Species-specific tissue culture media for the growth of cells

Abstract

Cell culture media are disclosed. The cell culture media include species-specific cell culture media containing species bFGF, species PDGF-BB, and species EGF, and species serum. Further, a canine cell culture medium is disclosed containing canine bFGF, canine PDGF-BB, and canine EGF, and canine serum. A composition is described, including a medium containing basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, species bFGF, species PDGF-BB, and species EGF, serum matching the species cells, and species MSC cells. A method for producing exosomes is described, the method comprising expanding mesenchymal stem cells and / or islet cells in culture, culturing the cells, and collecting exosomes released by the mesenchymal cells and / or adipose stem cells in the culture.

Description

[Technical Field]

[0001] The present invention relates to species-specific culture media for the culture and expansion of mesenchymal stromal cells, adipocyte-derived mesenchymal stromal cells, pancreatic islet cells and other cells, as well as the isolation of exosomes from various cells. [Background technology]

[0002] Cell culture media provide a controlled, artificial, and in vitro environment for growing, expanding, and maintaining cells. Humans and many other species are increasingly recipients of cell therapies, particularly mesenchymal stromal cells (MSCs), for the treatment of various disorders, including kidney disease, bone and joint disorders, diabetes, and immune disorders. These therapies require the isolation, purification, and often the culture of MSCs and / or other cells for many doublings. Such cultures require the use of serum-supplemented culture media. Given the susceptibility of humans and other animals, particularly dogs, to foreign protein reactions, ideally, the serum supplement is derived from the donor and recipient species so that the cells introduced into the recipient are not carriers of foreign proteins that could trigger an antigenic response in the recipient. Currently, standard culture media are typically supplemented with serum concentrations of 10–20% (v / v).

[0003] The culture medium formulation described herein is xeno-free, requires significantly less serum than standard culture media, and is designed and tailored for the culture of cells from multiple species (human, canine, feline, equine, etc.). To prevent xenogeneic immune responses, the medium contains species-specific or identical proteins at the amino acid level and species-specific serum, but does not contain xenogeneic animal proteins. Furthermore, the reduced serum requirement lowers both the ethical burden and the financial cost of cell culture. Summary of the Invention

[0004] In a first aspect, the present disclosure provides a species-specific cell culture medium comprising a basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, species bFGF, species PDGF-BB, species insulin, and species EGF, and species serum.

[0005] In a second aspect, the disclosure provides a canine cell culture medium comprising a basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, canine bFGF, canine PDGF-BB, and canine EGF, and canine serum.

[0006] In a third aspect, a composition is provided, the composition comprising a medium comprising a basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, species bFGF, species PDGF-BB, and species EGF, serum matching the species cells, and species mesenchymal stromal cells.

[0007] In a fourth aspect, the present disclosure provides a composition, comprising a medium comprising basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, species bFGF, species PDGF-BB, and species EGF, serum matching the species cells, and species islet cells.

[0008] In a fifth aspect, the present disclosure provides a method of preparing a species-specific cell culture medium, the method comprising providing a basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin; providing species bFGF, species PDGF-BB, and species EGF; and providing species serum.

[0009] In a sixth aspect, the present disclosure provides a method of preparing a cell culture medium, the method comprising providing a basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin; providing canine bFGF, canine PDGF-BB, and canine EGF; and providing canine serum.

[0010] In a seventh aspect, the present disclosure provides a method for culturing and growing cells, the method comprising: providing a cell culture medium comprising basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin; species-specific bFGF, species-specific PDGF-BB, and species-specific EGF; and species-specific serum; providing cells for growth; and providing conditions for growing the cells.

[0011] In an eighth aspect, the present disclosure provides a method of producing exosomes, the method comprising expanding cells in culture, wherein the culture medium comprises providing basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, providing species-specific bFGF, species-specific PDGF-BB, and species-specific EGF, providing species-specific serum, and collecting exosomes released by the cells in culture.

[0012] Further aspects and embodiments are provided in the foregoing drawings, detailed description and claims.

