Universal media systems for cell culture and methods of using the same

Abstract

Provided are cell culture media, additive compositions and supplements for cell culture media, and cell culture plate coatings. Methods of growing cells using the media, supplements, and plate coatings are also provided. Further provided are methods of determining optimal cell growth conditions, which may include methods of selecting a cell growth media, cell culture plate coating, and / or concentration of cell culture medium additives.

Description

UNIVERSAL MEDIA SYSTEMS FOR CELL CULTUREAND METHODS OF USING THE SAMESTATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 660,794, filed June 17, 2024, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under U.S. Army Medical Research and Materiel Command (USAMRMC) prototype Other Transaction Agreement W81XWH-15-9- 0001, awarded to Advanced Technology International ATI as the Consortium Manager of the Medical Technology Enterprise Consortium (MTEC). The U.S. Government has certain rights in this invention.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in XML format, entitled 9865-239-WO_ST26.xml, 9,805 in size, generated on June 16, 2025, and filed herewith, is hereby incorporated by reference in its entirety for its disclosures.FIELD

[0004] The present invention relates to cell culture media, cell culture media additive compositions and supplements, cell culture plate coatings, and methods of growing cells using the same. The present invention also relates to methods of determining optimal cell growth conditions.BACKGROUND

[0005] Experiments performed using cells collected from human primary tissues have assisted in disease modeling for decades dating back to the 1950s. Two-dimensional, single-cell culture later evolved into a more advanced method, paving the way to three-dimensional (3D) cell culture technologies that may more accurately represent the biological and cellular in vivo environment. This advancement has enabled cells to grow and interact within their surroundings such as that represented in spheroid development and tissue engineering. More recently, the field has migratedto even more complex designs involving numerous primary cell types, bioreactors and multilayer printed tissue constructs seeded with multiple human cell types collected from a patient or donor.

[0006] Cell culture media, however, has been a limitation due to the fact that the field has maintained the view that there is one optimized medium for each cell type rather than one medium that can sustain viability and proliferation across multiple cell types. While a single medium per cell type may be sufficient for in-lab cell culture, the desire to produce more complex human tissue appears to require the use of multiple media or accept compromise in the viability and / or performance of one of the cell types found in the multicell culture.

[0007] One aspect that has been hindering commercial media development is the common use of animal derived supplements such as fetal bovine serum (FBS), fetal calf serum (FCS), and bovine pituitary extract (BPE). Animal serum is a very beneficial component in media composition because it provides growth factors, macromolecules, carrier proteins, attachment factors, low molecular weight nutrients, and hormones. However, it is also a supplement comprised of complex and unknown components derived from different sources and containing a wide range of possible contaminants and pathogens, which can affect cell physiology, cellular metabolism, protein production, proliferation, and cell health. These contaminants could be a source of cellular reprogramming and differentiation in culture. Without proper screening, animal -derived additives could cause contamination within cells or tissues being developed for human transplant. Current methods to harvest animal derived supplements also are subject to lot-to-lot variations depending on the herd, and these differences impact the media quality and can introduce safety concerns.

[0008] Therefore, it would be beneficial to develop media that is chemically defined and xeno- free that has similar or improved growth support of primary cells, using standardized and reproducibly sourced supplements in the form of recombinant proteins, small molecules, etc., and chemically defined extracellular matrix, without the negative effects that using animal -derived serum provides. It would also be beneficial to determine optimal cell growth conditions so as to provide improved growth, differentiation, and / or survival over multiple passages for specific cell lines, cell types, or tissue types.SUMMARY

[0009] Provided herein according to some aspects of the present invention is a "universal" cell culture medium useful for culturing multiple primary cell types, said cell culture medium comprising: a) a basal medium (e.g., DMEM / F12, KSFM); b) a neuronal and / or stem cell growth supplement (e.g., a neuronal growth supplement such as xeno free B-27); c) non-essential aminoacids (NEAA) (e.g., 2, 3, 4, 5 or more of glycine, L-alanine, L-asparagine, L-aspartic acid, L- glutamic acid, L-proline, and L-serine) (e.g., present in the cell culture medium in a concentration of about 0.02 to about 0.5 mM); d) an antioxidant and / or essential vitamin (e.g., ascorbic acid, a tocopherol (e.g., a-tocopherol, P-tocopherol, y-tocopherol, and / or 5-tocopherol), glutathione, and / or N-acetyl-L-cysteine); e) epinephrine; f) a corticosteroid (e.g., hydrocortisone) and / or a glucocorticoid; and g) epidermal growth factor (EGF).

[0010] In some embodiments, the neuronal and / or stem cell growth supplement is present in a concentration from about 0.01% to about 1% by volume of the cell culture medium; the ascorbic acid is present in a concentration from about 0.08 to about 80 pg / mL; the epinephrine is present in a concentration from about 0.002 to about 20 pg / mL; the hydrocortisone is present in a concentration from about 0.01 to about 100 pg / mL; and the EGF is present in a concentration from about 0.2 to about 250 ng / mL.

[0011] In some embodiments, the cell culture medium is free of serum and / or animal products.

[0012] In some embodiments, the basal medium is Dulbecco's Modified Eagle Medium with Nutrient Mixture F 12 (DMEM / F12) or defined keratinocyte serum-free (KSF) medium.

[0013] In some embodiments, the cell culture medium further comprises: h) hepatocyte growth factor (HGF); i) platelet- derived growth factor (PDGF) (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); and j) fibroblast growth factor (FGF) (e.g., FGF-basic). In some embodiments, the HGF is present in a concentration from about 0.01 to about 100 ng / mL; the PDGF is present in a concentration from about 0.2 to about 25 ng / mL; and the FGF-basic is present in a concentration from about 0.2 to about 250 ng / mL.

[0014] In some embodiments, the cell culture medium further comprises human platelet lysate (PLT). In some embodiments, the PLT is devoid of heparin. In some embodiments, the PLT is present in a concentration from about 0.5% to about 10% by volume of the cell culture medium.

[0015] In some embodiments, the cell culture medium further comprises: k) vascular endothelial growth factor (VEGF) (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); 1) insulin-like growth factor-1 (IGF-1); and m) fibroblast growth factor-acidic (FGF-acidic). In some embodiments, the VEGF is present in a concentration from about 0.2 to about 500 ng / mL; the IGF- 1 is present in a concentration from about 0.2 to about 250 ng / mL; and the FGF-acidic is present in a concentration from about 0.2 to about 250 ng / mL.

[0016] In some embodiments, the cell culture medium further comprises: h) prolactin; i) prostaglandin El; and j) retinoic acid. In some embodiments, the prolactin is present in a concentration from about 0.2 to about 250 ng / mL; the prostaglandin El is present in aconcentration from about 0.025 to about 25 nmol / mL; and the retinoic acid is present in a concentration from about 0.01 to about 100 ng / mL.

[0017] In some embodiments, the cell culture medium further comprises: h) PDGF (e.g., PDGF- aa, PDGF-bb, PDGF-ab, or any combination thereof); i) FGF -basic; j) prolactin; k) prostaglandin El; 1) retinoic acid; m) VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); n) IGF-1; o) FGF-acidic; and p) optionally, heparin. In some embodiments, the PDGF is present in a concentration from about 0.2 to about 25 ng / mL; the FGF-basic is present in a concentration from about 0.2 to about 250 ng / mL; the prolactin is present in a concentration from about 0.2 to about 250 ng / mL; the prostaglandin El is present in a concentration from about 0.025 to about 25 nmol / mL; the retinoic acid is present in a concentration from about 0.01 to about 100 ng / mL; the VEGF is present in a concentration from about 0.2 to about 500 ng / mL; the IGF-1 is present in a concentration from about 0.2 to about 250 ng / mL; the FGF-acidic is present in a concentration from about 0.2 to about 250 ng / mL; and the heparin is present in a concentration from about 0.1 to about 5 units / mL.

[0018] Another aspect of the present invention is a cell culture plate coating comprising: serum or a serum replacement product; fibronectin; laminin (e.g., laminin-521); collagen (e.g., Type I collagen, Type II collagen, Type IV collagen, or any combination thereof); and tropoelastin, said coating optionally provided in an aqueous solution (e.g. comprising saline such as phosphate buffered saline (PBS) or other calcium-free and magnesium-free salt solution, and / or comprising alcohol such as ethanol or methanol) for application to the cell culture plate.

[0019] In some embodiments, the saline is present in a concentration from about 25% to about 75% by volume of the cell culture plate coating; the serum or serum replacement product is present in a concentration from about 25% to about 75% by volume of the cell culture plate coating; the fibronectin is present in a concentration from about 0.05 to about 2500 pg / mL; the laminin is present in a concentration from about 0.05 to about 2500 pg / mL; the Type I collagen is present in a concentration from about 0.05 to about 2500 pg / mL; the Type IV collagen is present in a concentration from about 0.05 to about 2500 pg / mL; and the tropoelastin is present in a concentration from about 0.05 to about 2500 pg / mL.

[0020] In some embodiments, the serum replacement product is knockout serum (e.g., Cell Therapy System (CTS™) KnockOut Serum Replacement (SR™)), engineered serum, and / or human platelet extract.

[0021] In some embodiments, the cell culture plate coating is free of serum and / or animal products.

[0022] Another aspect of the present invention is a method of culturing a mesoderm lineage primary cell, said method comprising incubating said cell with a cell culture medium of the present invention in a cell culture dish, flask, well, and / or plate. In some embodiments, the method further comprises coating the cell culture dish, flask, well, and / or plate with the cell culture plate coating of the present invention.

[0023] Another aspect of the present invention is a method of culturing an ectoderm lineage primary cell, said method comprising incubating said cell with a cell culture medium of the present invention in a cell culture dish, flask, well, and / or plate. In some embodiments, the method further comprises coating the cell culture dish, flask, well, and / or plate with the cell culture plate coating of the present invention.

[0024] Another aspect of the present invention is a method of culturing an endoderm lineage primary cell, said method comprising incubating said cell with a cell culture medium of the present invention in a cell culture dish, flask, well, and / or plate. In some embodiments, the method further comprises coating the cell culture dish, flask, well, and / or plate with the cell culture plate coating of the present invention.

[0025] Another aspect of the present invention is a method of culturing a tissue construct comprising cell types from multiple lineages (e.g., from two or more of a mesoderm, ectoderm and endoderm lineage), said method comprising incubating said tissue construct with a cell culture medium of the present invention in a cell culture dish, flask, well, and / or plate. In some embodiments, the method further comprises coating the cell culture dish, flask, well, and / or plate with the cell culture plate coating of the present invention.

[0026] Another aspect of the present invention is a method of culturing a 3D organoid comprising multiple primary cell types, said method comprising incubating said 3D organoid with a cell culture medium of the present invention in a cell culture dish, flask, well, and / or plate. In some embodiments, the method further comprises coating the cell culture dish, flask, well, and / or plate with the cell culture plate coating of the present invention.

[0027] Another aspect of the present invention is a method of measuring cell growth, said method comprising obtaining cells (e.g., primary cells) from a subject; incubating said cells with a cell culture medium (e.g., the cell culture medium of the invention) in a cell culture dish, flask, well, and / or plate for a predetermined amount of time; and counting the cell number after said amount of time thereby measuring the cell growth. In some embodiments, the method further comprises coating the cell culture dish, flask, well, and / or plate with the cell culture plate coating of the present invention.

[0028] Another aspect of the present invention is a method of selecting an optimal cell growth media, said method comprising measuring cell growth and / or other parameters according to a method of the present invention; repeating the measuring using a different cell culture medium and / or cell culture plate coating; and selecting the cell culture medium and / or the cell culture plate coating that provides a desired outcome (e.g., growth rate for the cells, proliferation rate, cell number, cell health, population doublings per day, etc.); thereby selecting the optimal cell growth media and or cell culture plate coating.

[0029] In some embodiments, the repeating step is carried out by providing a first array of different cell culture media and / or different cell culture plate coatings. In some embodiments, the method further comprises providing a second array comprising different concentrations of cell culture medium additives. In some embodiments, the method further comprises providing a third array comprising different passages of the cells.

[0030] Further provided is a cell culture medium additive composition, which additive composition (optionally in combination with one or more supplements as taught herein) can be added to a basal medium to produce a cell culture medium as taught herein. For example, the composition may comprise: a neuronal and / or stem cell growth supplement (e.g., B-27); NEAA; an antioxidant and / or essential vitamin (e.g., ascorbic acid, a tocopherol (e.g., a-tocopherol, P- tocopherol, y-tocopherol, and / or 5-tocopherol), glutathione, and / or N-acetyl-L-cysteine); epinephrine; a corticosteroid (e.g., hydrocortisone) and / or a glucocorticoid; and EGF.

