Large Scale Production of Bone marrow

1
Large Scale Production of Bone marrow derived
Mesenchymal stem Cell in cGMP facility
Introduction Mesenchymal stem cells (MSC) have
recently received a lot of attention in
biological research because of their self renewal
capability, to expand and trans- differentiate
into many different cell types. To meet this
demand, a fast MSC expansion method is required.
Taking into consideration the lack of a uniform
approach for MSC culture and expansion, we
attempted in this study, to identify the optimal
protocol for the large-scale production of MSCs
by various large cell culture containers (Nunc-
10CF and Corning- 10CS) while maintaining their
multilineage differentiation potential.
Culture protocol for MSC in large container At
day 0 MSC were seeded at 1X104 /cm2 in a one
chamber cell STACK with medium containing DMEM
low glucose with10FBS. After 6days of culture,
total cell number were Increased to three folds.
At confluence, the cells were passed and cultured
in a large container (10CellSTACK -10CS) which is
ten time more surface area in the condition
previously described. On day six 10-CS reach
confluence, the cells were harvested and frozen.
No of cells seeded
No of cells harvested
20X106
73X106
No of cells seeded
200X106
Population doubling of MSCs
650X106
610X106
No of cells harvested
No of cells harvested
Corning 10CS
NUNC 10CF
Adipocytes
Osteoblasts
Flow Cytometric analysis of cell surface markers
of MSCs
Differentiation of MSCs into Adipocytes
Osteoblasts
Conclusion Using this system and culture method,
it is easy to produce large amount of cells
required for cell therapy. After 6 days of
culture, total cell numbers were increased 3
folds in both. The large scale produced MSC were
frozen in serum free conditions. The quality of
MSC output after such a culture remained high.
Efforts to define an alternative serum-free
culture Condition for MSC expansion are under
way. Our data indicate that, using these large
culture containers it is easy to produce large
amount of MSC in cGMP condition and the
production process is simple, safe and
economical. In conclusion, in this report we
define the optimal culture conditions for the
successful expansion of human MSCs in high
numbers for subsequent cellular therapeutic
approaches.