US20130089928A1 - Serum-Free Chemically Defined Cell Culture Medium - Google Patents

Serum-Free Chemically Defined Cell Culture Medium Download PDF

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US20130089928A1
US20130089928A1 US13/703,296 US201113703296A US2013089928A1 US 20130089928 A1 US20130089928 A1 US 20130089928A1 US 201113703296 A US201113703296 A US 201113703296A US 2013089928 A1 US2013089928 A1 US 2013089928A1
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serum
cell culture
culture medium
chemically defined
defined cell
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Songzhu An
Yanan Zhu
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STEMRD Inc
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STEMRD Inc
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Definitions

  • a serum-free chemically defined cell culture medium for the culture of cells.
  • a serum-free chemically defined cell culture medium for the culture of mesenchymal stem cells which allows expansion while maintaining a pluripotent phenotype from which chondrocytes and osteocytes can be derived and methods of using such populations of expanded mesenchymal stem cell populations for the treatment of disorders benefitted by administration of a therapeutic amount of such expanded and differentiated mesenchymal stem cell populations.
  • Bone and cartilage transplantations are performed in reconstruction of bone and cartilage segments in plastic surgery, traumatic surgery or after the removal of neoplastic lesions.
  • human tissues from an autologous source, or from living or deceased donors have been used for this purpose.
  • stem cell research bone and cartilage cells derived from mesenchymal stem cells are becoming cellular sources for skeletal repair.
  • MSCs Mesenchymal stem cells
  • tissues of the body such as the bone marrow, blood, dermis and periosteum. They possess the ability to differentiate to other types of cells, and therefore may contribute to the healing of the tissues after injuries.
  • MSCs can be isolated and purified from the bone marrow and culturally expanded in-vitro.
  • bovine serum fetal bovine serum
  • human autologous serum substantially similar or equivalent serum
  • human serum substantially similar or equivalent serum
  • bovine serum may contain blood born pathogens, such as viruses and mad cow prions, bovine spongiform encephalopathy (“BSE”), or the like.
  • BSE bovine spongiform encephalopathy
  • bovine serum invokes antibody generation to xenobiotic proteins which may invoke immune responses in recipient patients.
  • bovine serum exhibits lot to lot variations which can result in inconsistent performance.
  • cell culture media containing only chemically defined substances and free of serum and xenobiotics may be highly desired for the culture of MSCs (and other cells) assuming that the inventive cell culture media and methods of utilizing the inventive cell culture media afford both expansion of cells including MSCs and further affords differentiation of MSCs in culture.
  • a broad object of the invention can be to provide one or more embodiments of an inventive chemically defined serum-free medium (also referred to as the “serum-free medium”) for cell culture and as to particular non-limiting embodiments of the inventive serum-free media for expanding populations of MSCs in vitro including but not limited to human MSCs (“hMSCs”).
  • an inventive chemically defined serum-free medium also referred to as the “serum-free medium”
  • hMSCs human MSCs
  • Another broad object of the invention can be to provide embodiments of a serum-free medium which contains a combination of chemical components capable of supporting MSC viability, proliferation and differentiation in ex-vivo in-vitro cell culture and in particular MSC in-vitro culture without containing any serum, such as fetal bovine serum, autologous serum, or other animal serum. Additionally, embodiments of the inventive media can be utilized to support MSC viability, proliferation and differentiation with substantially similar or greater effectiveness or results as compared to conventional cell culture media containing serum.
  • Another broad object of the invention can be methods of culturing cells such as MSCs and in particular human MSCs in the inventive media resulting in expansion of MSC populations and differentiation of MSCs to produce chondrocytes or osteocytes.
  • Another broad object of the invention can be a method of culturing cells such as MSCs on a negatively charged plastic surface such as the negatively charged surface of polystyrene plastic which avoids the step of coating the surface with fibronectin, or the like.
  • FIG. 1 provides images which compare cell culture morphology between hMSCs cultured in conventional medium containing 10% FBS and hMSCs cultured in a particular embodiment of the inventive serum-free medium.
  • FIG. 2 is a graph of cell number over days which compares the growth rate of hMSCs cultured in conventional medium containing 10% FBS and the growth rate of hMSCs cultured in a particular embodiment of the inventive serum-free medium.
  • FIG. 3 is a graph of cell number over passage number which compares total number of hMSCs at each split where the starting passage number is 4 from conventional medium containing 10% FBS into either a particular embodiment of the inventive serum-free medium or in conventional medium containing 10% FBS.
  • FIG. 4 provides images which compare colony forming ability of hMSCs cultured in a particular embodiment of the inventive serum-free medium to hMSCs cultured in conventional medium containing 10% FBS.
  • FIG. 5 provides images which compare multilineage differentiation potential of hMSCs after long term culture in a particular embodiment of the inventive serum-free medium to hMSCs cultured in conventional medium containing 10% FBS.
