ALLOGENEIC MESENCHYMAL STEM CELL COMPOSITIONS AND METHODS THEREOF

摘要

The present invention discloses a method of isolation, pooling and further culturing of Mesenchymal Stem cells (MSC) for clinical application. Present invention also discloses the method of establishing Master Cell bank, followed by Working Cell Bank from which the final therapeutic composition referred to as Investigational Product/Investigational Medicinal Product comprising of allogenic bone marrow-derived MSC is formulated for clinical applications.

主权项

1. A method of preparing a Master Cell Bank composition comprising Mesenchymal stem cells, Fetal Bovine Serum (FBS) and Dimethyl Sulphoxide (DMSO), the method comprising:
a) obtaining and diluting bone marrow cells in culture media and centrifuging the diluted cells to obtain a first pellet and re-diluting the first pellet with the culture media; b) adding density gradient solution to the re-diluted pellet and centrifuging to obtain a buffy layer at interface; c) re-adding equal volume of the culture media to the buffy layer and centrifuging to obtain a second pellet, followed by suspending the second pellet in the culture media and incubating to achieve about 80% to 85% confluency; d) aspirating the culture media and washing the confluent cells with Dulbecco's Phosphate Buffered Saline (DPBS), thereafter treating the cells with 0.25% trypsin and re-incubating the treated cells; and e) neutralizing the re-incubated cells with neutralization media and re-centrifuging the cells to obtain a third pellet, followed by suspending the third pellet in the culture media, culturing, harvesting and freezing the cells with freezing media comprising about 85% to 95% FBS and about 5% to 15% DMSO to obtain the Master Cell Bank composition.