Media and methods for sustainable cultivation of cells in suspension

Abstract

The present invention is directed to, inter alia, a method for culturing somatic cells with adherent growth properties in a suspension being support-free, wherein the somatic cells arc not genetically- or genomically-modified or -engineered, the method including contacting the somatic cells with an effective amount of a defined culture medium including: basal medium, serum replacement, and at least one non-essential amino acid (NEAA). Further provided are a defined culture medium, as well as a kit for culturing somatic cells with adherent growth propertied in a suspension being support-free.

Description

MEDIA AND METHODS FOR SUSTAINABLE CULTIVATION OF CELLS IN SUSPENSIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 632,013, titled “Media and Methods for Sustainable Cultivation of cells in Suspension”, filed 10 April 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0002] The present invention is in the field of culture media for culturing.BACKGROUND OF THE INVENTION

[0003] Cells play a crucial role in the diagnosis and cultivation of various medically significant microorganisms. They are integral to laboratory and diagnostic tests, forming the basis for studying interactions between pathogens and host cells, and are widely used as host cells for vaccine development and protein production. Given the importance of these cell lines in experimental and diagnostic laboratories, particularly for widely used cell cultures like CHO, HeLa, HEp-2, Vero, CaCo-2, 3T3, MDCK, many researchers are seeking platforms that do not involve serum or serum-derived compounds.

[0004] Today, there are several options available for different cell lines with serum or Xeno-free medium, each presenting unique features, advantages, and disadvantages.

[0005] For example, the McCoy cells line, a mouse fibroblast cell line are adherent cells with fibroblast-like morphology and can be cultured in various media, including minimum essential medium (MEM), Dulbecco’s modified eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, Medium 199, and others. The primary drawback of all these media is the inclusion of serum. To achieve suspension culture of the McCoy cell line and in other lines either co-culture with other cells is mandatory, or the cells are being genetically- modified.

[0006] There is still a great need for compositions and methods for mass growth of cells in suspension without the need for microcarriers and / or culturing transformed- or genetically modified- (or edited-) cell lines.SUMMARY OF THE INVENTION

[0007] According to the first aspect, there is provided a method for culturing somatic cells with adherent growth properties in a suspension being support-free, wherein the somatic cells arc not genetically- or genomically-modified or -engineered, the method comprising contacting the somatic cells with an effective amount of a defined culture medium comprising: basal medium, serum replacement, and at least one non-essential amino acid (NEAA).

[0008] According to another aspect, there is provided a kit comprising: (i) a defined medium comprising: (a) basal medium in an amount of 80-99% by volume of the defined medium; (b) serum replacement in an amount of 1-20% by volume of the medium; and (c) at least one NEAA in a concentration of 50 pM to 500 pM; and (ii) instructions for the culturing of somatic cells with adherent growth properties in a support-free suspension.

[0009] According to another aspect, there is provided a composition comprising the defined medium of the invention, and somatic cells with adherent growth properties.

[0010] According to another aspect, there is provided a defined medium consisting essentially of: (a) basal medium in an amount of 80-99% by volume of the defined medium; (b) serum replacement in an amount of 1-20% by volume of the medium; and (c) at least one NEAA in a concentration of 50 pM to 500 pM.

[0011] In some embodiments, the somatic cells are differentiated somatic cells.

[0012] In some embodiments, the somatic cells are not any one of: stem cells, pluripotent stem cells, and unipotent stem cells.

[0013] In some embodiments, the non-genetically- or -genomically-modified or -engineered somatic cells are adherent somatic cells when cultured under conditions of control culture medium.

[0014] In some embodiments, the somatic cells are transformed or transduced to produce at least one molecule or compound of interest.

[0015] In some embodiments, the at least one molecule or compound of interest is selected from: a recombinant protein, a micro-RNA (miRNA), an exosomes, or any combination thereof.

[0016] In some embodiments, support-free comprises scaffold-free, micro-carrier-free, or both.

[0017] In some embodiments, the method further comprises a preliminary step preceding the contacting of the somatic cells with the defined culture medium, comprising culturing the somatic cells under conditions of: (i) control medium; or (ii) a combination of the control medium andthe defined culture medium, and wherein the preliminary step comprises culturing the somatic cells on a support.

[0018] In some embodiments, the control medium comprises serum in a concentration ranging between 5% and 15%.

[0019] In some embodiments, the culturing of the somatic cells under conditions of control medium (i) being performed for a period of: 10 to 20 days, until 50-100% of the somatic cells being adhered to the support, or both.

[0020] In some embodiments, the control medium and the defined culture medium are present in the combination (ii) in a volume per volume ratio (v / v) ranging between 95:5 and 5:95.

[0021] In some embodiments, the control medium and the defined culture medium are present in the combination in (v / v) of any one of: 50:50, 25:75, 10:90, and any combination thereof.

