CROSS-REFERENCE TO RELATED APPLICATION

This disclosure claims priority to and benefit of U.S. Patent Application Serial No. 63/333,600, filed April 22, 2022, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to methods of culturing and manufacturing umbilical cord mesenchymal stem cells.

BACKGROUND

Mesenchymal stem cells (MCSs) can be isolated from newborn or perinatal tissues including the placenta and umbilical cord. The umbilical cord tissue is an attractive source of mesenchymal stem cells because of the non-invasive collection that takes place after birth from material that would otherwise be discarded as medical waste. There is a reduced risk of exposure to viral and environmental toxins using such tissue, and umbilical cord mesenchymal stem cells have an enhanced proliferative profile.

SUMMARY

Provided herein are methods of culturing umbilical cord mesenchymal stem cells that include: (a) disposing umbilical cord tissue segments into a volume of a first liquid culture medium within a culture dish coated with a first substrate, wherein the first liquid culture medium includes platelet lysate and heparin; (b) removing the umbilical cord tissue segments from the culture dish after about 7 days; and adding fresh first liquid culture medium to the culture dish to provide umbilical cord mesenchymal stem cells that are attached to the first substrate coating the culture dish at a density range of about 3 x 10 ³ cells/cm ² to about 58 x 10 ³ cells/cm ² ; (c) disposing the umbilical cord mesenchymal stem cells of step (b) into a vessel coated with a second substrate and incubating the umbilical cord mesenchymal stem cells to a cell population of about 50 x 10 ⁶ cells to about 91 x 10 ⁶ cells for a period of time to provide a seed train culture; (d) disposing a volume of the seed train culture into a third liquid culture medium comprised within a bioreactor coated with a third substrate, wherein the third liquid culture medium includes platelet lysate and heparin, to provide a bioreactor cell culture with an initial cell population in a range of between about 32 x 10 ⁶ cells to about 178 x 10 ⁶ cells; (e) perfusion culturing the bioreactor cell culture on a stirring platform; (f) removing a volume of the third liquid culture medium and adding a volume of fresh third liquid culture medium within about 12 hours to about 24 hours when a dissolved oxygen content of about 50% is reached; and (g) perfusion culturing the bioreactor cell culture to a cell population of between about 0.5 x 10 ⁹ cells to about 20 x 10 ⁹ cells.

In some embodiments, disposing the umbilical cord tissue segments in step (a) includes: thawing frozen umbilical cord tissue segments; and disposing the umbilical cord tissue segments into the first liquid culture medium within the culture dish. In some embodiments, the umbilical cord tissue segments includes about 7.5 g of umbilical cord tissue. In some embodiments of any of the methods described herein, the umbilical cord tissue segments includes about 3.7 x 10 ⁵ umbilical cord mesenchymal stem cells/g to about 5.3 x 10 ⁶ umbilical cord mesenchymal stem cells/g.

In some embodiments, wherein prior to step (d), the method includes repeating steps (b) and (c) until at least 55 x 10 ⁶ umbilical cord mesenchymal stem cells are obtained.

In some embodiments, the method includes, prior to step (c), collecting and freezing the umbilical cord mesenchymal stem cells.

Also provided herein are methods of culturing umbilical cord mesenchymal stem cells that include: (a) incubating umbilical cord mesenchymal stem cells into a volume of a first liquid culture medium within a vessel coated with a first substrate to a cell population of about 50 x 10 ⁶ cells to about 91 x 10 ⁶ cells for a period of time to provide a seed train culture, wherein the first liquid culture medium includes platelet lysate and heparin; (b) disposing a volume of the seed train culture into a second liquid culture medium comprised within a bioreactor coated with a second substrate, wherein the second liquid culture medium includes platelet lysate and heparin, to provide a bioreactor cell culture with an initial cell population in a range of between about 32 x 10 ⁶ cells to about 178 x 10 ⁶ cells; (c) perfusion culturing the bioreactor cell culture on a stirring platform; (d) removing a volume of the second liquid culture medium and adding a volume of fresh second liquid culture medium within about 12 hours to about 24 hours when a dissolved oxygen content of about 50% is reached; and (e) perfusion culturing the bioreactor cell culture to a cell population of between about 0.5 x 10 ⁹ cells to about 20 x 10 ⁹ cells.

In some embodiments, the method includes, prior to step (a): disposing umbilical cord tissue segments into a volume of the first liquid culture medium within a culture dish; removing the umbilical cord tissue segments from the culture dish after about 7 days; and adding fresh first liquid culture medium to the culture dish to provide umbilical cord mesenchymal stem cells that are attached to the first substrate coating the culture dish at a density range of about 3 x 10 ³ cells/cm ² to about 58 x 10 ³ cells/cm ² ; and disposing the umbilical cord mesenchymal stem cells into the vessel.

In some embodiments, disposing the umbilical cord tissue segments further include: thawing frozen umbilical cord tissue segments; and disposing the umbilical cord tissue segments into the first liquid culture medium within the culture dish.

In some embodiments of any of the methods described herein, the umbilical cord tissue segments includes about 7.5 g of umbilical cord tissue.

In some embodiments of any of the methods described herein, the umbilical cord tissue segments includes about 3.7 x 10 ⁵ umbilical cord mesenchymal stem cells/g to about 5.3 x 10 ⁶ umbilical cord mesenchymal stem cells/g.

In some embodiments of any of the methods described herein, the method includes, prior to step (a), enzymatically digesting umbilical cord tissue segments to provide a cell suspension including umbilical cord mesenchymal stem cells.

In some embodiments, the method includes, prior to step (a): thawing a frozen cell suspension of enzymatically digested umbilical cord tissue; and disposing a volume of the thawed cell suspension into the first liquid culture medium.

In some embodiments, the frozen cell suspension includes an umbilical cord mesenchymal cell density range of about 0.5 x 10 ⁶ cells/mL to about 3 x 10 ⁶ cells/mL.

In some embodiments, the frozen cell suspension includes a cord mesenchymal stem cell density range of about 1 x 10 ⁶ cells/mL.

In some embodiments of any of the methods described herein, the cell suspension contains a percentage of viable umbilical cord mesenchymal stem cells of at least 60%.

In some embodiments, the cell suspension contains a percentage of viable umbilical cord mesenchymal stem cells of at least 90%.

In some embodiments of any of the methods described herein, the first substrate, the second substrate and/or the third substrate is a human protein, e.g., a recombinant human protein.

In some embodiments of any of the methods described herein, the first substrate, the second substrate and/or the third substrate is a combination of between about 2 to about 20 extracellular matrix proteins.

In some embodiments of any of the methods described herein, the first substrate, the second substrate and/or the third substrate is a combination of about 2 to about 4 extracellular matrix proteins. In some embodiments, the first substrate, the second substrate and/or the third substrate is a combination of fibronectin and albumin.

In some embodiments of any of the methods described herein, the first substrate, the second substrate and/or the third substrate include at least 50% fibronectin or albumin.

In some embodiments of any of the methods described herein, the first substrate, the second substrate and/or the third substrate include at least 25% fibronectin and/or at least 25% albumin.

In some embodiments of any of the methods described herein, the vessel is coated with the first substrate or the second substrate at a volume to surface area ratio of about 50 pL/cm ² to about 300 pL/cm ² 2.0 cm ² .

In some embodiments, the vessel is coated with the first substrate, the second substrate and/or the third substrate at a volume to surface area ratio of about 67 pL/cm ² .

In some embodiments, the vessel is coated with the first substrate, the second substrate and/or the third substrate at a volume to surface area ratio of about 86 pL/cm ² .

In some embodiments, the vessel is coated with the first substrate, the second substrate and/or the third substrate at a volume to surface area ratio of about 278 pL/cm ² .

In some embodiments of any of the methods described herein, the vessel is a tissue culture flask or a cell culture plate.

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium and/or the third liquid culture medium includes a volume concentration of human platelet lysate between about 4% to about 6% of the final liquid culture medium.

In some embodiments, the first liquid culture medium, the second liquid culture medium and/or the third liquid culture medium includes a volume concentration of human platelet lysate of about 5% of the final liquid culture medium.

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium, and/or the third liquid culture medium includes a concentration of heparin between about 0.9 USP Unit/mL to about 1.1 USP Unit/mL.

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium, and/or the third liquid culture medium includes a xenogen-free liquid culture medium.

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium, and/or the third liquid culture medium includes about 4% to about 6% human platelet lysate and about 0.9 USP Unit/mL to about 1.1 USP Unit/mL heparin.

In some embodiments of any of the methods described herein, the period of time is about 1 day to about 14 days.

