Culture medium suitable for culturing ostrich cells

Abstract

The disclosure provides for the culture media particularly suitable to culture primary ostrich PGCs. The culture media contain specific growth factors and compounds that provide support for the growth and proliferation of ostrich PGCs. In certain embodiments, the culture media contain a basal culture medium and β-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In some embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and an antibiotic. The disclosure also includes methods and kits for producing such media as well as methods of culturing cells.

Description

Attorney Docket No. 069296.11163-33WO1CULTURE MEDIUM SUITABLE FOR CULTURING OSTRICH CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application 63 / 716,565 (filed on November 5, 2024) which is incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] This disclosure relates to culture media particularly suitable for culturing ostrich cells, such as e.g., ostrich primary primordial germ cells. The disclosure also relates to methods of culturing ostrich cells in the media as well as to methods of making such media.BACKGROUND OF THE INVENTION

[0003] The composition of the culture media is essential for the successful derivation of primary primordial germ cells (PGCs) as it provides the ability to culture PGCs in vitro. The specific growth factors impact the overall survival and proliferation which therefore has a substantial impact on both the overall survival and performance of these cells.

[0004] There is currently no defined media for the culture of ostrich PGCs. Media formulations to culture other avian species' PGCs, for example chicken, requires specific growth factors, such as e.g., Activin A, and do not require other factors, such as e.g, cholesterol and IGF1.

[0005] What is needed is a media that provides the growth factors and specific compounds required to support the growth of ostrich PGCs.SUMMARY OF THE INVENTION

[0006] This disclosure is directed to cell culture media supplemented with growth factors and specific compounds required to support the growth of ostrich PGCs. These growth factors are: (3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1.

[0007] One embodiment is a cell culture medium suitable for culturing ostrich cells containing a basal culture medium supplemented w ith [3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In certain embodiments, the basal culture medium is supplemented with from about 0.05% to about 0.6% (w / w) of (3-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF 1.4924-3504-1904.1 1Attorney Docket No. 069296.11163-33WO1

[0008] Another embodiment is a cell culture medium containing a basal culture medium and an ostrich supplement component, wherein the ostrich supplement component only contains growth factors and specific compounds to support growth of ostrich primary primordial germ cells, and wherein the ostrich supplement component comprises P- mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In certain embodiments, the ostrich supplement component contains from about 0.05% to about 0.6% (w / w) of (3- mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF1.

[0009] In certain embodiments, the cell culture medium has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg. In other embodiments, the cell culture medium has an osmolality7of about 265 mOsm / kg. In some embodiments, the osmolality is achieved by adding sodium chloride.

[0010] In some embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and an antibiotic. In one embodiment, the antibiotic includes penicillin and streptomycin.

[0011] In other embodiments, the cell culture medium is further supplemented with serum. In some embodiments, the cell culture medium is supplemented with from about 0.05% to about 0.6% (w / w) of serum. In one embodiment, the cell culture medium is supplemented with avian serum, such as e.g., chicken serum.

[0012] In other embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and avian serum. In some embodiments, the basal culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about lOng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of avian serum. In certain embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and chicken serum. In some embodiments, the basal culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about4924-3504-1904.1 2Attorney Docket No. 069296.11163-33WO10.6% (w / w) of chicken serum. In some embodiments, the knock-out DMEM contains calcium-free DMEM, water, pyruvate. MEM vitamin solution, and MEM amino acid solution.

[0013] In other embodiments, the cell culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, avian serum, P-mercaptoethanoL cholesterol, FGFL FGF2, BMP4, and IGF1. In some embodiments, the culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; from about 0.05% to about 0.6% (w / w) of avian serum; from about 0.05% to about 0.6% (w / w) of [3-mercaptoethanol; from about 0. 1 pg / mL to about 4 pg / mL of cholesterol; from about 1 ng / ml to about 8 ng / ml of FGF1 ; from about 1 ng / ml to about 8 ng / ml of FGF2; from about 5 ng / mL to about 50 ng / mL of BMP4; and from about 10 ng / mL to about 100 ng / ml of IGF1. In certain embodiments, the cell culture medium contains knock-out DMEM. B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, chicken serum, (3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In some embodiments, the culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; from about 0.05% to about 0.6% (w / w) of chicken semm; from about 0.05% to about 0.6% (w / w) of (3-mercaptoethanol; from about 0. 1 pg / mL to about 4 pg / mL of cholesterol; from about 1 ng / ml to about 8 ng / ml of FGF1; from about 1 ng / ml to about 8 ng / ml of FGF2; from about 5 ng / mL to about 50 ng / mL of BMP4; and from about 10 ng / mL to about 100 ng / ml of IGF 1. In other embodiments, the knock-out DMEM contains calcium- free DMEM, water, pyruvate, MEM vitamin solution, and MEM amino acid solution.

