalian cells and a cell culture medium, the method comprising dynamically agitating the cells in the bioreactor to produce an expanded cell culture.

2. The method of claim 1, wherein the dynamic agitation comprises agitating the cells at an agitation rate that varies during production of the expanded cell culture.

3. The method of claim 2, wherein the stirring speed is increased from a first stirring speed to a second stirring speed.

4. The method according to claim 2 or 3, wherein the stirring speed is in the range of 150RPM to 800RPM.

5. The method of claim 3 or 4, wherein the first stirring speed is increased to the second stirring speed at a rate of 6.25 RPM to 20 RPM per hour.

6. The method according to claim 4 or 5, wherein the first stirring speed is 150RPM to 300RPM, and the second stirring speed is 400RPM to 500RPM.

7. The method of claim 5, wherein the first stirring speed is 250 RPM, and optionally, the second stirring speed is 400 RPM.

8. The method according to claim 2 or 3, wherein the stirring power is 3W / m 3 Up to 600W / m 3 within the range.

9. The method according to claim 3 or 8, wherein the first stirring power is 2.35 W / m per hour. 3 Up to 23.53W / m 3 The rate is increased to the second stirring power.

10. The method according to claim 9, wherein the first stirring speed is 3 W / m 3 Up to 32W / m 3 And the second stirring speed is 70W / m 3 Up to 150W / m 3 .

11. The method according to claim 10, wherein the first stirring power is about 18 W / m 3 , and optionally the second stirring power is 75W / m 3 .

12. The method according to any preceding claim, wherein the dynamic agitation of the amplification method has a duration of 3 to 30 days, optionally 5 days.

13. The method according to any preceding claim, wherein the stirring speed is changed from day 2 to day 3, wherein the amplification method is initiated on day 0.

14. The method according to any preceding claim, wherein the cells used for expansion are human cells, and / or wherein the cells used for expansion are stem cells.

15. The method according to any one of claims 1 to 14, wherein the cells used for expansion are selected from any one of the following: pluripotent stem cells (PSC), muscle stem cells / satellite cells (MuSC / SC), adipose-derived stem cells (ADSC), adipocytes, epithelial cells, mesenchymal stem cells / stromal cells (MSC), fibroadipogenic progenitor cells (FAPS), induced pluripotent stem cells (iPSC), breast milk stem cells (BMSC), embryonic-like stem cells (ELC-C), chemically induced pluripotent stem cells (CiPSC) and chemically induced omnipotent stem cells (CiTotiSC), optionally wherein the cells used for expansion are iPSCs.

16. A method according to any preceding claim, wherein the method include: i) inoculating the bioreactor with a single cell sample, ii) dynamically agitating the cells in the bioreactor, and iii) isolating said expanded cell culture.

17. A method according to any preceding claim, wherein the method include: i) inoculating the bioreactor with a single cell sample, ii) dynamically agitating the cells in the bioreactor, wherein: a) agitating the cells from day 0 to day 2 at a first agitation speed of optionally 250 RPM, b) increasing the first stirring speed from day 2 to day 3, optionally at a rate of 6.25 RPM per hour, to a second stirring speed, and c) agitating the cells at a second agitation speed, optionally 400 RPM, from day 3 to day 5, iii) separating the expanded cell culture after 5 days.

18. A method according to any preceding claim, wherein the method include: i) inoculating the bioreactor with a single cell sample, ii) dynamically agitating the cells in the bioreactor, wherein: a) from day 0 to day 2 at optionally about 18 W / m 3 A first stirring power stirs the cells, b) from the 2nd day to the 3rd day, the first stirring power is optionally increased to 2.35 W / m per hour. 3 The rate is increased to the second stirring power, and c) from day 3 to day 5 at, optionally, about 75 W / m 3 The second stirring power stirs the cells, iii) separating the expanded cell culture after 5 days.

19. A method according to any preceding claim, wherein the method produces cell aggregates, wherein the method reduces the size of the cell aggregates compared to a method that does not include dynamic agitation, and / or wherein the method produces cell aggregates, wherein the method increases the circularity of the cell aggregates compared to a method not comprising dynamic agitation, and / or wherein the method increases cell viability compared to a method that does not include dynamic agitation, and / or wherein the cells used for expansion are pluripotent cells, and wherein the method increases the pluripotency of the cells compared to a method that does not include dynamic agitation.

20. A method according to any preceding claim, wherein the expanded cell culture is suitable for use as a food product or a pharmaceutical.

21. An expanded cell culture produced according to any preceding claim.

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