In some embodiments of any of the methods described herein, the dissolved oxygen content of about 50% is reached between about 3 days to about 8 days.

In some embodiments of any of the methods described herein, the method further includes harvesting and freezing the bioreactor cell culture at a cell density range of about 1 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL.

In some embodiments, harvesting includes incubating the bioreactor cell culture with an animal-origin free enzyme to detach the umbilical cord mesenchymal stem cells from the bioreactor.

In some embodiments, the animal origin-free enzyme is a recombinant enzyme obtained from microbial fermentation.

In some embodiments, the recombinant enzyme is a fungal serine protease with trypsin-like activity.

In some embodiments of any of the methods described herein, the stirring platform has a rotation of between about 5 rotations per minute (RPM) and about 80 RPM.

In some embodiments of any of the methods described herein, the bioreactor is a single-use multi-plate disposable bioreactor.

In some embodiments, the bioreactor has a volume of about IL to about 21.9L.

In some embodiments, the bioreactor has a volume about IL to about 6L.

In some embodiments of any of the methods described herein, the bioreactor includes 10 to 200 hydrophilized polystyrene plates. In some embodiments, the bioreactor includes 10 to 50 hydrophilized polystyrene plates. In some embodiments, when the bioreactor includes 100 to 200 hydrophilized polystyrene plates, the interior surfaces of the bioreactor not coated with a substrate.

As used herein, the word “a” before a noun represents one or more of the particular noun. For example “a cell” represents “one or more cells.”

As used herein, “obtain” or “obtaining” can be any means whereby one comes into possession of the sample by “direct” or “indirect” means. Directly obtaining a sample means performing a process (e.g., performing a physical method such as a biopsy or extraction) to obtain the sample. Indirectly obtaining a sample refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample). Directly obtaining a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting materials, such as a tissue biopsy, (e.g., umbilical cord biopsy that was previously isolated from a patient). Thus, obtain is sued to mean collection and/or removal of the sample from the subject.

The term “liquid culture medium” or “liquid medium” means a fluid that contains sufficient nutrients to allow a cell (e.g., an umbilical cord mesenchymal stem cell) to grow or proliferate in vitro. For example, a liquid can contain one or more of amino acids (e.g., 20 amino acids), a purine (hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol, thiamine, folic acid, biotic, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, a liquid culture medium does not contain serum from a non-human mammal. In some embodiments, a liquid culture medium does not contain serum or another extract from a mammal (a defined liquid culture medium). In some embodiments, a liquid culture medium can contain trace metals, a human growth factor, and/or a human growth factor. Another example of liquid culture medium is minimal medium (e.g., a medium containing only inorganic salts, a carbon source, and water). Nonlimiting examples of liquid culture medium are described herein. Additional examples of liquid culture medium are known in the art and are commercially available. A liquid culture medium can contain any number of mammalian cells (e.g., umbilical cord mesenchymal stem cells). For example, as used herein, a volume of liquid culture medium removed from a vessel (e.g., a bioreactor) can be substantially free of mammalian cells.

The term “substantially free” means a composition (e.g., a filtrate) that is at least or about 90% free, such as at least or about 95%, 96%, 97%, 98%, or at least about 99% free, or about 100% free of a specified substance.

The term “animal-derived component free liquid culture medium” means a liquid culture medium that does not contain components (e.g., proteins or serum) derived from a non-human mammal.

The term “serum-free liquid culture medium” means a liquid culture medium that does not contain a mammalian serum.

The term “serum-containing liquid culture medium” means a liquid culture medium that contains a mammalian serum.

The term “protein-free liquid culture medium” means a liquid culture medium that does not contain any protein (e.g., any detectable protein). The term “chemically-defined liquid culture medium” is a term of art and means a liquid culture medium in which all of the chemical components are known. For example, a chemically-defined liquid culture medium does not contain fetal bovine serum, bovine serum albumin, or human serum albumin, as these preparations typically contain a complex mix of albumins and lipids.

The term “clarified liquid culture medium” means a liquid culture medium obtained from a bacterial cell culture or a yeast cell culture that is substantially free (e.g., at least 75%, at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% free) of bacteria cells or yeast cells.

The term “agitation” or “stirring” means moving a portion of liquid culture medium in a bioreactor. This is performed in order to, e.g., increase dissolved O2 concentration in the liquid culture medium in a bioreactor. Agitation can be performed using any method known in the art, e.g., an instrument (e.g., a stirring platform) or propeller. Exemplary devices and methods that can be used to perform agitation of a portion of the liquid culture medium in a bioreactor are known in the art.

The term “O.lx volume” means about 10% of the volume. The term “0.5x volume” means about 50% of the volume. Likewise, 0.2x, 0.3x, 0.4x, 0.6x, 0.7x, 0.8x, lx, 2x, 5x means about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 100%, about 200%, or about 500% of the volume, respectively.

The term “cell culture” means a liquid culture medium containing a population of cells (e.g., umbilical cord mesenchymal stem cells) that is maintained or proliferated under a controlled set of environmental conditions.

The term “culturing” or “cell culturing” refers to the maintenance or proliferation of a cell under a controlled set of environmental conditions.

The term “vessel” is a term of art and means a device having an interior volume suitable for culturing a population of cells (e.g., umbilical cord mesenchymal stem cells) in a liquid culture medium under a controlled set of environmental conditions that allow for the maintenance or proliferation of the cells (e.g., umbilical cord mesenchymal stem cells). Nonlimiting examples of vessels are cell culture dishes (e.g., a 35 mm culture dish, a 60 mm culture dish, a 100 mm culture dish, a 150 mm culture dish, a multi -well plate (e.g., a 6-well culture plate, a 12-well culture plate, a 24-well culture plate, a 48-well culture plate, a 96- well culture plate), a tissue culture flask (e.g., a T-25 tissue culture flask, a T-75 tissue culture flask, a T-150 tissue culture flask, a T-175 tissue culture flask, a T-225 tissue culture flask), a bioreactor (e.g., any of the bioreactor described herein). The term “perfusion bioreactor” means a bioreactor containing a population of cells (e.g., umbilical cord mesenchymal stem cells), and having a means (e.g., an inlet, an outlet, a pump) for periodically or continuously removing a liquid culture medium from inside the bioreactor and having a means of adding a volume of replacement liquid culture medium (or fresh liquid culture medium) into the bioreactor. The fraction of media removed and replaced each day can vary depending on the particular cells being cultured, the initial seeding population, and the cell population at a particular time. Non-limiting examples of perfusion bioreactors that can be used in any of the methods described herein include: Xpansion® multiplate bioreactor (PALL) (e.g., Xpansion® 10, Xpansion® 50, Xpansion® 100, and Xpansion® 200). In some embodiments wherein the perfusion bioreactor is an Xpansion® multiplate bioreactor, the bioreactor can have 10 plates (e.g., a 10-layer bioreactor), 50 plates (e.g., a 50-layer bioreactor), 100 plates (e.g., a 100-lay er bioreactor) or 200 plates (e.g., a 200-layer bioreactor).

Unless otherwise defined, all technical and scientific terms used herein have the sample meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

Figure l is a bar graph showing MSC cell yield per gram of umbilical cord tissue explanted using different combination of basal medium, supplement, and substrates.

Figures 2A-B are light microscopy images showing the impact on MSC culture adherence of pretreatment of culture dishes with CELLstart™ attachment substrate. Figure 2A is a light microscopy image of MSCs that were seeded without a substrate at day 5 of culturing. Figure 2B is a light microscopy image of MSCs that were seeded onto a culture dish coated with CELLstart™ at day 5 of culturing.

Figure 3 is a bar graph showing activated MSC secretion of macrophage colonystimulating factor 1 (M-CSF1) with or without addition of 20 pM interferon-y. Figure 4 is a bar graph showing activated MSC secretion of vascular endothelial growth factor (VEGF) under culture 2% oxygen culture conditions (“low 02”) and at ambient oxygen (21% oxygen) culture conditions (“no treatment”).

Figures 5A-5C are images of MSCs staining showing tri-lineage differentiation: (A) osteogenic lineage with Alizarin Red; (B) adipogenic lineage with Oil Red O; and (C) chondrogenic lineage with Alcian Blue.

Figure 6 is a schematic representation of an exemplary process that initiates with disposing of umbilical cord tissue segments into a culture dish for explant outgrowth of MSCs through utilization of a seed train to inoculate a bioreactor.