[0022] In some embodiments, the somatic cells are cultured in any of the combinations comprising the control medium and the defined culture medium in (v / v) of any one of: 50:50, 25:75, and 10:90 for a period of at least 3 to 180 days.

[0023] In some embodiments, the basal medium is selected from the group consisting of: McCoy’s 5 A, Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Medium 199, and any combination thereof.

[0024] In some embodiments, the defined medium comprises the serum replacement in a concentration ranging between 1% and 20%.

[0025] In some embodiments, the at least one NEAA is selected from the group consisting of: glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine, tyrosine, and any combination thereof.

[0026] In some embodiments, the defined medium further comprises basal fibroblast growth factor (bFGF) in a concentration ranging between 1 ng / ml to 150 ng / ml.

[0027] In some embodiments, the somatic cells with adherent growth properties are of a cell-line selected from the group consisting of: CHO, HeLa, HEp-2, Vero, CaCo-2, 3T3, MDCK, and any combination thereof.

[0028] In some embodiments, the kit is for use in a method for culturing somatic cells with adherent growth properties in a suspension being support-free.

[0029] In some embodiments, the composition being in the form of suspension.

[0030] In some embodiments, the composition being support-free or devoid of a support.

[0031] In some embodiments, the support is a solid support.

[0032] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0033] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Fig. 1 includes a scheme of a non-limiting example of the proposed adaptation method from two-dimensional (2D) to three-dimensional (3D) suspension.

[0035] Figs. 2A-2B include graphs showing the growth rate (2A) and cell viability (2B) of adherent cells cultured over 20 days in a commercial (grey round marker) and the media of the invention (black triangle marker).

[0036] Fig. 3A-3C include representative light-microscopy images demonstrating cell morphology changes during adaptation from adherent into suspension culture, in serum-free media. (3A) Native adherent cell morphology; (3B) Loosely adherent morphology during adaptation; and (3C) Aggregates cell morphology in suspension culture. Scale bar = 100 pm.

[0037] Figs. 4A-4B include graphs showing long-term growth in suspension. (4A) Growth rate and (4B) cell viability of the McCoy cell line in micro-carrier- free suspension culture over 15 passages in 2 different recommended serum-free formulations. Media A (blue), and Media B (orange).

[0038] Figs. 5A-5B include graphs showing a suspension culture. (5A) Growth rate and (5B) cell viability of the McCoy cell line in micro-carrier-free suspension culture over 20 days in another 2 different recommended serum- free formulations. Media C (blue), and Media D (orange).

[0039] Figs. 6A-6B include graphs showing a suspension culture. (6A) Growth rate and (6B) cell viability of the McCoy cell line in micro-carrier-free suspension culture over 20 days in another 2 different recommended serum-free formulations. Media E (blue), and Media C (grey), and Media F (orange).

[0040] Figs. 7A-7F include representative light-microscopy images showing dynamic morphology changes of McCoy cell’s in the different suspension culture formulations (Media A- F). (7A-7B, and 7F) Cell aggregates; and (7C-7E) single cells / loosely aggregated. All cells were cultured in more than 16 passages. Scale bar = 100 pm.

[0041] Figs. 8A-8D include representative light-microscopy images and graphs showing cell population generated under adherent conditions in medium which consists of serum (8A), and in free-serum suspension conditions (8B). Following cell adaptation to suspension culture, cells were cultured for more than 20 days. (8C) Growth rate (8D) and cell viability of the McCoy cell line in microcarrier-free suspension culture after direct adaptation.

[0042] Figs. 9A-9D include representative light-microscopy images and graphs showing cell post thawing recovery and cell bank validation. The morphology of pre- and post-freezing McCoy cells were examined in two representative serum-free media. (9A) Cells morphology while grown in serum-free media A before freezing; (9B) after thawing. Cells were cultured over 16 passages. (9C) Growth rate; and (9D) cell viability. Media A (Blue), Media B (Orange). Scale bar = 100 pm.

[0043] Fig. 10 includes a scheme showing a non-limited proposed sequential adaptation method from 2D to 3D suspension.

[0044] Figs. 11A-11F include representative light-microscopy images showing sequential cell adaptation into suspension. (11A) Step 1 - cells cultured in treated-cultured plates with standard media (ST); adherent cell morphology was observed. (11B) Step 2 - cells cultured in untreated- cultured plates with ST media - aggregates attachment to the plate’ s surface was observed; (11C) Step 3 - cells cultured in untreated-cultured plates with 50% ST media and 50% of a medium of the invention - cells aggregates in suspension were observed); (11D) Step 4 - cells cultured in untreated-cultured plates with 25% ST and 75% of a medium of the invention - cell aggregate morphology in suspension was observed; (HE) Step 5 - cells cultured in untreated-culturedplates with 10% ST and 90% of a medium of the invention - cell aggregate morphology in suspension was observed; and (11F) Step 6 - cells cultured in untreated-cultured plates with 100% of a medium of the invention formulation; cell aggregate morphology in suspension was observed. Scale bar = 100 mm.