  • FIG. 6 is a graph of cell number over passage number which shows growth rates of hMSCs in a particular embodiment of the inventive serum-free medium are similar with or without use of a plate-coating on culture vessels.
  • FIG. 7 is a graph of cell number over passage number which compares growth rate of umbilical cord blood derived MSCs cultured in an embodiment of a base medium supplemented with 10% FBS, L-glutamine, and penicillin streptomycin, to umbilical cord blood derived MSCs cultured in a particular embodiment of the inventive serum-free medium.
  • FIG. 8 is a graph of cell number over passage number which compares growth rate of adipose tissue derived MSCs cultured in the base medium supplemented with 10% FBS, L-glutamine, and penicillin streptomycin, to adipose tissue derived MSCs cultured in a particular embodiment of the inventive serum-free medium.
  • FIG. 9 is a bar graph of total cell numbers over the type of culturing flask utilized in culturing hMSCs in a particular embodiment of the inventive serum-free medium.
  • a serum-free chemically defined cell culture media for the culture of cells Specifically, a serum-free chemically defined cell culture medium for the culture of mesenchymal stem cells which can be used for ex-vivo mononuclear cell expansion while maintaining a pluripotent phenotype in which chondrocytes and osteocytes can be derived from the mesenchymal stem cells.
  • Methods of use of a serum-free chemically defined cell culture media for differentiation of chondrocytes and osteocytes from in-vitro expanded mesenchymal stem cell populations and for treatment of disorders of the bone and cartilage benefited by a population of the derived chondrocytes or osteocytes.
  • serum-free means the absence of any blood serum of any species including, but not limited to, the absence of fetal bovine serum, calf bovine serum, human serum, or the like, or combinations thereof.
  • numeric values herein are assumed to be modified by the term “about”, whether or not explicitly indicated.
  • ranges may be expressed as from “about” one particular value to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value to the other particular value.
  • the recitation of numerical ranges by endpoints includes all the numeric values subsumed within that range.
  • a numerical range of one to five includes for example the numeric values 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. When a value is expressed as an approximation by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
  • the terms “combination” or “combining” refer to any method of putting two or more materials together. Such methods include, but are not limited to, mixing, blending, commingling, concocting, homogenizing, incorporating, intermingling, stirring, or the like.
  • an isolated compound refers to one or more of that entity; for example, “a protein” or “a peptide” refers to one or more of those compounds or at least one compound.
  • a protein or “a peptide” refers to one or more of those compounds or at least one compound.
  • the terms “a” or “an”, “one or more” and “at least one” can be used interchangeably herein.
  • a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including combinations of two or more of the compounds.
  • an isolated compound is a compound that has been removed from its natural milieu. As such, “isolated” does not necessarily reflect the extent to which the compound has been purified.
  • An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.
  • Embodiments of the inventive medium including the best mode of the invention can provide a serum-free base medium (also referred to as the “base medium”).
  • the serum-free base medium can include a mixture of Dulbecco's Modified Eagle Medium or a medium of the same, equivalent, or substantially similar composition (herein after referred to as “DMEM”) (a suitable DMEM for use in embodiments of the invention can be obtained from Invitrogen, 5791 Van Allen Way, Carlsbad, Calif., in liquid form PN 11965 or as a powder as PN 12100 and prepared in accordance with the manufacturer's instructions) and MCDB201 medium or a medium of the same, equivalent, or substantially similar composition (hereinafter referred to as “MCDB”) (a suitable MCDB for use in embodiments of the invention can be obtained from Sigma-Aldrich PN M6770).
  • DMEM Dulbecco's Modified Eagle Medium or a medium of the same, equivalent, or substantially similar composition
  • each of these conventional media also available with certain added components such as glutamine, sodium pyruvate, HEPES, phenol red, glucose, and without certain components such as methionine, cystine, or calcium, each such medium can be utilized in certain embodiments of the base medium depending on the application.
  • the base medium can include DMEM and MCDB combined in a ratio of DMEM:MCDB (v/v) in the range of about 0.75:1.25 to about 1.25:0.75; however, the invention is not so limited, and embodiments which confer advantages in particular applications can be prepared using ratios of DMEM:MCDB within a range selected from the group including: about 0.75:1.25 and about 0.85:1.15, about 0.80:1.20 and about 0.90:1.10, about 0.85:1.15 and about 0.95:1.05, about 90:110 and about 1.05:0.95, about 1.00:1.00 and about 1.10:0.90, about 1.05:0.95 and about 1.15:0.85, about 1.10:0.90 and about 1.20:0.80, and about 1.15:0.85 and about 1.25:0.75.