DETAILED DESCRIPTION

Provided herein are methods of culturing umbilical cord mesenchymal stem cells that include: (a) disposing umbilical cord tissue segments into a volume of a first liquid culture medium within a culture dish coated with a first substrate, wherein the first liquid culture medium includes platelet lysate and heparin; (b) removing the umbilical cord tissue segments from the culture dish after about 7 days; and adding fresh first liquid culture medium to the culture dish to provide umbilical cord mesenchymal stem cells that are attached to the first substrate coating the culture dish at a density range of about 3 x IO ³ cells/cm ² to about 58 x IO ³ cells/cm ² ; (c) disposing the umbilical cord mesenchymal stem cells of step (b) into a vessel coated with a second substrate and incubating the umbilical cord mesenchymal stem cells to a cell population of about 50 x 10 ⁶ cells to about 91 x 10 ⁶ cells for a period of time to provide a seed train culture; (d) disposing a volume of the seed train culture into a third liquid culture medium comprised within a bioreactor coated with a third substrate, wherein the third liquid culture medium includes platelet lysate and heparin, to provide a bioreactor cell culture with an initial cell population in a range of between about 32 x 10 ⁶ cells to about 178 x 10 ⁶ cells; (e) perfusion culturing the bioreactor cell culture on a stirring platform; (f) removing a volume of the third liquid culture medium and adding a volume of fresh third liquid culture medium within about 12 hours to about 24 hours when a dissolved oxygen content of about 50% is reached; and (g) perfusion culturing the bioreactor cell culture to a cell population of between about 0.5 x 10 ⁹ cells to about 20 x 10 ⁹ cells.

Also provided herein are methods of culturing umbilical cord mesenchymal stem cells that include: (a) incubating umbilical cord mesenchymal stem cells into a volume of a first liquid culture medium within a vessel coated with a first substrate to a cell population of about 50 x 10 ⁶ cells to about 91 x 10 ⁶ cells for a period of time to provide a seed train culture, wherein the first liquid culture medium includes platelet lysate and heparin; (b) disposing a volume of the seed train culture into a second liquid culture medium comprised within a bioreactor coated with a second substrate, wherein the second liquid culture medium includes platelet lysate and heparin, to provide a bioreactor cell culture with an initial cell population in a range of between about 32 x 10 ⁶ cells to about 178 x 10 ⁶ cells; (c) perfusion culturing the bioreactor cell culture on a stirring platform; (d) removing a volume of the second liquid culture medium and adding a volume of fresh second liquid culture medium within about 12 hours to about 24 hours when a dissolved oxygen content of about 50% is reached; and (e) perfusion culturing the bioreactor cell culture to a cell population of between about 0.5 x 10 ⁹ cells to about 20 x 10 ⁹ cells.

In some embodiments of any of the methods described herein, the period of time is about 1 day to about 14 days (e.g., about 1 to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 2 days to about 14 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 4 days, about 4 days to about 14 days, about 4 days to about 10 days, about 4 days to about 8 days).

In some embodiments of any of the methods described herein, the dissolved oxygen content of about 50% is reached between about 3 days to about 8 days (e.g., about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 4 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 5 days to about 8 days, about 5 days to about 7 days, about 6 days to about 8 days; about 4 days, about 5 days, about 6 days, about 7, or about 8 days).

Providing a Culture Dish and/or a Vessel

As can be appreciated in the art, the vessel that contains the umbilical cord mesenchymal stem cells can be any apparatus used in the art for the purpose of culturing cells (e.g., umbilical cord mesenchymal stem cells) e.g., a culture dish (e.g., any of the culture dishes described herein), a culture plate (e.g., any of the culture plates described herein), a flask (e.g., any of the flasks described herein). In some embodiments, the vessel can be externally agitated, e.g., through the use of a tilting platform or a stirring platform. In some embodiments, the vessel can be a 100 mm culture dish, a T-75 tissue culture flask, a T-150 tissue culture flask, a T-175 tissue culture flask, a T-225 tissue culture flask, or a Cell STACK® flask. The interior surface of the vessel (e.g., any of the vessels described herein) may have at least one substrate (e.g., fibronectin, collagen, poly-L-omithine, laminin), or a combination thereof. Non-limiting examples of substrates that can be used to coat the interior surface of the vessel include: CELLstart™, Matrigel® matrix, MesenCult™-SF Attachment Substrate, Biocoat® gelatin. CELLstart™ is a defined substrate, containing only components of human origin that is used for the attachment and expansion of human embryonic, mesenchymal, and neural stem cells. Matrigel® matrix is a substrate including approximately 60% laminin, 30% collagen IV, and 8% entactin. Matrigel® matrix also includes heparan sulfate proteoglycan (perlecan), transforming growth factor beta, epidermal growth factor, insulinlike growth factor, fibroblast growth factor, tissue plasminogen activator, and matrix metalloproteinases. MesenCult™-SF Attachment Substrate is a serum-free substrate that is used for the expansion of human mesenchymal stem cells and human progenitor cells. Biocoat® gelatin is a heterogenous mixture of collagen proteins.

In some embodiments, one or more cell culture dishes are used (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). The total surface area of the cell culture dishes can be about 450 cm ² to about 700 cm ² (e.g., about 450 cm ² about 700 cm ² , about 450 cm ² about 650 cm ² , about 450 cm ² about 600 cm ² , about 450 cm ² about 550 cm ² , about 450 cm ² about 500 cm ² , about 500 cm ² about 700 cm ² , about 500 cm ² about 650 cm ² , about 500 cm ² about 600 cm ² , about 500 cm ² about 550 cm ² , about 550 cm ² about 700 cm ² , about 550 cm ² about 650 cm ² , about 550 cm ² about 600 cm ² , about 600 cm ² about 700 cm ² , about 600 cm ² about 650 cm ² , or about 650 cm ² about 700 cm ² ).

As can be appreciated in the art, there are many ways that a population of cells can be disposed into a first liquid culture medium contained within a vessel. For example, disposing the population of cells can include the steps of thawing a frozen cell bank (e.g., any of the exemplary frozen cell banks described herein or known in the art) and disposing a volume of the thawed cell bank into the first liquid culture medium. A frozen cell bank can have a cell population range of, e.g., between about 0.5 x 10 ⁶ cells/mL to about 10 x 10 ⁶ cells/mL (e.g., about 0.5 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 0.5 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 0.5 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL, about 0.5 x 10 ⁶ cells/mL to about 4 x 10 ⁶ cells/mL, about 0.5 x 10 ⁶ cells/mL to about 2 x 10 ⁶ cells/mL, about 0.5 x 10 ⁶ cells/mL to about 1 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 10 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 4 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 10 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 4 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 10 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL, about 5 x 10 ⁶ cells/mL to about 10 x 10 ⁶ cells/mL, about 5 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 5 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 6 x 10 ⁶ cells/mL to about 10 x 10 ⁶ cells/mL, about 6 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 8 x 10 ⁶ cells/mL to about 10 x 10 ⁶ cells/mL; about 0.5 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL, about 5 x 10 ⁶ cells/mL, about 6 x 10 ⁶ cells/mL, about 8 x 10 ⁶ cells/mL, or about 10 x 10 ⁶ cells/mL).

Methods of generating a frozen cell bank are known in the art. For example, a frozen cell bank can be thawed by exposing the frozen cell bank to a block heater, a water-filled bath, or an aluminum bead-filled bath that is set at 37 °C or at 40 °C ± 3 °C, and quickly disposing (e.g., pipetting) a volume of the thawed cell bank into the first liquid culture medium. In some examples, the thawed cell bank is washed before disposing a volume of the thawed cell bank into the first liquid culture medium contained in the culture dish (e.g., to remove remnants of the freezing agent from the thawed cell bank). Thawing of the frozen cell bank can be performed over a period of between about 1 minute to about 10 minutes (e.g., between about 1 minute to about 8 minutes, about 1 minute to about 6 minutes, about 1 minute to about 5 minutes, about 1 minute to about 4 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 6 minutes, about 4 minutes to about 10 minutes, about 4 minutes to about 8 minutes, about 4 minutes to about 6 minutes, about 5 minutes to about 10 minutes, about 5 minutes to about 8 minutes, about 8 minutes to about 10 minutes). The thawed cell bank can contain a percentage of viable cells, e.g., at least 60%, (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%).

In some embodiments, disposing the umbilical cord tissue segments further include: thawing frozen umbilical cord tissue segments; and disposing the umbilical cord tissue segments into the first liquid culture medium within the culture dish.

In some embodiments of any of the methods described herein, the umbilical cord tissue segments includes between about 5 g to about 10 g of umbilical cord tissue (e.g., about 5g, about 6 g, about 7 g, about 7.5 g, about 8 g, or about 10 g). In some embodiments of any of the methods described herein, the umbilical cord tissue segments includes about 3.7 x 10 ⁵ umbilical cord mesenchymal stem cells/g to about 5.3 x 10 ⁶ umbilical cord mesenchymal stem cells/g (e.g., any subranges therein).

