WO2001048147A1

WO2001048147A1 - Cell culture medium containing growth factors and l-glutamine

Info

Publication number: WO2001048147A1
Application number: PCT/NL2000/000958
Authority: WO
Inventors: Joost Dick De Bruijn; Gerhardus Johannes Maria Tibbe; Sandra Claudia Da Silva Madureira Mendes
Original assignee: Iso Tis N.V.
Priority date: 1999-12-28
Filing date: 2000-12-27
Publication date: 2001-07-05
Also published as: DE60009956T2; AU3246101A; EP1242578A1; CA2395271A1; WO2001048147A9; AU780653B2; TR200401091T4; DE60009956D1; DK1242578T3; US6838284B2; ATE264385T1; JP2003518378A; EP1242578B1; ES2217031T3; US20030017588A1

Abstract

The invention relates to a culture medium for culturing cells, in particular human cells in a process for tissue engineering bone. The medium comprises glucose, a mineral, a vitamin, a growth factor and L-glutamine, wherein the L-glutamine is present in a concentration of at least 300 mg/L.

Description

CELL CULTURE MEDIM CONTAINING GROWTH FACTORS AND L-GLUTAMINE

The invention relates to the field of tissue engineering. In particular, the invention discloses a medium and a method for culturmg cells

The need for replacement parts for the human body, in combination with the shortage of donor tissue and/or organs has lead to the production of replacement tissue by seeding cells onto or into a scaffold. Eventually, this should lead to tissue engineered products ready to be implanted to take over the function of missing or injured body parts. The scaffold defines the construct shape and dimensions of the replacement to be implanted. Preferably, it is manufactured of a porous or fibrous biodegradable material, so that the degradation of the scaffold proceeds parallel with accumulation of tissue components (growth and synthesis of extracellular matrix (ECM)). Thus, the function of the scaffold, the provision of shape and strength, will gradually be taken over by the formed tissue components.

In view of the fact that cells from allogenic sources are generally rejected, autologous cells isolated from a tissue biopsy from the patient to be treated are preferably used. In order to minimize the size of the biopsy needed and to minimize the time required for cell expansion, the expanded cells have to be first applied m/onto the scaffold m an efficient manner. In addition, the cells should be distributed homogeneously throughout the scaffold, m order to enable continuous neo-tissue formation.

In general, the cells that have been harvested from the patient's body are cultured in vi tro for a certain period of time, either with or without a scaffold material present. During this culturmg period, proliferation and/or differentiation of the cells may take place, depending on the type of cells harvested and on the objective type of tissue

In the literature and on the market, various cell culture media are known. These usually contain glucose, inorganic salts (minerals), ammo acids, and vitamins. Other ingredients that are sometimes used include ribonucleosides, deoxyribonucleosides and antibiotics. Well-known, commercially available culture media are for instance Dulbecco's Modified Minimal Eagle's Medium (DMEM) and Alpha Minimal Eagle's Medium (α-MEM) .

In the US patent 5,197,985, a method is disclosed for enhancing the implantation and differentiation of marrow- derived mesenchymal cells. The method is stated to be particularly intended as a means for treating skeletal and other connective tissue disorders in humans. For the culturing of the mesenchymal cells, a medium was employed that comprised the commercially available BGJ_b medium (Fitton-Jackson modification) and selected lots of 10% fetal bovine serum. Further, the medium F-12 Nutrient Mixture (Ham) was used for selective marrow-derived mesenchymal cell separation.

The production of tissue engineered products will generally only commence once the type of injury or disorder is known and a specific treatment has been decided upon. While the tissue engineering is carried out, the patient is in the meantime suffering from his injuries or disorder. Thus, in order to minimize a patient's discomfort it is of great importance that the production of engineered tissues proceeds as fast as possible. A disadvantage of most of the known culture media is that they do not allow for a sufficiently fast proliferation and/or differentiation of the cells which are cultured in it. This disadvantage is particularly apparent in case human cells are cultured.