[0014] In other embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and avian serum. In some embodiments, the basal culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of avian serum. In certain embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin,4924-3504-1904.1 3Attorney Docket No. 069296.11163-33WO1 and chicken serum. In some embodiments, the basal culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of chicken serum. In some embodiments, the knock-out DMEM contains calcium-free DMEM, water, pyruvate, MEM vitamin solution, and MEM amino acid solution.

[0015] In other embodiments, the cell culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, avian serum, P-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In some embodiments, the culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; from about 0.05% to about 0.6% (w / w) of avian serum; from about 0.05% to about 0.6% (w / w) of P- mercaptoethanol; from about 0.1 pg / mL to about 4 pg / rnL of cholesterol; from about 1 ng / ml to about 8 ng / ml of FGF1; from about 1 ng / ml to about 8 ng / ml of FGF2; from about 5 ng / mL to about 50 ng / mL of BMP4; and from about 10 ng / mL to about 100 ng / ml of IGF 1. In certain embodiments, the cell culture medium contains knock-out DMEM, B27 Supplement, MEM- MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, chicken serum, P-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In some embodiments, the culture medium contains: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; from about 0.05% to about 0.6% (w / w) of chicken serum; from about 0.05% to about 0.6% (w / w) of P- mercaptoethanol; from about 0.1 pg / mL to about 4 pg / mL of cholesterol; from about 1 ng / ml to about 8 ng / ml of FGF1; from about 1 ng / ml to about 8 ng / ml of FGF2; from about 5 ng / mL to about 50 ng / mL of BMP4; and from about 10 ng / mL to about 100 ng / ml of IGF 1. In other embodiments, the knock-out DMEM contains calcium-free DMEM, water, pyruvate, MEM vitamin solution, and MEM amino acid solution.

[0016] Another aspect of the disclosure is directed to a method of culturing ostrich cells, which includes culturing the ostrich cells in a culture medium as described above. In certain embodiments, the ostrich cells are ostrich primary primordial germ cells.

[0017] In certain embodiments, the method includes removing one third of the culture medium after culturing and adding back one third of fresh culture medium. This may be4924-3504-1904.1 4Attorney Docket No. 069296.11163-33WO1 carried out every' second day of culturing. In other embodiments, the method includes culturing for 30 days.

[0018] Yet another aspect of the disclosure is directed to a kit for generating a cell culture medium suitable for culturing ostrich cells, such as e.g., ostrich PGCs, containing a basal medium component and ostrich supplement component, wherein the ostrich supplement component contains (3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In certain embodiments, the ostrich supplement component contains from about 0.05% to about 0.6% (w / w) of P-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF1. In other embodiments, the basal medium component contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and chicken serum.

[0019] In certain embodiments, basal medium component includes from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of chicken serum. In other embodiments of the kit, the cell culture medium has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg. In yet another embodiment of the kit, the cell culture medium has an osmolality of about 265 mOsm / kg.

[0020] Other features and advantages of the invention will be apparent from the detailed description and examples that follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing summary', as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] FIG. 1A and FIG. IB show ostrich PGC cultures using brightfield imaging and confocal microscopy with staining for DAZL, a PGC-specific marker.4924-3504-1904.1 5Attorney Docket No. 069296.11163-33WO1DETAILED DESCRIPTION

[0024] This disclosure is based on the discovery' that by supplementing culture media with a unique combination of growth factors and compounds it is possible to generate culture media that are able to provide support for the growth and proliferation of ostrich cells, such as e.g. primary primordial germ cells (PGCs). Specifically, this disclosure is based on the discovery' that a culture medium supplemented with P-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF 1 can support the growth and proliferation of ostrich cells, such as e.g., primary primordial germ cells (PGCs).

[0025] The general description and the following detailed description are exemplary and explanatory' only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.

[0026] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.Definitions

[0027] Unless defined otherwise, all 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods, and materials are now described.

[0028] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0029] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology' as4924-3504-1904.1 6Attorney Docket No. 069296.11163-33WO1“solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0030] Each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0031] As used herein, the term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0032] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0033] As used herein, the terms “comprising,” “including,” “containing” and “characterized by” are exchangeable, inclusive, open-ended and do not exclude additional, unrecited elements or method steps. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.

[0034] Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0035] As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0036] As used herein, the phrase “B-27 Supplement” refers to the commercially available B-27 supplement which contains vitamins (Biotin, DL Alpha Tocopherol Acetate, DL Alpha- Tocopherol, Vitamin A (acetate)), proteins (BSA, fatty acid free Fraction V, Catalase, Human Recombinant Insulin, Human Transferrin, Superoxide Dismutase), and other components (Corticosterone, D-Galactose, Ethanolamine HC1, Glutathione (reduced), L-Camitine HC1, Linoleic Acid, Linolenic Acid, Progesterone, Putrescine 2HC1, Sodium Selenite, T3 (triodo- I-thyronine)).4924-3504-1904.1 7Attorney Docket No. 069296.11163-33WO1

[0037] As used herein, the phrase “EmbryoMax® Nucleosides’" refers to the commercially available nucleosides sold under Sigma-Aldrich Catalogue Number ES-008-D. The components of 100 X EmbryoMax® Nucleosides are listed in the table below.

