Title: Cell culture medium containing growth factors and L-glutamine
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.
Patent Number: 6,838,284 Issued on 01/04/2005 to de Bruijn, et al.
| Inventors:
|
de Bruijn; Joost Dick (Amersfoort, NL);
Tibbe; Gerhardus Johannes M. (Amersfoort, NL);
da Silva Madureira Mendes; Sandra Claudia (Gouda, NL)
|
| Assignee:
|
IsoTis N.V. (Bilthoven, NL)
|
| Appl. No.:
|
178050 |
| Filed:
|
June 21, 2002 |
Foreign Application Priority Data
| Current U.S. Class: |
435/404; 435/325; 435/366; 435/405; 435/406; 435/408 |
| Intern'l Class: |
C12N 005/00 |
| Field of Search: |
435/404,405,406,408,366,325
|
References Cited [Referenced By]
U.S. Patent Documents
| 5733541 | Mar., 1998 | Taichman et al.
| |
| 5811094 | Sep., 1998 | Caplan et al. | 424/93.
|
| 5830682 | Nov., 1998 | Moore | 435/29.
|
| 5858783 | Jan., 1999 | Goodwin et al. | 435/373.
|
| 5908784 | Jun., 1999 | Johnstone et al.
| |
| 5942225 | Aug., 1999 | Bruder et al.
| |
| 6156570 | Dec., 2000 | Hu et al.
| |
| 6537782 | Mar., 2003 | Shibuya et al.
| |
| 6596274 | Jul., 2003 | Abatangelo et al.
| |
| 6617159 | Sep., 2003 | Cancedda et al.
| |
| Foreign Patent Documents |
| WO 98 16630 | Apr., 1998 | WO.
| |
Other References
ATCC Cell Lines and Hybridomas (8.sup.th). 1994. pp 516, 519-520 & 523.
|
Primary Examiner: Lankford, Jr.; Leon B.
Attorney, Agent or Firm: Banner & Witcoff, Ltd.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of prior application PCT/NL00/00958,
filed on Dec. 27, 2000; which claims priority from European patent
application number 99204569.0, filed Dec. 28, 1999.
Claims
What is claimed is:
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 and wherein the vitamin is L-ascorbic acid in an amount
between 75 and 1500 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 wherein L-ascorbic acid is added in
the form of L-ascorbic acid-2-phosphate.
4. Culture medium according to claim 1, wherein the growth factor is chosen
from the group of bone morphogenetic protein, epidermal growth factor,
basic fibroblast growth factor, nerve growth factor, bone derived growth
factor, transforming growth factor-.beta.1, and human growth hormone.
5. Culture medium according to claim 4, wherein the factor is basic
fibroblast growth factor.
6. Culture medium according to claim 5, wherein the basic fibroblast growth
factor is present in a concentration between 0.1 and 10 .mu.g/L.
7. Culture medium according to claim 1, wherein the mineral comprises one
or more ions chosen from the group of calcium, potassium, lithium,
magnesium, sodium, sulfate, chloride, bicarbonate, and dihydrogenphosphate
ions.
8. Culture medium according to claim 7, wherein the mineral is present in a
concentration between 5 and 15 g/L.
9. Culture medium according to claim 1, further comprising a vitamin chosen
from the group consisting of biotin, D-calcium pantothenate, choline
chloride, folic acid, i-inositol, nicotinamide, pyridoxal, riboflavin,
thiamine vitamin B.sub.12, vitamin A and combinations thereof.
10. Culture medium according to claim 1 comprising one or more amino acids
chosen from the group of L-alanine, L-arginine, L-asparagine, L-aspartic
acid, L-cysteine, 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 claim 1, 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 .mu.g/mL.
15. Culture medium according to claim 1 comprising dexamethasone.
16. Culture medium according to claim 1 comprising distilled water.
17. Culture medium according to claim 1 comprising a ribonucleoside and/or
a deoxyribonucleoside.
18. Culture medium according to claim 1 further comprising serum.
19. Use of a culture medium according to claim 1 for culturing human cells,
comprising the steps of:
obtaining human cells; and
culturing the human cells in the culture medium of claim 1.
20. Use according to claim 19, 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.
21. Use according to claim 20, wherein the cells are progenitor cells.
Description
FIELD OF THE INVENTION
The Invention relates to the field of tissue engineering. In particular,
the invention discloses a medium and a method for culturing cells.
BACKGROUND OF THE INVENTION
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 in/onto the scaffold in an
efficient manner. In addition, the cells should be distributed
homogeneously throughout the scaffold, in order to enable continuous
neo-tissue formation.
In general, the cells that have been harvested from the patient's body are
cultured in vitro for a certain period of time, either with or without a
scaffold material present. During this culturing 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), amino 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
(.alpha.-MEM).
In the U.S. Pat. No. 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.sub.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.
BRIEF SUMMARY OF THE INVENTION
The present invention seeks to provide a culture medium wherein cells
proliferate and/or differentiate very fast. The objective of the 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
in 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-glutamine is present in a concentration of at least 300
mg/L.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts cells grown in medium including 10% fetal bovine serum, 0.2
mM L-ascorbic acid 2-phosphate, 0.1 mg/ml penicillin G, 50 .mu.g/ml
gentamicin and 0.3 .mu.g/ml fungizone according to method A.
FIG. 2 depicts cells grown in improved medium according to method B.
FIG. 3 depicts de novo bone formation after subcutaneous implantation.
FIG. 4 graphically depicts growth curves of cells cultured using methods A
and B.
FIG. 5 graphically depicts bone formation in 22 patients using cells
cultured by method A and in 15 patients using cells cultured by method B.