The present seeks to provide a culture medium wherein cells proliferate and/or differentiate very fast. The objective culture medium is to allow for a reduction of the time needed for culturing cells in the treatment of a patient via a tissue engineering approach, when compared to conventional culture media. Surprisingly, it has now been found that the rate of proliferation and/or differentiation of cells can be significantly increased by culturing them m a medium which comprises a growth factor and an increased concentration of L-glutamine. The invention accordingly relates to a culture medium comprising glucose, a mineral, a vitamin, a growth factor and L-glutamine, wherein the L-glutamme is present a concentration of at least 300 mg/L.

Human cells have been found to proliferate much faster in a culture medium according to the invention than conventional culture media. Moreover, the differentiation of cells, which are capable of differentiation, has also been found to proceed faster than in conventional culture media. This increased cultivation rate is advantageous when the culture medium is employed for manufacturing a tissue engineered product for treatment of a patient. The culture medium comprises several components, which are dissolved or suspended m a suitable liquid, preferably distilled water. As cells will be cultured in the medium, it will be clear that it is of advantage to work under sterile conditions to prevent microbial contamination of the cultures.

In order for cells to be cultivated, it is necessary that sufficient nutrients are present m the culture medium. To this end, the present culture medium comprises glucose, minerals and vitamins. Glucose is an important nutrient in a culture medium.

In accordance with the invention it is preferred that the concentration of glucose is at least 750 mg/L, more preferred between 900 and 2000 mg/L.

The minerals may suitably be chosen from the group of calcium, potassium, lithium, magnesium, sodium, sulphate, chloride, bicarbonate, dihydrogenphosphate ions, and combinations thereof. Particularly suitable minerals are calcium chloride, preferably m an amount of 100-400 mg/L, potassium chloride, preferably m an amount of 200-600 mg/L, sodium chloride, preferably m an amount of 5500-8000 mg/L, magnesium sulphate, preferably an amount of 100-300 mg/L, sodium bicarbonate, preferably in an amount of 1500-3000 mg/L, and sodium dihydrogenphosphate, preferably in an amount of 100-200 mg/L. The concentrations of these minerals may be varied within rather wide ranges. Typically, the combined amount of minerals in a culture medium according to the invention is chosen such as to result in near physiological conditions (around 0.9%).

The vitamins may suitably be chosen from the group of L-ascorbic acid, biotin, D-calcium pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyridoxal, riboflavin, thiamine, vitamin B₁₂, vitamin A (retinoic acid) and combinations thereof. These may be present in the following, preferred concentrations:

L-ascorbic acid 75 - 1500 mg/L; biotin 0.03 - 0.5 mg/L;

D-calcium pantothenate 0.5 - 2 mg/L; choline chloride 0.5 - 2 mg/L; folic acid 0.5 - 2 mg/L; i-inositol 1 - 4 mg/L; nicotinamide 0.5 - 2 mg/L; pyridoxal 0.5 - 2 mg/L; riboflavin 0.03 - 0.5 mg/L; thiamine 0.05 - 2 mg/L; and vitamin B₁₂ 1 - 2 mg/L. It is further preferred that the present culture medium comprises amino acids. Suitably, these amino acids are selected from the following group in the specified concentrations :

L-alanine 10 - 50 mg/L; L-arginine 75 - 150 mg/L;

L-asparagine 30 - 80 mg/L,

L-aspartic acid 10 - 50 mg/L,

L-cystine 10 - 50 mg/L,

L-cysteine 75 - 130 mg/L; L-glutamic acid 50 - 100 mg/L;

L-alanyl -L-glutamine 5 - 20 mg/L; glycine 30 - 80 mg/L;

L-histidine 20 - 60 mg/L;

L-isoleucine 30 - 80 mg/L;

L-leucine 30 - 80 mg/L; L-lysine 50 - 100 mg/L;

L-methionine 5 - 30 mg/L;

L-phenylalanine 10 - 50 mg/L;

L-proline 20 - 60 mg/L;

L-serine 10 - 50 mg/L; L-threonine 30 - 80 mg/L;

L-tryptophan 5 - 15 mg/L;

L-tyrosine 10 - 60 mg/L; and

L-valine 30 - 80 mg/L.

