To produce clinical grade umbilical cord mesenchymal stromal cells (UC-MSCs) with a umbilical cord in a GLP lab.

The umbilical cord was collected after delivery and the arteries, veins and blood cells were removed. Different treatment conditions were applied to compare the yield of UC-MSCs. The Wharton's jelly was cut to 1 mm³ cubes, which were then either directly placed into an adhesive culture dish, or digested with different types of collagenases, followed by expansion in three types of serum free culture medium. After three passages in vitro, the cells were characterized and analyzed for cell viability, morphology, growth, sterility, endotoxin levels, chromosome karyotyping, surface antigen expression and differentiation capacity. Data analysis between different groups was performed with coupled t test.

Digestion with collagenaseⅡor collagenaseⅠachieved the highest yield of MSCs (5.3×10⁶±0.58/ml and 5.1×10⁶±0.57/ml, respectively, P = 0.07) , both higher than treatment with collagenaseⅠ+ 0.25﹪trypsin (2.53×10⁵±0.03/ml) and enzyme-free method. The culture medium appeared clear after collagenaseⅡdigestion. MSCs cultured in Stemcell MesenCult-ACF Medium showed optimal cell morphology. Before freezing, the cell viability was 99.8﹪, and 99﹪ after freezing and thawing. The obtained MSCs were negative for bacteria or mycoplasma contamination, hepatitis B virus (HBV) , hepatitis C virus (HCV) , human immunodeficiency virus (HIV) , Mycoplasma pneumonia (MP) , Treponema pallidum (TP) , EB virus (EBV) , and cytomegalovirus (CMV) . The endotoxin levels were less than 1 EU as tested by ELISA. FACS results showed that more than 98﹪of the obtained cells were positive for CD73, CD90 and CD105, and more than 98﹪of the cells negative for CD34, CD45 and HLA-DR. The cells showed a normal karyotype and could be differentiated into adipocytes, osteoblasts and cartilage cells.

Clinical grade MSCs are generated in a GLP lab with optimized isolation/culture conditions.