WO2014082759A2

WO2014082759A2 - Serum-free medium for human mesenchymal stem cells

Info

Publication number: WO2014082759A2
Application number: PCT/EP2013/061118
Authority: WO
Inventors: Gianni Soldati; Tiziano Tallone; Luca MARIOTTA
Original assignee: Swiss Stem Cell Foundation
Priority date: 2012-11-30
Filing date: 2013-05-29
Publication date: 2014-06-05
Also published as: WO2014082814A1; CN104837988A; WO2014082759A3; WO2014082685A1

Abstract

A highly effective serum-free growth medium, especially adapted for human MSCs and their subtypes,comprising,dissolved and/or suspended in a basis cell growth medium, at least five components selected from: human transferrin, human insulin, selenium or a salt thereof, human thyreoglobulin, human albumin, ascorbic acid or a salt thereof, pyruvic acid or a salt thereof; one or more human growth factors, and/or one or more additives selected from: a strontium salt, vitamin D, rosiglitazone, a prostaglandin, a BMP protein.

Description

TITLE

SERUM-FREE MEDIUM FOR HUMAN MESENCHYMAL STEM CELLS

FIELD OF THE INVENTION

The present invention relates to the field of artificial substrates for the growth and maintenance of human cells. We describe herein a new, highly safe medium, free of ingredients of animal origin, especially devised for growing and maintaining human mesenchymal stem cells (hMSCs).

STATE OF THE ART Cell culture media are widely used in the industrial production of biologically active products, such as hormones, enzymes, antigens, antibodies, etc. Cell growth media are usually a mixture of cell nutrients of organic and inorganic origin, dispersed or dissolved in a liquid or a semisolid system capable to support the growth of biological organisms.

Human mesenchymal stem cells (hMSCs) require especially adapted growth media: these must preserve/support the capacity of MSCs to differentiate into specific target tissues (vascular, neural, lipid, osseous, cartilage, etc.), and such capacity is quite sensitive to changes in the medium composition. The fine tuning of the media is also important in view of the fact that MSCs are used in clinical trials and will be used in the future as advanced cell therapy products (ACTPs), for delicate therapeutic/cosmetic applications, such preparation of cell suspensions /tissues/organs to be implanted in patients. These preparations require a precise and constant quality control of MSCs having reproducible characteristics under GMP guidelines. An insufficient growth/maintenance of MSCs within the culture media may result in a low-quality implant with potentially serious consequences to the patient, or it could cause the waste of the preparation with the associated costs. The MSC growth media should also be free of toxic components which could contaminate the stem cell culture and be transferred to the patient: in particular, the growth media should be free of ingredients of animal origin (often present in standard cell growth media) such as serum. Serum is a universal nutrient for the growth and maintenance of mammalian cells; growth media usually contain up to about 10% of animal serum, such as fetal bovine serum; although widely used, serum has the drawback of containing possible contaminants, e.g. virus such as BSE (Bovine Spongiform Encephalopathy), a transmissible neurodegenerative disease of cattle with a long latency or incubation period; this has raised regulatory concerns about using animal-derived sera in the production of biologically active products.

There is therefore a growing demand for the development of alternative media free from animal serum. This need is partially met by the commercial products currently available: in fact these are not fully satisfactory for the point of view of fine adaptation to MSCs and to the different subtypes thereof.

SUMMARY OF THE INVENTION

The present inventors have now obtained a group of highly effective serum-free growth media, especially adapted for MSCs and their subtypes, characterized by comprising, dissolved and/or suspended in a basis cell growth medium:

(a) at least five components selected from: human transferrin, human insulin, selenium or a salt thereof, human thyreoglobulin, human albumin, ascorbic acid or a salt thereof, pyruvic acid or a salt thereof,

(b') one or more human growth factors, and/or

(b") one or more additives selected from: a strontium salt, vitamin D, rosiglitazone, a

prostaglandin, a BMP protein.

The invention extends to the method of preparing the above medium, and its use in culturing human MSCs. DESCRIPTION OF THE FIGURES

Figure 1 : microscope images of human MSCs cultured in a growth medium in accordance with the present invention. Cells were photographed at two different confluences, with 4X, 10X or 20X magnifications.

Figure 2: Example of seeded and cultured SVF cells. A) 72 hours after seeding adherent cells can be observed. B) After washing the same cells reach the semi-confluence. C) Re-seeded cells arrange themselves forming so called pseudo-islands. D) In some instances cells can form spheroids or atypical groupings.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the basis cell growth medium is preferably a liquid system, i.e. a solution and/or suspension of different components; semi-solid media like gels and similar can also be used. The basis cell growth medium can be widely chosen among known standard cell culture media; these contain mixtures of nutrients such as inorganic salts, aminoacids, vitamins; preferred basis media are the commercially available products DMEM (Low- or High Glucose Dulbecco Modified Eagle Medium), HAM's F-12, IMDM (Iscove's Modified Dulbecco's Medium), and mixtures thereof preferaby in 1 :1 ratio; among the mixtures, especially preferred are: HAM's F12+IMDM and HAM's F12+DMEM.