[0045] Figs. 12A-12B include a representative light microscopy image and a graph showing cell population growth in the presence / absence of fetuin. (12A) A representative light-microscopy image demonstrating cell morphology in serum-free medium (SFM) with 0.5 mg / ml fetuin. Scalebar = 100 pm. (12B) A growth curve of cells cultured in SFM in the presence or absence of 0.5 mg / mL fetuin. In the presence of fetuin, cells were cultured for more than 21 days, while in the absence of fetuin, culture collapsed within 7 days.DETAILED DESCRIPTION OF THE INVENTIONMethods and composition

[0046] According to the first aspect, there is provided a method for culturing a somatic cell with adherent growth properties in a suspension being support-free.

[0047] In some embodiments, the cell is not genetically- or genomically-modified or - engineered. In some embodiments, genetically or genomically-modified or engineered is for the purpose of “non-adherence”. In some embodiments, the cell may comprise a genetic modification, a mutation, an aberration, an engineered construct, a transgene, and the like, as long as any one of: genetic modification, mutation, aberration, engineered construct, transgene, and the like, renders the cell non-adhering cell. In some embodiments, the cell is a wildtype cell. In some embodiments, the cell comprises an artificial or synthetic polynucleotide. In some embodiments, the artificial or synthetic polynucleotide is or comprises a transgene. In some embodiments, the artificial or synthetic polynucleotide is integrated in a plasmid, an expression vector, or the genome of the cell. In some embodiments, the artificial or synthetic polynucleotide comprises a nucleic acid sequence of a gene of interest. In some embodiments, the gene of interest encodes a protein product. In some embodiments, the cell heterologously expresses the protein produced encoded by the gene of interest.

[0048] In some embodiments, the somatic cells are characterized or comprise at least one copy or allele of an active, functional, wild-type, or any combination thereof, of at least one gene encoding: insulin-like growth factor 4 (Igfbp4), aquaporin 1 (Aqpl), dihydrofolate reductase (DHFR), Pirin, Caveolin, AMP-activated protein Kinase, Cadherin(s), or any combination thereof.

[0049] In some embodiments, the somatic cells comprise an active, functional, wild-type, or any combination thereof, of a protein selected from: IGFBP4, Aqpl, DHFR, Pirin, Caveolin, AMP- activated protein Kinase, Cadherin(s), or any combination thereof.

[0050] In some embodiments, the somatic cells comprise a protein selected from: IGFBP4, Aqpl, DHFR, Pirin, Caveolin, AMP-activated protein Kinase, Cadherin(s), or any combination thereof, in an amount, activity unit, or both, being essentially similar, equivalent, or identical to wild-type cells or the genetic background reference of the somatic cells.

[0051] As used herein, “essentially similar or equivalent” refers to being at least 90%, 95%, 97%, or 99% similar to the wild-type cells or the genetic background reference of the somatic cells, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0052] Methods for determining presence of mutated and / or wild-type allele(s) in a cell (or a genome thereof) are common and would be apparent to one of ordinary skill in the art. Nonlimiting examples for such methods, include, but are not limited to sequencing, next generation sequencing (NGS), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), or others.

[0053] Methods for determining amount or levels of a protein and / or of a transcript encoding thereof are common and would be apparent to one of ordinary skill in the art of molecular biology and biochemistry. Non-limiting examples for such methods, include, but are not limited to PCR, RT-PCR, real-time RT-PCR, western blot, and dot blot, to name a few.

[0054] The human IGFBP4 transcript and protein are disclosed under accession nos. NM_001552.3 and NP_001543.2, respectively.

[0055] The human Aqpl transcript and protein are disclosed under accession nos. NM_198098.4 and UQX14437.1, respectively.

[0056] The human DHFR transcript and protein are disclosed under accession nos. BC003584.1 and AAH03584.2, respectively.

[0057] The human Pirin transcript and protein are disclosed under accession nos. NM_003662.4 and NP_003653.1, respectively.

[0058] The human Caveolin transcript and protein are disclosed under accession nos. Z18951.1 and CAA79476.1, respectively.

[0059] The human AMP-activated protein Kinase transcript and protein are disclosed under accession nos. NM_001355028.2 (as well as NM_001355029.2, NM_001355034.2, NM_001355035.2, NM_001355036.2, and NM_001355037.2,) and NP_001341958.1, respectively.

[0060] The above-mentioned accession nos. are retrievable from the GenBank (of the national library of medicine of the national center for biotechnology information (NCBI).

[0061] The above human sequences are provided as mere examples. In view of these examples, a person of skill in the art would easily be able to identify or locate analogous and / homologous sequences of other species.

[0062] In some embodiments, the method comprises contacting the somatic cells with an effective amount of a defined culture medium comprising: basal medium, serum replacement, and at least one non-essential amino acid (NEAA).