  • MSCs mesenchymal stem cells
  • hMSCs human mesenchymal stem cells
  • Embodiments of the base medium can further include one or more of the components further described below which can be combined with the combination of DMEM and MCDB in various permutations, combinations, concentrations or amounts, depending upon the application.
  • the one or more components can be used within the range or in the amounts described and depending upon the application or combination of components certain advantages can be achieved using a selected portion of the ranges described for any particular component or element.
  • Embodiments of the base medium can further include a buffer such as an amount of sodium bicarbonate in the range of about 3.2 g/L and about 4.2 g/L of the base medium and as to particular embodiments about 3.7 g/L.
  • the pH of the base medium can be adjusted to a final pH in the range of about 7.3 to about 7.5 with an amount of sodium hydroxide with particular embodiments having a pH of about 7.4.
  • Embodiments of the base medium can further include a regulated iron source which releases an amount of iron upon binding with receptors of the cells being cultured.
  • a regulated iron source can be an amount of transferrin having a concentration in said base medium in the range of about 2 mg/L and about 10 mg/L.
  • the amount of transferrin can be selected from the group including: about 2 mg/L and about 4 mg/L, about 3 mg/L and about 5 mg/L, about 4 mg/L and about 6 mg/L, about 5 mg/L and about 7 mg/L, about 6 mg/L and about 8 mg/L, about 7 mg/L and about 9 mg/L, and about 8 mg/L and about 10 mg/L.
  • Particular embodiments of the base medium can include an amount of transferrin of about 5 mg/L.
  • Embodiments of the base medium can further include an electron transport activator.
  • the electron transport activator can comprise an amount of selenium or an amount of selenous acid; however, the invention is not so limited and other trace elements, metalloenzymes or proteins can be utilized to support electron transport.
  • the amount can be in a range of about 0.0000025 g/L and about 0.0000050 g/L with a particular embodiment including about 0.0000037 g/L.
  • Embodiments of the base medium can further include one or more antioxidants.
  • the antioxidants can include as non-limiting examples an amount of ⁇ -tocopherol acetate or an amount of ascorbic acid-2 phosphate, or an amount of both.
  • Particular embodiments can include an amount of ⁇ -tocopherol acetate in the range of about 0.0001 g/L and about 0.0003 g/L and an amount of ascorbic acid-2 phosphate in the range of about 0.02 g/L and about 0.04 g/L.
  • One non-limiting example includes an amount of ⁇ -tocopherol acetate of about 0.0002 g/L and an amount of ascorbic acid-2 phosphate of about 0.0322 g/L.
  • Embodiments of the base medium can further include one or more steroids.
  • the steroids can include as non-limiting examples an amount of dexamethasone or an amount of hydrocortisone, or an amount of both.
  • Particular embodiments can include an amount of dexamethasone in the range of about 2.0 m/L and about 5.0 ⁇ g/L in combination with an amount of hydrocortisone of about 0.5 mg/mL and about 1.5 mg/mL.
  • One non-limiting example of the base medium includes an amount of dexamethasone of about 0.000004 g/L in combination with an amount of hydrocortisone of about 0.001 g/L.
  • Embodiments of the base medium can further include an amount of 5-hydroxytryptamine in the range of about 0.001 g/L and about 0.003 g/L.
  • One non-limiting example of the base medium includes an amount of 5-hydroxytryptamine of about 0.002 g/L.
  • Embodiments of the base medium can further include an amount of human serum albumin (“HSA”)(which can be a recombinant human serum albumin (“rHSA”)).
  • HSA human serum albumin
  • Particular embodiments can include an amount of HSA in a range of about 0.1 g/L and about 0.3 g/L.
  • One non-limiting example of the base medium includes an amount of HSA of about 0.25 g/L.
  • a particular non-limiting embodiment of the base medium for the culture of MSCs or hMSCs can comprise, consist essentially of, or consist of the combination of ingredients each in the final concentration set out in Table 1.
  • this particular embodiment of the base medium is not intended to be limiting but rather exemplary of the numerous and varied embodiments which can be prepared using equivalent or substantially similar components, adjusted in concentration based upon the particular cells, cell lines, MSCs, or hMSCs being cultured.
  • the final concentrations listed for the embodiment of Table 1 may vary from the absolute value in a range related to the normal variations in production, depending upon the particular application, useful in culturing MSCs or hMSC.
  • certain embodiments of the invention may include fewer components than are listed in Table 1 and these embodiments are intended to be encompassed by the breadth of the invention.
  • Embodiments of the invention can further include a base medium supplement which can be combined with the base medium (the combination also referred to as the “supplemented medium”).
  • the base medium supplement can include one or more cell growth factors, one or more phospholipid growth factors, and one or more WNT signaling pathway activators, in various permutations and combinations. Understandably, embodiments of the inventive serum-free medium do not have to, but can include, the one or more of the cell growth factors, the one or more phospholipid growth factors, or the one or more WNT signaling pathway activators, in various combinations and permutations depending upon the application.