[0031] In some embodiments, the additive composition includes ascorbic acid, and the neuronal and / or stem cell growth supplement is present in the composition in a concentration from about 25% to about 75% by volume; the epinephrine is present in the composition in a weight ratio relative to the ascorbic acid from about 1 :50 to about 1 :500 (epinephrine : ascorbic acid); the hydrocortisone is present in the composition in a weight ratio relative to the ascorbic acid from about 1 :5 to about 1 : 100 (hydrocortisone : ascorbic acid); and / or the EGF is present in the composition in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 100,000 (EGF : ascorbic acid).

[0032] Further provided is a supplement useful for the cell culture medium additive composition, wherein the supplement comprises: HGF; PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); and FGF (e.g., FGF-basic). In some embodiments, the HGF is present in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 10,000 (HGF : ascorbic acid); the PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof) is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (PDGF : ascorbicacid); and the FGF -basic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF-basic : ascorbic acid).

[0033] In some embodiments, the supplement further comprises PLT. In some embodiments, the PLT is devoid of heparin. In some embodiments, the PLT is present in a concentration from about 20% to about 60% by volume of the additive composition.

[0034] Further provided is a supplement useful for the cell culture medium additive composition, comprising: VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); IGF-1; and FGF-acidic. In some embodiments, the VEGF is present in a weight ratio relative to the ascorbic acid from about 1 : 10,000 to about 1 : 150,000 (VEGF : ascorbic acid); the IGF-1 is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1:5,000 (IGF-1 : ascorbic acid); and the FGF-acidic is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 :5,000 (FGF-acidic : ascorbic acid).

[0035] Further provided is a supplement useful for the cell culture medium additive composition, comprising: prolactin; prostaglandin El; and retinoic acid. In some embodiments, the prolactin is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (prolactin : ascorbic acid); the prostaglandin El is present in a weight ratio relative to the ascorbic acid from about 1 :5,000,000 to about 1 :50,000,000 (prostaglandin El : ascorbic acid); and the retinoic acid is present in a weight ratio relative to the ascorbic acid from about 1 : 100,000 to about 1 :800,000 (retinoic acid : ascorbic acid).

[0036] Also provided is supplement useful for the cell culture medium additive composition, comprising: HGF; PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); FGF- basic; prolactin; prostaglandin El; retinoic acid; VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); IGF-1; FGF-acidic; and optionally, heparin. In some embodiments, the additive composition comprises ascorbic acid, and the HGF is present in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 10,000 (HGF : ascorbic acid); the PDGF (is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (PDGF : ascorbic acid); the FGF-basic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF-basic : ascorbic acid); the prolactin is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (prolactin : ascorbic acid); the prostaglandin El is present in a weight ratio relative to the ascorbic acid from about 1 :5,000,000 to about 1 :50,000,000 (prostaglandin El : ascorbic acid); the retinoic acid is present in a weight ratio relative to the ascorbic acid from about 1 : 100,000 to about 1 : 800,000 (retinoic acid : ascorbic acid); the VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof) is presentin a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (VEGF : ascorbic acid); the IGF-1 is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 : 10,000 (IGF-1 : ascorbic acid); the FGF-acidic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF-acidic : ascorbic acid); and / or the heparin is present in a concentration from about 0.1 to about 5 units / mL.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1A is an image showing the proliferation of bone marrow mesenchymal stem cells (BM-MSC) grown in UM1 and on UC3 for three days imaged by the Incucyte® S3 Analysis System and analyzed using the assisted machine learning software to create and apply a masking of the image.

[0038] FIG. IB is an image showing the proliferation of BM-MSC imaged by the Incucyte® S3 Analysis System with the aforementioned masking applied as an orange mask overlaying the cells; the masking area is then quantified to produce the % Phase Confluence (%PC) for that time point.

[0039] FIG. 2A is an image showing the proliferation of BM-MSC grown in UM1 with KnockOut Serum (KOS) Replacement (Thermo) and on UC3 for three days imaged by the Incucyte® S3 Analysis System and analyzed using the Cell-by-Cell software module to segment the phase confluence and count the objects that have been segmented per image.

[0040] FIG. 2B is an image showing the proliferation of BM-MSC imaged by the Incucyte® S3 Analysis System with the objects within the segmentation then quantified to produce the Phase Object Count Per Image (POC) for that time point.

[0041] FIG. 3A is a graph showing the proliferation of BM-MSC grown in various conditions for three days imaged by the Incucyte® S3 Analysis System quantified to produce the %PC for that time point.

[0042] FIG. 3B is a graph showing the proliferation of BM-MSC grown in various conditions for three days imaged by the Incucyte® S3 Analysis System quantified to produce the POC for that time point.

[0043] FIG. 4 is a schematic representation of a 96-well plate showing an example arrangement of a first array testing different combinations of basal media, supplements and / or small molecules (Additives), and cell culture plate coatings (Substrates or "Sub") as a primary cell growth screen. UM1, UM2, and UM3 indicates universal media formulations as described herein in some embodiments; SM indicates small molecule additives as described herein in some embodiments; UC indicates uncoated wells.

[0044] FIG. 5 is a schematic representation of a 96-well plate showing an example arrangement of a second array testing different concentrations of media additives (small molecules and / or growth supplements) and cell culture plate coatings / substrates (ECM) in designated sections of the plate as a secondary cell growth screen.

[0045] FIG. 6 is a schematic representation of a 96-well plate showing an example arrangement of a third array for comparing cell growth across different numbers of cell passages of selected combinations of basal media, supplements, small molecules, media additives, and growth substrates from the secondary growth screen (Sections 1-8) as a tertiary cell growth screen.

[0046] FIG. 7. is a graph showing the proliferation of normal human dermal fibroblasts (NHDF) grown in various conditions for 5 days imaged by the Incucyte® S3 Analysis System quantified to produce the %PC for that time point.

[0047] FIG. 8. is a graph showing the proliferation of bronchial smooth muscle cells (Brone SM) grown in various conditions for 5 days imaged by the Incucyte® S3 Analysis System quantified to produce the %PC for that time point.

[0048] FIG. 9. is a graph showing proliferation of adipose tissue mesenchymal stem cells (AT- MSC) grown in various conditions for 5 days imaged by the Incucyte® S3 Analysis System quantified to produce the %PC for that time point.DETAILED DESCRIPTION

[0049] Embodiments of the present invention are now described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

[0050] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0051] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaningin the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0052] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0053] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0054] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

[0055] Some embodiments are described herein with reference to the accompanying drawings, in which illustrative embodiments are shown. In the drawings, the relative sizes of regions or features may be exaggerated for clarity. This technology may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the technology to those skilled in the art.

[0056] It will be understood that, although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers, sections and / or arrays these elements, components, regions, layers, sections and / or arrays should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, section or array from another region, layer, section or array. Thus, a first element, component, region, layer, section or array discussed herein could be termed a second element, component, region, layer, section or array without departing from the teachings of the present disclosure.

[0057] Media, supplements, additives, coatings, or methods as taught herein may comprise, consists of, or consist essentially or the listed components or a subset thereof. As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel character! stic(s) of the claimed invention.

[0058] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X.

[0059] A range provided herein for a measurable value includes any range and / or individual value therein, which variations are encompassed by the instant disclosure. Recitation of ranges of values are intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0060] A "medium" or "media," sometimes referred to as a "culture medium" or "culture media," as used herein, refers to an aqueous based solution that is provided for the growth, viability, and / or maintenance of cells as described in the present invention. Components for the media or culture media may be natural or artificial. A medium or culture medium may include a base media and may be supplemented with nutrients and / or other components (e.g., one or more salts, amino acids, vitamins, trace elements, antioxidants, nucleobases, nucleosides, polyamines) to promote the desired cellular activity, such as cell viability, growth, proliferation, morphology, and / or differentiation of the cells cultured in the media.

[0061] A "base media," or "basal media" as used herein, refers to a basal salt nutrient or an aqueous solution of salts, amino acids, vitamins, buffers and other elements that provides cells with water and inorganic ions that are needed for normal cell metabolism and maintaining intracellular and / or extracellular osmotic balance. In some embodiments, a base media may include a buffering system to maintain the medium within a physiological pH range of about 7 to about 8, or a pH range of about 7.2 to about 7.8, or a pH of about 7.3 to about 7.5. Known base media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), DMEM with Nutrient Mixture F12 (DMEM / F12), defined Keratinocyte Serum -Free (KSF) medium, Minimum Essential Medium (MEM), Medium 200 (also known as Human Large Vessel Endothelial Cell Basal Medium), and CTS™ KnockOut SR™.

[0062] In some embodiments, a base media is supplemented with nutrients and / or other components (i.e., additives) such as growth factors, antimicrobial agents, etc., to provide a more complete media to support cell culture.

[0063] In some embodiments, the medium (e.g., base medium and / or complete medium) is free of serum and / or animal products (e.g., free of bovine albumin and / or other animal -derived proteins).

[0064] As used herein, "growth factors" include molecules that promote the regeneration, growth and survival of cells or tissue. Examples of growth factors include, but are not limited to, fibroblast growth factor (FGF) (e.g., FGF-basic and / or FGF-acidic), epidermal growth factor (EGF), insulinlike growth factor 1 (IGF-1), platelet-derived growth factor (PDGF) (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof). In some embodiments, the FGF-basic is present in the media in a concentration from about 0.2 to about 250 ng / mL (e.g., about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or about 250 ng / mL). In some embodiments, the FGF-acidic is present in the media in a concentration from about 0.2 to about 250 ng / mL (e.g., about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or about 250 ng / mL). In some embodiments, the EGF is present in the media in a concentration from about 0.2 to about 250 ng / mL (e.g., about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or about 250 ng / mL). In some embodiments, the PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof) is present in the media in a concentration from about 0.2 to about 25 ng / mL (e.g., about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 ng / mL). In some embodiments, the HGF is present in the media in a concentration from about 0.01 to about 100 ng / mL (e.g., about 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or about 100 ng / mL). In some embodiments, the VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof) is present in the media in a concentration from about 0.2 to about 500 ng / mL (e.g., about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or about 500 ng / mL). In some embodiments, the IGF-1 is present in the media in a concentration from about 0.2 to about 250 ng / mL (e.g., about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or about 250 ng / mL). In some embodiments, the FGF-acidic is present in the media in a concentration from about 0.2 to about 250 ng / mL (e.g., about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or about 250 ng / mL).

[0065] Examples of components (i.e., additives) that may be included to form a more complete media include, but are not limited to, amino acids, proteins, vitamins, inorganic salts, buffers,hormones, steroids, antioxidants, antibiotics, and other additives such as peptides and small molecules.

[0066] " Amino acids" include, but are not limited to, for example, Glycine, L-Alanine, L-arginine, L-Asparagine, L-Aspartic acid, L-Cysteine, L-Glutamic acid, L-Glutamine, L-Histidine, L- Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L- Threonine, L-Tryptophan, L-Tyrosine, and L-Valine. In some embodiments, the amino acids comprise non-essential amino acids (NEAA) (e.g., 2, 3, 4, 5 or more of glycine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic, L-proline, and L-serine). In some embodiments, each amino acid in the NEAA may be independently provided in the media in a concentration from about 0.02 to about 0.5 mM (e.g., from about 0.02, 0.03, 0.04, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, to about 0.5 mM).

[0067] " Vitamins" include, but are not limited to, for example, vitamin B12, d-biotin, choline chloride, D-pantothenic acid, D-calcium pantothenate, folinic acid, myo-inositol, niacinamide, pyridoxal hydrochloride, and thiamine hydrochloride.

[0068] "Inorganic salts" include, but are not limited to, ammonium metavanadate, ammonium molybdate, calcium chloride, cupric sulfate, ferric sulfate, magnesium sulfate, magnesium chloride, manganese sulfate, nickelous chloride, potassium phosphate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate, sodium meta silicate, sodium selenite, tin chloride, and zinc sulfate.

[0069] " Buffers" may be used in cell culture to maintain the pH of the media in the presence of about 4-10% carbon dioxide. Buffers include, for example, HEPES, sodium bicarbonate, MOPS, MES, tri cine, phosphate buffered saline (PBS), etc.

[0070] " Steroids" include, but are not limited to, progesterone, hydrocortisone, corticosterone, etc.

[0071] " Hormones" include, but are not limited to, triiodo-L-thyronine (e.g., T3), epinephrine, norepinephrine, prolactin, etc. In some embodiments, the epinephrine is present in the media in a concentration from about 0.002 to about 20 pg / mL (e.g., about 0.002, 0.004, 0.006, 0.008, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 pg / mL). In some embodiments, the hydrocortisone is present in the media in a concentration from about 0.01 to about 100 ng / mL (e.g., about 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or about 100 ng / mL). In some embodiments, the prolactin is present in the media in a concentration from about 0.2 to about 250 ng / mL (e.g., about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or about 250 ng / mL).