The combined concentration of the amino acids preferably lies between 800 and 5000 mg/L. It is to be noted that this concentration range does not include L-glutamine.

L-glutamine is an important component of a culture medium according to the invention. It has been found that a concentration L-glutamine of at least 300 mg/L, preferably between 400 and 800 mg/L leads to a particular high proliferation and/or differentiation rate of cells cultured in the present culture medium.

Another component of a culture medium according to the invention is a growth factor. The nature of the growth factor may suitably be chosen dependent on the type of cells to be cultured. Examples of preferred growth factors are Bone Morphogenetic Protein (BMP) , Epidermal Growth Factor (EGF) , basic Fibroblast Growth Factor (bFGF) , Nerve Growth Factor (NGF) , Bone Derived Growth Factor (BDGF) , Transforming Growth Factor-βl (TGF-βl) , and human Growth Hormone (hGH) . In a preferred embodiment, the culture medium is employed to culture cells which are used in tissue engineering bone. In accordance with this embodiment, the growth factor is preferably bFGF. The growth factor is preferably present in an amount between 0.1 μg/L and 10 μg/L. In a preferred embodiment, the present culture medium further comprises an antibiotic or a combination of antibiotics. Examples of suitable antibiotics include penicillin G, gentamic , fungizone, streptomycin. It has surprisingly been found that the nature of the antibiotic influences that cultivation rate of cells in the present culture medium. A highly preferred combination of antibiotics is penicillin G and streptomycin. These antibiotics are preferably each employed an amount of 50-150 μg/mL, more preferably they are both employed in an amount of 100 μg/mL. The total amount of antibiotics a culture medium according to the invention preferably lies between 75 and 300 μg/mL.

The culture medium may further comprise any ingredient conventionally employed m culture media. Examples of such ingredients include serum, such as fetal bovine serum, autologous serum or synthetic serum (e.g. Ultrocer^®) , thioctic acid, phenol red, sodium pyruvate, πbonucleosides and deoxyπbonucleosides. A πbonucleoside or a deoxyπbonucleoside is preferably present in a concentration of between 5 and 15 mg/L. It is to be noted that when serum is used, it is added after the formulation of the culture medium. In other words, the amount of serum added dilutes the concentrations mentioned herein. Fetal bovine serum may be added m such an amount that the composition of serum and culture medium comprises between 5 and 15 vol . % of serum.

Ultrocer^® may be added m such an amount that the composition of serum and culture medium comprises between 1 and 10 vol . % of serum.

As has been mentioned above, the present culture medium is particularly useful for culturmg human cells, e.g. m a process for manufacturing tissue engineered products, such as skin grafts, bone implants, cartilage implants. The present culture medium has particularly been found advantageous for the m vi tro production of bone tissue. More osteogenic cells are formed resulting a higher success of bone formation after implantation, when the cells are cultured in a culture medium in accordance with the invention when compared to conventional culture media.

In principle, cells of any type may be cultivated in a culture medium according to the invention. Preferred cell types include stem cells, progenitor cells, mesenchymal cells, epithelial cells, cartilaginous cells, osseous cells, muscular cells, gland cells, fat cells, pericytes, satellite cells and dermal cells. Highly preferred cells to be cultured in the present culture medium are progenitor cells which may differentiate into bone. Differentiation of the cells may be facilitated by the presence of growth factors, such as Bone Morphogenetic Protein, or dexamethasone , which is preferably used in an amount of between 1*10^"9 and 1*10^"7 μg/L. The cells may be cultured in the presence of a scaffold or without a scaffold. If the cells are cultured in the presence of a scaffold, they can first suitably be seeded onto or into the scaffold in any known manner.