[0013] The following drawings are provided to illustrate certain embodiments described herein. The drawings are merely exemplary and are not intended to limit the scope of the claimed invention, nor are they intended to show every possible feature or embodiment of the claimed invention. The drawings are not necessarily drawn to scale, and in some instances, certain elements of the drawings may be enlarged relative to other elements of the drawings for illustrative purposes. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows cell morphology of Canine 1 ASCs, comparing morphology when cultured in standard culture medium (left) with morphology in the species-specific medium disclosed herein (right). [Figure 2] FIG. 1 is a diagram of cell morphology of canine 2 ASCs comparing morphology when cultured in standard culture medium (left) with morphology in the species-specific medium disclosed herein (right). [Figure 3] FIG. 11 is a diagram of cell morphology of canine 3 ASCs comparing morphology when cultured in standard culture medium (left) with morphology in the species-specific medium disclosed herein (right). [Figure 4] FIG. 11 is a diagram of cell morphology of canine 4 ASCs comparing morphology when cultured in standard culture medium (left) with morphology in the species-specific medium disclosed herein (right). [Figure 5] FIG. 1 is a diagram of cell morphology of canine ASCs comparing morphology when cultured in standard culture medium (left) with morphology in the species-specific medium disclosed herein (right). [Figure 6] FIG. 1 shows cell morphology of canine ASCs, comparing morphology when cultured in standard culture medium (left) with that in the species-specific medium disclosed herein (right). [Figure 7]This graph shows the population doubling times (PDL) for canine MSCs from six different dogs cultured for 4 days in either standard culture medium containing 10% (v / v) canine serum or SCT canine culture medium. This figure shows that despite the significantly lower serum concentration in SCT medium, cell yields obtained with SCT medium are consistently higher than with standard medium. *, the difference between cells cultured in traditional serum-containing medium and cells cultured in SCT medium is statistically significant (P = 0.01, two-tailed paired t-test). [Figure 8] This is a graph of doubling time for canine ASCs from six different dogs cultured for 4 days in either standard culture medium containing 10% (v / v) canine serum or SCT canine culture medium. The figure shows that despite the significantly lower serum concentration in SCT medium, the growth rate obtained with SCT medium is consistently faster than with standard medium. *, the difference between cells cultured in traditional serum-containing medium and cells cultured in SCT medium is statistically significant (P<0.05, two-tailed paired t-test). [Figure 9] Figure 1 shows a graph of the doubling time of cells from the six donors in Table 1 cultured in SCT255 compared to standard medium, over a range of 5 to 16 doublings. Doubling times are faster and less variable for ASCs cultured in SCT255 than for the same ASCs cultured in standard 10% (v / v) serum-containing culture medium. [Figure 10A] The cytokine expression profile of canine ASCs cultured in SCT medium is similar to that of ASCs cultured in standard culture medium. Figure 10A shows the cytokine profile for each dog separately. Figure 10B shows the average cytokine profile for N = 6 dogs. In both Figures 10A and 10B, gene expression was normalized to that of cells cultured in DMEM + 10% (v / v) canine serum (standard medium). Expression of IGF-1 and IL-6 tended to be higher in cells cultured in SCT medium than in normal medium, but did not reach statistically significant levels (log10[RQ] ≥ ±2). IGF-1 expression by ASCs has pro-regenerative and immunomodulatory effects, and IL-6 is also immunomodulatory (1, 2). [Figure 10B] The cytokine expression profile of canine ASCs cultured in SCT medium is similar to that of ASCs cultured in standard culture medium. Figure 10A shows the cytokine profile for each dog separately. Figure 10B shows the average cytokine profile for N = 6 dogs. In both Figures 10A and 10B, gene expression was normalized to that of cells cultured in DMEM + 10% (v / v) canine serum (standard medium). Expression of IGF-1 and IL-6 tended to be higher in cells cultured in SCT medium than in normal medium, but did not reach statistically significant levels (log10[RQ] ≥ ±2). IGF-1 expression by ASCs has pro-regenerative and immunomodulatory effects, and IL-6 is also immunomodulatory (1, 2). [Figure 11A] These results show that canine ASCs cultured in SCT255c maintain gene upregulation of IDO-1 and other anti-inflammatory and immunomodulatory cytokines upon exposure to INF-γ, similar to cells cultured in standard medium plus 10% (v / v) canine serum, but the expression of the immunomodulatory gene PD-L2 is consistently and significantly upregulated when canine ASCs are cultured in SCT255c. In Figure 11A, canine ASCs from six different donors were cultured overnight in SCT255c plus INF-γ (10 mcg / ml). Gene expression was normalized to a control sample of the same cells cultured in the same medium but without INF-γ. A difference of log10(RQ) >±2 is considered statistically significant. Exposure of canine ASCs cultured in SCT255c to INF-γ results in the normal upregulation of immunomodulatory cytokine genes. In Figure 11B, canine ASCs from six different donors were cultured overnight in SCT255c + INF-γ. Expression was normalized to a control sample of the same cells cultured in medium + standard 10% (v / v) canine serum containing INF-γ. With the exception of PD-L2, differences were not statistically significant [log10(RQ) > ±2]. This figure shows that culture of canine ASCs in SCT255c provides the cells with a superior immunomodulatory cytokine profile than culture in standard medium + 10% (v / v) canine serum. [Figure 11B]These results show that canine ASCs cultured in SCT255c maintain gene upregulation of IDO-1 and other anti-inflammatory and immunomodulatory cytokines upon exposure to INF-γ, similar to cells cultured in standard medium plus 10% (v / v) canine serum, but the expression of the immunomodulatory gene PD-L2 is consistently and significantly upregulated when canine ASCs are cultured in SCT255c. In Figure 11A, canine ASCs from six different donors were cultured overnight in SCT255c plus INF-γ (10 mcg / ml). Gene expression was normalized to a control sample of the same cells cultured in the same medium but without INF-γ. A difference of log10(RQ) >±2 is considered statistically significant. Exposure of canine ASCs cultured in SCT255c to INF-γ results in the normal upregulation of immunomodulatory cytokine genes. In Figure 11B, canine ASCs from six different donors were cultured overnight in SCT255c + INF-γ. Expression was normalized to a control sample of the same cells cultured in medium + standard 10% (v / v) canine serum containing INF-γ. With the exception of PD-L2, differences were not statistically significant [log10(RQ) > ±2]. This figure shows that culture of canine ASCs in SCT255c provides the cells with a superior immunomodulatory cytokine profile than culture in standard medium + 10% (v / v) canine serum. [Figure 12] Figure 1 shows a graph showing PD-L2 gene expression in canine ASCs from six different donors cultured in either SCT255c or standard culture medium plus 10% (v / v) canine serum. PD-L2 was expressed in only one canine cell line cultured in standard medium, but was expressed in all six test lines cultured in SCT255c. Cycle thresholds normalized to an internal control (housekeeping gene) for ASCs from six dogs are shown. These data indicate that culture of canine ASCs in SCT255c results in cells with a superior immunomodulatory cytokine profile than culture in standard medium plus 10% (v / v) canine serum. [Figure 13]Figure 1 shows a graph showing PD-L1 protein expression in canine ASCs from six different donors cultured in either SCT255c + INF-γ or standard culture medium + 10% (v / v) canine serum + INF-γ. PD-L1 protein expression was assessed by FACS and graphed as the percentage of cells expressing the protein. These data also demonstrate that culture of canine ASCs in SCT255c provides the cells with a superior immunomodulatory cytokine profile than culture in standard medium + 10% (v / v) canine serum. [Figure 14A] Graph showing growth rate, cell yield of islet cells (IC) from N=6 dogs cultured in SCT255c compared to standard culture medium, demonstrating faster growth rate and superior yield of another cell type (canine islet cells) when cultured in SCT255c compared to standard culture medium + 20% (v / v) canine serum. [Figure 14B] Graph showing growth rate, cell yield of islet cells (IC) from N=6 dogs cultured in SCT255c compared to standard culture medium, demonstrating faster growth rate and superior yield of another cell type (canine islet cells) when cultured in SCT255c compared to standard culture medium + 20% (v / v) canine serum. [Figure 14C] Graph showing growth rate, cell yield of islet cells (IC) from N=6 dogs cultured in SCT255c compared to standard culture medium, demonstrating faster growth rate and superior yield of another cell type (canine islet cells) when cultured in SCT255c compared to standard culture medium + 20% (v / v) canine serum. [Figure 14D] Graph showing growth rate, cell yield of islet cells (IC) from N=6 dogs cultured in SCT255c compared to standard culture medium, demonstrating faster growth rate and superior yield of another cell type (canine islet cells) when cultured in SCT255c compared to standard culture medium + 20% (v / v) canine serum. [Figure 15A]Graph showing islet endocrine gene expression in P0 and P1 canine ICs (N=6) cultured in SCT255c compared to standard culture medium, demonstrating that islet cell identity is preserved when cells are cultured in SCT255c. [Figure 15B] Graph showing islet endocrine gene expression in P0 and P1 canine ICs (N=6) cultured in SCT255c compared to standard culture medium, demonstrating that islet cell identity is preserved when cells are cultured in SCT255c. [Figure 16A] 1A and 1B are photomicrographs and diagrams showing that normal capillaries (in the absence of underlying microvascular disease states) can accommodate relatively large mesenchymal stem cells (approximately 100 μm in diameter). [Figure 16B] FIG. 1 shows micrographs of mesenchymal stem cell-derived exosomes (approximately 40-100 nm in diameter) demonstrating that capillaries with microvascular disease (which are relatively occluded compared to normal capillaries) can accommodate relatively small exosomes containing MSC cargo better than they can accommodate MSCs. [Figure 17A] Protein concentration, nanoparticles per million, and nanoparticle mean and modal size for extracellular vesicles collected from canine ASCs cultured in SCT255c basal medium compared to standard basal medium. ASCs from four different dogs were cultured in each of two separate experiments. Differences in protein concentration (Figure 18A), extracellular vesicle reflectance, or particle number (Figure 18B) were significantly different between the two control medium experiments but not in the SCT255c medium experiment (Figure 18A). The mean size of particles collected from cells cultured in SCT255c was not significantly different from those cultured in standard medium (Figure 18C). The modal size was not different from the mean size of cells cultured in SCT255c, indicating that the collected extracellular vesicles were more consistent and uniform. However, the modal size of particles (extracellular vesicles) collected from standard medium was significantly different from the mean value, indicating substantial variation in extracellular vesicles (Figure 18C). [Figure 17B]Protein concentration, nanoparticles per million, and nanoparticle mean and modal size for extracellular vesicles collected from canine ASCs cultured in SCT255c basal medium compared to standard basal medium. ASCs from four different dogs were cultured in each of two separate experiments. Differences in protein concentration (Figure 18A), extracellular vesicle reflectance, or particle number (Figure 18B) were significantly different between the two control medium experiments but not in the SCT255c medium experiment (Figure 18A). The mean size of particles collected from cells cultured in SCT255c was not significantly different from those cultured in standard medium (Figure 18C). The modal size was not different from the mean size of cells cultured in SCT255c, indicating that the collected extracellular vesicles were more consistent and uniform. However, the modal size of particles (extracellular vesicles) collected from standard medium was significantly different from the mean value, indicating substantial variation in extracellular vesicles (Figure 18C). [Figure 17C] Protein concentration, nanoparticles per million, and nanoparticle mean and modal size for extracellular vesicles collected from canine ASCs cultured in SCT255c basal medium compared to standard basal medium. ASCs from four different dogs were cultured in each of two separate experiments. Differences in protein concentration (Figure 18A), extracellular vesicle reflectance, or particle number (Figure 18B) were significantly different between the two control medium experiments but not in the SCT255c medium experiment (Figure 18A). The mean size of particles collected from cells cultured in SCT255c was not significantly different from those cultured in standard medium (Figure 18C). The modal size was not different from the mean size of cells cultured in SCT255c, indicating that the collected extracellular vesicles were more consistent and uniform. However, the modal size of particles (extracellular vesicles) collected from standard medium was significantly different from the mean value, indicating substantial variation in extracellular vesicles (Figure 18C). [Figure 18]1 is a graph showing that IFG-1 mRNA cargo in extracellular vesicles collected from canine ASCs cultured in SCT255c is significantly upregulated compared to IFG-1 mRNA cargo in extracellular vesicles collected from canine ASCs cultured in standard medium. [Figure 19A] 1 is a photomicrograph showing the growth of human MSCs in SCT255h medium compared to control medium. [Figure 19B] 1 is a photomicrograph showing the growth of human MSCs in SCT255h medium compared to control medium. [Figure 20A] 1 is a photomicrograph showing the growth of human 041508 cells in SCT255h medium compared to control medium. [Figure 20B] 1 is a photomicrograph showing the growth of human 041508 cells in SCT255h medium compared to control medium. [Figure 21A] 1 is a photomicrograph showing the growth of human "Rooster" cells in SCT255h medium compared to control medium. [Figure 21B] 1 is a photomicrograph showing the growth of human "Rooster" cells in SCT255h medium compared to control medium. DETAILED DESCRIPTION OF THE INVENTION

[0015] The following description lists various aspects and embodiments of the invention disclosed herein. The specific embodiments are not intended to define the scope of the invention. Rather, the embodiments provide non-limiting examples of various compositions and methods that fall within the scope of the claimed invention. The description should be read from the perspective of one skilled in the art. Therefore, it does not necessarily include information that is familiar to one skilled in the art.

[0016] definition The following terms and phrases have the meanings indicated below, unless otherwise defined herein. This disclosure may use other terms and phrases not expressly defined herein. Such other terms and phrases shall have the meanings that they would have to one of ordinary skill in the art within the context of this disclosure. In some instances, terms or phrases may be defined in the singular or plural. In such instances, it is understood that any term in the singular may include its plural counterpart, and vice versa, unless a contradictory meaning is otherwise expressly stated.