[0072] " Proteins" that may be used include, but are not limited to, for example, holo-transferrin, insulin, growth factors, etc. In some embodiments, proteins are human proteins (e.g., hEGF). In some embodiments, proteins are recombinant proteins such as recombinant human (rh) proteins (e.g., rh EGF).

[0073] As used herein, the term "antioxidant" refers to those compounds capable of scavenging free radicals (e.g., reactive oxygen species (ROS) or free oxygen radicals such as superoxide and hydroxyl radicals), and those compounds that inactivate antioxidant enzymes (e.g., superoxide dismutase [SOD], catalase) under physiological conditions. Antioxidants include naturally- occurring as well as synthetic antioxidants, and further encompass both water-soluble and lipid- soluble antioxidants. Antioxidants include, but are not limited to, the tocopherols (especially a- tocopherol, or vitamin E), ascorbic acid (vitamin C), ascorbyl palmitate, vitamin A and the carotenes (including beta-carotene), lipoic acid, glutathione and its precursors, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), isopropyl citrate, riboflavin, manganese, selenium (e.g., sodium selenite), zinc, uric acid, bilirubin, the isoflavones, flavonoids, lycopene, and ubiquinol; and the chemical derivatives, analogs, and precursors thereof. In some embodiments, the antioxidant ascorbic acid is present in the media in a concentration from about 0.08 to about 80 pg / mL (e.g., about 0.08, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or about 80 pg / mL).

[0074] " Antibiotics" may be used in cell culture media for preventing contamination, maintaining aseptic conditions, and / or selecting for cells containing genetic modifications. Antibiotics include, but are not limited to, for example, penicillin, streptomycin, Amphotericin B, gentamicin, puromycin, etc.

[0075] Other media additives may include, but are not limited to, a nucleobase or a nucleoside (e.g., adenine, thymidine), nucleoside precursors and / or derivatives (e.g., hypoxanthine), sodium pyruvate, a polyamine (e.g., putrescine), albumin (e.g., human albumin), an anticoagulant (e.g., heparin), fatty acids (e.g., linoleic acid and linolenic acid), a sugar (e.g., galactose), an ethanolamine, a prostaglandin (e.g., prostaglandin El), retinoic acid, platelet lysate (PLT) (e.g., human platelet lysate such as PLT Gold®), etc.

[0076] In some embodiments, the media additive comprises one or more small molecules. In some embodiments, the small molecule is a protein inhibitor, such as a Rho-associated protein kinase inhibitor (ROCKi). In some embodiments, the ROCKi inhibits the biological function of ROCK1 (pl 60 ROCK) and / or ROCK2. In some embodiments, the ROCKi inhibits the biological functionof protein kinase A (PKA), protein kinase 2 (PRK2), protein kinase C (PKC), and / or myosin lightchain kinase (MLCK).

[0077] In some embodiments, the PLT is present in the media in a concentration from about 0.5% to about 10% by volume of the media (e.g., about 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or about 10%). In some embodiments, the prostaglandin (e.g., prostaglandin El) is present in the media in a concentration from about 0.005 to about 25 nmol / mL (e.g., about 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, or about 25 nmol / mL) or from about 0.0005 to about 10 ng / mL (e.g., about 0.0005, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or about 10 ng / mL). In some embodiments, the retinoic acid is present in the media in a concentration from about 0.01 to about 100 ng / mL (e.g., about 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or about 100 ng / mL). In some embodiments, the heparin is present in a concentration from about 0.1 to about 5 units / mL (e.g., about 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or about 5 units / mL).

[0078] The media may also include growth supplements specific to one or more cell types, including neuronal cell growth supplements (e.g., B-27 and derivatives), stem cell growth supplements (e.g., Mesenchymal Stem Cell Growth Supplement), fibroblast growth supplements (e.g., FibroLife®), epithelial growth supplements (e.g., mammary epithelial growth supplement), endothelial growth supplements (e.g., endothelial cell growth supplement from bovine neural tissue), etc. In some embodiments, the growth supplement is B-27 (e.g., xeno free B-27). See, e.g., Liu et al. (2017) Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition. PLoS ONE 12(2): e0172677; Chen et al. (2008) NS21 : re-defined and modified supplement B27 for neuronal cultures. J Neurosci Methods 171(2):239-47. In some embodiments, the growth supplement comprises Biotin, D,L alpha-tocopherol acetate, D,L alphatocopherol, vitamin A (acetate), vitamin A, catalase, insulin, holo transferrin, SOD, D-galactose, ethanolamine, reduced glutathione, L-carnitine, linoleic acid, linolenic acid, putrescine, sodium selenite, T3, progesterone, corticosterone, or any combination thereof.

[0079] The terms "coating", "plate coating", "cell culture plate coating", and "growth substrate" are used interchangeably herein to refer to compositions that comprise extracellular matrix (ECM) components provided to improve the adherence to the cell culture vessel, growth, viability, and / ormaintenance of cells as described in the present invention. In some embodiments, the plate coating may be added to a cell culture vessel prior to a cell culture media and / or cells. In some embodiments, the plate coating may be added to a cell culture vessel concurrently with a cell culture media and / or cells. A plate coating of the present invention may comprise said ECM components provided in an aqueous based solution (optionally including alcohol such as ethanol or methanol) for performing the coating, a hydrogel, or in a lyophilized powder. The plate coating may be supplemented with nutrients or other components (e.g., one or more salts, amino acids, vitamins, trace elements, antioxidants, nucleobases, nucleosides, polyamines, growth factors, peptides, glycosaminoglycans (gag)), as described above, to improve the adherence to the cell culture vessel, growth, viability, and / or maintenance of cells.

[0080] In some embodiments, the plate coating is provided in an aqueous based solution comprising a saline (e.g., PBS) and / or a basal cell culture media as described above. In some embodiments, the aqueous based solution may comprise the saline and basal cell culture media in a ratio of about 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, or about 1 :5, e.g., the saline may comprise from about 20% to about 80% by volume of the plate coating (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or about 80% by volume of the plate coating) and the basal cell culture media may comprise from about 20% to about 80% by volume of the plate coating (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or about 80% by volume of the plate coating).

[0081] In some embodiments, the ECM components comprise vitronectin, supervillin, neural cell adhesion molecules, fibronectin, laminin collagen (e.g., type I collage, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, and / or type XXVIII collagen), tropoelastin, elastin, integrin, fibrillin, tenascin, proteoglycans (e.g., glycosaminoglycans), polysaccharides (e.g., hyaluronic acid), synthetic peptides, poly-L-lysine (PLL), gelatin, cyclic arginylglycylaspartic acid (cyclic RGD), or any combination thereof. In some embodiments, the plate coating comprises fibronectin in a concentration from about 0.5 to about 10 pg / mL (e.g., from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 pg / mL). In some embodiments, the plate coating comprises laminin in a concentration from about 0.5 to about 10 pg / mL (e.g., from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 pg / mL). In some embodiments, the plate coating comprises Type I collagen in a concentration from about 0.5 to about 10 pg / mL (e.g., from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 pg / mL). In some embodiments, the plate coating comprises Type IV collagen in a concentration from about 0.5 to about 10 pg / mL (e.g., from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 pg / mL). In some embodiments, the plate coating comprises tropoelastin in a concentration from about 0.5 to about 10 pg / mL (e.g., from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 pg / mL). In some embodiments, the plate coating comprises PLL in a concentration from about 1 to about 20 pg / mL (e.g., from about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pg / mL). In some embodiments, the plate coating comprises gelatin in a range from about 0.01 to about 50% by weight of the coating (e.g., from about 0.05 or 0.1 to about 1 or 3%, or from about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, to about 50% by weight of the coating). In some embodiments, the plate coating comprises cyclic RGD in a concentration from about 0.05 to about 50 pg / mL (e.g., from about 0.05 or 0.1 to about 10, 15 or 20 pg / mL, or from about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 pg / mL).

[0082] In some embodiments, the ECM components comprise one or more polypeptide sequences comprising the amino acid sequence of KGRGDS (SEQ ID NO: 1), ADSQLH4GGLRS (SEQ ID NO:2), GKKQRFRHRNRKG (SEQ ID NO:3), GWQPPRARI (SEQ ID NO:4), KPHSRN (SEQ ID NO:5), GGGEVYVVAENQQGKSKA (SEQ ID NO:6), MHRMPSFLPTTL (SEQ ID NO:7), FHRRIKA (SEQ ID NO:8), MHRMPSFLPTTL (SEQ ID NOV), and / or GSDPGYIGSR (SEQ ID NO: 10).

[0083] " Cell" or "cells" as used herein are, in general, mammalian cells, such as dog, cat, cow, goat, horse, sheep, mouse, rabbit, rat, etc. cells. In some preferred embodiments the cells are human cells. "Isolated" as used herein signifies that the cells are placed into conditions other than their natural environment. Tissue or cells are "harvested" when initially isolated from living tissues, e.g., a biopsy, blood, bone marrow, etc., typically using enzymatic and / or mechanical methods to isolate the cells, and such cells and their progeny maintaining differentiation state are called "primary" cells. Primary cells or harvested cells may also be called "donor cells" herein. Cells are "autologous" with respect to a subject (e.g., a subject to which the cells may be administered for treatment) when initially isolated from the subject or a genetically identical donor. For example,autologous cells may be extracted from a patient and returned back to the same, genetically identical patient.

[0084] In some embodiments, a primary cell may be from a mesoderm lineage, such as renal epithelial cells, dermal fibroblasts, mesenchymal stem cells (MSC), bone marrow MSC (BM- MSC), adipose derived stem cell (AD-MSC), placental derived stem cell (PSC), skeletal muscle cells (e.g., skeletal muscle stem cells), chondrocytes, cardiac myocytes, osteoblasts, microvascular endothelial cells (MEC), umbilical vein endothelial cells (UVEC), bronchial smooth muscle cells (BSMC), etc.

[0085] In some embodiments, a primary cell may be from an ectoderm lineage such as mammary epithelial cells (MEC), follicle dermal papilla cells (FDPC-Hair), normal epidermal melanocytes (NEM), NEM-juvenile, normal epidermal keratinocytes (NEK), NEK-epidermis, astrocytes, etc.

[0086] In some embodiments, a primary cell may be from an endoderm lineage such as prostate epithelial cells, colonic epithelial cells, mammary epithelial cells, bladder epithelial cells, small airway epithelial cells, etc.

[0087] In some embodiments, the primary cells are human primary cells.

[0088] In some embodiments, harvested cells are disaggregated by enzymatic digestion before the primary culture, which may include incubation with a protease such as trypsin or trypLE; dispase, Dispase II, or BP Protease; a collagenase such as Collagenase HA, or a combination thereof.

[0089] " Cell culture" or "culture" is the growth or proliferation of cells in vitro. The "primary culture" is the first culture to become established after seeding disaggregated cells or primary explants into a culture vessel. "Expanding" or "expansion" as used herein refers to an increase in number of viable cells. Expanding may be accomplished by, e.g., "growing" the cells through one or more cell cycles, wherein at least a portion of the cells divide to produce additional cells. "Growing" as used herein includes the culture of cells such that the cells remain viable, and may or may not include expansion and / or differentiation of the cells. In some embodiments, the cell culture may comprise more than one differentiated cell type (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 differentiated cell types). In some embodiments, a cell is cultured in a cell culture media of the present invention, with use of a plate coating of the present invention, or a combination thereof.

[0090] " Three-dimensional tissue construct," "organoid," and "three-dimensional organoid" (3D organoid) refer to a composition of live cells, typically in a carrier media, arranged in a three- dimensional or multi-layered configuration (as opposed to a monolayer). Suitable carrier media include hydrogels, such as cross-linked hydrogels. Such constructs may comprise one differentiated cell type, or two or more differentiated cell types, depending upon the particulartissue or organ or system being modeled or emulated. In some embodiments, cells are mixed together with the extracellular matrix, or cross-linked matrix, to form the construct, while in other embodiments cell aggregates such as spheroids or organoids may be pre-formed and then combined with the extracellular matrix. In some embodiments, the tissue construct and / or organoid comprises cells that are human-derived cells, and, in some embodiments, the tissue construct and / or organoid comprises cells that consist of human-derived cells. In some embodiments, an organoid uses a cell culture media of the present invention, a plate coating of the present invention, or a combination thereof.