The invention will now be elucidated by the following, non-restrictive examples.

Example I

A culture medium was prepared by admixing the following ingredients in the specified concentrations in water :

Component lx Liquid mg/L

INORGANIC SALTS C CaaCCll₂₂ .. 2 2HH₂₂00 264 . 00

K KCC11 400 . 00

M MggSS00₄₄ .. 7 7HH₂₂00 200 . 00

N NaaCCll 6800 . 00

N NaaHHCC00₃₃ 2200 . 00

N NaaHH₂₂PP00₄₄ . . 22HH₂₂C0 ) 158 . 00

OTHER COMPONENTS D-Glucose 1000 . 00

DL- 68 Thioct ic Acid 0 . 20

Phenol Red 10 . 00

Sodium Pyruvate 110 . 00

AMINO ACIDS L-Alanine 25 . 00

L-Arginine . HC1 127 . 00

L-Asparagine . H₂0 50 . 00 L-Aspartic Acid 30.00

L-Cystine 24.00

L-Cysteine HCl 100.00

L-Glutamic Acid 75.00

Glycine 50.00

L-Histidine HCl . H₂0 42.00

L-Isoleucine 53.00

L-Leucme 52.00

L-Lysine . HCl 73.00

L-Methionme 15.00

L-Phenylalanme 32.00

L-Proline 40.00

L-Serine 25.00

L-Threonine 48.00

L-Tryptophan 10.00

L- Tyros ine 36.00

L-Valine 46.00

VITAMINS L-Ascorbic Acid 50.00

Biotin 0.10

D-Ca Pantothenate 1.00

Choline Chloride 1.00

Folic Acid 1.00

1-Inositol 2.00

Nicotinamide 1.00

Pyridoxal HCl 1.00

Riboflavin 0.10

Thiamine HCl 1.00

Vitamin B₁₂ 1.40

RIBONUCLEOSIDES Adenosme 10.00

Cytidine 10.00

Guanos me 10.00

Uridme 10.00

DEOXYRIBONUCLEOSIDES 2 'Deoxyadenosme 10.00

2 'Deoxycytidme HCl 11.00

2 ' Deoxyguanosme 10.00

2 ' Deoxythymidme 10.00 penicillin G 100 μg/mL streptomycin 100 μg/mL bFGF 1 ng/mL

L-glutamme 584 mg/L

Example II

Introduction.

In order to culture Human Bone Marrow Cells (HBMC's) efficiently it is necessary to develop a suitable cell culture medium. We compared two different media to investigate which type is most suitable for culturmg HBMC's and to see if there is a difference m, in vivo, bone formation after culturing these cells on a CaP scaffold. We performed several studies on HBMC's obtained of several different patients using either -MEM containing 10% Fetal Bovine Serum (FBS) , 0.2mM L-ascorbic acid 2-phosphate (AsAP) , 0. lmg/ml penicillin G, 50μg/ml gentamicin and 0.3μg/ml fungizone (AB) or in α-MEM containing 10% FBS, 0.2mM L- ascorbic acid 2-phosphate (AsAP) , lng/ml basic Fibroblast Growth Factor (bFGF) , 2mM L-glutamine (L-Glu) , lOOU/ml penicillin, lOOμg/ml streptomycin (pen/strep) and 1% (50U/ml) heparin (added in primary cultures only) and compared morphology, growth rates and bone formation. It was found that the combination of L-ascorbic acid, heparin, bFGF, L- Glutamine and penicillin/streptomycin in our culture medium enhanced cell -growth and showed a higher extent of bone formation.

Materials and Methods.