[0017] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a substituent" includes a single substituent, as well as two or more substituents, and the like.

[0018] As used herein, the words "for example," "for instance," "such as," or "including" are meant to introduce examples that further clarify a more general subject matter. Unless expressly indicated otherwise, such examples are provided merely as an aid in understanding the illustrated embodiments of the present disclosure and are not meant to be limiting in any way. Furthermore, these terms do not imply any kind of preference for the disclosed embodiments.

[0019] As used herein, "MSC" refers to mesenchymal stromal cells. These are multipotent cells found in nearly all tissues and can differentiate into many different cell types, including chondrocytes, osteoblasts, adipocytes, and potentially other cells. MSCs can also interact with immune cells and modulate immune responses, including enabling immunosuppression and tolerance induction. They have excellent proliferation, differentiation, and immunomodulatory capabilities. Mesenchymal stem cells can self-renew by division and differentiate into multiple tissues, including bone, cartilage, muscle, and adipocytes, as well as connective tissue.

[0020] As used herein, "ASC" refers to adipose stromal cells. ASCs are a type of mesenchymal stromal cell derived from adipocytes. ASCs can be easily harvested in large quantities from adipose tissue. Adipose stromal cells harvested from subcutaneous adipose tissue are often referred to as ASCs. ASCs are post-embryonic self-renewing cells that, when properly stimulated, can generate a variety of parenchymal cells in vitro. ASCs readily colonize in vitro, form stromal progeny, and, upon transplantation, express common fibroblast markers and can modulate the host immune system. Because ASCs are transient cells, they are generally thought to have a limited lifespan in the recipient. Potential benefits include affinity for sites of injury and inflammation, and secretion of trophic factors that influence the repair of damaged tissue. As used herein, M / ASC refers to MSCs and / or ASCs. As used herein, "diabetes" refers to a disease that causes too much sugar in the blood.

[0021] As used herein, "Type 1 diabetes" is meant to refer to a chronic condition in which the pancreas produces little or no insulin.

[0022] As used herein, "type 2 diabetes" is meant to refer to a chronic condition that affects the way the body processes blood sugar.

[0023] As used herein, "treating" or "treatment" means that the symptoms of the underlying disease are at least alleviated and / or one or more of the underlying cellular, physiological, or biochemical causes or mechanisms that cause the symptoms are reduced and / or eliminated. Reduction, as used in this context, is understood to refer relative to the state of the disease, including not only the physiological state of the disease, but also the molecular state of the disease. Treating or treatment does not require a complete cure.

[0024] As used herein, a "therapeutically effective amount" is an amount sufficient to act as a treatment as defined above, which can be determined, for example, by standard techniques used to monitor and / or diagnose a particular disease state.

[0025] As used herein, "extracellular vesicles" refers to a type of extracellular vesicle that contains particles (proteins, DNA, and RNA) from the cell that secretes them. Extracellular vesicles can fuse with other cells into which the particles are taken up, potentially affecting the function and behavior of the cells. Examples of extracellular vesicles include, but are not limited to, exosomes, mitosomes, and oncosomes.

[0026] In describing the composition and manufacture of reduced serotype xeno-free cell culture media, specific components are described with reference to the manufacturer and the specific products used. These specific products are intended to be exemplary of components in cell culture media and are not intended to limit the description of the use of similar components.

[0027] The present invention provides a reduced serum type, xeno-free cell culture medium for use in mammalian cell culture for the production of species-specific M / ASCs, islet cells, and other cell types.

[0028] Treatments for diseases and disorders continue to advance. Among the advances in disease treatment is cell therapy. Cell therapy is most often understood to be the transplantation or infusion of cells into a patient to repair or replace damaged or diseased tissue. Many types of cells are used in these therapies, including M / ASCs and islet cells. While cell therapies can use cells harvested from a donor subject and directly transplanted or infused into a recipient subject, the number of cells required for effective results is often greater than the number of cells harvested from the donor. Additionally, to provide cost-effective treatments, it is often more beneficial to culture and expand cells for use in these therapies rather than extracting cells from a donor subject and transplanting them into a recipient subject for each transplant, infusion, or treatment.

[0029] Many common acute and chronic diseases, both early and advanced, are characterized by the development of similar microvascular lesions that lead to progressive organ damage, organ loss, morbidity, and mortality. The unique capillary / microvascular network of all organs and tissues is crucial for their physiological function and overall health. Capillaries facilitate blood supply, oxygenation, and metabolic removal from all organs. Anatomically, capillaries are essentially composed of endothelial cells, smooth muscle cells, and pericytes. The latter "police" and maintain the function and anatomical integrity of all capillaries.

[0030] When pathological processes such as diabetes, inflammatory, autoimmune, degenerative, aging, trauma, and other injuries affect the function of the capillary complex, blood supply, tissue oxygenation, and removal of metabolites from the affected organ are impaired, translating into permanent loss of function and systemic morbidity and mortality.

[0031] Mesenchymal or stromal stem cells (MSCs) from bone marrow, adipose, umbilical cord, and other sources are non-embryonic "adult" stem cells with potent anti-inflammatory, anti-apoptotic, immunomodulatory, angiogenic and vasculoprotective, antifibrotic, and antithrombotic paracrine activities that have been used as promising treatments for a variety of acute and chronic diseases and organ injuries. MSCs in culture release beneficial cytokines and growth factors that mediate their pleiotropic effects.

[0032] Although it is possible to isolate MSCs from all postnatal tissues, adipose tissue and bone marrow are considered the primary sources of MSCs due to the ease of isolation from these tissues. MSCs isolated from adipose or adipose tissue are referred to as ASCs. As adult tissues cease differentiation, the number of M / ASCs in these tissues becomes low. Growing M / ASCs in culture helps produce sufficient quantities of M / ASCs for therapeutic applications. The heterogeneity of M / ASCs is affected by the isolation method and culture conditions (including culture reagents, culture vessels, and culture environment). Growth media for both the isolation and expansion of M / ASCs from adipose tissue and bone marrow have been described. Many commercially available basal culture media exist for use as cell culture media, typically requiring pH adjustment and supplementation with mammalian serum at concentrations of 10–20% (v / v). FBS is one of the most common serum supplements for culture media. FBS provides growth factors, attachment factors, and various other nutrients. The concentration, quality, and source or species of these factors and nutrients vary between suppliers, and even between batches from the same supplier. The aforementioned variations in nutrients and factors add to heterogeneity in the number and quality of M / ASCs and other cells. This heterogeneity is undesirable, especially within regulatory frameworks. Additionally, the introduction of proteins that are heterologous to the cells being cultured is problematic for several reasons. First, we hypothesize that cells grow best, most consistently, and most reproducibly when using proteins derived from their species of origin. Such proteins, as evolved, adapt to their intended target; proteins from other species may or may not adapt to their intended target, depending on the degree to which such proteins are naturally conserved. Second, proteins present in the culture medium are often taken up by the cultured cells and stored within them. Problems have been observed with human MSCs cultured using FBS as a serum source, which have subsequently been used therapeutically.MSCs themselves are typically immune privileged, but when FBS is used to culture cells for therapeutic use, bovine proteins are taken up by the cells and stored intracellularly, and subsequently, immune responses against the bovine proteins have been detected in individuals treated with the cells (3).

[0033] The present invention provides cell culture media comprising, in whole or in part, a modified basal medium. The modified basal cell culture medium can be derived from standard basal cell culture media known in the art. Suitable basal media include, but are not limited to, Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), DMEM / F12, Basal Medium Eagle's (BSE), or any of a variety of other media commercially available. In addition to the basal medium, the culture medium contains sodium bicarbonate, HEPES (4-(2-hydroxyethyl)-1-piperaziethanesulfonic acid), GlutaMax™ (L-glutamine, L-alanine), lipid concentrate (such as Gibco's chemically defined lipid concentrate), insulin, hydrocortisone (e.g., from a plant, wild yam), progesterone (e.g., synthetic), putrescine (e.g., microbial), ASC-2-P (vitamin C), bFGF (corresponding to the cell species), PDGF-BB (corresponding to the cell species), EGF (corresponding to the cell species), insulin, and serum (corresponding to the cell species). In certain embodiments, the insulin may be of the same species as the cultured cells.

[0034] Cells require nutrients and a favorable environment in order to grow. Necessary nutrients include both organic nutrients such as lipids, carbohydrates, amino acids, and vitamins, inorganic nutrients such as salts and minerals, and non-nutritional factors such as hormones and growth factors.