[0091] "Passaged in vitro" or "passaged" refers to the transfer or subculture of a cell culture to a second culture vessel, usually implying mechanical or enzymatic disaggregation, reseeding, and often division into two or more daughter cultures, depending upon the rate of proliferation. A cell culture may be referred to as the number of times it has been passaged (e.g., Pl for one passage). In some embodiments, the cells are passaged from 1 to 9 times, such as from 2 or 3 to 4, 5, 6 or 7 passages. If a cell population is selected for a particular genotype or phenotype, then the culture becomes a "cell strain" upon subculture, z.e., the culture is homogeneous and possesses desirable characteristics (e.g., the ability to express a certain protein or marker).

[0092] A cell culture vessel useful to the present invention includes, but is not limited to, a cell culture dish, cell culture plate, cell culture tube, cell culture flask, bioreactor, etc.

[0093] Cells may be grown, cultured, or passaged in a media and / or with a plate coating of the present invention for about 1 hour to about 23 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or about 23 hours); or for about 1 day to about 21 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or about 21 days). In some embodiments, the cells may be grown for about 1 week to about 8 weeks or more (e.g., about 1, 2, 3, 4, 5, 6, 7, or about 8 weeks). The rate of cell growth may be measured by any method known to those in the art including, but not limited to, cell imaging (e.g., brightfield imaging for percent phase confluence), cell staining (e.g., fluorescent tracking dye and Trypan blue), label free nuclear count (e.g., Cell-by-Cell counting), flow cytometry, nucleic acid staining (e.g., propidium iodide and 7-aminoactinomycin D), metabolic activity assays (e.g., 2 MTT, XTT, MTS, and WST1), antibody staining (e.g., Ki-67), adenosine triphosphate (ATP) concentration measurements, and nucleic acid synthesis labeling (e.g., radiolabeling, bromodeoxyuridine, and 5-ethynyl-2'- deoxyuridine). In some embodiments, the cell growth may be quantitated as a growth rate (e.g., the change in cell number over a predetermined amount of time), a total number of cell culturedoublings over a predetermined amount of time, and / or the time it takes to reach a predetermined cell number in the cell culture.

[0094] Cellular phenotype or cell morphology may be assessed by any method known to those in the art including, but not limited to, cell imaging (e.g., brightfield imaging), cell staining (e.g., fluorescent staining and Trypan blue), flow cytometry, etc. In some embodiments, assessing the phenotypic characteristics and / or cell morphology may include assessing the cell shape, cell size, cell form, cell texture, and / or cell pattern. In some embodiments, assessing the phenotypic characteristics and / or cell morphology may include assessing the shape, size, form, texture, pattern, presence, absence, and / or number of one or more different cellular compartments and / or structures (e.g., nuclei, endoplasmic reticulum, Golgi bodies, liposomes, chloroplasts, mitochondria, vesicles, flagella, endosomes, lysosomes, peroxisomes, cytosol, membranes, chromosomes, ribosomes, cytoskeletons, etc.).

[0095] "Subjects" are generally human subjects and include, but are not limited to, "patients." Subjects also include animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, non-human primates, etc., for, e.g., veterinary medicine, laboratory research and / or pharmaceutical drug development purposes.

[0096] Universal Medias and Coatings

[0097] Provided herein are medias with defined components that can support multiple cell types for in vitro growth and expansion. In some embodiments, the media may include some or all of: a) a basal medium (e.g., DMEM / F12, KSFM); b) a neuronal and / or stem cell growth supplement (e.g., a neuronal growth supplement such as B-27); c) non-essential amino acids (NEAA) (e.g., 2, 3, 4, 5 or more of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine, each present, for example, in a concentration of about 0.02 to about 0.5 mM); d) an antioxidant and / or essential vitamin (e.g., ascorbic acid, a tocopherol (e.g., a-tocopherol, P- tocopherol, y-tocopherol, and / or 5-tocopherol), glutathione, and / or N-acetyl-L-cysteine); e) epinephrine; f) a corticosteroid (e.g., hydrocortisone) and / or a glucocorticoid; and g) epidermal growth factor (EGF).

[0098] In some embodiments, the neuronal and / or stem cell growth supplement is present in a concentration from about 0.01% to about 1% by volume of the cell culture medium; the ascorbic acid is present in a concentration from about 0.08 to about 80 pg / mL; the epinephrine is present in a concentration from about 0.002 to about 20 pg / mL; the hydrocortisone is present in aconcentration from about 0.01 to about 100 pg / mL; and / or the EGF is present in a concentration from about 0.2 to about 250 ng / mL.

[0099] In some embodiments, the medium is free of serum and / or animal products (e.g., xeno- free).

[0100] In some embodiments, the medium may further include: h) hepatocyte growth factor (HGF); i) platelet-derived growth factor (PDGF) (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); and j) fibroblast growth factor (FGF, e.g., FGF-basic). In some embodiments, the HGF is present in a concentration from about 0.01 to about 100 ng / mL; the PDGF is present in a concentration from about 0.2 to about 25 ng / mL; and the FGF is present in a concentration from about 0.2 to about 250 ng / mL.

[0101] In some embodiments, the medium may further include human platelet lysate (PLT). In some embodiments, the PLT is devoid of heparin. In some embodiments, the PLT is present in a concentration from about 0.5% to about 10% by volume of the cell culture medium.

[0102] In some embodiments, the medium may further include: k) vascular endothelial growth factor (VEGF) (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); 1) insulinlike growth factor-1 (IGF-1); and m) fibroblast growth factor-acidic (FGF-acidic). In some embodiments, the VEGF is present in a concentration from about 0.2 to about 500 ng / mL; the IGF- 1 is present in a concentration from about 0.2 to about 250 ng / mL; and / or the FGF-acidic is present in a concentration from about 0.2 to about 250 ng / mL.

[0103] In some embodiments, the media may further include: h) prolactin; i) prostaglandin El; and j) retinoic acid. In some embodiments, the prolactin is present in a concentration from about 0.2 to about 250 ng / mL; the prostaglandin El is present in a concentration from about 0.025 to about 25 nmol / mL; and / or the retinoic acid is present in a concentration from about 0.01 to about 100 ng / mL.

[0104] In some embodiments, the media may further include some or all of: h) PDGF (e.g., PDGF- aa, PDGF-bb, PDGF-ab, or any combination thereof); i) FGF-basic; j) prolactin; k) prostaglandin El; 1) retinoic acid; m) VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); n) IGF-1; o) FGF-acidic; and p) heparin. In some embodiments, the PDGF is present in a concentration from about 0.2 to about 25 ng / mL; the FGF-basic is present in a concentration from about 0.2 to about 250 ng / mL; the prolactin is present in a concentration from about 0.2 to about 250 ng / mL; the prostaglandin El is present in a concentration from about 0.025 to about 25 nmol / mL; the retinoic acid is present in a concentration from about 0.01 to about 100 ng / mL; the VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof) is present in aconcentration from about 0.2 to about 500 ng / mL; the IGF-1 is present in a concentration from about 0.2 to about 250 ng / mL; the FGF-acidic is present in a concentration from about 0.2 to about 250 ng / mL; and / or the heparin is present in a concentration from about 0.1 to about 5 units / mL.

[0105] Example media compositions as described above are provided in the following tables. The UM1 example of Table 1 may find particular use with mesoderm lineage cells; UM2 of Table 2 with endoderm and ectoderm lineage cells; and UM3 of Table 3 with heterotypic cultures having cells from multiple lineages. However, an optimal or sufficient media type may vary for a given cell type, and thus the lineage cells are not limited to a particular universal media composition or compositions.Table 1: Example Universal Media 1 CompositionsTable 2: Example Universal Media 2 CompositionTable 3: Example Universal Media 3 Composition

[0106] Additional components may be included to aid in cell growth and viability. As a nonlimiting example, a supplemental composition is provided in Table 4 that may be added to the UM1 composition as provided in Table 1.Table 4: Example Universal Media 1 Composition Supplement

[0107] Also provided herein are cell culture plate coatings that can support multiple cell types for in vitro growth and expansion. In some embodiments, the coating may include some or all of: serum or a serum replacement product; fibronectin; laminin (e.g., laminin-521); collagen (e.g., Type I collagen, Type II collagen, Type IV collagen, or any combination thereof); and tropoelastin. The coating may be provided in an aqueous solution (e.g., comprising saline such as phosphate buffered saline (PBS) or other calcium-free and magnesium-free salt solution, and / or comprising alcohol such as ethanol or methanol) for application to the cell culture plate. In some embodiments,the serum replacement product is knockout serum (e.g., Cell Therapy System (CTS™) KnockOut Serum Replacement (SR™)), engineered serum, and / or human platelet extract (e.g., human platelet lysate (PLT)). In some embodiments, the cell culture plate coating is free of serum and / or animal products.

[0108] In some embodiments of a coating solution, the saline is present in a concentration from about 25% to about 75% by volume of the cell culture plate coating; the serum or serum replacement product is present in a concentration from about 25% to about 75% by volume of the cell culture plate coating; the fibronectin is present in a concentration from about 0.05 to about 2500 pg / mL; the laminin is present in a concentration from about 0.05 to about 2500 pg / mL; the Type I collagen is present in a concentration from about 0.05 to about 2500 pg / mL; the Type IV collagen is present in a concentration from about 0.05 to about 2500 pg / mL; and the tropoelastin is present in a concentration from about 0.05 to about 2500 pg / mL. A non-limiting example of a coating solution is provided in Table 5.Table 5: Example Universal Coating Composition

[0109] Methods of culturing primary cells may be performed by incubating the cells with a cell culture medium and / or coating as taught herein. For example, a mesoderm lineage primary cell may be incubated with a cell culture medium such as UM1 in a cell culture dish and / or plate. For example, an ectoderm lineage primary cell may be incubated with a cell culture medium such as UM2 in a cell culture dish and / or plate. For example, an endoderm lineage primary cell may be incubated with a cell culture medium such as UM2 in a cell culture dish and / or plate. For example, a tissue construct (e.g., a 3D organoid) with primary cells from two or more of a mesoderm, ectoderm and endoderm lineage may be incubated with a cell culture medium such as UM3 in a cell culture dish and / or plate.

[0110] In some embodiments, a particular primary cell type (e.g., a mesoderm lineage primary cell, an ectoderm lineage primary cell, or an endoderm lineage primary cell) may be cultured with a cell culture medium and / or coating that is selected as optimal using the methods as described herein (e.g., the cell matrix assay) in order to provide a desired outcome as described herein, which may or may not be that predicted based on cell lineage. In some embodiments, a primary cell (e.g., a mesoderm lineage primary cell, an ectoderm lineage primary cell, or an endoderm lineage primary cell) may be cultured with a cell culture medium and / or coating that is not selected as optimal (i.e., a non-optimal cell culture medium and / or coating) using the methods as described herein (e.g., the cell matrix assay) as long as said primary cell remains viable (e.g., does not die) throughout the culturing process.[OHl] In some embodiments, a non-optimal, but sufficient, cell culture medium and / or coating may be used, for example, if the cell culture comprises two or more different primary cells each with different optimal cell media and / or coatings, or the optimal cell medium and / or coating are undesirable to use for any reason (e.g., toxicity concerns, more expensive to source, etc.).

[0112] For example, a mesoderm lineage primary cell may be incubated with a cell culture medium such as UM2 or UM3 in a cell culture dish and / or plate. For example, an ectoderm lineage primary cell may be incubated with a cell culture medium such as UM1 or UM3 in a cell culture dish and / or plate. For example, an endoderm lineage primary cell may be incubated with a cell culture medium such as UM1 or UM3 in a cell culture dish and / or plate. For example, a tissue construct (e.g., a 3D organoid) with primary cells from two or more of a mesoderm, ectoderm and endoderm lineage may be incubated with a cell culture medium such as UM1 or UM2 in a cell culture dish and / or plate.

[0113] Cell Matrix Assays

[0114] Provided herein are assays useful in selecting an optimal media and / or coating for particular primary cells of interest. The methods may include: a) obtaining cells (e.g., primary cells) from a subject; b) incubating said cells with a cell culture medium (e.g., a cell culture medium and / or coating as taught herein) for a predetermined amount of time; and c) measuring a parameter indicating cell growth / expansion (e.g., growth rate for the cells, proliferation rate, cell number, cell health, population doublings per day, etc.) after said amount of time. The method may further include repeating the measuring using a different cell culture medium and / or different cell culture plate coating; and selecting the cell culture medium and / or the cell culture plate coatingthat provides an optional result in the measured parameter (e.g., the fastest growth rate, total cell number, and / or phenotypic characteristics such as marker expression or morphology).

[0115] Counting the cell number may be performed, for example, using bright field imaging (e.g., percent phase confluence) and / or label free nuclear counting (e.g., Cell-by-Cell counting).

[0116] Individual components of the medias and coatings taught herein may be tested in the assays to determine or confirm whether their inclusion would be beneficial for a particular primary cell culture. Other additives may also be tested. For example, agents such as R-Spondin, human noggin, human wnt, Y-27632, CHIR-99021, SB415286, etc., may be tested in the assays. The methods described herein may also be used to test other basal cell media, media supplements, and / or growth substrates.