[0038] As used herein, the phrase “IX MEM-MEAA” refers to a IX mixture of MEM Vitamin solution and MEM amino acid (MEAA). MEM vitamin solution contains choline chloride, D-calcium panthotenate, folic acid, nicotinamide, pyridoxial hydrochloride, thiamine hydrochloride, i-lnositol, and sodium chloride. MEAA contains the essential amino acids (except L-glutamine). MEAA contains L-Arginine hydrochloride, L-Cystine, L- Histidine hydrochloride-FEO, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L- Methionine, L-Phenylalanine, L-Threonine, L-Tr ptophan, L-Tyrosine, and L-V aline. The components of 100 X MEM Vitamin Solution are listed in the table below:4924-3504-1904.1 8Attorney Docket No. 069296.11163-33WO1The components of 50% MEM Amino Acids Solution are listed in the table below:

[0039] Before certain embodiments are described in greater detail, it is to be understood that this invention is not limited to certain embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherw ise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0041] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.4924-3504-1904.1 9Attorney Docket No. 069296.11163-33WO1

[0042] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Culture media

[0043] One aspect of the disclosure is directed to a culture medium containing a unique combination of growth factors and compounds to support the grow th of ostrich cells, such as e.g. ostrich PGCs. In particular, this aspect of the disclosure is directed to a culture medium supplemented with (3-mercaptoethanol, cholesterol, Fibroblast Growth Factor 1 (FGF1), Fibroblast Growth Factor 2 (FGF2), Bone Morphogenetic Protein 4 (BMP4), and Insulin-like growth factor 1 (IGF1).

[0044] One embodiment of the disclosure is a cell culture medium suitable for culturing ostrich cells compnsing a basal culture medium supplemented with (3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In certain embodiments, the culture media lack activin A and / or GDF-8.

[0045] In certain embodiments, the basal culture medium is supplemented with about 0.2% (w / w), alternatively from about 0.05% to about 0.6% (w / w), alternatively from about 0.05% to about 0.5% (w / w), alternatively from about 0.1% to about 0.35% (w / w) of [3- mercaptoethanol.

[0046] In other embodiments, the basal culture medium is supplemented with about 1 pg / rnL, alternatively from about 0. 1 pg / mL to about 4 pg / rnL, alternatively from about 0. 1 pg / rnL to about 3 pg / rnL, alternatively from about 0.5 pg / mL to about 1.5 pg / mL of cholesterol.

[0047] In further embodiments, the basal culture medium is supplemented with about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF1.4924-3504-1904.1 10Attorney Docket No. 069296.11163-33WO1

[0048] In other embodiments, the basal culture medium is supplemented with about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF2.

[0049] In additional embodiments, the basal culture medium is supplemented with about 25 ng / mL, alternatively from about 5 ng / mL to about 50 ng / mL, alternatively from about 5 ng / mL to about 40 ng / mL, alternatively from about 10 ng / mL to about 40 ng / mL of BMP4.

[0050] In alternate embodiments, the basal culture medium is supplemented with about 50 ng / mL, alternatively from about 10 ng / mL to about 100 ng / ml, alternatively from about 25 ng / mL to about 75 ng / ml, alternatively from about 40 ng / ml to about 60 ng / ml of IGF1.

[0051] In one embodiment, the basal culture medium is supplemented with: about 0.2% (w / w), alternatively from about 0.05% to about 0.6% (w / w), alternatively from about 0.05% to about 0.5% (w / w), alternatively from about 0.1% to about 0.35% (w / w) of (3-mercaptoethanol; about 1 pg / mL, alternatively from about 0. 1 pg / mL to about 4 pg / mL, alternatively from about 0.1 pg / mL to about 3 pg / mL. alternatively from about 0.5 pg / mL to about 1.5 pg / mL of cholesterol; about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF1; about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF2; about 25 ng / mL, alternatively from about 5 ng / rnL to about 50 ng / mL, alternatively from about 5 ng / mL to about 40 ng / mL, alternatively from about 10 ng / mL to about 40 ng / mL of BMP4; and about 50 ng / mL, alternatively from about 10 ng / mL to about 100 ng / ml, alternatively from about 25 ng / mL to about 75 ng / ml, alternatively from about 40 ng / ml to about 60 ng / ml of IGF 1.

[0052] In another embodiment, the basal culture medium is supplemented with from about 0.05% to about 0.6% (w / w) of [3-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF 1.

[0053] In certain embodiments, the culture medium supplemented with (3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1 has an osmolality of from about 250 mOSm / kg4924-3504-1904.1 11Attorney Docket No. 069296.11163-33WO1 to about 280 mOsm / kg, alternatively about 265 mOsm / kg. In some embodiments, the osmolality is achieved by adding sodium chloride.