[0035] There are several chemically defined lipid concentrates available on the market. These lipid concentrates are concentrated lipid emulsions designed to reduce or replace fetal bovine serum in cell culture media for a wide variety of applications. One example of a lipid concentrate is Gibco's chemically defined lipid concentrate, which can be purchased from ThermoFisher Scientific (catalog number 11905031). This lipid concentrate contains the following components: Arachidonic acid, 2.0 mg / L; cholesterol, 220.0 mg / L; DL-alpha-tocopherol acetate, 70.0 mg / L; ethyl alcohol; linoleic acid, 10.0 mg / L; linolenic acid, 10.0 mg / L; myristic acid, 10.0 mg / L; oleic acid, 10 mg / L; palmitic acid, 10.0 mg / L; palmitoleic acid, 10.0 mg / L; Pluronic F-68, 90,000.0 mg / L (non-ionic detergent to protect cells from hydrodynamic damage); stearic acid, 10.0 mg / L; Tween 80®, 2,200.0 mg / L (non-ionic detergent used for selective protein extraction and isolation of nuclei from mammalian cell lines).

[0036] Glutamine is an essential amino acid for protein and nucleic acid synthesis and energy production, and therefore its inclusion in culture media increases the function of many cell types. However, glutamine is difficult to include because it is highly unstable at physiological pH and decomposes non-enzymatically to ammonia. GlutaMax™ (L-glutamine, L-alanine; Invitrogen, Carlsbad, CA), a stabilized form of L-glutamine, is a glutamine substitute and is more stable in aqueous solution.

[0037] Growth factors must be included in the culture medium. These growth factors include, but are not limited to, fibroblast growth factor basic (bFGF), platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1), and epidermal growth factor (EGF) for the cell species being cultured. These growth factors are commercially available from a variety of sources. Typically, these growth factors are purchased, reconstituted in an appropriate liquid buffer, aliquoted, and stored according to the manufacturer's recommendations. The addition of bFGF to serum-free medium is known to promote the proliferation of human M / ASCs. Growth factor-defined culture medium for human mesenchymal stem cells.

[0038] Holo-transferrin (Sigma, St. Louis, MO) of the species of the cultured cells was added as an iron transport source. Holo-transferrin transports the trace mineral iron, and is an essential requirement for cells because it is a cofactor for metabolic enzymes and enzymes involved in DNA synthesis. Cells obtain iron from carrier proteins such as holo-transferrin. In some embodiments, bovine holo-transferrin is not used.

[0039] Adherence Factors: Adherence factors aid in the attachment of cells to culture surfaces. These factors are largely unknown. Recently, the glycoprotein fetuin-A has been shown to be a major serum cell attachment factor. Fetuin-A (α2HS-glycoprotein) is the major serum adhesion protein that mediates growth signaling in breast tumor cells. In some embodiments, 0.025% to 0.1% (w / v) fetuin from bovine serum (Sigma, St. Louis, MO) is used to promote the attachment of canine Ad-ASC to culture surfaces. In some embodiments, fetuin was not utilized.

[0040] Hormones influence growth and regulate cell function. Both steroid and non-steroid hormones exist. Hydrocortisone (HCN), an adrenal cortical hormone, supports M / ASC adhesion and growth. FBS typically contains 100 nM to 1 μM HCN. In some embodiments, synthetic HCN is used to supplement the medium. In other embodiments, the HCN of the cell species is used in the medium. Another hormone important for regulating M / ASC function is progesterone. In some embodiments, synthetic progesterone is also added to the medium. In other embodiments, the progesterone used is specific to the cell species in the medium.

[0041] Insulin is important in many cellular functions, including glucose and amino acid uptake, adipogenesis, intracellular transport, and protein and nucleic acid synthesis. Enhanced glycolysis promotes glucose uptake and is supported by insulin, making it a metabolic phenotype associated with the undifferentiated state of M / ASCs. Species-specific insulin was added to the culture medium at a concentration of 0.17 μM. In some embodiments, the insulin was derived from the species from which the cells were harvested. In some embodiments, the insulin was derived from a different species from which the cells were harvested, and the insulin is compatible between species.

[0042] Other components that support M / ASC and other cell growth include buffers, antioxidants, and polyamines.

[0043] In addition to being a metabolic precursor, sodium bicarbonate is commonly used as a buffer. Therefore, sodium bicarbonate was added to the medium at a concentration of 2.4 g / L. Additional buffering capacity was added to the medium in the form of HEPES, an organic buffer.

[0044] The diamine putrescine is a non-protein nitrogenous base and a precursor of the polyamines spermidine and spermine. Putrescine is associated with proliferation in several mammalian cell lines and is important for maintaining self-renewal in human embryonic stem (ES) cells. 56 μM putrescine was added to the medium.

[0045] Cell culture media is generated by starting with DMEM (Dulbelco Modified Eagle Medium) and adding F-12, a nutrient mixture that supports cell growth and proliferation. Additional nutrients are added to the media to create media for the growth of each species of M / ASC and islet cells.

[0046] The buffer system utilized in the medium is sodium bicarbonate, and HEPES assists in the buffering process. In some embodiments of the present invention, the concentration of sodium bicarbonate is at least about 18 mM, for example, at least about 24 mM, for example, at least about 28 mM, for example, at least about, for example, at least about 32 mM, for example, at least about 36 mM, for example, at least about 40 mM.

[0047] In some embodiments of the invention, the concentration of HEPES is at least about 1 mM, such as at least about 2 mM, for example, at least about 3 mM, for example, at least about 4 mM, for example, at least about 5 mM, for example, at least about 6 mM, for example, at least about 7 mM.

[0048] As mentioned above, glutamine, particularly L-glutamine, is an important nutrient for cell growth. GlutaMax™ (ThermoFisher) was selected as the L-glutamine additive. GlutaMax™ is a dipeptide of L-glutamine and L-alanine. In some embodiments of the present invention, the L-glutamine concentration is at least about 1.5 mM, e.g., at least about 2.0 mM, e.g., at least about 2.5 mM, e.g., at least about 3.0 mM, about 3.5 mM, e.g., at least about 4.0 mM. In some embodiments, the L-alanine concentration is at least about 0.2 mM, at least about 0.4 mM, at least about 0.6 mM, at least about 0.8 mM, at least about 1.0 mM, or at least about 1.2 mM.

[0049] In some embodiments, lipid is added to medium by volume to volume concentration.In these embodiments, the lipid concentration added to medium is at least about 4%, for example, at least about 6%, for example, at least about 8%, for example, at least about 10%, for example, at least about 12%, for example, at least about 14%, for example, at least about 16%.

[0050] In some embodiments of the invention, the concentration of insulin is at least about 0.10 μM, such as at least about 0.11 μM, for example, at least about 0.13 μM, for example, at least about 0.15 μM, such as at least about 0.15 μM, for example, at least about 0.17 μM, for example, at least about 0.19 μM, such as at least about 0.21 μM, for example, at least about 0.23 μM, for example, at least about 0.25 μM.

[0051] In some embodiments of the invention, the concentration of hydrocortisone is at least about 50 nM, such as at least about 60 nM, for example, at least about 80 nM, such as at least about 90 nM, for example, at least about 100 nM, such as at least about 110 nM, for example, at least about 120 nM, such as at least about 130 nM, for example, at least about 140 nM, for example, at least about 150 nM.

[0052] In some embodiments of the invention, the concentration of progesterone is at least about 11 nM, such as at least about 13 nM, for example, at least about 15 nM, such as at least about 17 nM, for example, at least about 19 nM, for example, at least about 21 nM, such as at least about 23 nM, for example, at least about 25 nM, for example, at least about 27 nM, for example, at least about 29 nM.

[0053] In some embodiments of the invention, the concentration of putrescine is at least about 35 μM, such as at least about 40 μM, for example, at least about 45 μM, for example, at least about 50 μM, for example, at least about 56 μM, such as at least about 60 μM, for example, at least about 65 μM, for example, at least about 70 μM, for example, at least about 75 μM, for example, at least about 80 μM.

[0054] In some embodiments of the invention, the concentration of Asc-2-p is at least about 100 μM, such as at least about 125 μM, for example, at least about 150 μM, for example, at least about 175 μM, for example, at least about 200 μM, for example, at least about 225 μM, for example, at least about 250 μM, for example, at least about 275 μM, for example, at least about 300 μM.

[0055] In some embodiments of the invention, the concentration of holo-transferrin is at least about 0.20 μM, such as at least about 0.25 μM, for example, at least about 0.30 μM, for example, at least about 0.35 μM, for example, at least about 0.40 μM, for example, at least about 0.45 μM, for example, at least about 0.50 μM, for example, at least about 0.55 μM, for example, at least about 0.60 μM. In some embodiments, holo-transferrin is not added to the medium.

[0056] In some embodiments of the invention, the concentration of each type of bFGF (e.g., bFGF-canine, bFGF-human, or bFGF-mouse) is at least about 1 ng / mL, such as at least about 2 ng / mL, for example, at least about 3 ng / mL, such as at least about 4 ng / mL, for example, at least about 5 ng / mL, such as at least about 6 ng / mL, for example, at least about 7 ng / mL, for example, at least about 8 ng / mL, such as at least about 9 ng / mL, for example, at least about 10 ng / mL, for example, at least about 11 ng / mL, for example, at least about 12 ng / mL, for example, at least about 13 ng / mL.