[0117] In some embodiments, the repeating step is carried out between 1 and 12 times (e.g., between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times) by providing a first array of different cell culture media and / or different cell culture plate coatings, and incubating the cells with each of the different cell culture media and / or cell culture plate coatings for a predetermined amount of time.

[0118] In some embodiments, the method of selecting an optimal media and / or coating further comprises: d) providing a second array comprising different concentrations of cell culture medium additives, optionally in combination with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) cell culture media and / or cell culture plate coatings selected from the first array; e) incubating the cells with each of the different concentrations of additives of the second array for a predetermined amount of time; and f) selecting one or more concentrations of additives that provide the fastest growth rate for the donor cells and / or another desired outcome (e.g., proliferation rate, cell number, cell health, population doublings per day, etc.).

[0119] In some embodiments, the method of selecting an optimal media and / or coating further comprises determining the cell growth across multiple cell passages. In some embodiments, determining the cell growth across multiple cell passages comprises passaging the cells from about 1 to about 12 times (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times) on a third array using the cell culture media, cell culture plate coatings, and / or concentrations of additives selected from the first and / or second array. In some embodiments, multiple cell passages on the third array can be used to determine the optimal concentration of supplements, mimics, and / or plate coatings to be used in a base culture media for achieving the optimal cell growth.

[0120] In some embodiments, the method of selecting an optimal media and / or coating comprises selecting one or more cell culture media, cell culture plate coatings, and / or the concentration of additives that provide an optional result in the measured parameter (e.g., the fastest growth rate,total cell number, and / or phenotypic characteristics such as marker expression or morphology). In some embodiments, the other desired outcome is the cell phenotype (e.g., cell size, cell shape, cell texture, organelle size, organelle number, organelle shape, uniformity of cell size in culture, uniformity of cell shape in culture, and / or arrangement of the cells in culture). In some embodiments, determining the cell phenotype may be performed, for example, using cell imaging (e.g., bright field imaging, fluorescent microscopy, and the like), proteomics, genomics, epigenomics, transcriptomics, and / or metabolomics. In some embodiments, the desired cell phenotype is an epithelial cell phenotype, an endothelial cell phenotype, an epidermal cell phenotype, a fibroblast cell phenotype, a mesenchymal cell phenotype, a stem cell phenotype, a muscle cell phenotype, an astrocyte cell phenotype, a neuronal cell phenotype, or a stem cell phenotype.

[0121] In some embodiments, measuring cell growth further comprises comparing the cell growth rate with the growth rate of a control cell grown in the cell growth medium and / or cell culture plate coating. In some embodiments, measuring cell growth further comprises comparing the cell growth rate with the growth rate of the cell grown in a control cell growth medium and / or control cell culture plate coating.

[0122] In some embodiments, the first array, second array, and / or third array is provided in a cell culture plate (e.g., a 4-well, 6-well, 8-well, 12-well, 24-well, 48-well, 96-well, or 384-well plate).

[0123] Cell Culture Additive Compositions (Packs)

[0124] Additive compositions, or "packs" useful to make the medias or coatings as taught herein are provided, which may be combined as appropriate and added to a basal medium.

[0125] For example, a cell culture medium additive composition may include some or all of: a neuronal and / or stem cell growth supplement (e.g., B-27); non-essential amino acids (NEAA) (e.g., 2, 3, 4, 5 or more of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L- proline, and L-serine); an antioxidant and / or essential vitamin (e.g., ascorbic acid, a tocopherol (e.g., a-tocopherol, P-tocopherol, y-tocopherol, and / or 5-tocopherol), glutathione, and / or N-acetyl- L-cysteine); epinephrine; a corticosteroid (e.g., hydrocortisone) and / or a glucocorticoid; and EGF. This example may provide a universal medium "starter pack" that can be added to a basal medium, in combination with one or more "add-on" packs to create a universal media as taught herein.

[0126] In some embodiments, the starter pack composition includes ascorbic acid, and the neuronal and / or stem cell growth supplement is present in the composition in a concentration from about 25% to about 75% by volume; the epinephrine is present in the composition in a weight ratiorelative to the ascorbic acid from about 1 :50 to about 1 :500 (epinephrine : ascorbic acid); the hydrocortisone is present in the composition in a weight ratio relative to the ascorbic acid from about 1 :5 to about 1 : 100 (hydrocortisone : ascorbic acid); and / or the EGF is present in the composition in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 100,000 (EGF : ascorbic acid).

[0127] As a non-limiting example, a universal media starter pack may include: 10 mL of 50x B27 5 mL of lOOx NEAA; 0.8 mL of 25 mg / mL ascorbic acid; 0.02 mL of 5 mg / mL epinephrine; 0.1 mL of 5 mg / mL hydrocortisone; and 0.0025 mL of 0.2 mg / mL recombinant human (rh) EGF, relative to about 500 mL of basal medium.

[0128] A universal medium starter pack may be used with additional add-on pack(s) as additives to basal media to form a medium as taught herein.

[0129] In some embodiments, a UM1 add-on pack may include: HGF; PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); and FGF (e.g., FGF-basic). This pack may be used along with the starter pack to form the UM1 media in basal medium such as DMEM / F12.

[0130] In some embodiments, the starter pack composition includes ascorbic acid, and the HGF of the UM1 add-on pack is present in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 10,000 (HGF : ascorbic acid); the PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof) is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (PDGF : ascorbic acid); and the FGF-basic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF-basic : ascorbic acid).

[0131] As a non-limiting example, a UM1 add-on pack may include: 0.05 mL of 0.1 ug / mL HGF; 0.025 mL of 100 mg / mL PDGF-ab; and 0.0125 mL of 0.2 mg / mL rhFGF basic. This may be added with a UM starter pack to DMEM / 12 to a total volume of about 500 mL to form UM1 medium.

[0132] In some embodiments, the UM1 add-on pack may further include PLT. In some embodiments, the PLT is devoid of heparin. In some embodiments, the PLT is present in a concentration from about 20% to about 60% by volume of the additive composition.

[0133] In some embodiments, a UM1 supplemental add-on pack (which may be added to the UM1 add-on pack) may include: VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); IGF-1; and FGF-acidic. In some embodiments, the starter pack composition includes ascorbic acid, and the VEGF is present in a weight ratio relative to the ascorbic acid from about 1 : 10,000 to about 1 : 150,000 (VEGF : ascorbic acid); the IGF-1 is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 :5,000 (IGF-1 : ascorbic acid); and the FGF-acidicis present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 :5,000 (FGF- acidic : ascorbic acid).

[0134] As a non-limiting example, a UM1 supplemental add-on pack may include: 10 pg recombinant human (rh) FGF acidic; 0.250 pg rh VEGF-121; and 10 pg rh IGF-1, to be added to about 500 mL of UM1 media.

[0135] In some embodiments, a UM2 add-on pack may include: prolactin; prostaglandin El; and retinoic acid. This pack may be used along with the starter pack to form the UM2 media in basal medium such as DKSFM. In some embodiments, the starter pack composition includes ascorbic acid, and the prolactin of the UM2 add-on pack is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (prolactin : ascorbic acid); the prostaglandin El is present in a weight ratio relative to the ascorbic acid from about 1 : 5,000,000 to about 1 : 50,000,000 (prostaglandin El : ascorbic acid); and the retinoic acid is present in a weight ratio relative to the ascorbic acid from about 1 : 100,000 to about 1 :800,000 (retinoic acid : ascorbic acid).

[0136] As a non-limiting example, a UM2 add-on pack may include: 0.0125 mL of 0.2 ug / mL Prolactin; 0.5 mL of 25 nmol / mL Prostaglandin El; and 0.5 mL of 1 mg / mL Retinoic acid, to be added with a UM starter pack to DKSFM to a total volume of about 500 mL.

[0137] In some embodiments, a UM3 add-on pack may include: HGF; PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); FGF -basic; prolactin; prostaglandin El; retinoic acid; VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); IGF-1; FGF -acidic; and heparin. This pack may be used along with the UM starter pack to form the UM3 media in basal medium such as DKSFM. In some embodiments, the starter pack composition includes ascorbic acid, and the HGF of the UM3 add-on pack is present in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 10,000 (HGF : ascorbic acid); the PDGF (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof) is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (PDGF : ascorbic acid); the FGF -basic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF- basic : ascorbic acid); the prolactin is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1:20,000 (prolactin : ascorbic acid); the prostaglandin El is present in a weight ratio relative to the ascorbic acid from about 1 :5,000,000 to about 1 :50,000,000 (prostaglandin El : ascorbic acid); the retinoic acid is present in a weight ratio relative to the ascorbic acid from about 1 : 100,000 to about 1 :800,000 (retinoic acid : ascorbic acid); the VEGF (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof) is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (VEGF : ascorbic acid); the IGF-1 is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 : 10,000 (IGF- 1 : ascorbic acid); the FGF-acidic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1:20,000 (FGF-acidic : ascorbic acid); and / or the heparin is present in a concentration from about 0.1 to about 5 units / mL.

[0138] Alternatively, a UM3 add-on pack may include VEGF (e.g., VEGF-165, VEGF-D, VEGF- 121, or any combination thereof); IGF-1; FGF-acidic; and heparin. This version of the UM3 addon pack can be combined with the UM1 and UM2 add-on packs and the UM starter pack to form the UM3 media in basal medium such as DKSFM.

[0139] As a non-limiting example, a UM3 add-on pack may include: 0.375 mL of 1,000 units / mL heparin; 0.0125 mL of 0.2 mg / mL rh FGF acidic; 0.025 mL of 0.1 mg / mL rh VEGF-121; and 0.0375 mL of 0.2 mg / mL rh IGF-1, to be added with a UM starter pack, a UM1 add-on pack and a UM2 add-on pack to DKSFM to a total volume of about 500 mL to form UM3 medium.

[0140] Also provided is a cell culture medium comprising a basal cell culture medium, a UM starter pack as taught herein, and a UM1, UM2 or UM3 add-on pack.

[0141] Mimetics

[0142] In some embodiments, the media or media additive / supplement comprises one or more mimetics. The term "mimetic" as used herein is intended to mean a compound, protein, nucleic acid, etc. that has a biological function that is similar to a particular compound, protein, nucleic acid, etc., and can thus be used as a replacement of some or all of that component used on the media, but wherein the mimetic is structurally distinct therefrom.

[0143] For example, in some embodiments, a cell culture medium of the present invention may comprise one or more of a neuronal growth supplement and / or a mimetic thereof; a stem cell growth supplement and / or a mimetic thereof; FGF and / or a mimetic thereof; EGF and / or a mimetic thereof; IGF and / or a mimetic thereof; PDGF and / or a mimetic thereof; HGF and / or a mimetic thereof; VEGF and / or a mimetic thereof; and heparin and / or a mimetic thereof. Thus, it is contemplated that wherever components such as a stem cell growth supplement, heparin, or growth factors such as FGF, EGF, IGF, PDGF, HGF, VEGF, etc., are provided in the compositions taught herein, these can be replaced wholly or partially by one or more corresponding mimetics.

[0144] In some embodiments, the one or more mimetics may be selected from Table 6.Table 6. Small molecule mimetics.Table 6 (cont'd). Small molecule mimetics.

[0145] The present invention is further described in the following non-limiting examples.EXAMPLES

[0146] Example 1: Universal Media (UM) Compositions and Testing

[0147] Three Universal Medias were created using human growth factors and select basal mediums (Table 7). Universal Media One (UM1) was developed for cells derived from the mesoderm origin and supports growth on cells such as mesenchymal stem cells (MSC), osteoblasts (OSB), and cardiac myocytes (CM). Universal Media Two (UM2) was developed for cells derived from the endoderm and ectoderm origin and cover many types of epithelial cells. UM1 and UM2 are intended to be used as monotypic expansion medias due to the high cell yields they produced, whereas Universal Media Three (UM3) was created with the intent of supporting heterotypic human constructs which are more similar the native tissue environment.Table 7: UM1, UM2, and UM3 formulations.

[0148] Extracellular matrices located in the microenvironment immediately surrounding cells are also of interest for standardization. Universal Cell Culture Coating (UC) was developed to recapitulate the complex structure of the ECM microenvironment (Table 8).Table 8: Formulation of the UC (also referred to as "UC3").

[0149] Example 2: Assay for Media Formulation Screening

[0150] Cellular population doublings (PDL) and doubling time (DT) are of specific interest due to the planning requirements during a clinical procedure. When cells are removed from a patient the clock begins ticking both genetically and phenotypically as cells divide. Senescence is a major concern during cellular expansion. Prior to tissue engineered constructs being developed, cells that are no longer able to divide or have terminally differentiated prior to their incorporation will reduce the efficacy and safety of the construct. Understanding the PDL, DT and cellular yield can guide a clinician down the right path while designing and implementing a patient's procedure.