[0054] Another embodiment of the disclosure is a cell culture medium containing a basal culture medium and an ostrich supplement component, wherein the ostrich supplement component contains growth factors and specific compounds to support growth of ostrich primary primordial germ cells, and wherein the ostrich supplement component contains [3- mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In certain embodiments, the ostrich supplement component only contains growth factors and specific compounds to support growth of ostrich primary primordial germ cells. In other embodiments, the ostrich supplement component only contains [3-mercaptoethanol. cholesterol, FGF1. FGF2, BMP4, and IGF1 .

[0055] In certain embodiments, the ostrich supplement component contains about 0.2% (w / w), alternatively from about 0.05% to about 0.6% (w / w), alternatively from about 0.05% to about 0.5% (w / w), alternatively from about 0.1% to about 0.35% (w / w) of (3- mercaptoethanol.

[0056] In other embodiments, the ostrich supplement component contains about 1 pg / mL. alternatively from about 0. 1 pg / mL to about 4 pg / mL, alternatively from about 0. 1 pg / mL to about 3 pg / mL. alternatively from about 0.5 pg / mL to about 1.5 pg / mL of cholesterol.

[0057] In alternative embodiments, the ostrich supplement component contains about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF1.

[0058] In further embodiments, the ostrich supplement component contains about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF2.

[0059] In alternate embodiments, the ostrich supplement component contains about 25 ng / mL, alternatively from about 5 ng / mL to about 50 ng / mL, alternatively from about 5 ng / mL to about 40 ng / mL, alternatively from about 10 ng / mL to about 40 ng / mL of BMP4.

[0060] In some embodiments, the ostrich supplement component contains about 50 ng / rnL, alternatively from about 10 ng / mL to about 100 ng / ml, alternatively from about 25 ng / mL to about 75 ng / ml, alternatively from about 40 ng / ml to about 60 ng / ml of IGF 1.

[0061] In certain embodiments, the ostrich supplement component contains:4924-3504-1904.1 12Attorney Docket No. 069296.11163-33WO1 about 0.2% (w / w), alternatively from about 0.05% to about 0.6% (w / w), alternatively from about 0.05% to about 0.5% (w / w), alternatively from about 0. 1% to about 0.35% (w / w) of P-mercaptoethanol; about 1 pg / mL, alternatively from about 0.1 g / mL to about 4 pg / L. alternatively from about 0.1 pg / mL to about 3 pg / mL. alternatively from about 0.5 pg / mL to about 1.5 pg / mL of cholesterol; about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF1; about 4 ng / mL, alternatively from about 1 ng / ml to about 8 ng / ml, alternatively from about 2 ng / ml to about 6 ng / ml, alternatively from about 3 ng / ml to about 5 ng / ml of FGF2; about 25 ng / mL, alternatively from about 5 ng / mL to about 50 ng / mL, alternatively from about 5 ng / mL to about 40 ng / mL, alternatively from about 10 ng / mL to about 40 ng / mL of BMP4; and about 50 ng / mL, alternatively from about 10 ng / mL to about 100 ng / ml, alternatively from about 25 ng / mL to about 75 ng / ml, alternatively from about 40 ng / ml to about 60 ng / ml of IGF 1.

[0062] In one embodiment, the ostrich supplement component contains from about 0.05% to about 0.6% (w / w) of P-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF 1.

[0063] In certain embodiments, the culture medium containing the basal culture medium and the ostrich supplement component has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg, alternatively about 265 mOsm / kg. In some embodiments, the osmolality is achieved by adding sodium chloride.

[0064] A variety of different basal culture media can be used in the cell culture medium supplemented with P-mercaptoethanol, cholesterol. FGF1, FGF2, BMP4, and IGF1 and in the cell culture medium supplemented with the ostrich supplement component.

[0065] In certain embodiments, the basal culture media are based on knock-out DMEM. In one embodiment, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, nucleosides (cytidine, guanosine, uridine, adenosine, and thymidine), sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and an antibiotic.4924-3504-1904.1 13Attorney Docket No. 069296.11163-33WO1

[0066] In certain embodiments, the nucleosides are provided in the form of commercially available mixture such as EmbryoMax® Nucleosides. In certain embodiments, cytidine, guanosine, uridine, adenosine, and thymidine are the only nucleosides added to the basal culture medium.

[0067] As noted above, MEM-MEAA is a mixture of MEM Vitamin Solution and MEM amino acid. MEM-MEAA contains choline chloride, D-calcium panthotenate, folic acid, nicotinamide, pyridoxial hydrochloride, thiamine hydrochloride, i-Inositol, sodium chloride, and the essential amino acids (except L-glutamine). In certain embodiments, MEM-MEAA contains choline chloride, D-calcium panthotenate, folic acid, nicotinamide, pyridoxial hydrochloride, thiamine hydrochloride, i-Inositol, sodium chloride, L-Arginine hydrochloride, L-Cystine, L-Histidine hydrochloride-LLO, L-Isoleucine, L-Leucine, L- Lysine hydrochloride, L-Methionine, L-Phenyl alanine, L-Threonine, L-Tr ptophan, L- Tyrosine, and L-V aline. In certain embodiment, the vitamins, inorganic salts, and essential amino acids in MEM-MEAA can be supplied individually or as different mixture of components.