[0057] In some embodiments of the invention, the concentration of each species of PDGF-BB (e.g., canine or human) is at least about 1 ng / mL, such as at least about 2 ng / mL, for example, at least about 3 ng / mL, such as at least about 4 ng / mL, for example, at least about 5 ng / mL, such as at least about 6 ng / mL, for example, at least about 7 ng / mL, such as at least about 8 ng / mL, for example, at least about 9 ng / mL, such as at least about 10 ng / mL, for example, at least about 11 ng / mL, such as at least about 12 ng / mL, for example, at least about 13 ng / mL.

[0058] In some embodiments of the invention, the concentration of EGF (e.g., EGF canine or EGF human) is at least about 1 ng / mL, such as at least about 2 ng / mL, for example, at least about 3 ng / mL, such as at least about 4 ng / mL, for example, at least about 5 ng / mL, such as at least about 6 ng / mL, for example, at least about 7 ng / mL, such as at least about 8 ng / mL, for example, at least about 9 ng / mL, such as at least about 10 ng / mL, for example, at least about 11 ng / mL, such as at least about 12 ng / mL, for example, at least about 13 ng / mL.

[0059] In some embodiments of the present invention, the concentration of TGF-β1 is at least about 0.25 ng / mL, such as at least about 0.5 ng / mL, for example, at least about 0.75 ng / mL, for example, at least about 1.0 ng / mL, for example, at least about 1.25 ng / mL, for example, at least about 1.50 ng / mL, for example, at least about 1.75 ng / mL, for example, at least about 2.0 ng / mL. In some embodiments, TGF-β1 is not utilized in the culture medium.

[0060] Albumin and other serum proteins may be included in embodiments. In some embodiments of the invention, the concentration of canine serum is at least about 1%, such as at least about 2%, for example, at least about 3%, such as at least about 4%, for example, at least about 5%, such as at least about 6%, for example, at least about 7%, such as at least about 8%, for example, at least about 9%, such as at least about 10%, for example, at least about 11%, for example, at least about 15%.

[0061] In some embodiments of the invention, the concentration of SA (species-specific serum albumin - see comments related to paragraph 60) is at least about 0.001 g / L, such as at least about 0.002 g / L, for example, at least about 0.003 g / L, for example, at least about 0.004 g / L, for example, at least about 0.005 g / L, for example, at least about 0.006 g / L, for example, at least about 0.007 g / L, for example, at least about 0.008 g / L, for example, at least about 0.009 g / L, for example, at least about 0.010 g / L. In some embodiments, SA is absent from the medium.

[0062] In some embodiments of the invention, the concentration of fetuin is at least about, for example, at least about 0.25 ng / mL, for example, at least about 0.50 ng / mL, for example, at least about 0.75 ng / mL, 1 ng / mL, for example, at least about 2 ng / mL, for example, at least about 3 ng / mL, for example, at least about 4 ng / mL, for example, at least about 5 ng / mL, for example, at least about 6 ng / mL, for example, at least about 7 ng / mL, for example, at least about 8 ng / mL, for example, at least about 9 ng / mL, for example, at least about 10 ng / mL, for example, at least about 11 ng / mL, for example, at least about 12 ng / mL, for example, at least about 13 ng / mL. In some embodiments, fetuin is not present in the culture medium.

[0063] Example 1 Canine-specific media Dogs are increasingly becoming recipients of cell therapies, particularly mesenchymal stromal cells (MSCs), for the treatment of various disorders, including kidney disease, bone and joint disorders, diabetes, and immune disorders. These therapies require the isolation, purification, and often culture of MSCs for many doublings. Such cultures require the use of serum-supplemented culture media, and given dogs' susceptibility to foreign protein reactions, ideally the serum supplement is canine-derived so that the cells introduced into the dog are not carriers of xenogeneic proteins that could trigger an antigenic response in the recipient. A xenogeneic, reduced-serum culture medium (SCT255c) has been developed for the culture of canine cells. To prevent xenogeneic immune responses, this medium contains only canine-derived proteins and canine serum, but no other animal proteins. Two cell types, adipose- or bone marrow-derived mesenchymal stromal cells (A / MSCs) and pancreatic islet cells (ICs), have been tested using this medium versus standard commercially available culture media. A / MSCs cultured in SCT255c exhibit faster growth rates and higher yields, and also have a superior immunomodulatory profile compared to those cultured in standard 10% canine serum-containing medium. For cells derived from canine islets of Langerhans, culture in SCT255c allows for greater doubling potential, superior growth rates, and preserved islet endocrine gene expression than cultures in standard growth medium. This medium does not require attachment factors such as fetuin A, which have been shown by others to be necessary to support canine MSC culture. Furthermore, the serum required for culture of M / ASCs and ICs is significantly reduced to 1% for M / ASCs and P0 ICs and to 5% for primary islets, reducing both the ethical and financial costs of cell culture. See Tables 1 and 2.

[0064] The SCT-reduced serum-based medium for canine M / ASC and islet cells was directly compared to standard culture media of either DMEM / F-12 + 10% (v / v) canine serum or low-glucose DMEM + 10% (v / v) canine serum. Cells were evaluated for various beneficial cytokine phenotypes, growth rates, and gene expression profiles.

[0065] Several variations of reduced serotype have been tested. The composition of the most effective variant of reduced serotype medium is listed in Table 1. In this variation, bFGF is 2 ng / mL, PDGF-BB is 5 ng / mL, and EGF is 5 ng / mL. This variation has been named SCT255 and will be referred to as such from now on. [Table 1]

[0066] Alternative formulations of reduced serotype media were also tested and the compositions of these are also included in Tables 2 and 3. [Table 2] [Table 3]

[0067] Cells to be evaluated Canine M / ASC MSCs isolated from subcutaneous fat (also known as ASCs) from six dogs were studied. The donor dogs were of various ages, either sex, and either healthy or had heart failure (atrial fibrillation or ventricular tachycardia) for 6 weeks to 2 years at the time of fat donation (see Table 4). [Table 4]

[0068] cell growth As shown in Figures 1-6, all tested canine-derived M / ASCs exhibit a more cobblestone-like appearance when cultured in SCT formulations than in standard culture medium. Canine M / ASCs cultured for 4 days in standard culture medium (DMEM or DMEM low glucose) + 10% (v / v) canine serum underwent an average of 4.9 population doublings. In contrast, the same canine M / ASCs cultured in SCT medium underwent a statistically significantly higher 6.2 doublings over the same time frame (Figure 7), due to the significantly higher doubling time of cells cultured in SCT medium (Figure 8).

[0069] proliferation Canine M / ASCs from the donors listed in Table 3 were cultured for 4 to 6 passages in either SCT formulation or standard culture medium. The faster doubling time and resulting increased PDL numbers observed for cells cultured in SCT formulation and shown in Figures 7 and 8 were maintained through 16 doublings (see Figure 9). Indeed, doubling times were significantly shorter and more consistent for canine M / ASCs cultured in SCT255 than for cells cultured in standard medium + 10% (v / v) serum (Figure 9). Figures 7–9 demonstrate that culturing cells in this medium reduces variability introduced by cell differences, often resulting from different donors. Producing cells with uniform characteristics is often desirable, resulting in more consistent results in both testing and using the cells for cell therapy products, etc.

[0070] identity Canine M / ASCs are characterized in part by the expression of cell surface markers, with positive expression of CD90 and CD44 and lack of expression of CD34, CD45, and class II antigen (DLA-DR). As shown in Table 5, canine M / ASCs cultured in SCT medium exhibit positive expression of CD90 and CD44, but negative expression of CD34, CD45, and DLA-DR. Expression of DLA-DR after overnight exposure to IFN-γ (10 ng / ml) was also assessed by FACS and found to be negative.

[0071] Passaging the cells through PDLS for more than 20 times did not affect the expression of these markers (see Table 5). [Table 5]

[0072] Gene expression Gene expression of beneficial paracrine cytokines in M / ASCs cultured in SCT medium is similar to that of the same cells cultured in standard 10% (v / v) serum-containing medium (Figures 10A-10B). Expression levels of IGF-1 and IL-6 (both involved in immunoregulation by MSCs) tend to be higher in cells cultured in SCT medium than in normal medium, but do not reach statistically significant levels (log10[RQ] ≥ ±2). IGF-1 expression by MSCs has pro-regenerative and immunomodulatory effects, and IL-6 is also immunomodulatory.

[0073] immunomodulation As shown in Figures 11A-11B, cells cultured in SCT medium respond to overnight INF-γ exposure by inducing the expression of IDO-1 and other genes involved in immune regulation. Culture in SCT255 results in a consistent and highly significant upregulation of the potent anti-inflammatory cytokine CXCL10.