  • embodiments of the base medium supplement can include the one or more cell growth factors such as: insulin which can be obtained from Sigma-Aldrich PN 16634 (CAS NO.: 11070-73-8), basic fibroblast growth factor human (bFGF) which can be obtained from Sigma-Aldrich PN F0291-25UG (CAS No.: 106096-93-9), Platelet-Derived Growth Factor bb (PDGF-bb) which can be obtained from Sigma-Aldrich PN P3201, Epiderman Growth Factor (EGF) which can be obtained from Sigma-Aldrich PN E9644 (CAS NO.: 62253-63-8), and Insulin Like Growth Factor-1 (IGF-1) (CAS NO.: 7733-29-1) which can be obtained from Sigma-Aldrich PN 18779 or PN 13769.
  • cell growth factors such as: insulin which can be obtained from Sigma-Aldrich PN 16634 (CAS NO.: 11070-73-8), basic fibroblast growth factor human (
  • Embodiments of the base medium supplement can include an amount of insulin.
  • Particular embodiments can have a concentration in the serum-free medium in the range of about 0.004 g/L and about 0.006 g/L.
  • a concentration in the serum-free medium in the range of about 0.004 g/L and about 0.006 g/L.
  • One non-limiting example includes an amount of insulin of about 0.005 g/L.
  • Embodiments of the base medium supplement can include an amount of bFGF.
  • Particular embodiments can have a concentration of bFGF in the serum-free medium in the range of about 5 ng/mL and about 20 ng/mL.
  • One non-limiting example includes an amount of bFGF of about 10 ng/mL.
  • Embodiments of the base medium supplement can include an amount of PDGF-bb.
  • Particular embodiments can have a concentration of PDGF-bb in the serum-free medium in the range of about 5 ng/mL and about 20 ng/mL.
  • One non-limiting example includes an amount of PDGF-bb in the serum-free medium of about 10 ng/mL.
  • Embodiments of the base medium supplement can include an amount of EGF.
  • Particular embodiments can have a concentration of EGF in the serum-free medium in the range of about 15 ng/mL and about 25 ng/mL.
  • One non-limiting example includes an amount EGF in the serum-free medium of about 20 ng/mL.
  • Embodiments of the base medium supplement can include an amount of IGF-1.
  • Particular embodiments can have a concentration of IGF-1 in the serum-free medium in the range of about 2 ng/mL and about 10 ng/mL.
  • One non-limiting example includes an amount IGF-1 in the serum-free medium of about 5 ng/mL.
  • PDGF-bb As to each of the above-described cell growth factors: PDGF-bb, bFGF, EGF, and IGF-1, there can be advantages to concentrations in the serum-free medium selected from the group including: about 2 ng/mL to about 4 ng/mL, about 3 ng/mL to about 5 ng/mL, about 4 ng/mL to about 6 ng/mL, about 5 ng/mL to about 7 ng/mL, about 6 ng/mL to about 8 ng/mL, about 7 ng/mL to about 9 ng/mL, about 8 ng/mL to about 10 ng/mL, about 9 ng/mL to about 11 ng/mL, about 10 ng/mL to about 12 ng/mL, about 11 ng/mL to about 13 ng/mL, about 12 ng/mL to about 14 ng/mL, about 13 ng/mL to about 15 ng/mL, about 14 ng
  • Embodiments of the base medium supplement can include one or more phospholipid growth factors, such as: lysophosphatidic acid (“LPA”)(CAS NO.:22022-87-5) which can be obtained from Sigma-Aldrich PN L7260, and sphingosine 1-phosphate (“S1P”)(CAS NO.: 26993-30-6) which can be obtained from Sigma-Aldrich PN 59666.
  • LPA lysophosphatidic acid
  • S1P sphingosine 1-phosphate
  • Particular embodiments can have a concentration of each of LPA or S1P, or of both, in the serum-free medium in the range of about 80 nM and about 200 nM.
  • One non-limiting example includes an amount LPA in the serum-free medium of about 100 nM combined with an amount of S1P in the serum-free medium of about 150 nM.
  • concentrations in the serum-free medium selected from the group including: about 80 nM and about 100 nM, about 90 nM and about 110 nM, about 100 nM and about 120 nM, about 110 nM and about 130 nM, about 120 nM and about 140 nM, about 130 nM and about 150 nM, about 140 nM and about 160 nM, about 150 nM and about 170 nM, about 160 nM and about 180 nM, about 190 nM and about 200 nM.
  • Embodiments of the base medium supplement can include one or more WNT signaling pathway activators.