[0151] Small molecules (Table 9) may be used to prevent apoptosis by blocking kinase inhibitors in cells recently harvested from a patient, with a goal to reduce cell death and increase overall cellular yield. By creating a rapid screening assay focused on the UM formulations as well as the required ECM substrates and small molecules, a unique media-substrate-small molecule combinations can be identified for human primary cells collected from a donor or patient and used to optimize cellular expansion and tissue construct viability.Table 9: Small molecule supplements.

[0152] Methods

[0153] Using a 96-well plate, vessel coatings were added as 150 pl of substrate per well and incubated at 37° C at 5% CO2 for 1 hour followed by a rinse using 150 pl of DPBS. Cells were thawed from LN2, counted using the NC-200 and 150 pl of cell suspension was added to 3 wells (N = 3) as per the seeding densities in Table 10. These seeding densities were based on manufacturers recommendations as well as previous expansion efforts for each cell type. The assay was then placed into the Incucyte® S3 Analysis System (Sartorius, Gottingen, Germany) andscanned for % Phase Confluence (%PC) (FIG. 1) as well as a label free nuclear count using the Cell-by-Cell software module (available for Incucyte® Devices). Scans were conducted every 24 hours with 5 images per well and analyzed using on-board software provided with the Incucyte® S3 Analysis System for confluence and nuclear count. For the duration of the experiment the plates were cultured in the Incucyte® S3 Analysis System which was placed inside an incubator at 37° C at 5% CO2 with a fresh media change at 48 hours and monitored until confluence reached >80%.Table 10: Tested cell lines and seeding density.^Standard coatings based on pilot studies comparing Fibronectin, Gelatin 0.1 %, Poly-L-Lysine, CollagenType I, Collagen Type IV, Tropoelastin and Laminin 521.

[0154] After 72 hours, BM-MSC had the highest %PC in the UM1 media formulation and grown on the UC substrate (Table 7) with small molecules (SM) listed in Table 9 added (FIG. 2). The highest phase object count per image (POC) was obtained using UM1 media formulation with KnockOut Serum (KOS) Replacement (Thermofisher, cat#10828028) grown on UC3 w / SM. When measuring growth using %PC, the same UM1 / KOS on UC3-SM conditions also proved to be the second-best growth condition, indicating that the cells are truly doubling as the confluence increases. Similarly, the second-best growth conditions identified with POC is the same set of conditions as the best growth conditions identified by %PC, lending a similar conclusion of this pair of metrics. Conversely, the third-best growth conditions identified by %PC, UM1 grown on UC3, is interesting due to the fact that the cells performed similarly in these conditions evenwithout the KOS in the media or SM in the vessel coating. This indicates that those additives are not essential but may be helpful in enhancing proliferative performance.

[0155] Similar analysis and graphs were completed for each cell type, and the results of the top growth conditions can be seen in Table 11 and in FIG. 3. A summary of each cell type is provided below.Table 11: Top 3 growth conditions as identified by two measuring protocols.Media / Additive : Coating / Additive eg: UM1 / SM:UC3 is cells grown using universal media one with small molecules on UC3 vessel coating. STND is the coating condition indicated in Table 9 above. HBM is HumaMatrix native human derived ECM solution (Humabiologics, Inc.).

[0156] Osteoblasts

[0157] Osteoblasts showed UM1 :UC3 / SM as the best culture condition after three days for both %PC and POC, showing these conditions are the optimized growth conditions for this cell type. This was similar for the second-best growth conditions, UM1 :UC3, which may imply that the SM additives are not required but do, however, enhance the cell proliferation. The third-best conditions seem to confirm these suspicions since KOS, SM again, and HBM vessel coating are present but may not produce a great enough increase in cellular proliferation to merit adding them to the culture conditions.

[0158] Human Cardiac Myocytes (HCM)

[0159] HCM had overlapping success with UM1 / KOS / SM:UC3 being the top culture condition after five days for both %PC and POC. The second-best growth conditions also aligned with the conditions of UM1 / KOS / SM media formulation grown on their standard vessel coating of fibronectin or gelatin. The third best growth conditions also were the same for %PC and POC being UM1 / KOS / SM grown on UC3 / SM added. UM1 / KOS / SM grown on fibronectin or gelatin would suffice for this cell type and cellular proliferation would likely not suffer in the absence of more complex coatings.

[0160] Human Umbilical Vein Endothelial Cells (HUVEC)

[0161] HUVEC best conditions proved to be UM3 / SM media formulation grown on fibronectin. The second best %PC is shown to be UM3 / SM grown on UC3 however the second best POC was UM3 grown on fibronectin. The third-best conditions were also different from one another with %PC being UM1 on UC3 and POC being UM3 / SM grown on HBM vessel coating. These findings are interesting for a few reasons, the first being that there are growth factors absent in the UM1 media formulation that are present in UM3, however, in the presence of the UC3 vessel coating these deficient media growth factors are superseded by the presence of a more complex and robust vessel coating. The second insight comes from the simplicity of the vessel coating in the first- and second-best growth conditions, implying that a more robust media formulation is required for a simpler vessel coating. In other words, a simpler media formulation can be used in the presence of a more complex vessel coating or vice-versa, a more robust media formulation can be used in thepresence of a simpler vessel coating. However, the cells require these factors in some form, be they soluble or insoluble.

[0162] Colonic Epithelial Cells

[0163] Colonic epithelial cells best growth conditions, for both %PC and POC, emerged after seven days as UM3 / SM media formulation grown on their standard vessel coating of collagen type IV. The second and third best conditions are inverse to one another involving UM3 / SM on UC3 and UM2 / SM on collagen type IV (COL IV). This may imply that the extra growth factors added to UM3 enhance proliferation but are not required. The same can be said regarding the vessel coating considering the cells doubled on both the simpler COL IV solution as well as the more complex UC3 vessel coating. This could be a time dependent observation since, as the cells proliferate, they continually attempt to produce the most optimized growth conditions through mechanisms such as endogenous growth factor release as well as remodeling of the ECM proteins for greater cellular adhesion to the culture vessel.

[0164] Small Airway Epithelial Cells (SAEC)

[0165] Both %PC and POC showed similar top three optimal growth conditions for SAEC after three days. The best growth conditions were UM2 / SM media grown on UC3, second-best were UM2 / SM media grown on HBM and third-best being UM2 / SM media grown on COL IV. As seen with some of the previously discussed cell types, there seems to be a trend toward simplicity in the fact that similar growth conditions were achieved using COL IV alone as a vessel coating rather than the more complex coatings of HBM and UC3. Although the media conditions of UM2 seemed to perform better with SM were standard across all three scenarios showing the proliferative benefits of using the SM cocktail with this cell type even regardless of vessel coating.

[0166] Normal Human Epidermal Keratinocytes (NHEK)

[0167] NHEK were consistent across conditions after four days, with the best conditions being UM2 / SM grown on UC3; the second best were UM2 / SM grown on COL IV; and third best were UM2 / SM grown on UC3 / SM added. Again, we found the trend of having similar growth conditions across all three scenarios even though some of the conditions were far more complex than others, implying not everything the cells are exposed to is required for their proliferation.

[0168] Pancreatic Epithelial Cells

[0169] The best growth conditions for pancreatic epithelial cells were seen after seven days as being UM3 media and grown on UC3. The second-best conditions proved to be UM3 / SM and grown on UC3. Thus, the addition of SM may actually inhibit growth in some fashion since its introduction produced less desirable conditions than UM3:UC3 alone. The third best set of conditions were UM3 grown on HBM vessel coating, suggesting that while the use of SM may not be required, a vessel coating with more additives such as UC3 or HBM is beneficial to cellular proliferation.

[0170] Human Amniotic Stem Cells (HASC)

[0171] After four days, the HASCs first and second conditions reached the confluence threshold using UM1 on HBM and similar results using UM1 on UC3 substrate. The third set of conditions involved UM1 / KOS on HBM for %PC and UM1 on UC3 / SM for POC. Based on these results, the UM1 formulation is the best culture media and either the HBM or the UC3 substrate would be recommended and considered for scale up to determine which substrate would provide the most impact as cell yields increase.

[0172] Placental Stem Cells

[0173] The confluence threshold of 80% was reached for placental stem cells after 24 hours, which was quite surprising, using UM1 / KOS media on both the HBM and UC3 / SM substrates. The final optimum condition was achieved with UM1 / KOS on UC3 with and without SM. These results demonstrate the benefits of UM1 / KOS on placental stem cells, and further that the cells seemed to favor the HBM and variations of UC3 both with and without SM added.

[0174] Conclusion

[0175] Three chemically defined and xeno-free media formulations were developed for use across multiple human primary cell types. The formulations were then used in a combinatory fashion with human extracellular matrix (ECM) substrates and tested across multiple human primary cell types as a high throughput screening tool to determine the optimal growth conditions for each cell type's expansion. Metrics of success were based on % phase confluence and nuclear count, which allowed for selection of multiple media and substrate combinations that maximize monotypic proliferation and can provide insights into heterotypic tissue engineered construct viability.

[0176] Without wishing to be bound by any particular theory, a universal proliferation assay, such as the one developed here, could prove useful in a clinical manufacturing setting where a donor biopsy is received and tested for expansion and construct viability. The results of the assay are useful for the determination of custom media-substrate combinations that could then be scaled up for rapid cellular expansion. This scalability is beneficial in meeting the high demands often encountered in clinical settings, especially for therapies that require a large number of cells, such as regenerative medicine or cell-based therapies.

[0177] Example 3: Development of supplemented UM1 (UM1+) and additional media supplements

[0178] After rigorous testing, it was confirmed that UM1 supported the growth of almost all mesoderm cell types. Additionally, UM1 performs equal to, or outperforms, serum containing media for most cell types. In order to further improve the performance of this medium for all cell lines, including those that are more difficult to culture, cell culture supplements were tested. Cell culture supplements are used to maintain the growth and phenotype of cells in vitro that have traditionally been deemed "difficult" to culture. MSC, for example, are reticulo-endothelial cells that are found around endothelial cells in the pericytic niche surrounding blood vessels. These cells are constantly bathed in serum and, as such, have been traditionally grown in serum-containing media. There are a number of "serum -free" BM-MSC media, and all contain platelet lysates to act in a manner similar to serum by providing supraphy si ologi cal amounts of beta-fibroblast growth factor (B-FGF), platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Moreover, several recent studies have used human platelet lysate (PLT) as a better- defined replacement for FBS in culturing mesenchymal stem cells (reviewed in Guitto et al., Journal of Translational Medicine, 2020). Accordingly, we have formulated a supplemented UM1 media with PLT, which has been processed to remove a portion of the factors but retains a well characterized selection of growth factors, protease inhibitors, and adhesion molecules.

[0179] Additionally or alternatively to the PLT, we have identified growth factors (Table 12) that can be added to the UM1 media as a supplement for cells that do not have optimal growth in UM1 (e.g., some mesoderm cells).Table 12: UM1 growth factor supplements.

[0180] Example 4: Cell Matrix Assay

[0181] To aid in identifying optimal cell growth conditions for particular cell types or combinations of cell types, we developed a cell matrix assay. This assay utilizes cell culture plates to efficiently test multiple combinations and concentrations of basal cell media, growth supplements, cell culture plate coatings, and other media additives. It may also include controls for comparisons to other cell types and / or commercially available media.

[0182] With reference to FIG. 4, the first array is prepared using a 96-well plate that may be coated in different areas (e.g., different columns) with different substrates (Sub 1 - Sub 11) such as collagen, tropoelastin, gelatin, and the like, and a Universal Coating (UC) such as UC3 (Table 8), as described in Example 2, and may optionally include an uncoated column. The plate is loaded with different medias in different areas (e.g., along different rows), such as UM1, UM2, and UM3 described above in Table 7, media supplements such as growth factors (Table 12) and / or small molecules (Table 9). Cells may be washed and isolated from any residual ECM proteins. Cells are counted using the NC-200, and 150 pl of cell suspension is added to each of the wells as per the seeding densities in Table 10. These seeding densities can be based on manufacturers recommendations and / or empirically determined for each cell type. The assay is then placed into the Incucyte® S3 Analysis System and scanned for % Phase Confluence (%PC) as well as a label free nuclear count using the Cell-by-Cell software module (available for Incucyte® Devices). Scans are conducted every 24 hours with 5 images per well and analyzed using on-board software provided with the Incucyte® S3 Analysis System for confluence and nuclear count. For the duration of the experiment, the plates are cultured in the Incucyte® S3 Analysis System which is placed inside an incubator at 37° C at 5% CO2 with a fresh media change at 48 hours and monitored until confluence reached a pre-determined level for the given cell type (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% confluency or more). The cells can be further visualized using microscopy (e.g., bright field imaging and / or fluorescent microscopy) for other phenotypic markers as needed. The results from this array can be used to determine optimal basal cell media, additives, and plate coatings for the selected cell type.