DETAILED DESCRIPTION OF THE INVENTION
Human cells have been found to proliferate much faster in a culture medium
according to the invention than in 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 in 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 in 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 in an amount of 100-400 mg/L,
potassium chloride, preferably in an amount of 200-600 mg/L, sodium
chloride, preferably in an amount of 5500-8000 mg/L, magnesium sulphate,
preferably in 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 B.sub.12, 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 B.sub.12 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-.beta.1 (TGF-.beta.1), 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 .mu.g/L and 10 .mu.g/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, gentamicin, 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 in an amount of 50-150
.mu.g/mL, more preferably they are both employed in an amount of 100
.mu.g/mL. The total amount of antibiotics in a culture medium according to
the invention preferably lies between 75 and 300 .mu.g/mL.
The culture medium may further comprise any ingredient conventionally
employed in culture media. Examples of such ingredients include serum,
such as fetal bovine serum, autologous serum or synthetic serum (e.g.
Ultrocer.RTM.), thioctic acid, phenol red, sodium pyruvate,
ribonucleosides and deoxyribonucleosides. A ribonucleoside or a
deoxyribonucleoside 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 in such an amount that the composition of serum and culture
medium comprises between 5 and 15 vol. % of serum. Ultrocer.RTM. may be
added in 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 culturing human cells, e.g. in 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 in vitro production of bone tissue. More osteogenic
cells are formed resulting in 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.sup.-9 and
1*10.sup.-7 .mu.g/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 1 .times. Liquid mg/L
INORGANIC SALTS
CaCl.sub.2.2H.sub.2 O 264.00
KCl 400.00
MgSO.sub.4.7H.sub.2 O 200.00
NaCl 6800.00
NaHCO.sub.3 2200.00
NaH.sub.2 PO.sub.4.2H.sub.2 O 158.00
OTHER COMPONENTS
D-Glucose 1000.00
DL-68 Thioctic Acid 0.20
Phenol Red 10.00
Sodium Pyruvate 110.00
AMINO ACIDS
L-Alanine 25.00
L-Arginine.HCl 127.00
L-Asparagine.H.sub.2 O 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.H.sub.2 O 42.00
L-Isoleucine 53.00
L-Leucine 52.00
L-Lysine.HCl 73.00
L-Methionine 15.00
L-Phenylalanine 32.00
L-Proline 40.00
L-Serine 25.00
L-Threonine 48.00
L-Tryptophan 10.00
L-Tyrosine 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 B.sub.12 1.40
RIBONUCLEOSIDES
Adenosine 10.00
Cytidine 10.00
Guanosine 10.00
Uridine 10.00
DEOXYRIBONUCLEOSIDES
2'Deoxyadenosine 10.00
2'Deoxycytidine HCl 11.00
2'Deoxyguanosine 10.00
2'Deoxythymidine 10.00
penicillin G 100 .mu.g/mL
streptomycin 100 .mu.g/mL
bFGF 1 ng/mL
L-glutamine 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 culturing
HBMC's and to see if there is a difference in, 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
.alpha.-MEM containing 10% Fetal Bovine Serum (FBS), 0.2 mM L-ascorbic
acid 2-phosphate (AsAP), 0.1 mg/ml penicillin G, 50 .mu.g/ml gentamicin
and 0.3 .mu.g/ml fungizone (AB) or in .alpha.-MEM containing 10% FBS, 0.2
mM L-ascorbic acid 2-phosphate (AsAP), 1 ng/ml basic Fibroblast Growth
Factor (bFGF), 2 mM L-glutamine (L-Glu), 100 U/ml penicillin, 100 .mu.g/ml
streptomycin (pen/strep) and 1% (50 U/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 .alpha.-MEM containing 50
U/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
.alpha.-MEM containing 10% FBS, 0.2 mM L-ascorbic acid 2-phosphate (AsAP),
0.0 mg/ml penicillin G, 50 .mu.g/ml gentamicin and 0.3 .mu.g/ml fungizone
(AB). The obtained mononucleated cells are then plated at a density of
.+-.500.000 cells/cm.sup.2 in tissue culture flasks. Cells were grown at
37.degree. C. with 5% CO.sub.2 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.degree. C. for at least 10 Minutes. The released cells are then
thoroughly resuspended and replated at a density of 5000-10.000
cells/cm.sup.2 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 .alpha.-MEM containing 50
U/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
.alpha.-MEM containing 10% FBS, 0.2 mM AsAP, 1 ng/ml bFGF, 2 mM
L-glutamine (L-Glu), 100 U/ml penicillin and 100 .mu.g/ml streptomycin
(pen/strep) and 1% (50 U/ml) heparin (added in primary cultures only). The
obtained mononucleated cells are then plated at a density of .+-.500.000
cells/cm.sup.2 in tissue culture flasks. Cells were grown at 37.degree. C.
with 5% CO.sub.2 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.degree. C.
for at least 10 Minutes. The released cells are then thoroughly
resuspended and replated at a density of 5000-10.000 cells/cm.sup.2
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 .alpha.-MEM containing 10% FBS, 0.2 mM ASAP, 10 nM
Dexamethason (Dex) and 10 mM beta glycerophosphate (.beta.GP) before
implantation.
Shortly before implantation the samples were soaked in .alpha.-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 10
.mu.m 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.
Morphology.
Medium with AB (Method A): FIG. 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): FIG. 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).
FIG. 4 shows a comparison of the growth curves of the cells cultured with
methods A and B.
FIG. 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 .alpha.-MEM containing 10% FBS, 0.2 mM AsAP, 0.1
mg/ml penicillin G, 50 .mu.g/ml gentamicin and 0.3 .mu.g/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 50 U/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.
*