In some embodiments of any of the methods described herein, the method includes enzymatically digesting umbilical cord tissue segments to provide a cell suspension including umbilical cord mesenchymal stem cells. In some embodiments, the method includes thawing a frozen cell suspension of enzymatically digested umbilical cord tissue; and disposing a volume of the thawed cell suspension into the first liquid culture medium.

In some embodiments, the frozen cell suspension includes an umbilical cord mesenchymal cell density range of about 0.5 x 10 ⁶ cells/mL to about 3 x 10 ⁶ cells/mL (e.g., about 0.5 x 10 ⁶ cells/mL to about 2 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 3 x 10 ⁶ cells/mL; about 1 x 10 ⁶ cells/mL; about 2 x 10 ⁶ cells/mL; or about 3 x 10 ⁶ cells/mL).

In some embodiments of any of the methods described herein, the cell suspension contains a percentage of viable umbilical cord mesenchymal stem cells of at least 60% (e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%).

In some examples of any of the methods described herein, the disposing of a population of cells (e.g., umbilical cord mesenchymal stem cells) into the first liquid culture medium to generate a first cell culture in a cell culture dish can include the step of disposing a volume of a seed train cell culture comprising a population of cells into the first liquid culture medium. The cell population of the seed train cell culture can be any of the exemplary cell population described herein. As can be appreciated by one skilled in the art, the volume of the seed train cell culture sufficient to generate a first cell culture in a cell culture dish with an initial cell population between about 50 x 10 ⁶ cells to about 91 x 10 ⁶ cells (e.g., about 50 x 10 ⁶ cells to about 90 x 10 ⁶ cells, about 50 x 10 ⁶ cells to about 85 x 10 ⁶ cells, about 50 x 10 ⁶ cells to about 80 x 10 ⁶ cells, about 50 x 10 ⁶ cells to about 75 x 10 ⁶ cells, about 50 x 10 ⁶ cells to about 70 x 10 ⁶ cells, about 50 x 10 ⁶ cells to about 65 x 10 ⁶ cells, about 50 x 10 ⁶ cells to about 60 x 10 ⁶ cells, about 50 x 10 ⁶ cells to about 55 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 91 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 90 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 85 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 80 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 75 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 70 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 65 x 10 ⁶ cells, about 55 x 10 ⁶ cells to about 60 x 10 ⁶ cells, about 60 x 10 ⁶ cells to about 91 x 10 ⁶ cells, about 60 x 10 ⁶ cells to about 90 x 10 ⁶ cells, about 60 x 10 ⁶ cells to about 85 x 10 ⁶ cells, about 60 x 10 ⁶ cells to about 80 x 10 ⁶ cells, about 60 x 10 ⁶ cells to about 75 x 10 ⁶ cells, about 60 x 10 ⁶ cells to about 70 x 10 ⁶ cells, about 60 x 10 ⁶ cells to about 65 x 10 ⁶ cells, about 65 x 10 ⁶ cells to about 91 x 10 ⁶ cells, about 65 x 10 ⁶ cells to about 90 x 10 ⁶ cells, about 65 x 10 ⁶ cells to about 85 x 10 ⁶ cells, about 65 x 10 ⁶ cells to about 80 x 10 ⁶ cells, about 65 x 10 ⁶ cells to about 75 x 10 ⁶ cells, about 65 x 10 ⁶ cells to about 70 x 10 ⁶ cells, about 70 x 10 ⁶ cells to about 91 x 10 ⁶ cells, about 70 x 10 ⁶ cells to about 90 x 10 ⁶ cells, about 70 x 10 ⁶ cells to about 85 x 10 ⁶ cells, about 70 x 10 ⁶ cells to about 80 x 10 ⁶ cells, about 70 x 10 ⁶ cells to about 75 x 10 ⁶ cells, about 75 x 10 ⁶ cells to about 91 x 10 ⁶ cells, about 75 x 10 ⁶ cells to about 90 x 10 ⁶ cells, about 75 x 10 ⁶ cells to about 85 x 10 ⁶ cells, about 75 x 10 ⁶ cells to about 80 x 10 ⁶ cells, about 80 x 10 ⁶ cells to about 91 x 10 ⁶ cells, about 80 x 10 ⁶ cells to about 90 x 10 ⁶ cells, about 80 x 10 ⁶ cells to about 85 x 10 ⁶ cells, about 85 x 10 ⁶ cells to about 91 x 10 ⁶ cells, or about 85 x 10 ⁶ cells to about 90 x 10 ⁶ cells) can be determined from the cell population of the seed train cell culture and the volume of the first liquid culture medium present in the vessel (prior to disposing the seed train cell culture into the first liquid culture medium).

Fed Batch Culturing

The culturing step in the methods described herein can include fed batch culturing. Fed batch culturing includes periodic or continuous addition of a liquid culture medium to an initial cell culture, which includes a first liquid culture medium, without significant or substantial removal of the first liquid culture medium from the cell culture. In some embodiments, the feed liquid culture medium is a concentrated form of the first liquid culture medium. In some embodiments, the feed liquid culture medium comprises adding both a first liquid culture medium and a second liquid culture medium to the initial first liquid culture medium. In some examples, the addition of the first feed liquid culture medium and the addition of the second feed liquid culture medium is conducted at about the same time. In some embodiments, the total volume of the first feed liquid culture medium and the second feed liquid culture medium added to the cell culture over time are about the same.

Fed batch culturing does not include the removal of a significant or substantial volume of a liquid culture medium during culturing. The batch culturing can be performed using any of the exemplary temperatures and/or CO2 gas exposures described herein.

Addition of the feed liquid culture medium can be held constant or can be increased (e.g., steadily increased) over the culturing period. Addition of the feed liquid culture medium can be initiated at day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, or day 10 of the culturing period. In some examples, the addition of the feed liquid culture medium can be started at a specific time point during the culturing period (e.g., when the umbilical mesenchymal stem cells reach a target population of about 50 x 10 ⁶ cells to about 91 x 10 ⁶ cells). Addition of the feed liquid culture medium can occur at regular intervals (e.g., every 2 hours, every 4 hours, every 6 hours, every 8 hours, every 12 hours, every day, every 2 days, every 3 days, or every 4 days). In some examples, the amount of feed liquid culture medium added can progressively increase between the first incremental addition of feed liquid culture medium and subsequent additions of feed liquid culture medium. The volume of a feed liquid culture medium added to the initial cell culture can be a fraction of the initial volume of the vessel containing the culture (e.g., O.Olx, 0.02x, 0.04x, 0.05x, O.lx, 0.2x, 0.4x, 0.5x, lx, 2x, 3x, 4x, 5x). In some examples, the volume of a feed liquid culture medium added to the initial cell culture can be between about 1% to about 50% of the volume of the initial culture (e.g., about 1%, about 2%, about 5%, about 10%, about 20%, about 25%, about 40%, about 50%).

For example, the addition of the feed liquid culture medium can occur at a period of time that is between about 6 hours and about 10 days (e.g., between about 6 hours and about 8 days, between about 6 hours and about 7 days, between about 6 hours and about 6 days, between about 6 hours and about 4 days, between about 6 hours and about 2 days, between about 6 hours and about 1 day, between about 6 hours and about 12 hours, between about 12 hours and about 10 days, between about 12 hours and about 8 days, between about 12 hours and about 6 days, between about 12 hours and about 4 days, between about 12 hours and about 2 days, between about 12 hours and about 1 day, between about 1 day and about 10 days, between about 1 day and about 8 days, between about 1 day and about 6 days, between about 1 day and about 4 days, between about 1 day and about 2 days; or about 6 hours, about 12 hours, about 1 day, about 2 days, about 4 days, about 5 days, about 6 days, about 7 days).

Other exemplary parameters of batch culturing that can be used in the present methods are described herein.

A variety of different methods for determining cell population (including cell density) are known in the art (e.g., use of a light microscope and a hemocytometer; or use of an automated cell counter, e.g., ViCell® Cell Viability Analyzer, Scepter™ 2.0 Handheld Cytometer, Scepter™ 3.0 Handheld Cytometer, NucleoCounter®, Coulter Counter, or flow cytometer.

Perfusion Culturing The culturing step in the methods described herein can include perfusion culturing. Perfusion culturing includes removing from a vessel (e.g., a bioreactor) a first volume of a first liquid culture medium, and adding to the vessel (e.g., a bioreactor) a second volume of a first liquid culture medium and/or a second liquid culture medium, wherein the first volume and the second volume are about the same. During perfusion culturing, the cells (e.g., umbilical cord mesenchymal stem cells) are retained in the vessel. Removal and addition of the liquid culture medium can be performed sequentially or simultaneously, or in a combination of the two. In some embodiments, the entire volume of the first liquid culture medium is completely removed and a similar volume of fresh first liquid culture medium and/or second liquid culture medium is added.