  • the WNT signaling pathway activators can be selected from the non-limiting group including: a human WNT-3a protein (“WNT3A”) which can be obtained from StemRD Inc., Burlingame, Calif. PN W3A-H005 and R-spondin-1 (“RSPO 1 ”) which can be obtained from StemRD Inc., Burlingame, Calif. PN RSPO-005.
  • Particular embodiments can include each of the one or more WNT signaling pathway activators in a concentration in the serum-free medium in a range of about 10 ng/mL and about 50 ng/mL.
  • One non-limiting example includes an amount WNT3A in the serum-free medium of about 20 ng/mL combined with an amount of RSPO 1 in the serum-free medium of about 40 ng/mL.
  • a particular non-limiting embodiment of the base medium supplement for the culture of MSCs or hMSCs can comprise, consist essentially of, or consist of the combination of ingredients each in the final concentration in the serum-free medium set out in Table 2.
  • MSC Medium Growth Factors.
  • Growth Factor Final Concentration insulin 0.005 g/L PDGF-bb 10 ng/mL bFGF 10 ng/mL EGF 20 ng/mL IGF-l 5 ng/mL LPA 100 nM S1P 150 nM WNT3A 20 ng/mL RSPO1 40 ng/mL
  • Table 2 lists the final concentration of the various cell growth factors, lipid growth factors, and WNT signaling pathway activators utilized in a particular embodiment of the inventive serum-free medium for the expansion of MSCs in culture; the invention is not so limited, and additional embodiments of the invention can be achieved for the culture of cells including, but not limited to, deriving chondrocytes and osteocytes from MSCs or hMSCs, using one or more than one, or all of the growth factors listed in the table in various combinations and permutations with the concentration of each component or element varied as above described depending upon the application.
  • Embodiments of the serum-free medium can further include an amount of transforming growth factor beta 1 (“TGFB1”) which can be obtained from StemRD Inc., Burlingame, Calif. PN TGF-b-005.
  • the amount of TGFB1 can be an amount sufficient to derive chondrocytes from mesenchymal stem cells cultured in the serum-free chemically defined cell culture medium above described.
  • Particular embodiments of the serum-free medium can include a concentration of TGFB1 in the serum-free medium in the range of about 0.5 ng/mL and about 5 ng/mL.
  • One non-limiting example includes an amount TGFB1 having a concentration in the serum-free medium of about 1 ng/mL.
  • Embodiments of the serum-free medium can further include an amount of bone morphogenic protein 2 (“BMP2”) which can be obtained from Sigma-Aldrich PN B3555.
  • BMP2 bone morphogenic protein 2
  • the amount of BMP2 can be an amount sufficient to derive osteocytes from mesenchymal stem cells cultured in the serum-free chemically defined cell culture medium above described.
  • Particular embodiments of the serum-free medium can include a concentration of BMP2 in the serum-free medium in the range of about 0.5 ng/mL and about 5 ng/mL.
  • One non-limiting example includes an amount TGFB1 having a concentration in the serum-free medium of about 1 ng/mL.
  • embodiments can include one or both of the TGFB1 or BMP2 sufficient to derive a population of chondrocytes or a population of osteocytes or a population including both a population chondrocytes and a population of osteocytes from the MSCs or hMSCs.
  • a particular embodiment of the serum-free medium suitable for deriving chondrocytes or osteocytes from hMSCs can comprise, consist essentially of, or consist of a combination of the ingredients enumerated in Table 1 and Table 2 and then admixing an amount of either of or both of TGFB1 and BMP2 into the combination.
  • inventive serum-free chemically defined cell culture media including certain embodiments of the inventive base media and the inventive supplemented media
  • Frozen cells including, but not limited to MSCs, and in particular hMSCs, can be adapted to the inventive serum-free medium above-described, regardless of the medium prior used to grow or freeze cells.
  • the stepwise procedure includes thawing frozen cells, such as hMSCs, in a water bath at about 37° C.
  • the hMSCs can be transferred into a 50 mL conical tube or other suitable vessel.
  • For each 1 mL of hMSC suspension add drop-wise about 10 mL of the inventive serum-free medium pre-warmed to about 37° C. while gently swirling. Transfer the contents of the conical tube into a tissue culture flask or plated multiple wells of a tissue culture plate.
  • hMSCs can also be centrifuged at about 250 ⁇ g (1200 rpm) for about 10 minutes, re-suspended in the inventive serum-free medium (the base medium or the supplemented medium depending on the application) and then transferred or plated.
  • the hMSCs can be incubate at about 36° C. to about 38° C. in a humidified atmosphere containing about 4% to about 6% carbon dioxide. After about 24 hours, replace the inventive serum-free medium in which the hMSCs were transferred or plated with fresh inventive serum-free medium. Maintain the hMSC cell culture by changing the inventive serum-free medium every 2 days until cell expansion requires passaging or splitting.