[0074] Unexpectedly, PD-L2, a PD-1 ligand that, together with PD-L1, confers immune tolerance, is expressed in canine M / ASCs cultured in SCT medium, even in the absence of INF-γ exposure. This immunoregulatory gene is often not expressed when cells are cultured in standard medium without INF-γ (Figure 12). Furthermore, induction of PD-L2 upon overnight exposure of cells to INF-γ is significantly stronger when cells are cultured in SCT medium compared with standard culture medium (either DMEM or DMEM low glucose; Figure 11B).

[0075] Secretion of PD-L1 and PD-L2 by human M / ASCs has been shown to directly affect T cell behavior, induce immune tolerance, and mediate antifibrotic effects. As shown in Figure 13, upon exposure to INF-γ, PD-L1 expression is significantly higher in cells cultured in SCT255 than in standard medium. PD-L1 expression by cancer cells is known to induce tolerance, allowing such cells to evade destruction by the immune system. Blockade of the PD-1 / PD-L1 pathway is currently used as an anticancer therapeutic in humans and is being developed in canines due to the similarity of this pathway between the two species. Therefore, culturing canine M / ASCs in SCT medium under standard conditions is likely to result in cells with better immunomodulatory capabilities than cells cultured using standard culture medium.

[0076] Canine islet cells Islets isolated from pancreata from six dogs were cultured and evaluated. The donor dogs were of various ages, either sex, and either healthy or had heart failure for 6 weeks to 2 years at the time of pancreas donation (see Table 6). [Table 6]

[0077] Cell Growth and Proliferation Islets were isolated and cultured in either the SCT medium formulation as described above or standard culture medium (DMEM-low glucose formulation + 20% (v / v) canine serum). Because islet cells require more serum in their culture medium, SCT medium for primary islet culture was formulated with a serum concentration of 5% (v / v) rather than the 1% (v / v) required for A / MSCs.

[0078] P0 islet cells were harvested from such cultures when they were approximately 90% confluent using 2x trypsin-EDTA, assessed for yield, growth rate, and doublings, and then passaged to P1, which were also similarly grown, harvested, and assessed to determine performance differences between SCT255 and standard media.

[0079] As shown in Figures 14A-14D, SCT medium consistently supported significantly better and more rapid growth of primary islets, resulting in an average of approximately 20,000 more P0 islet cells per cm2 (primary culture) in approximately 2 fewer days than standard culture medium (Figures 14A and B). Similarly, doubling time was an average of 2 hours faster when P0 cells were passaged and cultured to P1 (Figures 14C and D).

[0080] Gene expression Endocrine gene expression was assessed by rtPCR for P0 and P1 islet cells cultured in either SCT media formulations or standard culture medium. Results for SCT media were normalized to the results of the same passage of cells cultured in standard culture medium using the delta-delta ct method, and a log10[RQ] of ≥ ±2 was considered statistically significant. As shown in Figures 15A-15B, islet cells cultured in SCT medium underwent more doublings, but endocrine gene expression was well preserved, with a trend toward increased expression levels for all evaluated genes except glucagon at P1 (Figure 15B).

[0081] Exosomes Cell-derived extracellular vesicles, such as exosomes from MSCs, are increasingly being envisioned as potential therapeutic agents for situations in which cellular therapeutic cargo is desired, but the cells themselves are compromised, potentially further impairing or obstructing the microvasculature due to their larger size (Figures 16A and 16B).

[0082] To determine whether SCT255c basal medium (SCT255c without serum additives) is suitable for exosome collection and harvesting, and whether exosomes derived from this medium are comparable to or different from exosomes derived from cells incubated in DMEM (standard basal medium), the following experiment was performed.

[0083] ASCs from dogs 1, 2, 3, and 4 were cultured in T75 flasks using either DMEM + 10% (v / v) canine serum or SCT255c + 1% (v / v) canine serum until 85% confluence. Once the cells reached 85% confluence, the medium of both sets of cells was replaced with basal medium (serum-free), and the cells were cultured for an additional 24 hours. Afterward, the medium was collected, and the cells were harvested and counted. Exosomes from each set were isolated using the Exoquick TC kit. Exosomes from each set were analyzed for protein content using the Bradford assay, associated mRNA cargo by rtPCR, and size and particle number using Nanosite. Consistent with previous findings reported above, cells grew significantly faster and underwent significantly more PDL in SCT255c than in standard culture medium. Cells cultured in SCT255c yielded more consistent results in terms of protein concentration and modal particle size than cells cultured in standard medium (Figures 17A-17B). The size and particle number of microvesicles collected from SCT255c were not significantly different from those collected from MSCs cultured in standard medium (Figure 17C). Until now, inconsistent yield and quality of extracellular vesicles have been considered one of the barriers to the therapeutic use of MSC-derived exosomes. Therefore, SCT255c basal medium may offer significant advantages for extracellular vesicle isolation over other basal media. With the exception of IGF-1, mRNA cargo was not significantly different in exosomes derived from cells cultured in SCT255c compared to control medium (Figure 18), again reflecting the potentially superior immunomodulatory capabilities of MSCs and their extracellular vesicles when cultured in this medium formulation compared to other media formulations.

[0084] Example 2 Human-specific growth medium Because the serum supplement is human-derived, cells introduced into humans are not carriers of xenogeneic proteins that could trigger an antigenic response in the recipient. For the culture of human cells, we developed a xenogeneic, reduced-serum culture medium (SCT255h). To prevent xenogeneic immune responses, this medium contains only human-derived proteins and human serum, but no other animal proteins. Two cell types, adipose- or bone marrow-derived mesenchymal stromal cells (A / MSCs) and pancreatic islet cells (ICs), have been tested using this medium versus standard commercially available culture media.

[0085] The reduced serum medium variants for human M / ASC and islet cells were directly compared to standard culture media, and cells were evaluated for various beneficial cytokine phenotypes, growth rates, and gene expression profiles.

[0086] Several variations of reduced serum type media have been generated and tested. Table 7 shows the human-specific equivalents of the most effective canine sera. [Table 7] [Table 8]

[0087] Cells to be evaluated Human M / ASC Bone marrow-derived human MSCs were cultured in SCT255h and 1% (v / v) human platelet lysate (hPLA) and compared to bone marrow-derived MSCs cultured in alphaMEM + 10% (v / v) hPLA, which served as a control. Additional variations in cell culture media were also evaluated, including one utilizing SCT255h + 2% (v / v) hPLA. This variation in media had similar results to SCT255h + 1% (v / v) hPLA.

[0088] Two additional MSC lines were also evaluated: the cell lines were hMSCs purchased from RoosterBio P1, designated "Rooster," and hMSC-041508-P2 from a previous clinical trial, designated 041508.

[0089] cell growth There was little difference in growth rate and cell morphology between cells grown in SCT255h medium and control medium. Figure 19A shows bone marrow-derived MSCs in SCT255h + 1% (v / v) hPLA medium, and Figure 19B shows the same cells in control medium. As can be seen from the micrographs, cells in both media appear similar, with the only slight difference being that cells cultured in SCT255h medium are longer and more spindle-shaped. Additional cell lines also show little difference between cells grown in SCT255h medium and the control. Figure 20A shows a micrograph of 041508 cells grown in SCT255h medium, and Figure 20B shows 041508 cells grown in control medium. Figure 21A shows a micrograph of Rooster cells grown in SCT255h medium, and Figure 21B shows Rooster cells grown in control medium.

[0090] proliferation Cells in both media underwent 3.8 doublings, with each doubling time being approximately 37 hours. Table 9 shows the Rooster and 041508 cell lines cultured in various media. [Table 9]

[0091] identity Human MSCs are partially identified by the expression of cell surface markers. Cells from both culture medium types did not have significantly different expression of cell surface markers.

[0092] Passaging cells through PDLS for 20 or more times is not expected to affect the expression of these markers (see Table 5).

[0093] Gene expression Gene expression of beneficial paracrine cytokines in MSCs cultured in SCT medium is expected to be similar to that of the same cells cultured in standard 10% (v / v) serum-containing medium. Expression levels of IGF-1 and IL-6 (both involved in immunoregulation by MSCs) are expected to tend to be higher in cells cultured in SCT medium than in normal medium, but not to reach statistical significance (log10[RQ] ≥ ±2). IGF-1 expression by MSCs has pro-regenerative and immunomodulatory effects, and IL-6 is also immunomodulatory.

[0094] immunomodulation Cells cultured in SCT255 medium are expected to respond to overnight INF-γ exposure by inducing the expression of IDO-1 and other genes involved in immune regulation (Note: induction of CCL8 and CXCL10 has been published in humans but not for canine MSCs). Culture in SCT255 results in a consistent and highly significant upregulation of the potent anti-inflammatory cytokine CXCL10.