Among components (a): the human albumin is preferably an injectable human albumin; the ascorbic acid salt is preferably a phosphate salt, e.g ascorbic acid-2-phosphate salt; the pyruvic acid salt is preferably a sodium salt.

Among components (b'): the human growth factors are preferably selected from bFGF (basic fibroblast growth factor), PDGF (platelet-derived growth factor, e.g. PDGF-AB, PDGF-BB), TGF (transforming growth factor, e.g. TGF-βΙ , TGF- 3), EGF (epidermal growth factor).

Among components (b"): the prostaglandin is preferably selected from PGE₂; the BMP protein is preferably BMP-6.

The interaction of components (a) with components [(b') and/or (b")] has a special importance for:

(i) adapting the basis growth medium to the growth of MSCs;

(ii) adapting the growth medium to specific subtypes of MSCs, e.g.

MSCs of lipid tissue source, or for adipogenic, osteogenic or chondrogenic induction;

(iii) obtaining the objectives (i) and (ii) in serum-free condition.

In particular, growth media adapted for MSCs of lipid tissue source (e.g. from lipoaspirates) should preferably comprise the components (a) combined with the human growth factors (b'). In particular growth media adapted for MSCs of lipid tissue source preferably include: bFGF, PDGF, TGF. A particularly preferred composition for MSCs of lipid tissue source is the following:

Basis growth medium: HAM's F12/IMDM 1 :1

Components (a): human transferrin (5 pg/ml)

human insulin (5 pg/ml)

selenium (5 ng/ml)

human thyreoglobulin (5 pg/ml)

injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (50 μΜ)

Components (b'): bFGF (1 ng/ml)

PDGF-AB (1 ng/ml)

PDGF-BB (1 ng/ml)

TGF-βΙ (0.4 ng/ml). In another embodiment, the composition comprises the following. A growth medium comprising, in a HAM's F12/IMDM 1 :1 basic growth medium, the following:

components (a): glutamin

primocin

human transferrin (5 pg/ml)

human insulin (5 pg/ml)

selenium (5 ng/ml)

human thyreoglobulin (50 ng/ml)

injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (50 μΜ)

components (b'): bFGF (10 ng/ml)

PDGF-AB (10 ng/ml)

PDGF-BB (10 ng/ml)

TGF-βΙ (0.6 ng/ml).

A particularly preferred composition is the following:

Component Final concentration Stock

solution

Ham's F12/IMDM 1 :1 50 ml

Glutamin 2 mM 500 μΙ

Primocin 0.1 g/ml 100μΙ

A. Ascorbic acid 2-phosphate 50 μΜ 50 μΙ

B. Human transferrin 5 g/ml

B. Human Insulin 5 g/ml 50 ul ITS

B. Selenium 5 ng/ml

C. Human Thyreoglobulin 50 ng/ml 50 μΙ

D. Injectable Human Albumin 250 pg/ml 250 μΙ

1 . bFGF 10 ng/ml 10 μΙ

2. PDGF-AB 10 ng/ml 10 μΙ

3. PDGF-BB 10 ng/ml 10 μΙ

4. TGF-βΙ 0.6 ng/ml 10 μΙ Stock solutions preparation and storing conditions: A - Ascorbic acid 2-phosphate 50 mM (1000x)

2-Phospho-L-ascorbic acid trisodium salt (Sigma 49752), MW 322.05 g/mol. Dissolve 160 mg in 10 ml of culture medium.

Store at -20°C away from light.

B - ITS Universal Culture Supplement Premix 5 ml (1000x)

Dissolve the content of a vial (BD cat. no. 354351 ) in 5 ml of ddH2O.

Stable for 3 months at 4°C in a freeze-dried condition or at -20°C dissolved. Avoid thawing cycles.

C - Human Thyreoglobulin (1000x)

Dissolve 1 mg of human Thyreoglobulin (Calbiochem 609312) in 20 ml of PBS.

Store at 4°C in a freeze-dried condition or at -20°C in PBS.

D - Injectable Human Albumin 50 mg / ml (200x)

Human Albumin Solution 5% (50 g/L) of CLS Behring AG ready for use. Store at 4°C.

1 - bFGF 50 pg / ml (5000x)

10 pg bFGF (Prospec cyt-218) in 100 μΙ of ddH2O and 100 μΙ PBS:Albumin

(50ϊτιΙ:250μΙ).