[0063] In some embodiments, the at least one NEAA comprises a plurality of NEAA. In some embodiments, the at least one NEAA comprises one or more NEAA.

[0064] As used herein, the term “plurality” refers to any integer being equal to or greater than 2.

[0065] In some embodiments, the somatic cells are differentiated somatic cells. In some embodiments, the somatic cells are fully differentiated somatic cells. In some embodiments, the somatic cells are not totipotent cells, pluripotent cells, multipotent cells, or unipotent cells. In some embodiments, the somatic cells are not stem cells. In some embodiments, stem cells comprises induced stem cells, such as, but not limited to, induced pluripotent stem cells (iPSC). In some embodiments, the somatic cells are not mesenchymal stem cells. In some embodiments, stem cells comprise mesenchymal stem cells.

[0066] In some embodiments, a stem cell comprises totipotent stem cell, pluripotent stem cell, unipotent stem cell, or any combination thereof.

[0067] In some embodiments, the differentiated or fully differentiated somatic cells are committed to a particular cell lineage. In some embodiments, the differentiated or fully differentiated somatic cells do not express a gene known to be a biomarker or characteristic of a stem cell. In some embodiments, the differentiated or fully differentiated somatic cells are committed cells. In some embodiments, committed is to a particular cell lineage.

[0068] In some embodiments, the differentiated or fully differentiated somatic cells are devoid of a transcript (and / or a protein product thereof) transcribed from a gene known to be a biomarker or characteristic of a stem cell, a protein product encoded / translated therefrom, or both.

[0069] In some embodiments, the non-genetically- or -genomically-modified or -engineered somatic cells are known to be adherent cells. In some embodiments, the non-genetically- or - genomically-modified or -engineered somatic cells are adherent cells when cultured under conditions of a control culture medium.

[0070] In some embodiments, the somatic cells are transformed or transduced to produce at least one product of interest. In some embodiments, the at least one product of interest is selected from: a recombinant protein or polypeptide, a transcript or a plurality thereof, an exosome, or any combination thereof.

[0071] In some embodiments, a transcript comprises an encoding RNA and / or a non-coding RNA. In some embodiments, a non-coding RNA comprises a micro RNA (miRNA).

[0072] In some embodiments, support-free comprises any scaffold-free, micro-carrier- free, or both.

[0073] As used herein, the term “support- free” refers to any method and / or type of cell culture which does not involve culturing cells on a solid support. In some embodiments, support-free culturing comprises culturing somatic cells in a suspension wherein the somatic cells are not adhered and / or bound to a surface, e.g., a solid surface, such as, but not limited to a scaffold material, a micro-carrier, or both. In some embodiments, support-free comprises a cell culture wherein cells are suspended and are free swimming, solitary cells, in loosely attached aggregates, or the like.

[0074] In some embodiments, a somatic cell cultured according to the method of the invention is in a state selected from: solitary, loosened, unattached, unfixed, unlinked, or the like.

[0075] In some embodiments, the method further comprises a step comprising culturing the somatic cells under conditions of: (i) control medium; or (ii) a combination of a control medium and the defined culture medium. In some embodiments, the step comprising culturing the somatic cells under conditions of: (i) control medium; or (ii) a combination of a control medium and the defined culture medium is a preliminary step preceding the culturing step with the defined medium of the invention. In some embodiments, the preliminary step comprises culturing the somatic cells on a support. In some embodiments, a support comprises a solid support. In someembodiments, a solid support comprises a well, a well plate, a scaffold material, a micro-carrier, or any combination thereof.

[0076] Types of solid support for in vitro or ex vivo culturing of cells in adherence are common and would be apparent to one of ordinary skill in the art.

[0077] In some embodiments, a control medium comprises serum in a concentration ranging between 1% and 20%, 2% and 20%, 3% and 20%, 5% and 20%, 5% and 15%, 10% and 20%, 1% and 15%, 1% and 10%, 7% and 20%, or 4% and 18%. Each possibility represents a separate embodiment of the invention.

[0078] In some embodiments, culturing of somatic cells under conditions of control medium is performed for a period of: 10 to 20 days, 12 to 20 days, 14 to 20 days, 16 to 20 days, 18 to 20 days, 10 to 18 days, 5 to 25 days, 7 to 20 days, or 11 to 19 days, until 50-100%, 60-100%, 70- 100%, 80-100%, 90-100%, 95-100%, or 97-100% of the somatic cells being adhered to the support, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0079] In some embodiments, the control medium and the defined culture medium are present in the combination (ii) in a volume per volume ratio (v / v) ranging between 95:5 and 5:95, 90:10 and 10:90, 85:15 and 15:85, 80:20 and 20:80, 75:25 and 25:75, 70:30 and 30:70, 65:35 and 35:65, 60:40 and 40:60, 55:45 and 45:55, or 50:50. Each possibility represents a separate embodiment of the invention.