  • Coating or other treatment to the surface of cell culture vessels may not be necessary when culturing cells, including, but not limited to MSCs and in particular human MSCs in embodiments of the inventive serum-free medium (such as the “base medium” or the “supplemented medium”).
  • Sufficient and typically optimal cell attachment and growth of cells including MSCs and hMSCs can be achieved utilizing conventional tissue culture vessels without coating.
  • negatively charged polystyrene vessels such as 25 cm 2 tissue culture flask available through BD Primaria, 1 Becton Drive, Franklin Lakes, N.J.
  • BD Cat#353808 and BD Primaria, Falcon 6-well plates (BD Cat#353046) for cell passage can be utilized with embodiments of the inventive medium without plate-coating; however, the invention is not so limited, and if desired the surface of the vessels can be coated with Fibronectin at about 0.5-1.0 microgram/cm 2 surface area for about 1 hour at about 37° C., followed by plating of cells in the inventive serum-free medium.
  • Cells, including MSCs and hMSCs cultured in serum-containing or serum-free media can be quickly and easily adapted into embodiments of the inventive serum-free medium. In most cases, a one-step transition from serum-containing medium into embodiments of the inventive serum-free medium can be sufficient. If so desired, step-wise adaptation with a gradual increase of the amount of the inventive serum-free medium (e.g. 25%, 50%, etc) can also be performed, as follows.
  • MSCs can be passaged when they reach about 70% confluency. If the MSC or hMSC culture reaches confluency of about 80% or higher, the cells may stop proliferating after passage. Therefore, MSCs and hMSCs can be passaged prior to reaching confluency of 80% to avoid this result.
  • MSC or hMSC or cells under a microscope. When cells start to detach, gently tap the side of the vessel to help loosen the remaining cells. The time required for the cells to detach should be about 1 minute to about 3 minutes, if the MSCs or hMSCs have been cultured in embodiments of the inventive serum-free medium. Interestingly, cells grown in conventional serum-containing media may require a longer incubation time to detach.
  • MSCs Once the cells have detached, proceed to the following step. Do not leave MSCs in cell dissociation enzyme, such as Trypsin or TrypLETM Express, for an extended amount of time after the cells have detached, as this will adversely affect the growth of MSCs.
  • cell dissociation enzyme such as Trypsin or TrypLETM Express
  • DPBS Dulbeccos Phosphate Buffered Saline
  • Centrifuge cells at 1200 rpm (250 ⁇ g) for 10 minutes. Aspire and discard substantially all the supernatant. Resuspend cells in DPBS or the inventive serum-free medium, and centrifugate again. Aspire the supernatant and resuspend cells in pre-warmed inventive medium. Take an aliquot from the cell suspension for cell counting.
  • cryopreservation solution by admixing the base medium with about 10% supplemented medium and 10% Dimethyl Sulfoxide (DMSO).
  • Pellet cells such as MSCs, by centrifugation, gently re-suspend cells in cryopreservation solution to about 1.0 ⁇ 10 6 cells/mL, and transfer to cryovials.
  • Place cryovials in a freezing container such as a Nalgene, 5100 Cryo Freezing Container Product No 5100-0001
  • place in a ⁇ 70° C. freezer overnight Transfer cryovials to liquid nitrogen for long-term storage.
  • the differentiation potential of cells such as MSCs or hMSCs expanded in the inventive serum-fee medium can be tested in-vitro.
  • the expanded cells may be induced to form chondrocytes under the experimental conditions described.
  • the expanded MSCs can be transferred in anchorage-independent conditions and maintained as a pellet culture for about 1 to about 4 weeks in the inventive serum free base medium otherwise using the method previously described by Johnstone et al., Exp. Cell Res. 238, 265-272 (1998) to induce chondrogenesis of MSCs expanded in the inventive serum-free.
  • results indicate that the MSCs expanded in embodiments of the inventive serum-free base medium formed a cartilaginous structure, which can stain positive for alcian blue and type II collagen, and can stain mostly negative for type I collagen.
  • the extent of chondrogenesis may be enhanced by the addition of TGFB1 and WNT pathway inhibitors to the inventive base medium, or use of the supplemented medium, above described.
  • inventive base medium the inventive base medium further including TGFB1 and WNT pathway inhibitors, or the supplemented medium, may induce chondrogenesis to the same or greater extent, rate, or efficiency than conventional medium containing FBS.
  • the differentiation potential of cells such as MSCs or hMSCs expanded in the inventive medium can be tested in-vitro.
  • the expanded cells may be induced to form osteocytes under the experimental conditions described.
  • the extent of osteogenesis may be enhanced by the addition of BMP2 and WNT pathway activators to embodiments of the inventive base medium or by use of the supplemented medium, as above described.
  • results may evidence that methods which utilize the inventive base medium, the base medium, or the supplemented medium, may induce osteogenesis at the same or greater extent, rate, or efficiency than conventional medium containing FBS.