Human Bone Marrow cell collection and culture

Method A

Bone marrow aspirate was obtained from the iliac crest of a patient . In short 5 ml of aspirate was resuspended in 20 ml α-MEM containing 50U/ml heparin and 10% fetal bovine serum. The cell suspension is then resuspended using a 20G needle and centrifuged for 10 minutes at.300 g. The supernatant is discarded and the cell pellet is resuspended in α-MEM containing 10%FBS, 0.2mM L-ascorbic acid 2-phosphate (AsAP), 0. lmg/ml penicillin G, 50μg/ml gentamicin and 0.3μ g/ml fungizone (AB) . The obtained mononucleated cells are then plated at a density of ±500.000 cells/cm² in tissue culture flasks. Cells were grown at 37°C with 5% C0₂ in a humid atmosphere. The culture medium is refreshed twice a week and at near confluency the adherent cells are washed with phosphate buffered saline solution (PBS) and enzymatically released by incubating the cells with a 0.25% Trypsin-EDTA solution at 37°C for at least 10 Minutes. The released cells are then thoroughly resuspended and replated at a density of 5000-10.000 cells/cm² subsequent passages (up to the fifth passage) are performed when cells reach near confluency and cell morphology is monitored with light microscopy.

Method B

Bone marrow aspirate was obtained from the iliac crest of a patient. In short 5 ml of aspirate was resuspended in 20 ml α-MEM containing 50U/ml heparin and 10% fetal bovine serum. The cell suspension is then resuspended using a 20G needle and centrifuged for 10 minutes at 300 g. The supernatant is discarded and the cell pellet is resuspended in α-MEM containing 10%FBS, 0.2mM AsAP, lng/ml bFGF, 2mM L- glutamine (L-Glu) , lOOU/ml penicillin and lOOμg/ml streptomycin (pen/strep) and 1% (50U/ml) heparin (added in primary cultures only) . The obtained mononucleated cells are then plated at a density of ±500.000 cells/cm² in tissue culture flasks. Cells were grown at 37°C with 5% C0₂ in a humid atmosphere. The culture medium is refreshed twice a week and at near confluency the adherent cells are washed with phosphate buffered saline solution (PBS) and enzymatically released by incubating the cells with a 0.25%Trypsin-EDTA solution at 37°C for at least 10 Minutes. The released cells are then thoroughly resuspended and replated at a density of 5000-10.000 cells/cm² subsequent passages (up to the fifth passage) are performed when cells reach near confluency and cell morphology is monitored with light microscopy.

In Vivo experiments and Histology.

Porous CaP particles of size 2 by 3 mm are used for culturing the released HBMC's on. In short harvested HBMC's of several passages were seeded in a density of 100.000-

200.000 cells/particle. The cells were cultured during one week using α-MEM containing 10%FBS, 0.2mM AsAP, 10 nM Dexamethason (Dex) and lOmM beta glycerophosphate (βGP) before implantation.

Shortly before implantation the samples were soaked in α-MEM, washed in PBS and subcutaneously implanted into nude mice and kept in vivo for 4-6 weeks. Control samples incubated in both media, without cells were also implanted. At the end of the in vivo period the implanted samples were removed and immediately fixated in 1.5% glutaraldehyde in 0.14 M cacodylic acid buffer, pH 7.2-7.4.

After dehydration in an alcohol series and embedding in methyl methacrylate, the samples are sectioned on a Histological diamond innerlock saw (Leyden Microtome cutting system) . Sections of around 10μm are stained with basic fuchsin and methylene blue, in order to study bone formation. The sections were then scored per patient for bone formation.

Results .

Morpho1ogy .

Medium with AB (Method A) : figure 1 shows that often larger, flatter cell morphology observed. Cell growth was limited, and there was only occasionally bone formation observed after subcutaneous implantation. Improved medium (Method B) : figure 2 shows that cells with spindle shaped and fibroblastic morphology were obtained. Rapid cell proliferation was observed. Also, De Novo bone formation after subcutaneous implantation was widespread (see fig. 3) . Figure 4 shows a comparison of the growth curves of the cells cultured with methods A and B.