[0080] In some embodiments, the control medium and the defined culture medium are present in the combination in (v / v) of 50:50.

[0081] In some embodiments, the control medium and the defined culture medium are present in the combination in (v / v) of 25:75.

[0082] In some embodiments, the control medium and the defined culture medium are present in the combination in (v / v) of 10:90.

[0083] In some embodiments, the somatic cells are cultured in any combination comprising the control medium and the defined culture medium as disclosed herein for a period of at least 3 days, 5 days, 7 days, 14 days, 28 days, 5 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0084] In some embodiments, the somatic cells are cultured in any combination comprising the control medium and the defined culture medium as disclosed herein for a period of 3 to 180 days, 3 to 140 days, 3 to 120 days, 3 to 90 days, 3 to 60 days, 7 to 180 days, 14 to 180 days, 30 to 180 days, 60 to 180 days, 90 to 180 days, 20 to 150 days, 14 to 180 days.

[0085] Types of basal medium for culturing somatic cells are common and would be apparent to one of ordinary skill in the art.

[0086] In some basal medium is selected from: McCoy’s 5A, Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Medium 199, Glasgow Modified Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), F12, L15 (Leibovitz) media or any combination thereof.

[0087] In some embodiments, a defined medium comprises serum replacement in a concentration ranging between 1% and 20%, 1% and 8%, 1% and 10%, 1% and 15%, 3% and 14%, 2% and 15%, or 5% and 17%. Each possibility represents a separate embodiment of the invention.

[0088] In some embodiments, the at least one NEAA is selected from: glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine, tyrosine, or any combination thereof.

[0089] In some embodiments, the defined medium further comprises basal fibroblast growth factor (bFGF). In some embodiments, the defined medium further comprises bFGF in a concentration ranging between 1 ng / ml to 150 ng / ml, 10 ng / ml to 150 ng / ml, 20 ng / ml to 150 ng / ml, 50 ng / ml to 150 ng / ml, 75 ng / ml to 150 ng / ml, 90 ng / ml to 150 ng / ml, 100 ng / ml to 150 ng / ml, 110 ng / ml to 150 ng / ml, 120 ng / ml to 150 ng / ml, 135 ng / ml to 150 ng / ml, 1 ng / ml to 10 ng / ml, 1 ng / ml to 15 ng / ml, 1 ng / ml to 25 ng / ml, 1 ng / ml to 40 ng / ml, 10 ng / ml to 100 ng / ml, 5 ng / ml to 50 ng / ml, 20 ng / ml to 100 ng / ml, 35 ng / ml to 120 ng / ml, or 2 ng / ml to 70 ng / ml.

[0090] In some embodiments, the defined medium further comprises a compound being an inhibitor of the formation of a calcium comprising crystal. In some embodiments, the inhibitor of the formation of a calcium comprising crystal comprises a polypeptide or a protein. In some embodiments, the polypeptide or protein being an inhibitor of the formation of a calcium comprising crystal comprises fetuin. In some embodiments, the defined medium further comprises fetuin. In some embodiments, the defined culture medium further comprises fetuin in a concentration ranging between 0.01 mg / ml to 10 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.1 mg / ml to 1 mg / ml, 0.3 mg / ml to 5 mg / ml, 0.2 mg / ml to 0.8 mg / ml. Each possibility represents a separate embodiment of the invention.

[0091] Types of cells with adherent growth properties are common and would be apparent to one of ordinary skill in the art.

[0092] In some embodiments, somatic cells with adherent growth properties are of a cell-line selected from: CHO, HeLa, HEp-2, Vero, CaCo-2, 3T3, MDCK, PER.C6, McCoy, CEF, HEK 293, BHK-21, MDBK.PK-15, MA104, or any combination thereof.

[0093] According to another aspect, there is provided a defined medium comprising, consisting essentially of, or consisting of: (a) basal medium in an amount of 80-99% by volume of the defined medium; (b) serum replacement in an amount of 1-20% by volume of the medium; and (c) at least one NEAA in a concentration of 50 pM to 500 pM.

[0094] In some embodiments, the defined medium further comprises P-mercaptoethanol. In some embodiments, the defined medium further comprises bFGF. In some embodiments, the defined medium further comprises Penicillin and / or Streptomycin.

[0095] In some embodiments the basal medium further comprises P-mercaptoethanol, bFGF, Penicillin and / or Streptomycin.

[0096] In some embodiments, the defined culture medium is serum-free.

[0097] In some embodiments, the defined culture medium is suitable for culturing somatic cells with adherent growth properties in a suspension being support-free.

[0098] In some embodiments, the defined culture medium comprises serum replacement in a concentration of between 1-15%, 2-14%, 5-15%, 7-13%, 6-14%, or 8-12%. Each possibility represents a separate embodiment of the invention.

[0099] In some embodiments, serum replacement comprises or is knockout serum (Ko-SR).

