Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM.
BACKGROUND
Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements.


METHODS
As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation.


RESULTS
We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP).


CONCLUSIONS
Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.
Müller
I
I
University Children's Hospital Tübingen, Tübingen, Germany. Ingo.Mueller@med.uni-tuebingen.de
Kordowich
S
S
Holzwarth
C
C
Spano
C
C
Isensee
G
G
Staiber
A
A
Viebahn
S
S
Gieseke
F
F
Langer
H
H
Gawaz
M P
MP
Horwitz
E M
EM
Conte
P
P
Handgretinger
R
R
Dominici
M
M
D000943:Antigens, Differentiation / Q000096:biosynthesis; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D006801:Humans; D008648:Mesoderm / Q000166:cytology* / Q000378:metabolism; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism
D000943:Antigens, Differentiation; D016895:Culture Media, Serum-Free; D010982:Platelet-Derived Growth Factor