[0068] In one embodiment, the L-alanyl-L-glutamine dipeptide is Glutamax. Accordingly, in certain embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and an antibiotic.

[0069] In one embodiment, the basal culture medium contains about 0. 15 mM, alternatively from about 0.01 mM to about 0.5 mM, alternatively from about 0.05 mM to about 0.3 mM, alternatively from about 0. 1 mM to about 0.2 mM of calcium chloride.

[0070] In another embodiment, the basal culture medium contains about 50 ng / mL, alternatively from about 10 ng / mL to about 100 ng / ml, alternatively from about 25 ng / mL to about 75 ng / ml, alternatively from about 40 ng / ml to about 60 ng / ml of ovotransferrin.

[0071] In alternate embodiments, the basal culture medium contains about 0.15 mM, alternatively from about 0.01 mM to about 0.5 mM, alternatively from about 0.05 mM to about 0.3 mM, alternatively from about 0.1 mM to about 0.2 mM of calcium chloride and about 50 ng / mL, alternatively from about 10 ng / mL to about 100 ng / ml, alternatively from about 25 ng / mL to about 75 ng / ml, alternatively from about 40 ng / ml to about 60 ng / ml of ovotransferrin.

[0072] In certain embodiments, the basal medium is supplemented with serum, such as e. , avian serum. In some embodiments, the basal medium is supplemented with about 0.2 %4924-3504-1904.1 14Attorney Docket No. 069296.11163-33WO1(w / w), alternatively from about 0.05% to about 0.6% (w / w), alternatively from about 0.05% to about 0.5% (w / w), alternatively from about 0.1% to about 0.35% (w / w) of serum (e.g, avian serum).

[0073] In one embodiment, the basal culture medium is supplemented with chicken serum. In certain embodiments, the basal culture medium is supplemented with about 0.2 % (w / w), alternatively from about 0.05% to about 0.6% (w / w). alternatively from about 0.05% to about 0.5% (w / w), alternatively from about 0. 1% to about 0.35% (w / w) of chicken serum. In other embodiments, the basal culture medium is supplemented with chicken serum and also contains about 0.15 mM, alternatively from about 0.01 mM to about 0.5 mM, alternatively from about 0.05 mM to about 0.3 mM. alternatively from about 0. 1 mM to about 0.2 mM of calcium chloride and about 50 ng / mL, alternatively from about 10 ng / mL to about 100 ng / ml, alternatively from about 25 ng / mL to about 75 ng / ml, alternatively from about 40 ng / ml to about 60 ng / ml of ovotransferrin.

[0074] A variety of different antibiotics suitable to prevent bacterial contamination can be used. In some embodiments, the antibiotic is penicillin and streptomycin.

[0075] Accordingly, in certain embodiments, the basal culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, an antibiotic, and chicken serum. In other embodiments, the basal culture medium contains knock-out DMEM. B27 Supplement, MEM-MEAA. Glutamax. cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, penicillin, streptomycin, and chicken serum.

[0076] In certain embodiments, the cell culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, an antibiotic (e.g. penicillin and streptomycin), serum, 0-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In other embodiments, knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, an antibiotic (e.g., penicillin and streptomycin), avian serum, 0- mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In further embodiments, the cell culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl- L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate,4924-3504-1904.1 15Attorney Docket No. 069296.11163-33WO1 ovalbumin, sodium heparin, calcium chloride, ovotransferrin, an antibiotic (e.g. penicillin and streptomycin), chicken serum. P-mercaptoethanol, cholesterol, FGF1, FGF2. BMP4, and IGF1. In certain embodiments, the cell culture medium components are present in the amounts listed in the Table below. Any of the various amounts listed in each of embodimentsA1-A4 can be combined.4924-3504-1904.1 16Attorney Docket No. 069296.11163-33WO1

[0077] In alternate embodiments, the cell culture medium contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, an antibiotic (e.g. penicillin and streptomycin), serum, [3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In other embodiments, knock-out DMEM, B27 Supplement, MEM- MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, an antibiotic e.g, penicillin and streptomycin), avian serum, [3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In further embodiments, the cell culture medium contains knock-out DMEM, B27Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, an antibiotic (e.g. penicillin and streptomycin), chicken serum, [3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1. In certain embodiments, the cell culture medium components are present in the amounts listed in the Table below. Any of the various amounts listed in each of embodiments B1-B4 can be combined.4924-3504-1904.1 17Attorney Docket No. 069296.11163-33WO1

[0078] In certain embodiments, the media of the disclosure contain knock-out DMEM as a base media, sodium chloride to increase the osmolality to 265 mOsm / kg, IX B27 supplement, IX MEM-MEAA, IX Glutamax, IX EmbryoMax® Nucleosides (cytidine, guanosine, uridine, adenosine, and thymidine), sodium pyruvate, ovalbumin, sodium heparin, pen / strep, about 0.15 mM calcium chloride, about 50 ng / mL ovotransferrin, about 0.2 % chicken serum, about 0.2% P-mercaptoethanol, about 1 pg / mL cholesterol, about 4 ng / mL FGF1, about 4 ng / mL FGF2, about 25 ng / mL BMP4, and about 50 ng / mL IGF1.