[0100] According to some embodiments, there is provided a composition comprising: (i) a defined culture medium of the invention; and (b) somatic cells with adherent growth properties as disclosed herein.

[0101] In some embodiments, the composition is in the form of a suspension. In some embodiments, the somatic cells are suspended in the composition. In some embodiments, the composition, suspension, or both, are support-free. In some embodiments, the composition, suspension, or both, are devoid of a support. In some embodiments, the support is a support for cells, as disclosed herein. In some embodiments, a support is a cell support, such as, for adherence in a culture in vitro or ex vivo.

[0102] As used herein, the term “consists essentially of’ denotes that a given compound or substance, constitute(s) the vast majority of the active ingredient’s portion or fraction of the composition.Kits

[0103] According to another aspect, there is provided a kit for use in culturing somatic cells with adherent growth properties in a suspension being support-free.

[0104] In some embodiments, the kit comprises: (i) a defined medium comprising, consisting essentially of, or consisting of: (a) basal medium in an amount of 80-99% by volume of the defined medium; (b) serum replacement in an amount of 1-20% by volume of the medium; and (c) at least one NEAA in a concentration of 50 pM to 500 pM; and instructions for the culturing of somatic cells with adherent growth properties in a support-free suspension.

[0105] In some embodiments, the composition further comprises an acceptable carrier.

[0106] In some embodiments, the carrier comprises a pharmaceutically acceptable carrier and / or a nutraceutically acceptable carrier.

[0107] The term “pharmaceutically acceptable carrier” as used herein refers to any of the standard pharmaceutical carriers known in the field such as sterile solutions, tablets, coated tablets, and capsules. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acids or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Examples of pharmaceutically acceptable carriers include, but are not limited to, the following: water, saline, buffers, inert, nontoxic solids (e.g., mannitol, talc). Compositions comprising such carriers are formulated by well-known conventional methods. Depending on the intended mode of administration and the intended use, the compositions may be in the form of solid, semi-solid, or liquid dosage forms, such, for example, as powders, granules, crystals, liquids, suspensions, liposomes, nano-particles, nanoemulsions, pastes, creams, salves, etc., and may be in unit-dosage forms suitable for administration of relatively precise dosages.

[0108] In some embodiments, there is provided a use of the composition of the invention.

[0109] In some embodiments, the use is selected from: therapeutics, diagnosis, recombinant protein production, transcript overexpression, exosome production and / or secretion and / or harvest, drug screening, drug / vaccine / therapeutic agent production.General

[0110] Any number range recited herein relating to any physical feature, such as sequence homology or identity, are to be understood to include any integer within the recited range, unless otherwise indicated.

[0111] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0112] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.

[0113] About refers to ±10%.

[0114] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

[0115] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0116] In the description and claims of the present application, each of the verbs, “comprise”, “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0117] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0118] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below finds experimental support in the following examples.EXAMPLES

[0119] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, bioengineering, bioprocessing, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methodsin Cellular Immunology", W. H. Freeman and Co., New York (1980); Molecular Cell Biology Berk A. et al. 8thedition; Molecular Biotechnology : Principles and Applications of Recombinant DN, Glick BR. 5thedition; Culture of Animal Cells : A Manual of Basic Technique and Specialized Applications Freshney IR, 7thedition;; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0120] The present invention is divided into several parts, as further disclosed hereinbelow.

[0121] There are provided new serum-free and micro-carrier- free media formulations for cell growth in suspension and adherent culture (McCoy cells; Figs. 4-7). Table 1 includes a list of the new serum-free and micro-carrier free media formulations of the invention. The components of media A-H are presented in Tables 2-9 below, respectively. Standard control media are presented in Tables 10-11.Method for Adapting Cell Morphology in Suspension Culture

[0122] The invention includes a method for adapting cell morphology in suspension culture by adding a minimal concentration of bFGF (4-10 ng / ml). Adaption of McCoy cell line to low-bFGF media resulted in morphology change from aggregate to single cells. This change in morphology is reversible (Fig. 7).Morphology adaption method from aggregate to single cells morphology in suspension

[0123] Cells were cultured on treated plates in standard control media containing 10% FBS (Fig. 8A), and enzymatically treated with Trypsin solution contain 0.25% EDTA (BioWest) for subsequent splitting.

[0124] Approximately 150,000 cells / ml were transferred into untreated plates and cultured in serum- free media - Media E, at 37 °C and 5% CO2. Cell aggregate morphology was observed after at least 2 passages (Fig. 7E).

[0125] Following the addition of at least 4 ng / ml bFGF to the growth media (Media F formulation), after at least 2 passages, cell morphology was transformed into a single cell in suspension (Fig. 7F).