  • FIG. 1 provides images that compare cell cultures of hMSCs grown in conventional medium containing 10% FBS and cell cultures of hMSCs grown in the inventive serum-free medium (also referred to in the Figures as “MesenGro”) at each of 40% confluence and at 80% confluence.
  • the comparison of the images evidences that hMSC cultures in the inventive serum-free medium do not appear substantively different than hMSC cultures in conventional medium containing 10% FBS.
  • the inventive serum-free medium provides the advantage of not having to utilize FBS which can vary to a greater degree in composition, and cannot be traced back to the donor animal, and may inhibit or prevent MSC or hMSC differentiation.
  • the inventive serum-free medium can afford other advantages as further described below.
  • a graph of cell number over time in days compares cell growth of hMSCs in conventional medium containing 10% FBS to cell growth in the inventive serum-free medium (“MesenGro”).
  • Cell numbers per well (24-well plate) at each time point were counted with a medium change every 2 days.
  • the graph evidences that the rate of cell growth of hMSCs in the inventive serum-free medium can be greater than in conventional medium containing 10% FBS. In certain applications this affords the advantage of allowing passage at an earlier point in time or afford the advantage of obtaining a desired number of cells in a lesser amount of time.
  • a graph of total hMSC numbers over passage number compares cell growth of hMSCs in conventional medium containing 10% FBS to cell growth in the inventive serum-free medium (referred to as “MesenGro”). Total cell numbers per well (6-well plate) at each split (every 3 days) were counted.
  • the starting passage number is 4 (from conventional medium containing 10% FBS into either the inventive serum-free medium or into conventional medium containing 10% FBS), with an initial cell density of about 20,000/well.
  • the graph evidences a similar exponential expansion of hMSC in the inventive serum-free medium as in the conventional medium.
  • FIG. 4 provides images of the colony forming ability of hMSCs grown in conventional medium containing 10% FBS as compared with the colony forming ability of hMSCs grown in the inventive serum-free medium (referred to as “MesenGro”).
  • the images evidence no substantive difference in the colony forming ability of those hMSCs grown in the inventive serum-free medium as compared to those hMSCs grown in conventional medium containing 10% FBS.
  • FIG. 5 provides images which compare the multilineage differentiation potential of hMSCs after long-term culture in the inventive serum-free medium (referred to as “MesenGro”) to hMSCs after long-term culture in conventional medium containing 10% FBS.
  • MesenGro inventive serum-free medium
  • cells were plated in 6-well plate, and differentiation achieved in the inventive serum-free medium was compared to differentiation achieved in the conventional medium containing 10% FBS after 18-24 days.
  • the results evidence that hMSCs cultured in the inventive serum-free medium retain a multilineage differentiation potential comparable and which may be greater than hMSCs cultured in conventional medium containing 10% FBS.
  • FIG. 6 is a graph of cell number over passage number. Growth rates of bone marrow derived hMSCs (Cellular Engineering Technologies) in the inventive serum-free medium are similar on BD Primaria Falcon 6-well plates (BD Cat#353046) with or without use of a fibronectin coating as above described. This affords an advantage over several other commercially available serum-free media, such as StemPro MSC SFM from Invitrogen (PN A10332-01) and MesenCult-XF from StemCell Technologies (PN 05420), which requires the step of plate-coating to achieve similar cell number over passage number results.
  • StemPro MSC SFM from Invitrogen
  • MesenCult-XF from StemCell Technologies
  • FIG. 7 is a graph of cell number over passage number which compares growth rate of umbilical cord blood derived MSCs (Cellular Engineering Technologies) cultured in an embodiment of the base medium supplemented with 10% FBS, L-glutamine, and penicillin streptomycin to umbilical cord blood derived MSCs cultured in an embodiment of the inventive serum-free medium (referred to as “MesenGro”).
  • Umbilical cord blood derived MSCs were cultured for two to six passages in medium containing 10% FBS. Cultured MSCs were then split into two groups and each of the two groups cultured in either of: the base medium supplemented with 10% FBS, L-glutamine, and penicillin streptomycin, or the inventive serum-free medium (referred to as “MesenGro”).
  • FIG. 8 is a graph of cell number over passage number which compares growth rate of adipose tissue derived MSCs (Cellular Engineering Technologies) cultured in an embodiment of the base medium supplemented with 10% FBS, L-glutamine, and penicillin streptomycin to adipose tissue derived MSCs cultured in an embodiment of the inventive serum-free medium (referred to as “MesenGro”).
  • Adipose tissue derived MSCs were cultured for two to six passages in medium containing 10% FBS. Cultured MSCs were then split into two groups and each of the two groups cultured in either of: the base medium supplemented with 10% FBS, L-glutamine, and penicillin streptomycin, or the inventive serum-free medium (referred to as “MesenGro”).