The rate at which the first volume of the first liquid culture medium is removed (volume/unit of time) and the rate at which the second volume of the first and/or second liquid culture medium is added (volume/unit of time) can be varied depending on the conditions of the particular vessel and/or size of the vessel. In some examples, the rate at which the first volume of the first liquid culture medium is removed (volume/unit of time) and the rate at which the second volume of the first and/or second liquid culture medium is added (volume/unit of time) can be about the same. In other examples, the rate at which the first volume of the first liquid culture medium is removed (volume/unit of time) and the rate at which the second volume of the first and/or second liquid culture medium is added (volume/unit of time) can be different.

In some examples, the first volume that is removed and the second volume that is added can be constant or can be increased (e.g., steadily increased) over the culturing period.

In some examples, after the first 24 hours to 96 hours of the culturing period, over a 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the first liquid culture medium and/or the second liquid culture medium added is about 1% to about 50% of the volume of the initial culture (e.g., about 1%, about 2%, about 5%, about 10%, about 20%, about 25%, about 40%, about 50%) of the volume of the first liquid culture medium.

Skilled practitioners will appreciate that the first liquid culture medium and the second liquid culture medium can be the same liquid culture media. In other embodiments, the first liquid culture medium and the second liquid culture medium are different liquid culture media.

The methods described herein include a step of perfusion culturing the bioreactor cell culture to a cell population of between about 0.5 x 10 ⁹ cells to about 20 x 10 ⁹ cells (e.g., about 0.5 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 6 x 10 ⁹ cells, about

0.5 x 10 ⁹ cells to about 5 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 4 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 2 x 10 ⁹ cells, about 0.5 x 10 ⁹ cells to about 1 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 6 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 5 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 4 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 2 x 10 ⁹ cells, about 0.6 x 10 ⁹ cells to about 1 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 6 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 5 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 4 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 2 x 10 ⁹ cells, about 0.8 x 10 ⁹ cells to about 1 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 6 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 5 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 4 x 10 ⁹ cells, about 1 x 10 ⁹ cells to about 2 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 6 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 5 x 10 ⁹ cells, about 2 x 10 ⁹ cells to about 4 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 6 x 10 ⁹ cells, about 4 x 10 ⁹ cells to about 5 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 5 x 10 ⁹ cells to about 6 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 6 x 10 ⁹ cells to about 8 x 10 ⁹ cells, about 8 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 8 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 8 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 8 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 8 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 8 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 8 x 10 ⁹ cells to about 10 x 10 ⁹ cells, about 10 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 10 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 10 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 10 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 10 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 10 x 10 ⁹ cells to about 12 x 10 ⁹ cells, about 12 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 12 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 12 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 12 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 12 x 10 ⁹ cells to about 14 x 10 ⁹ cells, about 14 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 14 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 14 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 14 x 10 ⁹ cells to about 15 x 10 ⁹ cells, about 15 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 15 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 15 x 10 ⁹ cells to about 16 x 10 ⁹ cells, about 16 x 10 ⁹ cells to about 20 x 10 ⁹ cells, about 16 x 10 ⁹ cells to about 18 x 10 ⁹ cells, about 18 x 10 ⁹ cells to about 20 x 10 ⁹ cells).

The bioreactor used in any of the methods described herein can have an internal volume of, e.g., between about IL to about 22L (e.g., about IL to about 21.9L, about IL to about 20L, about IL to about 18L, about IL to about 16L, about IL to about 15L, about IL to about 14L, about IL to about 12L, about IL to about 11.2L, about IL to about 10L, about IL to about 8L, about IL to about 6L, about IL to about 5.6L, about IL to about 5L, about IL to about 4L, about IL to about 2L, about IL to about l.L, about 1.5L to about 22L, about 1.5L to about 21.9L, about 1.5L to about 20L, about 1.5L to about 18L, about 1.5L to about 16L, about 1.5L to about 15L, about 1.5L to about 14L, about 1.5L to about 12L, about 1.5L to about 11.2L, about 1.5L to about 10L, about 1.5L to about 8L, about 1.5L to about 6L, about 1.5L to about 5.6L, about 1.5L to about 5L, about 1.5L to about 4L, about 1.5L to about 2L, about 1.6L to about 20L, about 1.6L to about 18L, about 1.6L to about 16L, about 1.6L to about 15L, about 1.6L to about 14L, about 1.6L to about 12L, about 1.6L to about 11.2L, about 1.6L to about 10L, about 1.6L to about 8L, about 1.6L to about 6L, about 1.6L to about 5.6L, about 1.6L to about 5L, about 1.6L to about 4L, about 1.6L to about 2L, about 2L to about 20L, about 2L to about 18L, about 2L to about 16L, about 2L to about 15L, about 2L to about 14L, about 2L to about 12L, about 2L to about 11.2L, about 2L to about 10L, about 2L to about 8L, about 2L to about 6L, about 2L to about 5.6L, about 2L to about 5L, about 2L to about 4L, about 4L to about 20L, about 4L to about 18L, about 4L to about 16L, about 4L to about 15L, about 4L to about 14L, about 4L to about 12L, about 4L to about 11.2L, about 4L to about 10L, about 4L to about 8L, about 4L to about 6L, about 4L to about 5.6L, about 5L to about 20L, about 5L to about 18L, about 5L to about 16L, about 5L to about 15L, about 5L to about 14L, about 5L to about 12L, about 5L to about 11.2L, about 5L to about 10L, about 5L to about 8L, about 5L to about 6L, about 5L to about 5.6L, about 5.6L to about 20L, about 5.6L to about 18L, about 5.6L to about 16L, about 5.6L to about 15L, about 5.6L to about 14L, about 5.6L to about 12L, about 5.6L to about 11.2L, about 5.6L to about 10L, about 5.6L to about 8L, about 5.6L to about 6L, about 6L to about 20L, about 6L to about 18L, about 6L to about 16L, about 6L to about 15L, about 6L to about 14L, about 6L to about 12L, about 6L to about 11.2L, about 6L to about 10L, about 6L to about 8L, about 8L to about 20L, about 8L to about 18L, about 8L to about 16L, about 8L to about 15L, about 8L to about 14L, about 8L to about 12L, about 8L to about 11.2L, about 8L to about 10L, about 10L to about 20L, about 10L to about 18L, about 10L to about 16L, about 10L to about 15L, about 10L to about 14L, about 10L to about 12L, about 10L to about 11.2L, about 11.2L to about 20L, about 11.2L to about 18L, about 11.2L to about 16L, about 11.2L to about 15L, about 11.2L to about 14L, about 11.2L to about 12L, about 12L to about 20L, about 12L to about 18L, about 12L to about 16L, about 12L to about 15L, about 12L to about 14L, about 14L to about 20L, about 14L to about 18L, about 14L to about 16L, about 15L to about 20L, about 15L to about 18L, about 15L to about 16L, about 16L to about 20L, about 16L to about 18L, about 18L to about 20L). In some embodiments, the internal volume of the bioreactor is about 1.6L (e.g., a 10-plate bioreactor). In some embodiments, the internal volume of the bioreactor is about 5.6L (e.g., a 50-plate bioreactor). In some embodiments, the internal volume of the bioreactor is about 11.2L (e.g., a 100-plate bioreactor). In some embodiments, the internal volume of the bioreactor is about 21.9L (e.g., 200-plate bioreactor).