[0126] By forcing the cells to grow in suspension, cell splitting was performed using mechanical force, without the addition of enzymatic additives.Morphology flexibility of cultured cells

[0127] The current method encompasses two main types of cultures: (i) suspension cell culture, wherein cells float freely in the culture medium, and (ii) adherent cell culture. Both conditions allow cells to grow in a vessel without microcarriers (Figs. 1-3).Serum free cell banking process

[0128] Cells were collected and centrifuged at 800 rpm for 3 minutes and resuspended with 95% serum-free growth media (Tables 2-9) with the addition of 5% DMSO. Cells were transferred into cryogenic storage vials and frozen at -80 °C in a freezing container. Following 24 hours, vials were transferred into liquid nitrogen and cryopreserved until thawing. Following at least two weeks of storage in liquid nitrogen (LN2), cells were thawed in a 37 °C water bath and resuspended with fresh media, the same serum-free media wherein they were cultured before freezing. Cells were then seeded in untreated plates and cultures for at least 5 passages (Fig. 9). The cells were grown in optimal conditions (37 °C and 5% CO2).A method of culture adaptation

[0129] Sequential adaptation was performed by reducing the serum concentration gradually (mixing standard control media with media H - 50%:50%, 25%:75%, and 10%:90%, respectively), and cells’ adaptation to low serum-free media was observed (Fig. 10).

[0130] For example, sequential adaptation from serum-containing media to low serum media was performed using CEF / DF1 cell line, as follows.

[0131] Approximately 0.5xl06of CEF cells were seeded in untreated plates in standard control media - 10% FBS in DMEM, as recommended by ATCC. The cells were grown in optimal conditions (39 °C and 5% CO2).

[0132] When aggregate attachment to the surface of the plate was observed, 50% of the cell media was replaced with 50% of the defined media formulation as disclosed herein (Media H).

[0133] After 24 hours, detachment of cell aggregates was observed.

[0134] Aggregates were split by enzymatic treatment as follows. After cell collection and centrifugation, the media was discarded. Cells were washed with PBS (Phosphate-Buffer-Saline) and 0.5 ml TrypLE was added. Cells were then incubated for 2-3 minutes in a 37 °C water bath.

[0135] Size of cell aggregate(s) can be controlled by prolonging the incubation time of enzymatic treatment (as incubation time increases, cell aggregate size decreases, data not shown).

[0136] Following enzyme inhibition, cells were resuspended with fresh media and seeded in untreated plates with 50%:50% media configuration. The cells were grown for at least 5-7 days until adapted to the new media configuration.

[0137] Cells were re-cultured every 3-5 days or when the diameter of the aggregates reached above -200 microns.

[0138] The same method was used for the next steps for the media’s adaptation (Fig. 10). Stepl - 50%:50% ratio of standard control and Media H, Step 2 - 25%:75%, Step 3 - 10%:90%, and Step 4 - 100% Media H- serum-free media.Direct Adaptation (Figs. 1-3, and 8)

[0139] Cells were cultured as adherent population on treated plates with media comprising at least 10 % FBS. McCoy cell line maintenance in 2D: as recommended by ATCC.

[0140] Splitting cells was performed using enzymatic reaction including Trypsin, after which the cells were resuspended directly into serum-free media. The cells can be seeded into untreated or treated plate directly into serum free-media without gradually decreasing serum concentration.McCoy cell line Growth in 2D culture in serum free media

[0141] Approximately 0.2xl06McCoy cells / well were seeded in 6 well-plate and suspended with G or B Media. The cells were grown in optimal conditions (37 °C and 5% CO2).

[0142] When reaching 80%-100% confluency, the cells were sub -cultivated into a new 6 well plate. As further described. The cell media was removed and discarded. The cells were rinsed with PBS (Phosphate-Buffer-Saline). Following PBS discard, 0.5 ml / well of Trypsin-EDTA solution was added to each well and incubated in 37 °C, for 1-2 minutes. Until cell detachment was observed under a microscope. One (1) ml of fresh media was added to each well to inhibit Trypsin’s activity. Following a centrifugation at 800 RPM for 3 minutes, cells were reseeded in fresh media.McCoy adaption to 3D suspension culture in serum-free media

[0143] After - 14-17 days of culturing in 2D, cells were transferred to untreated petri dishes in the same media and grown under optimal conditions. After 24 hours, aggregate cell morphology was observed.

[0144] The cells were adapted to lower serum replacement concentration by gradually replacing media with decreasing % of the KoSR serum replacement in the media, from 15% to 5%, and from 5% to 2%.

[0145] Following 24 hr of culturing, -80% of the cells were in suspension appeared either as single cells (singles morphology; Figs. 7A-7B, and 7F) or in clusters (Figs. 7C-7E).