Store at -20°C.

2 - PDGF-AB 50 μg / ml (5000x)

10 μg PDGF-AB (Prospec cyt-342) in 100 μΙ ddH2O and 100 μΙ PBS:Albumin (50 ηηΙ:250μΙ).

Store at -20°C. 3 - PDGF-BB 50 pg / ml (5000x)

10 pg PDGF-AB (Prospec cyt-501 ) in 100 μΙ ddH2O and 100 μΙ PBS:Albumin (50ηηΙ:250μΙ).

Store at -20°C.

4 - TGF-βΙ 3 pg / ml (5000x)

5 μg PDGF-AB (Prospec cyt-716) in 833 μΙ ddH2O and 833 μΙ PBS:Albumin (50ιτιΙ:250μΙ).

Store at -20°C.

Cell seeding and culture protocol: Seed the cells on plastic using human collagen coating I at an optimal density of 30Ό00 cells/cm2 for SVF cells or 15'000/cm2 for expanded mesenchymal cells.

Lay the cells in an incubator and let them adhere for 3-4 days. After approximatively 72 hours it will be possible to observe adherent cells (fig.

I A) .

4 days after seeding a first washing shall be carried out: transfer the surnatant in a centrifuge tube and centrifuge at full speed; add 50% fresh SF medium and 50% old centrifuged surnatant to the cells.

Wash daily using the same procedure (50% SF and 50% centrifuged surnatant). Approximatively 6 days after seeding the cells will be semi-confluent (fig.

I B) . Once confluence is obtained, a passage of the cells shall be carried out: remove the surnatant and wash with PBS, apply a minimum dose of trypsin (TrypLE) and incubate at 37°C for 5 min; check cell detachment; dilute in SF and re-seed the cells at 15Ό00 cells/cm2 in 50% SF and 50% centrifuged surnatant. 24 hours after re-seeding the cells will show an homogeneous appearance and will form so called pseudo-islands (fig. 1 C).

Keep culturing the cells with daily washes (50% SF and 50% centrifuged surnatant).

Should cells form spheroids or atypical groupings (fig. 1 D) immediately perform their passage and re-seeding.

Growth media for adipogenic induction of MSCs preferably comprise the components (a) combined with EGF as a component (b') and with rosiglitazone as a component (b").

A particularly preferred composition for adipogenic induction of MSCs is the following:

Basis growth medium: HAM's F12/DMEM 1 :1

Components (a): human transferrin (5 pg/ml)

human insulin (10 pg/ml)

selenium (5 ng/ml)

human thyreoglobulin (50 ng/ml)

injectable human albumin (250 pg/ml)

Components (b'): EGF (1 ng/ml)

Components (b") rosiglitazone (5 μΜ)

Growth media adapted for osteogenic induction of MSCs should preferably comprise the components (a) in combination with a strontium salt and/or vitamin D as a component (b"). The components (b') are not present in this case. A particularly preferred composition for osteogenic induction of MSCs is the following:

Basis growth medium: DMEM Low Glucose

Components (a): human transferrin (5 pg/ml)

human insulin (5 pg/ml)

selenium (5 ng/ml)

human thyreoglobulin (50 ng/ml)

injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (50 μΜ)

Components (b") Sr²⁺ salt (3 mM)

Vitamin D (10-200 nM)

Growth media adapted for chondrogenic induction of MSCs should preferably comprise the components (a) (including the pyruvic acid salt and the ascorbic acid salt), in combination TGF and bFGF as components (b') and a BMP protein and a prostaglandin as components (b").

A particularly preferred composition for chondrogenic induction of MSCs is the following:

Basis growth medium: DMEM High Glucose

Components (a): human transferrin (5 pg/ml)

selenium (5 ng/ml)

injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (0.1 mM)

sodium pyruvate (1 mM);

Components (b'): bFGF (10 ng/ml)

TGF- 3 (10 ng/ml);

Components (b"): BMP-6 (10 ng/ml)

PGE₂ : (10^"7 M) All the above growth media can be used as a solution/suspension, or can be coated onto a solid support (plate), as well-known in the art. In the latter case, the solution/suspension of the growth medium is supplemented with a suitable thickening agent, making the medium capable to stably adhere to the support, and then the obtained mixture is coated onto the support. Alternatively, the support is preventively coated with the thickening agent, and then the solution/suspension of the growth medium is applied onto the thus treated support; in a further alternative, the solution/suspension of the growth medium is applied to the support, and then the thickening agent is applied onto the thus treated support. The thickening agent is preferably collagen, more preferably human collagen, in particular human collagen I; it is preferably used in form of solution or suspension.