[0079] In other embodiments, the knock-out DMEM can be commercially purchased. Alternatively, it can be prepared in-house. The in-house knock-out DMEM contains calcium4924-3504-1904.1 18Attorney Docket No. 069296.11163-33WO1 free DMEM, approximately 23% tissue culture grade water, pyruvate, MEM vitamin solution, and MEM Amino Acid Solution.

[0080] In certain embodiments, L-glutamine is used instead of Glutamax / L-alanyl-L- glutamine dipeptide.Kits

[0081] The disclosure also includes kits for generating the cell culture media of the disclosure. One embodiment is a kit for generating a cell culture medium suitable for culturing ostrich containing a basal medium component and ostrich supplement component, whereby the ostrich supplement component contains 0-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1.

[0082] In certain embodiments, the ostrich supplement component comprises from about 0.05% to about 0.6% (w / w) of (3-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2. from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF1. In other embodiments, any of the other amounts of [I- mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1 described above can be used.

[0083] In further embodiments, the basal medium component contains knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and chicken serum. In certain embodiments, the chicken serum is replaced with other serum such as e.g, other avian serum. In some embodiments, the basal medium component contains from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of chicken serum. In certain embodiments, the cell culture medium has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg. In other embodiments, the cell culture medium has an osmolality of about 265 mOsm / kg. In certain embodiments, any of the other amounts of calcium chloride, ovotransferrin, and serum described above can be used.

[0084] Another aspect of the disclosure is related to a kit containing ostrich PGCs and the culture media of the disclosure.Method of seneratins the culture media

[0085] Yet another aspect of the disclosure is directed to methods of generating the culture media of the disclosure. The methods of generating the culture media of the disclosure generally include generating the basal medium and the ostrich supplement, each of which has4924-3504-1904.1 19Attorney Docket No. 069296.11163-33WO1 the features described above. The basal medium and ostrich supplement are mixed together to form a culture medium of the disclosure. In other embodiments, the components of the ostrich supplement are added individually to the basal medium.

[0086] In certain embodiments, the method of generating the culture media includes first preparing a basal medium which contains knock-out DMEM as abase media, sodium chloride to increase the osmolality to 265 mOsm / kg, IX B27 supplement, IX MEM-MEAA, IX Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, pen / strep, about 0.15 mM calcium chloride, about 50 ng / mL ovotransferrin, and about 0.2 % chicken serum. In certain embodiments, this basal medium can be stored for up to one month. The media of the disclosure can be made from the basal medium, to which about 0.2% (3-mercaptoethanol, about 1 pg / mL cholesterol, about 4 ng / mL FGF1, about 4 ng / mL FGF2, about 25 ng / mL BMP4, and about 50 ng / mL IGF1 are added. In certain embodiments, the medium can be stored for up to two weeks.Methods of Culturing Ostrich Cells

[0087] Yet another aspect of the disclosure is directed to method of culturing ostrich cells using the culture media of the disclosure. In certain embodiments, the ostrich cells are PGCS.

[0088] In certain embodiments, the method includes removing one third of the culture medium after culturing and adding back one third of fresh culture medium.

[0089] The cells can be cultured for a suitable amount of time. In certain embodiments, the culturing includes culturing the cells for 30 days.

[0090] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.4924-3504-1904.1 20Attorney Docket No. 069296.11163-33WO1EXAMPLESExample 1 - Methods of Culturing Primary PGC

[0091] In this example, the suitability of an example of a culture media of the disclosure to culture primary PGC was investigated.Generation of Culture medium

[0092] The culture medium is generated by separately preparing an Avian Base medium which is turned into an Ostrich Working Medium by adding a unique combination of growth factors and specific compounds to the Avian Base medium.

[0093] First the Avian Base medium which contains Knock-out DMEM as a base media, sodium chloride to increase the osmolality to 265 mOsm / kg, IX B27 supplement, IX MEM- MEAA, IX Glutamax, 1 X EmbryoMax® Nucleosides (cytidine, guanosine, uridine, adenosine, and thymidine) , sodium pyruvate, ovalbumin, sodium heparin, pen / strep. 0.15 mM calcium chloride, 50 ng / mL ovotransferrin, and 0.2 % chicken serum, was prepared. This can be stored for up to one month. The Avian Based medium can be turned into Ostrich Working Medium by adding 0.2% (3-mercaptoethanol, 1 pg / mL cholesterol, 4 ng / mL FGF1, 4 ng / mL FGF2. 25 ng / mL BMP4, and 50 ng / mL IGF1 to the Avian Base medium.Culturing of Primary Ostrich PGC

[0094] Primary ostrich PGCs were obtained by collecting blood from an Hl 4- 17 ostrich embryo. Specifically, 2 pL blood from an HH14-17 ostrich embryo was collected and cultured in 300 pL Ostrich Working Media in a 48 well tissue culture treated tissue culture plate. Every other day, 100 pL of media was removed and 100 pL of fresh Ostrich Working Media was added. The cells were cultured at 37 °C, 5% CO2, in a humidified incubator. The culturing was continued for one month, after which PGCs were observed to be the primary cell type in the well.