[0146] The cells were grown in suspension over 16 passages.Table 1. List of media of the inventionTable 2. Media A of the inventionTable 3. Media B of the inventionTable 4. Media C of the inventionTable 5. Media D of the inventionTable 6. Media E of the inventionTable 7. Media F of the inventionTable 8. Media G of the inventionTable 9. Media H of the inventionTable 10. McCoy standard control mediaTable 11. CEF standard control media

[0147] Further, the inventors have cultured cells in the defined culture medium of the invention in the presence or absence of fetuin. The results show that supplementation of fetuin resulted in cells having an aggregated morphology in suspension (Fig. 12A). Further, cell cultures being deprived of or cultured in the absence of fetuin have collapsed within about 7 days (Fig. 12B). In sharp contrast, cells cultured in the defined culture medium of the invention, which was further supplemented with fetuin, were cultured for more than 21 days (Fig. 12B). Therefore, it is concluded that fetuin may further enhance or contribute to the positive attributes of the defined culture medium on cells cultured in a suspension, according to the method of the invention.

[0148] While the present invention has been particularly described, people skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims which follow.

Claims

CLAIMSWhat is claimed:

1. A method for culturing somatic cells with adherent growth properties in a suspension being support-free, wherein said somatic cells are not genetically- or genomically-modified or -engineered, the method comprising contacting said somatic cells with an effective amount of a defined culture medium comprising: basal medium, serum replacement, and at least one non-essential amino acid (NEAA).

2. The method of claim 1, wherein said somatic cells are differentiated somatic cells.

3. The method of claim 1 or 2, wherein said somatic cells are not any one of: stem cells, pluripotent stem cells, and unipotent stem cell.

4. The method of any one of claims 1 to 3, wherein said non-genetically- or - genomically-modified or -engineered somatic cells being adherent somatic cells when cultured under conditions of control culture medium.

5. The method of any one of claims 1 to 4, wherein said somatic cells are transformed or transduced to produce at least one molecule or compound of interest.

6. The method of claim 5, wherein said at least one molecule or compound of interest is selected from: a recombinant protein, a micro-RNA (miRNA), an exosomes, or any combination thereof.

7. The method of any one of claims 1 to 6, wherein said support-free comprises scaffold- free, micro-carrier-free, or both.

8. The method of any one of claims 1 to 7, further comprising a preliminary step preceding said contacting of said somatic cells with said defined culture medium, comprising culturing said somatic cells under conditions of: (i) control medium; or (ii) a combination of said control medium and said defined culture medium, and wherein said preliminary step comprises culturing said somatic cells on a support.

9. The method of claim 8, wherein said control medium comprises serum in a concentration ranging between 5% and 15%.

10. The method of claims 8 or 9, wherein said culturing of said somatic cells under conditions of control medium (i) being performed for a period of: 10 to 20 days, until 50- 100% of said somatic cells being adhered to said support, or both.

11. The method of any one of claims 8 to 10, wherein said control medium and said defined culture medium are present in said combination (ii) in a volume per volume ratio (v / v) ranging between 95:5 and 5:95.

12. The method of claim 11, wherein said control medium and said defined culture medium are present in said combination in (v / v) of any one of: 50:50, 25:75, 10:90, and any combination thereof.

13. The method of claim 12, wherein said somatic cells are cultured in any of said combinations comprising said control medium and said defined culture medium in (v / v) of any one of: 50:50, 25:75, and 10:90 for a period of at least 3 to 180 days.

14. The method of any one of claims 1 to 13, wherein said basal medium is selected from the group consisting of: McCoy’s 5A, Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Medium 199, and any combination thereof.

15. The method of any one of claims 1 to 14, wherein said defined medium comprises said serum replacement in a concentration ranging between 1% and 20%.

16. The method of any one of claims 1 to 15, wherein said at least one NEAA is selected from the group consisting of: glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine, tyrosine, and any combination thereof.

17. The method of any one of claims 1 to 16, wherein said defined medium further comprises basal fibroblast growth factor (bFGF) in a concentration ranging between 1 ng / ml to 150 ng / ml.

18. The method of any one of claims 1 to 17, wherein said somatic cells with adherent growth properties are of a cell-line selected from the group consisting of: CHO, HeLa, HEp- 2, Vero, CaCo-2, 3T3, MDCK, and any combination thereof.

19. A kit comprising: (i) a defined medium comprising: (a) basal medium in an amount of 80-99% by volume of said defined medium; (b) serum replacement in an amount of 1-20% by volume of said medium; and (c) at least one NEAA in a concentration of 50 pM to 500pM; and (ii) instructions for the culturing of somatic cells with adherent growth properties in a support-free suspension.

20. The kit of claim 19, for use in a method for culturing somatic cells with adherent growth properties in a suspension being support-free.

21. A defined medium consisting essentially of: a. basal medium in an amount of 80-99% by volume of said defined medium; b. serum replacement in an amount of 1-20% by volume of said medium; and c. at least one NEAA in a concentration of 50 pM to 500 pM.

22. A composition comprising the defined medium of claim 21, and somatic cells with adherent growth properties.

23. The composition of claim 22, being in the form of suspension.

24. The composition of claim 23, being support-free or devoid of a support.

25. The composition of claim 24, wherein said support is a solid support.

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