[0095] Secretion of PD-L1 and PD-L2 by human MSCs has been shown to directly affect T cell behavior, induce immune tolerance, and mediate antifibrotic effects. Upon exposure to IFN-γ, PD-L1 expression is predicted to be higher in cells cultured in SCT255 than in standard medium. PD-L1 expression by cancer cells is known to induce tolerance, allowing such cells to evade destruction by the immune system. Blockade of the PD-1 / PD-L1 pathway is currently used as an anticancer therapeutic agent in humans.

[0096] human islet cells Islets isolated from pancreatic human subjects are cultured and evaluated. The donor humans were of various ages, either gender, and varied in a variety of other clinical factors.

[0097] Cell Growth and Proliferation Islets were isolated and cultured in either the SCT medium formulation as described above or standard culture medium (DMEM-low glucose formulation + 20% (v / v) human serum). Because islet cells require more serum in their culture medium, SCT medium for primary islet culture was formulated with a serum concentration of 5% (v / v) rather than the 1% (v / v) required for A / MSCs.

[0098] P0 islet cells are harvested from such cultures when they are approximately 90% confluent using 2x trypsin-EDTA, assessed for yield, growth rate, and doublings, and then passaged to P1, which are also similarly grown, harvested, and assessed to determine performance differences between SCT255 and standard media.

[0099] SCT medium consistently supports significantly better and more rapid growth of primary islets, and is expected to result in an average of approximately 20,000 more P0 islet cells per cm2 (primary culture) in approximately 2 fewer days than standard culture medium. Similarly, doubling times are expected to be an average of 2 hours faster when P0 cells are passaged and cultured to P1.

[0100] Gene expression Endocrine gene expression is assessed by rtPCR for P0 and P1 islet cells cultured in either SCT media formulations or standard culture medium. SCT media results are normalized to the results of the same passage of cells cultured in standard culture medium using the delta-delta ct method, and a log10[RQ] of ≥ ±2 is considered statistically significant.

[0101] Example 3 Feline-specific growth medium Because the serum supplement is feline-derived, cells introduced into felines are not carriers of xenogeneic proteins that could trigger an antigenic response in the recipient. A xenogeneic, reduced-serum culture medium (SCT255f) variant is currently under development for culturing feline cells. To prevent xenogeneic immune responses, this medium contains only feline-derived proteins and feline serum, but no other animal proteins. Two cell types, adipose- or bone marrow-derived mesenchymal stromal cells (A / MSCs) and pancreatic islet cells (ICs), are being tested using this medium versus standard commercially available culture media.

[0102] Reduced serum media for feline M / ASC and islet cells were directly compared to standard culture media of either DMEM / F-12 + 10% (v / v) feline serum or low glucose DMEM + 10% (v / v) feline serum. Cells were evaluated for various beneficial cytokine phenotypes, growth rates, and gene expression profiles.

[0103] Several variations of reduced serum type media are generated and tested. Table 9 shows the feline-specific sera that correspond to the most effective canine sera. [Table 10] [Table 11]

[0104] Cells to be evaluated Feline M / ASC Feline MSCs were isolated and studied from subcutaneous fat (also known as ASCs) or bone marrow. Donor felines varied in age, sex, and other clinical factors.

[0105] cell growth Because feline cells share many properties with canine cells, the same feline M / ASCs cultured in SCT255f medium would be expected to undergo statistically significantly higher doublings over the same time frame compared to doublings in standard medium.

[0106] proliferation Feline MSCs are cultured for 4-6 passages in either SCT formulation or standard culture medium. Those cultured in SCT medium are expected to exhibit a faster doubling time, resulting in an increased number of PDLs observed and maintained through 16 doublings.

[0107] identity Feline MSCs are identified in part by the expression of cell surface markers, and feline MSCs cultured in SCT medium are expected to show positive expression of certain cell markers and negative expression of other cell markers.

[0108] Passaging cells through PDLS for 20 or more times is not expected to affect the expression of these markers.

[0109] Gene expression Gene expression of beneficial paracrine cytokines in MSCs cultured in SCT medium is expected to be similar to that of the same cells cultured in standard 10% (v / v) serum-containing medium. Expression levels of IGF-1 and IL-6 (both involved in immunoregulation by MSCs) are expected to tend to be higher in cells cultured in SCT medium than in normal medium, but not to reach statistical significance (log10[RQ] ≥ ±2). IGF-1 expression by MSCs has pro-regenerative and immunomodulatory effects, and IL-6 is also immunomodulatory.

[0110] immunomodulation Cells cultured in SCT medium are expected to respond to overnight INF-γ exposure by inducing expression of IDO-1 and other genes involved in immune regulation. Culture in SCT255 results in a consistent and highly significant upregulation of the potent anti-inflammatory cytokine CXCL10.

[0111] Secretion of PD-L1 and PD-L2 by human MSCs has been shown to directly influence T cell behavior, induce immune tolerance, and mediate antifibrotic effects. The response in feline cells upon exposure to IFN-γ is expected to be similar. PD-L1 is expected to be more highly expressed in cells cultured in SCT255 than in standard medium. PD-L1 expression by cancer cells is known to induce tolerance, allowing such cells to avoid destruction by the immune system. Blockade of the PD-1 / PD-L1 pathway is currently used as an anticancer therapeutic in humans.

[0112] feline island cells Islets isolated from pancreatic feline subjects are cultured and evaluated. Donor felines vary in age, sex, and other clinical factors.

[0113] Cell Growth and Proliferation Islets were isolated and cultured in either the SCT medium formulation as described above or standard culture medium (DMEM-low glucose formulation + 20% (v / v) feline serum). Because islet cells require more serum in their culture medium, SCT medium for primary islet culture was formulated with a serum concentration of 5% (v / v) rather than the 1% (v / v) required for A / MSCs.

[0114] P0 islet cells are harvested from such cultures when they are approximately 90% confluent using 2x trypsin-EDTA, assessed for yield, growth rate, and doublings, and then passaged to P1, which are also similarly grown, harvested, and assessed to determine performance differences between SCT255f and standard media.

[0115] SCT medium consistently supports significantly better and more rapid growth of primary islets, and is expected to result in an average of approximately 20,000 more P0 islet cells per cm2 (primary culture) in approximately 2 fewer days than standard culture medium. Similarly, doubling times are expected to be an average of 2 hours faster when P0 cells are passaged and cultured to P1.

[0116] Gene expression Endocrine gene expression is assessed by rtPCR for P0 and P1 islet cells cultured in either SCT media formulations or standard culture medium. SCT media results are normalized to the results of the same passage of cells cultured in standard culture medium using the delta-delta ct method, and a log10[RQ] of ≥ ±2 is considered statistically significant.

[0117] Example 4 Equine-specific growth medium Because the serum supplement is feline-derived, cells introduced into equine cells are not carriers of xenogeneic proteins that could trigger an antigenic response in the recipient. A xenogeneic, reduced-serum culture medium (SCT255e) variant is currently under development for the culture of equine cells. To prevent xenogeneic immune responses, this medium contains only equine-derived proteins and equine serum, but no other animal proteins. Two cell types, adipose- or bone marrow-derived mesenchymal stromal cells (A / MSCs) and pancreatic islet cells (ICs), are being tested using this medium versus standard commercially available culture media.

[0118] Reduced serum media for feline M / ASC and islet cells were directly compared to standard culture media of either DMEM / F-12 + 10% (v / v) equine serum or low glucose DMEM + 10% (v / v) equine serum. Cells were evaluated for various beneficial cytokine phenotypes, growth rates, and gene expression profiles.

[0119] Several variations of reduced serum type media are generated and tested. Table 11 shows the feline-specific sera that correspond to the most effective canine sera. [Table 12]

[0120] Alternative formulations of reduced serotype media were also tested and these compositions are also included in Table 13. [Table 13]

[0121] Cells to be evaluated Equine M / ASC Equine MSCs were isolated and studied from subcutaneous fat (also known as ASCs) or bone marrow. Donor horses varied in age, sex, and other clinical factors.

[0122] cell growth Because equine cells share many properties with canine cells, the same equine M / ASCs cultured in SCT255e medium would be expected to undergo statistically significantly higher doublings over the same time frame compared to doublings in standard medium.

[0123] proliferation Equine MSCs are cultured for 4-6 passages in either SCT formulation or standard culture medium. Those cultured in SCT medium are expected to exhibit a faster doubling time, resulting in an increased number of PDLs observed and maintained through 16 doublings.

[0124] identity Equine MSCs are identified in part by the expression of cell surface markers. Feline MSCs cultured in SCT medium are expected to show positive expression of certain cell markers and negative expression of other cell markers.

[0125] Passaging cells through PDLS for 20 or more times is not expected to affect the expression of these markers.

[0126] Gene expression Gene expression of beneficial paracrine cytokines in MSCs cultured in SCT medium is expected to be similar to that of the same cells cultured in standard 10% (v / v) serum-containing medium. Expression levels of IGF-1 and IL-6 (both involved in immunoregulation by MSCs) are expected to tend to be higher in cells cultured in SCT medium than in normal medium, but not to reach statistical significance (log10[RQ] ≥ ±2). IGF-1 expression by MSCs has pro-regenerative and immunomodulatory effects, and IL-6 is also immunomodulatory.