  • MesenGro adipose tissue derived MSCs in the inventive serum-free medium
  • FIG. 9 provides a bar graph of total cell numbers over the type of culturing flask utilized in culturing hMSCs.
  • Frozen bone marrow derived MSCs (Cellular Engineering Technologies) were thawed in accordance with the procedure of Example 1. Bone marrow derived MSCs that had been passed for 6 passages in the inventive serum-free medium (“MesenGro”) on uncoated Primaria T25 flasks were then seeded at 0.09 ⁇ 10 6 per flask on to two different kinds of flasks: BD Primaria T25 and Corning CellBIND T25.
  • the inventive serum-free medium confers a variety of advantages over culturing MSCs or hMSCs in a medium supplemented with fetal bovine serum or with human autologous serum, or other serum containing medium.
  • embodiments of the inventive serum-free medium do not contain bovine serum, human serum, or other animal serum. Accordingly, the embodiments of the inventive serum-free medium cannot contain any corresponding blood born pathogens, such as viruses and mad cow prions, bovine spongiform encephalopathy (“BSE”), or the like.
  • BSE bovine spongiform encephalopathy
  • embodiments of the inventive serum-free medium do not invoke antibody generation to xenobiotic proteins which can invoke immune responses in patients into which populations of ex-vivo expanded populations of MSCs can be transferred to in treatment of disorders of the cartilage and bone.
  • embodiments of the inventive serum-free medium have a substantially lesser lot to lot variation in composition and thereby lot to lot performance of the inventive serum-free medium can be utilized with a greater consistency.
  • embodiments of the inventive serum-free medium can be utilized with uncoated culture flasks manufactured by a variety of different manufactures.
  • other commercially available serum-free media such as Invitrogen's StemPro MSC SFM, or StemCell Technologies' MesenCult-XF, require pre-coating the culture flasks with attachment material.
  • embodiments of the inventive serum-free medium exhibit unexpectedly good results in supporting expansion of MSCs derived from a variety of MSC sources such as umbilical cord matrix, umbilical cord blood, bone morrow and adipose tissue-derived MSCs as compared to conventional medium supplemented with fetal bovine serum as evidenced by FIG. 7 and FIG. 8 and the above description.
  • a first non-limiting use of embodiments of the inventive serum-free medium can be provision of a cell culture kit which includes a portion of or all of the components of the inventive serum-free medium whether combined or combinable in various permutations and combinations to prepare various embodiments of the serum-free medium for the purpose of ex-vivo expansion of cells, MSC, or hMSC populations.
  • a population of mesenchymal stem cells expanded ex-vivo using the inventive serum-free medium can be obtained as above described.
  • a therapeutically effective amount of the population of mesenchymal stem cells expanded as above described can be administered to an individual.
  • the individual may be suffering from a disorder of the cartilage tissue and a population of osteocytes derived from a population of mesenchymal stem cells expanded ex-vivo using the inventive serum-free medium can be obtained and a therapeutically effective or sufficient amount of such population of osteocytes can be administered to the individual to benefit, to assist in reconstituting, or reconstituting cartilage tissue.
  • the individual may be suffering from a disorder of the bone and a population of chondrocytes derived from a population of mesenchymal stem cells expanded ex-vivo using the inventive serum-free medium can be obtained and a therapeutically effective or sufficient amount of such population of chondrocytes can be administered to the individual to benefit, to assist in reconstituting or reconstituting bone.
  • the basic concepts of the present invention may be embodied in a variety of ways.
  • the invention involves numerous and varied embodiments of a serum-free medium including the best mode useful in the ex-vivo expansion and differentiation of cells, MSCs and hMSCs and treatment of disorders benefitted by administration of populations of MSCs or differentiated populations derived from such MSCs.
  • each element of an apparatus or each step of a method may be described by an apparatus term or method term. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all steps of a method may be disclosed as an action, a means for taking that action, or as an element which causes that action. Similarly, each element of an apparatus may be disclosed as the physical element or the action which that physical element facilitates.
  • the disclosure of a “cell culture” should be understood to encompass disclosure of the act of “culturing cells”—whether explicitly discussed or not—and, conversely, were there effectively disclosure of the act of “culturing cells”, such a disclosure should be understood to encompass disclosure of a “cell culture” and even a “means for culturing cells.”
  • Such alternative terms for each element or step are to be understood to be explicitly included in the description.
  • the applicant(s) should be understood to claim at least: i) a serum-free cell culture medium as herein disclosed and described, ii) the related methods disclosed and described, iii) similar, equivalent, and even implicit variations of each of these devices and methods, iv) those alternative embodiments which accomplish each of the functions shown, disclosed, or described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying examples, x) the various combinations and permutations of each of the previous elements disclosed.

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