[0183] With reference to FIG. 5, a second array may be used to assess different concentrations of the selections from the first array. As such, a second array is prepared from selected combinations of cell media, additives, and plate coatings from the first array. A 96-well plate can be partitioned, for example, into 8 sections of 12-wells each, as seen in FIG. 5. Each section is coated with increasing concentrations, e.g., across the columns, of one selected substrate as described previously. Each section is then loaded with a selected cell medium and with increasing concentrations, e.g., across the rows, of selected additives. As above, cells are seeded into the wells and the assay is placed into the Incucyte® S3 Analysis System and scanned for % Phase Confluence (%PC) as well as a label free nuclear count using the Cell-by-Cell software module (available for Incucyte® Devices). Scans are conducted every 24 hours with 5 images per well and analyzed using on-board software provided with the Incucyte® S3 Analysis System for confluence and nuclear count. For the duration of the experiment the plates are cultured in the Incucyte® S3 Analysis System which is placed inside an incubator at 37° C at 5% CO2 with a fresh media change at 48 hours and monitored until confluence reached a pre-determined level for the given cell type. The cells can be further visualized using microscopy (e.g., bright field imaging and / or fluorescent microscopy) for other phenotypic markers as needed. The results from this array determine the optimal concentration of additives and / or plate coatings for the selected cell type.

[0184] With reference to FIG. 6, a next step may be to assess the effect of the selected concentration of additives and plate coatings from the second array across multiple passages. As such, a third array may be prepared with the concentrations of additives and plate coatings from the second array. A 96-well plate is partitioned into 8 sections of 12-wells each, as seen in FIG. 6. Each section is coated with a selected substrate concentration from the second array, as described previously, and loaded with a selected cell medium and concentration of selected additives. Alternatively, one row may be used to include a commercially available cell medium as a control or comparison. Cells are seeded into the wells of a particular portion, such as columns 1-3 (Passage 1). The assay is placed into the Incucyte® S3 Analysis System and scanned for % Phase Confluence (%PC) as well as a label free nuclear count using the Cell-by-Cell software module (available for Incucyte® Devices). Scans are conducted every 24 hours with 5 images per well and analyzed using on-board software provided with the Incucyte® S3 Analysis System for confluence and nuclear count. For the duration of the experiment the plates are cultured in the Incucyte® S3 Analysis System which is placed inside an incubator at 37° C at 5% CO2 with a fresh media change at 48 hours and monitored until confluence reached a pre-determined level for the given cell type. The cells can be further visualized using microscopy (e.g., bright field imaging and / or fluorescentmicroscopy) for other phenotypic markers as needed. Once the cells have reached a desired confluency, they are split and seeded into the wells of a different area, e.g., columns 4-6 (Passage 2) such that cells in well Al are moved into well A4; A2 into A5; A3 into A6; Bl into B4; and so on. The growth and passaging steps can be repeated twice more, into columns 7-9 (Passage 3) and subsequently columns 10-12 (Passage 4). The results from this array demonstrate how the selected concentrations of additives and plate coatings affect the cells as they are passaged.

[0185] Example 5: Small molecule and peptide mimetics

[0186] Small molecules can be used as synthetic analogs to human recombinant growth factors and supplements that are currently used in commercially available medias. Without wishing to be bound by any particular theory, the small molecules chosen for this example were intended to mimic the proliferative effects of EGF, hGH, FGF2, PDGFab, human platelet lysate and heparin in cell culture.

[0187] Six human primary cell types were chosen based on previous optimization experiments. Each cell type was tested with a selection of small molecules and at a range of concentrations to determine any morphological or proliferation trends. The small molecule synthetic mimetics are shown in Table 12. Proliferation was determined using the Incucyte® S3 Analysis System where images were captured at 8-hour intervals over 5 days, masked and plotted as Phase Confluence over Time.Table 12. Tested small molecule mimetics

[0188] The cell growth results for the medias tested with the above mimetics are shown in FIGS. 7-9 and Tables 13-19. Tables 14-19 show the growth rate of multiple cells lines as measured by phase object confluence (%). The p-value was determined using a t-test comparing the indicated growth condition to a basal media control. For Tables 13-19, UMS indicates the universal media synthetic; HP indicates high performance small molecule combinations; Cl indicates concentration 1 of the small molecules; and C2 indicates concentration 2 of the small molecules.Table 13. Quantification of the cell growth rates compared to a basal media controlTable 14. Phase Object Confluence (%) for human bronchial smooth muscle cells grown in the indicated conditions.Table 15. Phase Object Confluence (%) for human adipose tissue MSCs grown in the indicated conditions.Table 18. Phase Object Confluence (%) for human dermal fibroblasts grown in the indicated conditions.

[0189] UM1 showed consistently high proliferation rates across all cell types. All synthetic formulas provided significant positive effects across most cell types, as PLT 2.5% enhanced proliferation across all cell types. While there were significant results using some of the synthetic formulas among individual cell types, no one formulation stood out across all cell types.

[0190] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

That which is claimed is:

1. A cell culture medium useful for culturing multiple primary cell types, said cell culture medium comprising: a) a basal medium (e.g., DMEM / F12, KSFM); b) a neuronal and / or stem cell growth supplement (e.g., B-27), or a mimetic thereof; c) non-essential amino acids (NEAA); d) an antioxidant and / or essential vitamin (e.g., ascorbic acid, a tocopherol (e.g., a- tocopherol, P-tocopherol, y-tocopherol, and / or 5-tocopherol), glutathione, and / or N-acetyl-L- cysteine); e) epinephrine; f) a corticosteroid (e.g., hydrocortisone) and / or a glucocorticoid; and g) epidermal growth factor (EGF), or a mimetic thereof.

2. The cell culture medium of claim 1, wherein the NEAA comprises 2, 3, 4, 5 or more of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

3. The cell culture medium of claim 2, wherein each amino acid in the NEAA is present in the cell culture medium in a concentration of about 0.02 to about 0.5 mM.

4. The cell culture medium of any one of claims 1-3, wherein the neuronal and / or stem cell growth supplement or a mimetic thereof is a neuronal growth supplement (e.g., xeno free B-27).

5. The cell culture medium of any one of claims 1-4, wherein the antioxidant and / or essential vitamin is ascorbic acid.

6. The cell culture medium of any one of claims 1-5, wherein the corticosteroid and / or glucocorticoid is hydrocortisone.

7. The cell culture medium of claim 6, wherein: the neuronal and / or stem cell growth supplement or a mimetic thereof is present in a concentration from about 0.01% to about 1% by volume of the cell culture medium; the ascorbic acid is present in a concentration from about 0.08 to about 80 pg / mL;the epinephrine is present in a concentration from about 0.002 to about 20 pg / mL; the hydrocortisone is present in a concentration from about 0.01 to about 100 pg / mL; and the EGF or mimetic thereof is present in a concentration from about 0.2 to about 250 ng / mL.

8. The cell culture medium of any one of claims 1-7, wherein the medium is free of serum and / or animal products.

9. The cell culture medium of any one of claims 1-8, wherein the basal medium is Dulbecco's Modified Eagle Medium with Nutrient Mixture F12 (DMEM / F12).

10. The cell culture medium of any one of claims 1-9, wherein the medium further comprises: h) hepatocyte growth factor (HGF) or a mimetic thereof; i) platelet-derived growth factor (PDGF) (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof) or a mimetic thereof; and j) fibroblast growth factor (FGF) or a mimetic thereof.

11. The cell culture medium of claim 10, wherein the FGF or a mimetic thereof is FGF-basic.

12. The cell culture medium of claim 11, wherein: the HGF or a mimetic thereof is present in a concentration from about 0.01 to about 100 ng / mL; the PDGF or a mimetic thereof is present in a concentration from about 0.2 to about 25 ng / mL; and the FGF-basic is present in a concentration from about 0.2 to about 250 ng / mL.

13. The cell culture medium of any one of claims 10-12, wherein the medium further comprises human platelet lysate (PLT).

14. The cell culture medium of claim 13, wherein the PLT is devoid of heparin.

15. The cell culture medium of claim 13 or 14, wherein the PLT is present in a concentration from about 0.5% to about 10% by volume of the cell culture medium.

16. The cell culture medium of any one of claims 10-15, wherein the medium further comprises: k) vascular endothelial growth factor (VEGF) (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof) or a mimetic thereof; l) insulin like growth factor or a mimetic thereof (e.g., insulin-like growth factor-1 (IGF- 1)); and m) fibroblast growth factor-acidic (FGF-acidic).

17. The cell culture medium of claim 16, wherein: the VEGF or a mimetic thereof is present in a concentration from about 0.2 to about 500 ng / mL; the IGF or a mimetic thereof (e.g., IGF-1) is present in a concentration from about 0.2 to about 250 ng / mL; and the FGF-acidic is present in a concentration from about 0.2 to about 250 ng / mL.

18. The cell culture medium of any one of claims 1-8, wherein the basal medium is defined keratinocyte serum-free (KSF) medium.

19. The cell culture medium of any one of claims 1-8 or 18, wherein the medium further comprises: h) prolactin; i) prostaglandin El; and j) retinoic acid.

20. The cell culture medium of claim 19, wherein: the prolactin is present in a concentration from about 0.2 to about 250 ng / mL; the prostaglandin El is present in a concentration from about 0.025 to about 25 nmol / mL; and the retinoic acid is present in a concentration from about 0.01 to about 100 ng / mL.

21. The cell culture medium of any one of claims 1-8 or 18, wherein the medium further comprises:h) PDGF or a mimetic thereof (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); i) FGF or a mimetic thereof (e.g., FGF-basic); j) prolactin; k) prostaglandin El; l) retinoic acid; m) VEGF or a mimetic thereof (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof); n) IGF or a mimetic thereof (e.g., IGF-1); o) FGF-acidic; and p) optionally, heparin or a mimetic thereof.

22. The cell culture medium of claim 21, wherein: the PDGF or a mimetic thereof is present in a concentration from about 0.2 to about 25 ng / mL; the FGF or a mimetic thereof (e.g., FGF-basic) is present in a concentration from about 0.2 to about 250 ng / mL; the prolactin is present in a concentration from about 0.2 to about 250 ng / mL; the prostaglandin El is present in a concentration from about 0.025 to about 25 nmol / mL; the retinoic acid is present in a concentration from about 0.01 to about 100 ng / mL; the VEGF or a mimetic thereof is present in a concentration from about 0.2 to about 500 ng / mL; the IGF or a mimetic thereof (e.g., IGF-1) is present in a concentration from about 0.2 to about 250 ng / mL; the FGF-acidic is present in a concentration from about 0.2 to about 250 ng / mL; and the heparin or a mimetic thereof is present in a concentration from about 0.1 to about 5 units / mL.

23. A cell culture plate coating comprising: serum or a serum replacement product; fibronectin; laminin (e.g., laminin-521);collagen (e.g., Type I collagen, Type II collagen, Type IV collagen, or any combination thereof); and tropoelastin, said coating optionally provided in an aqueous solution (e.g., comprising saline such as phosphate buffered saline (PBS) or other calcium-free and magnesium-free salt solution, and / or comprising alcohol such as ethanol or methanol) for application to the cell culture plate.

24. The cell culture plate coating of claim 23, wherein the serum replacement product is knockout serum (e.g., Cell Therapy System (CTS™) KnockOut Serum Replacement (SR™)), engineered serum, and / or human platelet extract.

25. The cell culture plate coating of claim 23 or 24, wherein the cell culture plate coating is free of serum and / or animal products.

26. The cell culture plate coating of any one of claims 23-25, wherein the collagen comprises Type I collagen and Type IV collagen.

27. The cell culture plate coating of claim 26, wherein: the saline is present in a concentration from about 25% to about 75% by volume of the cell culture plate coating; the serum or serum replacement product is present in a concentration from about 25% to about 75% by volume of the cell culture plate coating; the fibronectin is present in a concentration from about 0.05 to about 2500 pg / mL; the laminin is present in a concentration from about 0.05 to about 2500 pg / mL; the Type I collagen is present in a concentration from about 0.05 to about 2500 pg / mL; the Type IV collagen is present in a concentration from about 0.05 to about 2500 pg / mL; and the tropoelastin is present in a concentration from about 0.05 to about 2500 pg / mL.

28. A method of culturing a mesoderm lineage primary cell, said method comprising incubating said mesoderm cell with the cell culture medium of any one of claims 1-17 in a cell culture dish, flask, well, and / or plate.