In some embodiments, the bioreactor is a single-use multi-plate disposable bioreactor. In some embodiments, the bioreactor has a volume about IL to about 22L (e.g., about IL to about 21.9L, about IL to about 20L, about IL to about 18L, about IL to about 16L, about IL to about 15L, about IL to about 14L, about IL to about 12L, about IL to about 10L, about IL to about 8L, about IL to about 6L, about IL to about 5L, about IL to about 4L, about IL to about 3L, about IL to about 2L, about 2L to about 22L, about 2L to about 21.9L, about 2L to about 18L, about 2L to about 16L, about 2L to about 15L, about 2L to about 14L, about 2L to about 12L, about 2L to about 10L, about 2L to about 8L, about 2L to about 6L, about 2L to about 5L, about 2L to about 4L, about 3L to about 22L, about 3L to about 21.9L, about 3L to about 18L, about 3L to about 16L, about 3L to about 15L, about 3L to about 14L, about 3L to about 12L, about 3L to about 10L, about 3L to about 8L, about 3L to about 6L, about 4L to about 22L, about 4L to about 21.9L, about 4L to about 18L, about 4L to about 16L, about 4L to about 15L, about 4L to about 14L, about 4L to about 12L, about 4L to about 10L, about 4L to about 8L, about 4L to about 6L, about 5L to about 22L, about 5L to about 21.9L, about 5L to about 18L, about 5L to about 16L, about 5L to about 15L, about 5L to about 14L, about 5L to about 12L, about 5L to about 10L, about 5L to about 8L, about 5L to about 6L, about 6L to about 22L, about 6L to about 21.9L, about 6L to about 18L, about 6L to about 16L, about 6L to about 15L, about 6L to about 14L, about 6L to about 12L, about 6L to about 10L, about 6L to about 8L, about 8L to about 22L, about 8L to about 21.9L, about 8L to about 18L, about 8L to about 16L, about 8L to about 15L, about 8L to about 14L, about 8L to about 12L, about 8L to about 10L, about 10L to about 22L, about 10L to about 21.9L, about 10L to about 18L, about 10L to about 16L, about 10L to about 15L, about 10L to about 14L, about 10L to about 12L, about 12L to about 22L, about 12L to about 21.9L, about 12L to about 18L, about 12L to about 16L, about 12L to about 15L, about 12L to about 14L, about 14L to about 22L, about 14L to about 21.9L, about 14L to about 18L, about 14L to about 16L, about 15L to about 22L, about 15L to about 21.9L, about 15L to about 18L, about 15L to about 16L, about 16L to about 22L, about 16L to about 21.9L, about 16L to about 18L, about 18L to about 22L, about 18L to about 21.9L, about 18L to about 20L, about 20L to about 22L, or about 20L to about 21.9L). In some embodiments, the bioreactor comprises 10 to 200 hydrophilized polystyrene plates (e.g., 10, 50, 100 or 200). In some embodiments, the bioreactor comprises 10 hydrophilized polystyrene plates (e.g., a 10-layer bioreactor). In some embodiments, the bioreactor comprises 50 hydrophilized polystyrene plates (e.g., a 50- layer bioreactor). In some embodiments, the bioreactor comprises 100 hydrophilized polystyrene plates (e.g., a 100-layer bioreactor). In some embodiments, the bioreactor comprises 200 hydrophilized polystyrene plates (e.g., a 200-layer bioreactor). In some embodiments, when the bioreactor comprises 100 to 200 hydrophilized polystyrene plates, the interior surfaces of the bioreactor is not coated with a substrate.

The bioreactor used in any of the methods described herein can be placed on a stirring platform or a tilting platform with an agitation rate of between about 40 rotations per minute (RPPM) to about 100 RPM (e.g., about 40 RPM to about 95 RPM, about 40 RPM to about 90RPM, about 40 RPM to about 85 RPM, about 40 RPM to about 80 RPM, about 40 RPM to about 75 RPM, about 40 RPM to about 60 RPM, about 40 RPM to about 50 RPM, about 42 RPM to about 100 RPM, about 42 RPM to about 95 RPM, about 42 RPM to about 90RPM, about 42 RPM to about 85 RPM, about 42 RPM to about 80 RPM, about 42 RPM to about 75 RPM, about 42 RPM to about 60 RPM, about 42 RPM to about 50 RPM, about 45 RPM to about 100 RPM, about 45 RPM to about 95 RPM, about 45 RPM to about 90RPM, about 45 RPM to about 85 RPM, about 45 RPM to about 80 RPM, about 45 RPM to about 75 RPM, about 45 RPM to about 60 RPM, about 45 RPM to about 50 RPM, about 50 RPM to about 100 RPM, about 50 RPM to about 95 RPM, about 50 RPM to about 90RPM, about 50 RPM to about 85 RPM, about 50 RPM to about 80 RPM, about 50 RPM to about 75 RPM, about 50 RPM to about 60 RPM, about 50 RPM to about 50 RPM, about 53 RPM to about 100 RPM, about 53 RPM to about 95 RPM, about 53 RPM to about 90RPM, about 53 RPM to about 85 RPM, about 53 RPM to about 80 RPM, about 53 RPM to about 75 RPM, about 53 RPM to about 60 RPM, about 55 RPM to about 100 RPM, about 55 RPM to about 95 RPM, about 55 RPM to about 90RPM, about 55 RPM to about 85 RPM, about 55 RPM to about 80 RPM, about 55 RPM to about 75 RPM, about 55 RPM to about 60 RPM, about 60 RPM to about 100 RPM, about 60 RPM to about 95 RPM, about 60 RPM to about 90RPM, about 60 RPM to about 85 RPM, about 60 RPM to about 80 RPM, about 60 RPM to about 75 RPM, about 65 RPM to about 100 RPM, about 65 RPM to about 95 RPM, about 65 RPM to about 90RPM, about 65 RPM to about 85 RPM, about 65 RPM to about 80 RPM, about 65 RPM to about 75 RPM, about 70 RPM to about 100 RPM, about 70 RPM to about 95 RPM, about 70 RPM to about 90RPM, about 70 RPM to about 85 RPM, about 70 RPM to about 80 RPM, about 70 RPM to about 75 RPM, about 75 RPM to about 100 RPM, about 75 RPM to about 95 RPM, about 75 RPM to about 90RPM, about 75 RPM to about 85 RPM, about 75 RPM to about 80 RPM, about 80 RPM to about 100 RPM, about 80 RPM to about 95 RPM, about 80 RPM to about 90RPM, about 80 RPM to about 85 RPM, about 42 RPM to about 100 RPM, about 42 RPM to about 95 RPM, about 42 RPM to about 90RPM, about 42 RPM to about 85 RPM, about 85 RPM to about 100 RPM, about 85 RPM to about 95 RPM, about 85 RPM to about 90RPM, about 90 RPM to about 100 RPM, about 90 RPM to about 95 RPM, or about 95 RPM to about 100 RPM).

In some embodiments, the method further comprises harvesting and freezing the bioreactor cell culture at a cell density range of about 1 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL (e.g., about 1 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 1 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 4 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 2 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 2 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 2 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL, about 2 x 10 ⁶ cells/mL to about 4 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 4 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 4 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL, about 5 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 5 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL, about 5 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 5 x 10 ⁶ cells/mL to about 6 x 10 ⁶ cells/mL, about 6 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 6 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL, about 6 x 10 ⁶ cells/mL to about 8 x 10 ⁶ cells/mL, about 8 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 8 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL, about 1 x 10 ⁶ cells/mL to about 1 x 10 ⁷ cells/mL, about 1 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL, about 8 x 10 ⁶ cells/mL to about 9 x 10 ⁶ cells/mL.)

In some embodiments, harvesting includes incubating the bioreactor cell culture with an animal-origin free enzyme to detach the umbilical cord mesenchymal stem cells from the bioreactor In some embodiments, the animal origin-free enzyme is a recombinant enzyme obtained from microbial fermentation. In some embodiments, the recombinant enzyme is a fungal serine protease with trypsin-like activity.

First, Second and/or Third Liquid Culture Medium

The methods described herein can include a first liquid culture medium, a second liquid culture medium, and/or a third liquid culture medium. In some embodiments of any of the methods described herein, the fist liquid culture medium, the second liquid culture medium, and the third liquid culture medium are the same. In some embodiments of any of the methods described herein, the fist liquid culture medium, the second liquid culture medium, and the third liquid culture medium are different. In some embodiments of any of the methods described herein, the fist liquid culture medium and the second liquid culture medium are the same. In some embodiments of any of the methods described herein, the fist liquid culture medium and the second liquid culture medium are different. In some embodiments of any of the methods described herein, the fist liquid culture medium and the third liquid culture medium are the same. In some embodiments of any of the methods described herein, the fist liquid culture medium and the third liquid culture medium are different. In some embodiments of any of the methods described herein, the second liquid culture medium and the third liquid culture medium are the same. In some embodiments of any of the methods described herein, the second liquid culture medium and the third liquid culture medium are different.

Any of the liquid culture media described herein can be selected from the group consisting of: animal-derived component free liquid culture medium, serum-free liquid culture medium, serum-containing liquid culture medium, chemically-defined liquid culture medium, and protein-free liquid culture medium.

A liquid culture medium can typically include an energy source (e.g., a carbohydrate, such as glucose), free fatty acids, vitamins, and/or trace elements. The first, second, and/or third liquid culture medium used in any of the methods described herein can be supplemented with growth hormones and/or growth factor (e.g., fibroblast growth factor-2 (FGF2), platelet- derived growth factor (PDGF), epidermal growth factor (EGF), ascorbic acid, insulin, transferrin), and/or amino acid (e.g., L-glutamine), and/or polysaccharide (e.g. heparin), salts and/or buffers (e.g., calcium, magnesium, and phosphate salts).