[0095] The results of the culturing are shown in FIG. 1 A and FIG. IB. FIG. 1A shows brightfield images at day 19 in culture (Panel I and Panel II). FIG. IB shows immunocytochemistry and confocal imaging of DAZL (green) in ostrich PGCs at day 22 in culture. The testing establishes that is possible to culture ostrich PGCs using the media of disclosure.

[0096] While the invention has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in4924-3504-1904.1 21Attorney Docket No. 069296.11163-33WO1 the art. It is intended that the specification and examples be considered as exemplary', only, with the true scope and spirit of the invention being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.EMBODIMENTS

[0097] The invention provides also the following non-limiting embodiments.

[0098] Embodiment 1 is a cell culture medium suitable for culturing ostrich cells comprising a basal culture medium supplemented with (3-mercaptoethanol, cholesterol, FGF1, FGF2. BMP4. and IGF1.

[0099] Embodiment 2 is the cell culture medium of embodiment 1, wherein the basal culture medium is supplemented with from about 0.05% to about 0.6% (w / w) of 0- mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF 1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF1 .

[0100] Embodiment 3 is a cell culture medium comprising a basal culture medium and an ostrich supplement component, wherein the ostrich supplement component contains grow th factors and specific compounds to support growth of ostrich primary primordial germ cells, and wherein the ostrich supplement component comprises [3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1.

[0101] Embodiment 4 is the cell culture medium of embodiment 3. wherein the ostrich supplement component comprises from about 0.05% to about 0.6% (w / w) of 0- mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF1.

[0102] Embodiment 5 is the cell culture medium of any one of embodiments 1 to 4, wherein the cell culture medium has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg.

[0103] Embodiment 6 is the cell culture medium of embodiment 5, wherein the cell culture medium has an osmolality of about 265 mOsm / kg.

[0104] Embodiment 7 is the cell culture medium of embodiment 6, wherein the osmolality is achieved by adding sodium chloride.

[0105] Embodiment 8 is the cell culture medium of any one of embodiments 1 to 7, wherein the basal culture medium comprises knock-out DMEM, B27 Supplement, MEM-MEAA, L-4924-3504-1904.1 22Attorney Docket No. 069296.11163-33WO1 alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and an antibiotic.

[0106] Embodiment 9 is the cell culture medium of embodiment 8, wherein the antibiotic comprises penicillin and streptomycin.

[0107] Embodiment 10 is the cell culture medium of embodiment 9, wherein the cell culture medium is further supplemented with serum.

[0108] Embodiment 11 is the cell culture medium of embodiment 10, wherein the cell culture medium is supplemented with from about 0.05% to about 0.6% (w / w) of serum.

[0109] Embodiment 12 is the cell culture medium of embodiments 10 or 11, wherein the cell culture medium is supplemented with avian serum.

[0110] Embodiment 13 is the cell culture medium of embodiment 12, wherein the cell culture medium is supplemented with chicken serum.[OHl] Embodiment 14 is the cell culture medium of any one of embodiments 1 to 6, wherein the basal culture medium comprises knock-out DMEM, B27 Supplement, MEM- MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and chicken serum.

[0112] Embodiment 15 is the cell culture medium of embodiment 8, wherein the basal culture medium comprises: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of chicken serum.

[0113] Embodiment 16 is the cell culture medium of any one of embodiments 8 to 15, wherein the knock-out DMEM comprises calcium-free DMEM, water, pyruvate, MEM vitamin solution, and MEM amino acid solution.

[0114] Embodiment 17 is a method of culturing ostrich cells comprising culturing the ostrich cells in a culture medium of any one of embodiments 1 to 16.

[0115] Embodiment 18 is the method of embodiment 17, wherein the ostrich cells are ostrich primary primordial cells.

[0116] Embodiment 19 is the method of embodiments 17 or 18, wherein the method comprises removing one third of the culture medium after culturing and adding back one third of the culture medium of any one of embodiments 1 to 16.

[0117] Embodiment 20 is the method of any one of embodiments 17 to 19, wherein the method comprises culturing for 30 days.

[0118] Embodiment 21 is a kit for generating a cell culture medium suitable for culturing ostrich comprising a basal medium component and ostrich supplement component, wherein4924-3504-1904.1 23Attorney Docket No. 069296.11163-33WO1 the ostrich supplement component comprises [3-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and 1GF1.

[0119] Embodiment 22 is the kit of embodiment 21, wherein the ostrich supplement component comprises from about 0.05% to about 0.6% (w / w) of (3-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF1.

