Optimization and identification of in vitro isolation and culture condition of human umbilical cord blood mesenchymal stem cells

Chunsheng ZHAO; Gengyin WANG; Junxian LI

ABSTRACT

BACKGROUND:

Compared with the bone marrow mesenchymal stem cells, umbilical cord blood mesenchymal stem cells (UCB-MSCs) is an ideal source of tissue engineered seed cells, but the culture success rate is low.

OBJECTIVE:

To explore establish a stable reliable method to isolate and culture UCB-MSCs by optimizing medium choice,centrifugation speed and time, incubation density, choice of growth factor, first time of medium change.DESIGN, TIME AND

SETTING:

The cytological in vitro study was performed at the Department of Blood Transfusion, Bethune International Peace Hospital of Chinese PLA from January to October 2008.MATERIALS A total of 20 samples of neonatal UCB by full-term uterine-incision delivery were supplied by Stem Cell Center,Bethune International Peace Hospital of Chinese PLA. Parturient and their family member signed the informed consent.

METHODS:

Under sterile conditions, UCB-MSCs were isolated by combination of density gradient centrifugation (1 500 r/min, totally 15 minutes) and different adherent time method. UCB-MSCs were incubated in DMEM/F12 medium, supplemented with 10% human UCB serum, 5 μg/L granulocyte-macrophage colony-stimulating factor (GM-CSF), 15 pg/L interleukin-3. MSCs at 1 ×1010/L were incubated in plastic flask coated with human UCB serum at 37℃ and 5% CO2 saturated humidity. The medium was changed following 3 days of culture. Non-adhered cells were removed. Subsequently, the medium was changed once every other 24 hours. When 80% confluence, UCB-MSCs were digested by the mixture of pancreatin-athylenediamine tetraacatic acid.MAIN OUTCOME

MEASURES:

Morphological changes of UCB-MSCs were observed by inverted phase contrast microscopy. Cell immunophenotypes were determined by flow cytometry.

RESULTS:

A small quantity of adherent round cells were determined after 24 hours, and adherent cells became more at 48 hours,with a few monopole spindle cells. Cell colonies were detected at day 7. Fibroblast-like cells arranged parallelly, presented whirlpool-shape and unclear boundary, with 80% 80% confluence 2-3 weeks following culture. At the second passage, these calls adhered at hour 12, and reached 80% 90% confluence at day 10. Flow cytometry showed that these calls were positive for CD29 and CD44, but negative for hematopoietic lineage marker CD34.

CONCLUSION:

MSCs can be successfully isolated from human UCB by using this modified method in vitro, with short culture cycle and high cell purification. Adherent cells have the same immunophenotype as bone marrow mesenchymal stem cells.