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium and/or the third liquid culture medium comprises a volume concentration of human platelet lysate between about 4% to about 6% (e.g., about 4% to about 5%; about 5% to about 6%, about 4%, about 5%, or about 6%) of the final liquid culture medium.

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium, and/or the third liquid culture medium comprises a concentration of heparin between about 0.9 USP Unit/mL to about 1.1 USP Unit/mL (e.g., about 0.9 USP Unit/mL, about 1.0 USP Unit/mL, or about 1.1 USP Unit/mL).

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium, and/or the third liquid culture medium comprises a xenogen-free liquid culture medium.

In some embodiments of any of the methods described herein, the first liquid culture medium, the second liquid culture medium, and/or the third liquid culture medium comprises about 4% to about 6% (e.g., about 4% to about 5%; about 5% to about 6%, about 4%, about 5%, or about 6%) human platelet lysate and about 0.9 USP Unit/mL to about 1.1 USP Unit/mL heparin (e.g., about 0.9 USP Unit/mL, about 1.0 USP Unit/mL, or about 1.1 USP Unit/m).

First, Second and/or Third Substrate

The methods described herein can include a culture dish and/or a vessel coated with a first substrate and/or a bioreactor coated with a second substrate. In some embodiments of any of the methods described herein, the fist substrate, the second substrate and the third substrate are the same. In some embodiments of any of the methods described herein, the first substrate, the second and/or the third substrate are different. In some embodiments of any of the methods described herein, the first substrate and the second substrate are the same. In some embodiments of any of the methods described herein, the first substrate and the third substrate are the same. In some embodiments of any of the methods described herein, the second substrate and the third substrate are the same.

In some embodiments, the first substrate, the second substrate and/or the third substrate is a human protein, e.g., a recombinant human protein. In some embodiments, the first substrate, the second substrate and/or the third substrate is a combination of between about 2 to about 20 extracellular matrix proteins (e.g., about 2 to about 18, about 2 to about 16, about 2 to about 14, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 4, about 4 to about 20, about 4 to about 18, about 4 to about 16, about 4 to about 14, about 4 to about 12, about 4 to about 10, about 4 to about 8, about 4 to about 6, about 5 to about 20, about 5 to about 18, about 5 to about 16, about 5 to about 14, about 5 to about 12, about 5 to about 10, about 5 to about 8, about 10 to about 20, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 12, about 12 to about 20, about 12 to about 18, about 12 to about 16, about 12 to about 14, about 14 to about 20, about 14 to about 18, about 14 to about 16, about 16 to about 20, about 16 to about 18, about 18 to about 20, about 2, about 4, about 5, about 6, about 7, about 8, about 10, about 12, about 14, about 15, about 16, about 18, or about 20).

The interior surface of the culture dish and/or the vessel (e.g., any of the vessels described herein) may have at least one substrate (e.g., fibronectin, albumin, nidogen-1, collagen, poly-L-omithine, laminin), or a combination thereof. Non-limiting examples of substrates that can be used to coat the interior surface of the culture dish and/or the vessel include: CELLstart™, Matrigel® matrix, MesenCult™-SF Attachment Substrate, Biocoat®. In some embodiments, the first substrate, the second substrate and/or the third substrate is a combination of fibronectin, albumin, and nidogen-1 (Hughes, et. al., Proteomics 2011, 11(20):3983-3991). In some embodiments, the first substrate, the second substrate and/or the third substrate is a combination of fibronectin and albumin. In some embodiments, the first substrate, the second substrate and/or the third substrate comprise at least 50% fibronectin or at least 50% albumin. In some embodiments, the first substrate, the second substrate and/or the third substrate comprise at least 25% fibronectin (e.g., at least 30%, at least 35%, at least 40%, at least 45%) and/or at least 25% albumin (e.g., at least 30%, at least 35%, at least 40%, at least 45%).

In some embodiments, the first substrate, the second substrate and/or the third substrate is a combination of fibronectin and nidogen-1. In some embodiments, the first substrate, the second substrate and/or the third substrate comprise at least 50% fibronectin or at least 50% nidogen-1. In some embodiments, the first substrate, the second substrate and/or the third substrate comprise at least 25% fibronectin (e.g., at least 30%, at least 35%, at least 40%, at least 45%) and/or at least 25% nidogen-1 (e.g., at least 30%, at least 35%, at least 40%, at least 45%).

In some embodiments, the first substrate, the second substrate and/or the third substrate is a combination of nidogen-1 and albumin. In some embodiments, the first substrate, the second substrate and/or the third substrate comprise at least 50% nidogen-1 or at least 50% albumin. In some embodiments, the first substrate, the second substrate and/or the third substrate comprise at least 25% nidogen-1 (e.g., at least 30%, at least 35%, at least 40%, at least 45%) and/or at least 25% albumin (e.g., at least 30%, at least 35%, at least 40%, at least 45%).

In some embodiments, the first substrate, the second substrate and/or the third substrate is a combination of fibronectin, albumin, and nidogen-1. In some embodiments, the first substrate, the second substrate and/or the third substrate comprise at least 25% fibronectin (e.g., at least 30%, at least 35%, at least 40%, at least 45%), at least 25% albumin (e.g., at least 30%, at least 35%, at least 40%, at least 45%), and at least 25% nidogen-1 (e.g., at least 30%, at least 35%, at least 40%, at least 45%).

In some embodiments, the vessel is coated with the first substrate, the second substrate, or the third substrate at a volume to surface area ratio of about 50 pL/cm ² to about 300 pL/cm ² (e.g., about 50 pL/cm ² to about 200 pL/cm ² , 50 pL/cm ² to about 100 pL/cm ² , about 100 pL/cm ² to about 300 pL/cm ² , about 100 pL/cm ² to about 200 pL/cm ² , or 200 pL/cm ² to about 300 pL/cm ² ). In some embodiments, the vessel is coated with the first substrate, the second substrate or the third substrate at a volume to surface area ratio of about 67 pL/cm ² . In some embodiments, the vessel is coated with the first substrate, the second substrate or the third substrate at a volume to surface area ratio of about 86 pL/cm ² . In some embodiments, the vessel is coated with the first substrate, the second substrate or the third substrate at a volume to surface area ratio of about 278 pL/cm ² .

Culturing Parameters

The methods described herein can be performed at a temperature of about 31 °C to about 40 °C (e.g., about 34 °C, about 37 °C). The methods described herein can include exposing the liquid culture medium contained in the culture dish and/or the vessel (e.g., any of the vessels described herein) to an atmosphere containing at most or about 15% CO2 (e.g., at most or about 14% CO ₂ , 12% CO ₂ , 10% CO ₂ , 8% CO ₂ , 6% CO ₂ , 5% CO ₂ , 4% CO ₂ , 2% CO2, or at most about 1% CO2). The culture dish and/or the vessel can be contained in a humidified atmosphere (e.g., at a humidity of greater than 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or a humidity of 100%). The surfaces of any culture dish and/or vessel described herein can be coated with a substrate (e.g., any of the exemplary substrates described herein or a combination thereof).

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1. Experimental optimization of cell culture media

Optimization of medium for cord tissue (CT) explantation and mesenchymal stem cell (MSC) expansion culture was performed iteratively over multiple studies. The initial formulation for explantation consisted of MesenCult™ MSC Basal Medium, supplemented with MesenCult™ Stem Cell Stimulatory Supplement, and L-Glutamine (all manufactured by Stemcell™ Technologies). Significant improvement in per-gram cell yield was seen with MesenCult™ MSC Basal Medium supplemented with MesenCult™-XF Growth Supplement used in combination with MesenCult™-SF Attachment Substrate, but not with MesenCult™- ACF Supplement used in combination with MesenCult™-SF Attachment Substrate (see “Study 1” in Figure 1). MesenCult™ MSC Basal Medium supplemented with MesenCult™- XF Growth Supplement used in combination with MesenCult™-SF Attachment Substrate was used for subsequent experiments.

Upon discontinuation of the MesenCult™-XF product, multiple alternatives were tested, with the best-performing being the RoosterNourish™ kit (RoosterBasal™-MSC medium supplemented with RoosterBooster™-MSC Supplement, by RoosterBio) used in combination with MesenCult™-SF Attachment Substrate. The RoosterNourish™ kit used without attachment substrate yielded inferior results (see “Study 2”, in Figure 1).

To move towards clinical utility, a cGMP -grade attachment substrate was desirable. The RoosterNourish™ medium kit used in combination with CELLstart™ attachment substrate (Gibco), provided results comparable to the MesenCult™-XF medium/MesenCult™-SF Attachment substrate combination that had been employed in R&D protocols (see “Study 2” in Figure 1).