[0127] immunomodulation Cells cultured in SCT medium are expected to respond to overnight INF-γ exposure by inducing expression of IDO-1 and other genes involved in immune regulation. Culture in SCT255 results in a consistent and highly significant upregulation of the potent anti-inflammatory cytokine CXCL10.

[0128] Secretion of PD-L1 and PD-L2 by human MSCs has been shown to directly influence T cell behavior, induce immune tolerance, and mediate antifibrotic effects. The response in equine cells upon exposure to IFN-γ is expected to be similar. PD-L1 is expected to be more highly expressed in cells cultured in SCT255 than in standard medium. PD-L1 expression by cancer cells is known to induce tolerance, allowing such cells to avoid destruction by the immune system. Blockade of the PD-1 / PD-L1 pathway is currently used as an anticancer therapeutic in humans.

[0129] equine islet cells Islets isolated from pancreatic equine subjects are cultured and evaluated. Donor equines vary in age, sex, and other clinical factors.

[0130] Cell Growth and Proliferation Islets were isolated and cultured in either the SCT medium formulation as described above or standard culture medium (DMEM-low glucose formulation + 20% (v / v) equine serum). Because islet cells require more serum in their culture medium, SCT medium for primary islet culture was formulated with a serum concentration of 5% (v / v) rather than the 1% (v / v) required for A / MSCs.

[0131] P0 islet cells are harvested from such cultures when they are approximately 90% confluent using 2x trypsin-EDTA, assessed for yield, growth rate, and doublings, and then passaged to P1, which are also similarly grown, harvested, and assessed to determine performance differences between SCT255e and standard media.

[0132] SCT medium consistently supports significantly better and more rapid growth of primary islets, and is expected to result in an average of approximately 20,000 more P0 islet cells per cm2 (primary culture) in approximately 2 fewer days than standard culture medium. Similarly, doubling times are expected to be an average of 2 hours faster when P0 cells are passaged and cultured to P1.

[0133] Gene expression Endocrine gene expression is assessed by rtPCR for P0 and P1 islet cells cultured in either SCT media formulations or standard culture medium. SCT media results are normalized to the results of the same passage of cells cultured in standard culture medium using the delta-delta ct method, and a log10[RQ] of ≥ ±2 is considered statistically significant.

[0134] While the invention has been described with reference to various specific and preferred embodiments and techniques, it will be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0135] References (incorporated herein by reference): There were also other references in the submitted documents that may be useful here. 1.Silva DN,Souza BSF,Azevedo CM,Vasconcelos JF,de Jesus PG,Feitoza MS,et al.IGF-1-Overexpressing Mesenchymal Stem / Stromal Cells Promote Immunomodulatory and Proregenerative Effects in Chronic Experimental Chagas Disease.Liu J(editor)Stem Cells Int[Internet].2018;2018:9108681. Available from https: / / doi.org / 10.1155 / 2018 / 9108681 2. Chen L, Tredget EE, Wu PYG, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One [Internet]. 2008 Apr 2;3(4):e1886. Available from: http: / / www.ncbi.nlm.nih.gov / pubmed / 18382669 3. Spees JL, Gregory CA, Singh H, Tucker HA, Peister A, Lynch PJ, et al. Internalized Antigens Must Be Removed to Prepare Hypoimmunogenic Mesenchymal Stem Cells for Cell and Gene Therapy.2004;9(5):747-56. Protocol references for MSC / ASC isolation and expansion (incorporated herein by reference): 5.Toegel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C:Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.Am J Physiol Renal Physiol.2005 Jul;289(1):F31-42.Epub 2005 Feb 15 6.Togel F,Weiss K,Yang Y,Hu Z,Zhang P,Westenfelder C:Vasculotropic,paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury.Am J Physiol Renal Physiol.2007 May;292(5):F1626-35.Epub 2007 Jan 9. Protocol references for isolation of MSC / ASC-derived exosomes (incorporated herein by reference): 7.Lotvall J, Hill AF, Hochberg F et al.: Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.Journal of Extracellular Vesicles 2014,3:26913-http: / / dx.doi.org / 10.3402 / jev.v3.26913 8. Bang C, Thum T: Exosomes: New players in cell=cell communication. Int J Biochem Cell Biol 2012; 44:2060-2064. 9.Kholina S,Ranghino A,Garnieri P et al.:Extracellular vesicles as new players in angiogenesis.Vasc Pharmacol 2016;S1537-1891:30105-3-101。

Claims

1. 1. A species-specific cell culture medium comprising: Basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, Insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, bFGF of said species, PDGF-BB of said species, and EGF of said species; and serum of said species.

2. 10. The species-specific cell culture medium of claim 1, wherein said species-specific cell culture medium comprises from about 1 percent to about 20 percent by volume of said serum of said species.

3. 3. The species-specific cell culture medium of claim 2, wherein the amount of serum is about 5 percent by volume.

4. 10. The species-specific cell culture medium of claim 1, wherein the amount of serum is about 10 percent by volume.

5. 2. The species-specific cell culture medium of claim 1, wherein the insulin is an insulin of the species.

6. 1. A canine cell culture medium comprising: Basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, Insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin, canine bFGF, canine PDGF-BB, and canine EGF; A canine cell culture medium comprising: canine serum.

7. 7. The medium of claim 6, wherein the concentration of the canine serum is about 1 percent by volume.

8. 7. The medium of claim 6, wherein the concentration of the canine serum is about 5 percent by volume.

9. 7. The medium of claim 6, wherein the concentration of the canine serum is about 10 percent by volume.

10. 1. A composition comprising: The medium according to any one of claims 1 to 4, and mesenchymal stem cells of said species.

11. The composition of claim 10, wherein the species is Homo sapiens.

12. The composition of claim 10 , wherein the mesenchymal stem cells comprise adipose stromal cells.

13. The composition of claim 10, wherein the species is canine.

14. The composition of claim 10 , wherein the composition further comprises islet cells of said species.

15. 1. A composition comprising: The medium according to any one of claims 1 to 4, and islet cells of said species.

16. 1. A method for preparing a species-specific cell culture medium, comprising: Basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, providing insulin, hydrocortisone, progesterone, Asc-2-p, and holo-transferrin; providing said species of bFGF, said species of PDGF-BB, and said species of EGF; providing serum of said species.

17. 17. The cell culture medium of claim 16, wherein the amount of serum is from about 1 percent to about 20 percent by volume.

18. 17. The cell culture medium of claim 16, wherein the amount of serum is about 5 percent by volume.

19. 17. The cell culture medium of claim 16, wherein the amount of serum is about 10 percent by volume.

20. 1. A method for preparing a cell culture medium, comprising: Basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, providing insulin, hydrocortisone, progesterone, Asc-2-p, and holo-transferrin; providing canine bFGF, canine PDGF-BB, and canine EGF; providing canine serum.

21. 1. A method for culturing and growing cells, comprising:

1. A cell culture medium comprising: Basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, Insulin, hydrocortisone, progesterone, Asc-2-p, holo-transferrin; species-specific bFGF, species-specific PDGF-BB, and species-specific EGF; and providing a cell culture medium comprising a species-specific serum; providing species-specific cells for propagation; providing conditions for the cells to grow.

22. 22. The method of claim 21, wherein the cells comprise mesenchymal stromal cells.

23. 23. The method of claim 22, wherein the mesenchymal stromal cells comprise canine mesenchymal stromal cells.

24. 24. The method of claim 23, wherein the canine mesenchymal stromal cells comprise canine adipose stromal cells.

25. 23. The method of claim 22, wherein the mesenchymal stromal cells comprise human mesenchymal stromal cells.

26. 26. The method of claim 25, wherein the human mesenchymal stromal cells comprise human adipose stromal cells.

27. 22. The method of claim 21, wherein the cells comprise islet cells.

28. 28. The method of claim 27, wherein the islet cells comprise canine islet cells.

29. 28. The method of claim 27, wherein the islet cells comprise human islet cells.

30. 1. A method for producing exosomes, said method comprising: expanding the cells of the species in culture, wherein the culture medium comprises: Basal medium, sodium bicarbonate, HEPES, L-glutamine, L-alanine, providing insulin, hydrocortisone, progesterone, Asc-2-p, and holo-transferrin; providing a bFGF, a species-specific PDGF-BB, and a species-specific EGF of said species; providing a species-specific serum; and collecting exosomes released by said cells in culture.

31. 31. The method of claim 30, wherein the cells are mesenchymal cells.

32. 31. The method of claim 30, wherein the cells are islet cells.

33. 31. The method of claim 30, further comprising treating a subject of said species with said collected exosomes.

34. 32. The method of claim 31, wherein the mesenchymal cells and / or islet stem cells are allogeneic to the subject.

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