Figure 5 shows a comparison of 22 patients cultured with method A and 15 patients cultured with method B, in relation to bone formation. Discussion.

HBMC's cultured according to method B showed a linear culture expansion compared to HBMC's cultured according to method A. The growth rate of the two methods observed show that culturing HBMC's according to method B, results in a higher growth rate than observed for method A. In time culturing HBMC's with α-MEM containing 10%FBS, 0.2mM AsAP, 0. lmg/ml penicillin G, 50μg/ml gentamicin and 0.3μg/ml fungizone (method A) shows a decrease in cell growth. Use of culture medium comprising of bFGF, L-Glu, pen/strep, AsAP, with the addition of 50U/ml heparin in primary cultures has shown an improved morphology, growth rate and in vivo osteogenic character of the cells, after they have been stimulated to osteoprogenitor differentiation with dexamethasone .

Claims

1. Culture medium comprising glucose, a mineral, a vitamin, a growth factor and L-glutamine, wherein the L- glutamine is present in a concentration of at least 300 mg/L.

2. Culture medium according to claim 1, wherein the L- glutamine is present in a concentration between 400 and 800 mg/L .

3. Culture medium according to claim 1 or 2 , wherein the growth factor is chosen from the group of Bone Morphogenetic Protein (BMP) , Epidermal Growth Factor (EGF) , basic Fibroblast Growth Factor (bFGF) , Nerve Growth Factor (NGF) , Bone Derived Growth Factor (BDGF) , Transforming Growth Factor-βl (TGF-βl) , and human Growth Hormone (hGH) .

4. Culture medium according to claim 3, wherein the growth factor is bFGF.

5. Culture medium according to claim 4, wherein the bFGF is present in a concentration between 0.1 and 10 μg/L.

6. Culture medium according to any of the preceding claims, wherein the mineral comprises one or more ions chosen from the group of calcium, potassium, lithium, magnesium, sodium, sulphate, chloride, bicarbonate, and dihydrogenphosphate ions .

7. Culture medium according to claim 6, wherein the mineral is present in a concentration between 5 and 15 g/L.

8. Culture medium according to any of the preceding claims, wherein the vitamin is chosen from the group of L- ascorbic acid, biotin, D-calcium pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyridoxal, riboflavin, thiamine, vitamin B₁₂ , vitamin A and combinations thereof .

9. Culture medium according to claim 8 comprising L- ascorbic acid in a concentration between 75 and 1500 mg/L.

10. Culture medium according to any of the preceding claims comprising one or more amino acids chosen from the group of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-cysteine, L-glutamic acid, L-alanyl-L- glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L- Lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

11. Culture medium according to claim 10, wherein the amino acids are present in a concentration between 0.8 and 5 g/L.

12. Culture medium according to any of the preceding claim comprising an antibiotic.

13. Culture medium according to claim 12, wherein the antibiotic is chosen from the group of penicillin G, gentamicin, fungizone, and streptomycin.

14. Culture medium according to claim 13 comprising as antibiotic a combination of penicillin G and streptomycin, wherein both penicillin G and streptomycin are present in an amount of 50-150 μg/mL.

15. Culture medium according to any of the preceding claims comprising distilled water.

16. Culture medium according to any of the preceding claims comprising a ribonucleoside and/or a deoxyribonucleoside .

17. Culture medium according to any of the preceding claims to which serum is added.

18. Use of a culture medium according to any of the preceding claims for culturing human cells.

19. Use according to claim 18, wherein the cells are chosen from the group of stem cells, progenitor cells, mesenchymal cells, epithelial cells, cartilaginous cells, osseous cells, muscular cells, gland cells, fat cells, pericytes, satellite cells and dermal cells.

20. Use according to claim 19, wherein the cells are progenitor cells.