Transition to a cGMP-grade platelet lysate supplement was determined to be desirable. cGMP-grade MEMa Basal Medium (Gibco) supplemented with PLTMax® (Mill Creek) and heparin (Hospira) used in combination with an attachment substrate provided results that were comparable to RoosterNourish™ medium and MesenCult™-XF medium (see “Study 3” in Figure 1).

In all studies, superior results were produced when the liquid culture medium was combined with use of an attachment substrate. The final liquid culture medium formulation, selected for its combination of performance and cGMP-grade components, was MEMa Basal Medium supplemented with PLTMax® and heparin and used in combination with CELLstart™ attachment substrate.

Example 2. Effect of different substrates on cell proliferation and/or viability

Two 10-layer bioreactors were each seeded with a population of 53 x 10 ⁶ MSCs. One 10-layer bioreactor was treated with a 1 :50 dilution of CELLstart™. The other 10-layer bioreactor remained untreated. MSCs were cultured under identical operating parameters for 5 days, with full media exchanges on Days 3 and 4. MSCs were observed and harvested on Day 5. As seen in Figure 2A, MSC cell cultures in the untreated bioreactor were observed to exhibit poor adherence to the culture plastic, with evidence of cells lifting from the plastic in sheets or clumps of cells. In contrast, MSCs cultured in the CELLstart™-treated bioreactor exhibited typical small, spindle shaped morphology with colony growth that expanded rapidly outward in monolayer (Figure 2B). Approximately 330 x 10 ⁶ MSCs were recovered from the untreated bioreactor, while approximately 1 x 10 ⁹ MSCs were recovered from the CELLstart™-treated bioreactor.

Additional experiments will be carried out to evaluate the efficacy of specific extracellular matrix proteins and combinations of extracellular matrix proteins for the isolation of MSCs from explanted umbilical cord tissue and/or expansion of MSCs in culture. MSCs will be seeded at a similar density across multiple cell culture plates. Each cell culture plate will be coated with a different substrate, e.g., only fibronectin, only albumin, only nidogen-1, combinations of fibronectin and albumin (e.g., 1 : 1, 1:2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :8, 1 : 10, 2: 1, 3: 1, 4: 1, 5: 1, 6:1, 8: 1, or 10: 1 ), combination of fibronectin and nidogen-1 (e.g., 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :8, 1 : 10, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 8: 1, or 10: 1 ), combinations of fibronectin, albumin and nidogen-1 (e.g., 1 : 1 : 1, 1 :2: 1). All other operating parameters will be identical between each cell culture plate. As described above, MSCs will be culture for 5 days, with full media exchanges on Days 3 and 4. MSCs will be observed and harvested on Day 5. Cell culture morphology, cell viability and cell harvest yield will be determined using methods known in the art.

Example 3. Effect of liquid culture medium and culturing conditions on MSC secretion

One characteristic function of MSCs is their secretion of factors that can influence inflammation and cell survival. MSCs may be primed to produce various factors with these activities by adding activating factors to the medium or by altering the oxygen conditions in which the cells are cultured.

MSCs cultured on cell culture plates treated with MesenCult™- SF Attachment Substrate in MesenCult™ Basal Medium supplemented with MesenCult™-XF supplement and L-glutamine into which 20 pM interferon y was added exhibited a nearly 6-fold increase in secretion of macrophage colony-stimulating factor 1 (M-CSF1) after two days of culture compared to cells not cultured in medium with no interferon-y (Figure 3).

MSCs cultured on cell culture plates treated with MesenCult™- SF Attachment Substrate in MesenCult™ Basal Medium supplemented with MesenCult™-XF supplement and L-glutamine and incubated in 2% oxygen exhibited over 400-fold increase in vascular endothelial growth factor (VEGF) secretion compared to MSCs incubated at ambient oxygen conditions (21% oxygen) (Figure 4).

Additional experiments will be carried out to evaluate the effect of the addition of TNF-a to the liquid culture medium at concentrations ranging between about 10 pM and about 50 pM. MSCs will be seeded at a similar density across multiple cell culture plates that will be coated with the same substrate. MSCs will be cultured in liquid culture media including a concentration of TNF-a ranging between about 10 pM and about 50 pM. As a negative control, MSCs will also be cultured in a liquid culture medium that does not include the addition of TNF-a. All other operating parameters will be identical between each cell culture plate. As described above, MSCs will be culture for 5 days, with full media exchanges on Days 3 and 4. 48-hours after the addition of TNF-a, liquid culture medium from each cell culture plate will be sampled and analyzed by enzyme-linked immunosorbent assay (ELISA) to detect the secreted levels of several secreted factors, such as IL-6, IL-10, and TGFp.

Example 4. Analysis of umbilical cord MSC cell surface markers

Umbilical cord MSCs can be characterized by analysis of their expressed cell surface proteins. Umbilical cord MSCs can be identified by flow cytometric analysis of a combination of the cell surface expression of the markers shown in Table 1. Positive expression is typically considered at >95%. Negative expression is typically considered at <5%. 7AAD can be used to quantify the viability percentage of the population of MSCs.

Upon harvesting of MSCs, MSCs will be stained with fluorescent antibodies and analyzed by flow cytometry to identify populations of umbilical cord MSCs. A combination of cell surface markers will be identified to distinguish between populations of umbilical cord MSCs and other MSCs. An “umbilical cord MSC” cell surface expression signature will be determined.

Table 1. Characteristic Cell Surface Marker Panel for MSCs

Example 5. Differentiation of Umbilical Cord MSCs

Umbilical cord MSCs can be driven to differentiate towards three distinct lineages, e.g., the osteogenic lineage, the adipogenic lineage, and the chondrogenic lineage.

For example, umbilical cord MSCs can be driven to differentiate towards the osteogenic lineage by culturing the cells in fibronectin-coated (Biocoat, Corning Life Sciences) wells of a multi-welllplate in osteogenic medium (AdipoMax, Millipore Sigma) for 4 weeks with full liquid culture medium exchanges every three days. At the end of this period, the cells can be fixed in 4% paraformaldehyde and stained with Alizarin Red S, which stains deposited calcium. The presence of deposited calcium indicates an osteoblast-like phenotype. Additionally, at the end of the four week period, the cells can be assessed via colorimetric assay for alkaline phosphatase activity (MBL International), the presence of which also indicates an osteoblast-like phenotype (Figure 5 A).

Next, umbilical cord MSCs can be driven to differentiate towards the adipogenic lineage by culturing umbilical cord MSCs in individual wells of a multi-well plate in an adipogenic medium (AdipoMax, Millipore Sigma) for 4 weeks with full liquid culture medium exchanges every three days. At the end of this period, the cells can be fixed in 4% paraformaldehyde and stained with Oil Red O, which stains lipids. The presence of lipids in cells indicates an adipocyte-like phenotype (Figure 5B).

Lastly, umbilical cord MSCs can be driven to differentiate towards the chondrogenic lineage by pelleting the cells and culturing the cell mass in chondrogenic medium (ChondroMax, Millipore Sigma) for 4 weeks with full liquid culture medium exchanges every three days, during which time the cells will condense into a spheroid. At the end of this period, the spheriods can be fixed in 4% paraformaldehyde, paraffin embedded and sectioned on a microtome, and stained with Alcian Blue, which stains glycosaminoglycans. The presence of glycosaminoglycans indicates a chondrocyte-like phenotype (Figure 5C).

Example 6. Removal of substrate as a culture requirement

Culturing MSCs in vessels coated with a substrate has been shown to be important to optimizing MSC cell culture outcomes for early passage MSCs. However, for later passages, there may be benefits to culturing MSCs in vessels without a substrate. To investigate whether MSCs require culturing in vessels coated with a substrate beyond the third passage, the following study will be conducted.

Passage 4 MSCs, that were cryopreserved at the end of passage 3 after expansion in a 10-layer bioreactor treated with a substrate, will be thawed, counted, and rinsed. The thawed MSCs will be re-suspended in a MSC-supportive liquid culture medium. One million MSCs will be seeded into each of two T-150 tissue culture flasks in 20 mL of liquid culture medium. One T-150 tissue culture flask will be coated with CELLstart™ substrate and the other T-150 tissue culture flask will not be treated. MSCs will be observed daily for adherence, growth, and morphology. MSCS will be harvested and counted on Day 5, following a full liquid culture medium exchange on Day 3. These conditions will be tested in duplicate. A comparison of the substrate-treated MSC culture and the untreated MSC culture will indicate whether a substrate is required for continued MSC culture beyond passage 3. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.