[0120] Embodiment 23 is the kit of embodiments 21 or 22, wherein the basal medium component comprises knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and chicken serum.

[0121] Embodiment 24 is the kit of any one of embodiments 21 to 23, wherein the basal medium component comprises from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of chicken serum.

[0122] Embodiment 25 is the kit of any one of embodiments 21 to 24, wherein the cell culture medium has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg.

[0123] Embodiment 26 is the kit of embodiment 25, wherein the cell culture medium has an osmolality of about 265 mOsm / kg.4924-3504-1904.1 24

Claims

Attorney Docket No. 069296.11163-33WO1CLAIMSWhat is claimed is:

1. A cell culture medium suitable for culturing ostrich cells comprising a basal culture medium supplemented with P-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1.

2. The cell culture medium of claim 1, wherein the basal culture medium is supplemented with from about 0.05% to about 0.6% (w / w) of P-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF 1.

3. A cell culture medium comprising a basal culture medium and an ostrich supplement component, wherein the ostrich supplement component contains growth factors and specific compounds to support growth of ostrich primary primordial germ cells, and wherein the ostrich supplement component comprises -mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1.

4. The cell culture medium of claim 3, wherein the ostrich supplement component comprises from about 0.05% to about 0.6% (w / w) of P-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / rnL to about 100 ng / ml of IGF 1.

5. The cell culture medium of any one of claims 1 to 4. wherein the cell culture medium has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg.

6. The cell culture medium of claim 5, wherein the cell culture medium has an osmolality of about 265 mOsm / kg.

7. The cell culture medium of claim 6, wherein the osmolality is achieved by adding sodium chloride.

8. The cell culture medium of claims 1 or 3, wherein the basal culture medium comprises knock-out DMEM, B27 Supplement, MEM-MEAA, L-alanyl-L-glutamine dipeptide, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and an antibiotic.4924-3504-1904.1 25Attorney Docket No. 069296.11163-33WO19. The cell culture medium of claim 8, wherein the antibiotic comprises penicillin and streptomycin.

10. The cell culture medium of claim 9, wherein the cell culture medium is further supplemented with serum.

11. The cell culture medium of claim 10, wherein the cell culture medium is supplemented with from about 0.05% to about 0.6% (w / w) of serum.

12. The cell culture medium of claim 10, wherein the cell culture medium is supplemented with avian serum.

13. The cell culture medium of claim 12, wherein the cell culture medium is supplemented with chicken serum.

14. The cell culture medium of claims 1 or 3, wherein the basal culture medium comprises knock-out DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and chicken serum.

15. The cell culture medium of claim 8, wherein the basal culture medium comprises: from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / rnL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of chicken serum.

16. The cell culture medium of claim 8, wherein the knock-out DMEM comprises calcium-free DMEM, water, pyruvate, MEM vitamin solution, and MEM amino acid solution.

17. A method of culturing ostrich cells comprising culturing the ostrich cells in a culture medium of any one of claims 1 to 4.

18. The method of claim 17, wherein the ostrich cells are ostrich primary primordial cells.

19. The method of claim 17. wherein the method comprises removing one third of the culture medium after culturing and adding back one third of the culture medium of any one of claims 1 to 4.

20. The method of claim 17, wherein the method comprises culturing for 30 days.4924-3504-1904.1 26Attorney Docket No. 069296.11163-33WO121. A kit for generating a cell culture medium suitable for culturing ostrich comprising a basal medium component and ostrich supplement component, wherein the ostrich supplement component comprises P-mercaptoethanol, cholesterol, FGF1, FGF2, BMP4, and IGF1.

22. The kit of claim 21 , wherein the ostrich supplement component comprises from about 0.05% to about 0.6% (w / w) of P-mercaptoethanol, from about 0.1 pg / mL to about 4 pg / mL of cholesterol, from about 1 ng / ml to about 8 ng / ml of FGF1, from about 1 ng / ml to about 8 ng / ml of FGF2, from about 5 ng / mL to about 50 ng / mL of BMP4, and from about 10 ng / mL to about 100 ng / ml of IGF 1.

23. The kit of claims 21 or 22, wherein the basal medium component comprises knockout DMEM, B27 Supplement, MEM-MEAA, Glutamax, cytidine, guanosine, uridine, adenosine, thymidine, sodium pyruvate, ovalbumin, sodium heparin, calcium chloride, ovotransferrin, and chicken serum.

24. The kit of claims 21 or 22, wherein the basal medium component comprises from about 0.01 mM to about 0.5 mM of calcium chloride; from about 10 ng / mL to about 100 ng / ml of ovotransferrin; and from about 0.05% to about 0.6% (w / w) of chicken serum.

25. The kit of claims 21 or 22, wherein the cell culture medium has an osmolality of from about 250 mOSm / kg to about 280 mOsm / kg.

26. The kit of claim 25, wherein the cell culture medium has an osmolality of about 265 mOsm / kg.4924-3504-1904.1 27

Images