In a specific embodiment, an exemplified composition in accordance with the present invention was thickened with human collagen I and coated onto a solid support. After incubation, the plates were observed under the microscope to assess the growth and viability of the MSCs. The microscope pictures at different magnifications (Figure 1 ) showed highly viable MSCs and a widespread diffusion denoting an extended growth.

Claims

1 . Serum-free growth medium adapted for human MSCs, characterized by comprising, dissolved and/or suspended in a basis cell growth medium:

(a): at least five components selected from: human transferrin, human insulin, selenium or a salt thereof, human thyreoglobulin, human albumin, ascorbic acid or a salt thereof, pyruvic acid or a salt thereof;

(b'): one or more human growth factors, and/or

(b"): one or more additives selected from: a strontium salt, vitamin D, rosiglitazone, a prostaglandin, a BMP protein.

2. The growth medium of claim 1 , wherein the human albumin is an injectable human albumin and/or the ascorbic acid salt is a phosphate salt, and/or the pyruvic acid salt is a sodium salt.

3. The growth medium of claims 1 -2, wherein the components (b') are one or more among: bFGF (basic fibroblast growth factor), PDGF (platelet- derived growth factor, e.g. PDGF-AB, PDGF-BB), TGF (transforming growth factor, e.g. TGF-βΙ , TGF- 3), EGF (epidermal growth factor).

4. The growth medium of claims 1 -3, wherein the BMP protein is BMP-6 and/or the prostaglandin is PGE₂.

5. The growth medium of claim 1 -4, for MSCs of lipid tissue source, comprising bFGF, PDGF, TGF as components (b').

6. The growth medium of claim 5 comprising, in a HAM's F12/IMDM 1 :1 basic growth medium, the following:

components (a): human transferrin (5 pg/ml) human insulin (5 pg/ml)

selenium (5 ng/ml)

human thyreoglobulin (5 pg/ml)

injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (50 μΜ)

components (b'): bFGF (1 ng/ml)

PDGF-AB (1 ng/ml)

PDGF-BB (1 ng/ml)

TGF-βΙ (0.4 ng/ml).

7. The growth medium of claim 5 comprising, in a HAM's F12/IMDM 1 :1 basic growth medium, the following:

components (a): glutamin

primocin

human transferrin (5 pg/ml)

human insulin (5 pg/ml)

selenium (5 ng/ml)

human thyreoglobulin (50 ng/ml)

injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (50 μΜ)

components (b'): bFGF (10 ng/ml)

PDGF-AB (10 ng/ml)

PDGF-BB (10 ng/ml)

TGF-βΙ (0.6 ng/ml).

8. The growth medium of claims 1 -4, for adipogenic induction, comprising EGF as a component (b') and rosiglitazone as a component (b").

9. The growth medium of claim 8 comprising, in HAM's F12/DMEM 1 :1 basic growth medium, the following:

components (a): human transferrin (5 pg/ml)

human insulin (10 pg/ml) selenium (5 ng/ml)

human thyreoglobulin (50 ng/ml) injectable human albumin (250 pg/ml)

components (b'): EGF (1 ng/ml)

components (b") rosiglitazone (5 μΜ)

10. The growth medium of claims 1 -4, for osteogenic induction of MSCs, comprising a strontium salt and/or vitamin D as a component (b"), and not comprising the components (b').

1 1 . The growth medium of claim 10 comprising, in DMEM Low Glucose basic growth medium, the following:

components (a): human transferrin (5 pg/ml)

human insulin (5 pg/ml)

selenium (5 ng/ml)

human thyreoglobulin (50 ng/ml) injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (50 μΜ)

components (b") Sr²⁺ salt (3 mM)

Vitamin D (10-200 nM)

12. The growth medium of claims 1 -4, for chondrogenic induction of MSCs, comprising a pyruvic acid salt and an ascorbic acid salt among the components (a), TGF and bFGF as components (b'), and a BMP protein and a prostaglandin as components (b").

13. The growth medium of claim 12 comprising, in DMEM High Glucose basic growth medium, the following:

components (a): human transferrin (5 pg/ml)

selenium (5 ng/ml)

injectable human albumin (250 pg/ml)

ascorbic acid 2-phosphate (0.1 mM) sodium pyruvate (1 mM);

components (b'): bFGF (10 ng/ml)

TGF- 3 (10 ng/ml);

components (b"): BMP-6 (10 ng/ml)

PGE₂ : (10^"7 M)

14. The growth medium of claims 1 -13, coated onto a solid support.

15. A method to prepare the growth medium of claims 1 -14, comprising combining the components (a) with components (b') and/or (b").

16. Use of the growth medium of claims 1 -14 for culturing MSCs, in particular for MSCs of lipid tissue source, or for adipogenic, osteogenic or chondrogenic induction.