29. The method of claim 28, wherein the mesoderm lineage primary cell is a renal epithelial cell, a dermal fibroblast, a mesenchymal stem cell (MSC) (e.g., bone marrow MSC (BM-MSC)), a skeletal muscle cell (e.g., skeletal muscle stem or progenitor cell), a chondrocyte, a cardiac myocyte, an osteoblast, a microvascular endothelial cell (MEC), an umbilical vein endothelial cell (UVEC), or a smooth muscle cell (e.g., bronchial smooth muscle cell (BSMC)).

30. A method of culturing an ectoderm lineage primary cell, said method comprising incubating said ectoderm cell with the cell culture medium of any one of claims 1-8 or 18-20 in a cell culture dish, flask, well, and / or plate.

31. The method of claim 30, wherein the ectoderm lineage primary cell is an epithelial cell (e.g., mammary epithelial cell (MEC)), a follicle dermal papilla cell (FDPC-Hair), a melanocyte (e.g., normal epidermal melanocytes (NEM), NEM-juvenile), an epidermal cell (e.g., normal epidermal keratinocytes (NEK), NEK-epidermis), or an astrocyte.

32. A method of culturing an endoderm lineage primary cell, said method comprising incubating said endoderm cell with the cell culture medium of any one of claims 1-8 or 18-20 in a cell culture dish, flask, well, and / or plate.

33. The method of claim 32, wherein the endoderm lineage primary cell is an epithelial cell (e.g., prostate epithelial cell, colonic epithelial cell, mammary epithelial cell, bladder epithelial cell, small airway epithelial cell.

34. A method of culturing a tissue construct comprising primary cell types from multiple lineages (e.g., from two or more of a mesoderm, ectoderm and endoderm lineage), said method comprising incubating said tissue construct with the cell culture medium of any one of claims 1- 8, 18, 21, or 22 in a cell culture dish, flask, well, and / or plate.

35. A method of culturing a 3D organoid or tissue comprising multiple primary cell types, said method comprising incubating said 3D organoid or tissue with the cell culture medium of any one of claims 1-8, 18, 21, or 22.

36. The method of any one of claims 28-31, wherein the cell culture dish and / or plate is coated with the cell culture plate coating of any one of claims 23-27.

37. A method of measuring cell growth, said method comprising a) obtaining cells (e.g., primary cells) from a subject; b) incubating said cells with a cell culture medium (e.g., the cell culture medium of any one of claims 1-22) for a predetermined amount of time; and c) counting the cell number after said amount of time; thereby measuring the cell growth.

38. The method of claim 37, wherein the cell culture dish and / or plate is coated with the cell culture plate coating of any one of claims 23-27.

39. The method of claim 37 or 38, wherein counting the cell number comprises counting the cell number using bright field imaging (e.g., percent phase confluence) and / or label free nuclear counting (e.g., Cell-by-Cell counting).

40. A method of selecting an optimal cell growth media and / or cell culture plate coating for donor cells, said method comprising: a) measuring cell growth according to any one of claims 37-39, and / or other cell culture parameters; b) repeating step a) using a different cell culture medium and / or different cell culture plate coating; and c) selecting one or more cell culture media and / or the cell culture plate coatings that provide the fastest growth rate for the donor cells and / or another desired outcome (e.g., proliferation rate, cell number, cell health, population doublings per day, etc.); thereby selecting the optimal cell growth media and / or cell culture plate coating.

41. The method of claim 40, wherein the repeating step is carried out between 1 and 12 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times) by providing a first array of different cell culture media and / or different cell culture plate coatings, and incubating the cells with each of the different cell culture media and / or cell culture plate coatings for a predetermined amount of time.

42. The method of claim 41, wherein the method further comprises: d) providing a second array comprising different concentrations of cell culture medium additives, optionally in combination with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) cell culture media and / or cell culture plate coatings selected from the first array; e) incubating the cells with each of the different concentrations of additives of the second array for a predetermined amount of time; and f) selecting one or more concentrations of additives that provide the fastest growth rate for the donor cells and / or another desired outcome (e.g., proliferation rate, cell number, cell health, population doublings per day, etc.).

43. The method of any one of claims 40-42, wherein step a) further comprises counting the cell number using bright field imaging (e.g., percent phase confluence) and label free nuclear counting (e.g., Cell-by-Cell counting).

44. The method of claim 43, wherein selecting the cell culture medium, the cell culture plate coating, and / or the concentration of additives that provides the fastest growth rate for the donor cells further comprises selecting the cell culture medium and / or the cell culture plate coating that provides the fastest growth rate for the donor cells based on the average of counting the cell number using bright field imaging (e.g., percent phase confluence) and label free nuclear counting (e.g., Cell-by-Cell counting).

45. The method of any one of claims 40-44, wherein the other desired outcome is the cell phenotype (e.g., cell size, cell shape, cell texture, organelle size, organelle number, organelle shape, uniformity of cell size in culture, uniformity of cell shape in culture, and / or arrangement of the cells in culture).

46. The method of claim 45, wherein the cell phenotype is determined using cell imaging, proteomics, genomics, epigenomics, transcriptomics, and / or metabolomics.

47. The method of claim 45 or 46, wherein the desired cell phenotype is an epithelial cell phenotype, an endothelial cell phenotype, an epidermal cell phenotype, a fibroblast cell phenotype, a mesenchymal cell phenotype, a stem cell phenotype, a muscle cell phenotype, an astrocyte cell phenotype, a neuronal cell phenotype, or a stem cell phenotype.

48. A cell culture medium additive composition comprising: a neuronal and / or stem cell growth supplement (e.g., B-27) or a mimetic thereof;NEAA; an antioxidant and / or essential vitamin (e.g., ascorbic acid, a tocopherol (e.g., a-tocopherol, P-tocopherol, y-tocopherol, and / or 5-tocopherol), glutathione, and / or N-acetyl-L-cysteine); epinephrine; a corticosteroid (e.g., hydrocortisone) and / or a glucocorticoid; andEGF or a mimetic thereof.

49. The cell culture medium additive composition of claim 48, wherein the NEAA comprises 2, 3, 4, 5 or more of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

50. The cell culture medium additive composition of claim 49, wherein each amino acid in the NEAA is present in the additive composition in a concentration of about 1 to about 10 mM.

51. The cell culture medium additive composition of any one of claims 48-50, wherein the neuronal and / or stem cell growth supplement is a neuronal growth supplement (e.g., xeno free B- 27).

52. The cell culture medium additive composition of any one of claims 48-51, wherein the antioxidant and / or essential vitamin is ascorbic acid.

53. The cell culture medium additive composition of any one of claims 48-52, wherein the corticosteroid and / or glucocorticoid is hydrocortisone.

54. The cell culture medium additive composition of any one of claims 48-53, wherein the ascorbic acid is present in a concentration from about 0.5 to about 5 mg / mL;55. The cell culture medium additive composition of claim 54, wherein: the neuronal and / or stem cell growth supplement or a mimetic thereof is present in the composition in a concentration from about 25% to about 75% by volume;the epinephrine is present in the composition in a weight ratio relative to the ascorbic acid from about 1 :50 to about 1 :500 (epinepherine : ascorbic acid); the hydrocortisone is present in the composition in a weight ratio relative to the ascorbic acid from about 1 :5 to about 1 : 100 (hydrocortisone : ascorbic acid); and / or the EGF or a mimetic thereof is present in the composition in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 100,000 (EGF : ascorbic acid).

56. The cell culture medium additive composition of any one of claims 48-55, wherein the composition is free of serum and / or animal products.

57. A supplement useful for the cell culture medium additive composition of any one of claims 48-56, wherein the supplement comprises:HGF or a mimetic thereof;PDGF or a mimetic thereof (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof); andFGF or a mimetic thereof.

58. The supplement of claim 57, wherein the FGF is FGF-basic.

59. The supplement of claim 58, wherein, when added to the cell culture medium additive composition: the HGF or a mimetic thereof is present in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 10,000 (HGF : ascorbic acid); the PDGF or a mimetic thereof (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof) is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (PDGF : ascorbic acid); and the FGF-basic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF-basic : ascorbic acid).

60. The supplement of any one of claims 57-59, wherein the supplement further comprises PLT.

61. The supplement of claim 60, wherein the PLT is devoid of heparin.

62. The supplement of claim 60 or 61, wherein the PLT is present in a concentration from about 20% to about 60% by volume of the additive composition.

63. A supplement useful for the cell culture medium additive composition of any one of claims 48-56, comprising:VEGF or a mimetic thereof (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof);IGF or a mimetic thereof (e.g., IGF-1); and F GF -acidic.

64. The supplement of claim 63, wherein, when added to the cell culture medium additive composition: the VEGF or a mimetic thereof is present in a weight ratio relative to the ascorbic acid from about 1 : 10,000 to about 1 : 150,000 (VEGF : ascorbic acid); the IGF or a mimetic thereof (e.g., IGF-1) is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 :5,000 (IGF-1 : ascorbic acid); and the FGF-acidic is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 :5,000 (FGF-acidic : ascorbic acid).

65. A supplement useful for the cell culture medium additive composition of any one of claims 48-56, comprising: prolactin; prostaglandin El; and retinoic acid.

66. The supplement of claim 65, wherein, when added to the cell culture medium additive composition: the prolactin is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (prolactin : ascorbic acid); the prostaglandin El is present in a weight ratio relative to the ascorbic acid from about 1 :5,000,000 to about 1 :50,000,000 (prostaglandin El : ascorbic acid); andthe retinoic acid is present in a weight ratio relative to the ascorbic acid from about 1 : 100,000 to about 1 :800,000 (retinoic acid : ascorbic acid).

67. A supplement useful for the cell culture medium additive composition of any one of claims 48-56, comprising:HGF or a mimetic thereof;PDGF or a mimetic thereof (e.g., PDGF-aa, PDGF-bb, PDGF-ab, or any combination thereof);FGF or a mimetic thereof (e.g., FGF-basic); prolactin; prostaglandin El; retinoic acid;VEGF or a mimetic thereof (e.g., VEGF-165, VEGF-D, VEGF-121, or any combination thereof);IGF or a mimetic thereof (e.g., IGF-1);FGF -acidic; and optionally, heparin or a mimetic thereof.

68. The supplement of claim 67, wherein, when added to the cell culture medium additive composition: the HGF or a mimetic thereof is present in a weight ratio relative to the ascorbic acid from about 1 : 1,000 to about 1 : 10,000 (HGF : ascorbic acid); the PDGF or a mimetic thereof is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (PDGF : ascorbic acid); the FGF or a mimetic thereof (e.g., FGF-basic) is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF-basic : ascorbic acid); the prolactin is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (prolactin : ascorbic acid); the prostaglandin El is present in a weight ratio relative to the ascorbic acid from about 1 :5,000,000 to about 1 :50,000,000 (prostaglandin El : ascorbic acid); the retinoic acid is present in a weight ratio relative to the ascorbic acid from about 1 : 100,000 to about 1 :800,000 (retinoic acid : ascorbic acid);the VEGF or a mimetic thereof is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (VEGF : ascorbic acid); the IGF or a mimetic thereof (e.g., IGF-1) is present in a weight ratio relative to the ascorbic acid from about 1 :500 to about 1 : 10,000 (IGF-1 : ascorbic acid); the FGF-acidic is present in a weight ratio relative to the ascorbic acid from about 1 :2,000 to about 1 :20,000 (FGF-acidic : ascorbic acid); and / or the heparin or a mimetic thereof is present in a concentration from about 0.1 to about 5 units / mL.

69. A cell culture medium comprising: a basal cell culture medium (e.g., Dulbecco's Modified Eagle Medium with Nutrient Mixture F 12 (DMEM / F12)); and the additive composition of any one of claims 48-56.

70. A cell culture medium comprising: a basal cell culture medium (e.g., DMEM / F12); the additive composition of any one of claims 48-56; and the supplement of any one of claims 57-59.

71. A cell culture medium comprising: a basal cell culture medium (e.g., DMEM / F12); and the additive composition of any one of claims 48-56; and the supplement of any one of claims 60-62.

72. A cell culture medium comprising: a basal cell culture medium (e.g., DMEM / F1)); and the additive composition of any one of claims 48-56; the supplement of any one of claims 57-59; and the supplement of claim 63 or 64.

73. A cell culture medium comprising: a basal cell culture medium (e.g., defined keratinocyte serum-free (KSF) medium); and the additive composition of any one of claims 48-56; andthe supplement of claim 67 or 68.

74. A cell culture medium comprising: a basal cell culture medium (e.g., defined KSF medium); and the additive composition of any one of claims 48-56; and the supplement of claim 65 or 66.

Images