Ex Vivo Expansion Of Human Umbilical Cord Mesenchymalstem Cells Using Microcarrier Culture Systems

Stem cell therapy is promising in solving a series of complicated diseased including degenerative cells or tissue disorders; muscle, bone and cartilage defects; cancer etc. Umbilical cord mesenchymal stem cells (hUCMSCs) are some type of stem cells with high degree of self-renewal and differentiation potential that have recently been discovered. hUCMSCs have variety of sources, easy to isolation, low in immune rejection, and are considered to be the ideal cells for stem cell therapy.However, 109 cells are required by a successful treatment, which means that isolated stem cells must be effectively expanded in vitro before clinically used. hUCMSCs proliferation using the traditional two-dimensional culture system is hard to meet the need of quantity of cells in a short period of time, and the excessive passage times may cause damage to stem cells. Culture using three-dimensional microcarriers can provide enough growth area, and simulate a 3D environment in order to maintain the characteristics of the undifferentiated stem cells. Our work discussed the features and value of application of three-dimensional microcarrier culture system.To study the culture features of human umbilical cord mesenchymal stem cells (hUCMSCs) on 2D plate, 3D Cytodex-3 and Cultispher-S. Primary hUCMSCs were passaged for use. Immunophenotypic characteristics of cells were identified by flow cytometry. 4×104/ml hUCMSCs were seeded in plate, spinner flask with Cytodex-3 and spinner flask with Cultispher-S, respectively. In the following 7 days, microcarrier structures and cell morphology were observed by Scanning Electron Microscopic (SEM). Cell number was calculated by cck-8 assay and the growth curve was drawn. Cell distribution on microcarriers was observed by DAPI staining. Metabolic kinetics was measured. Cells under three culture systems were harvested and differentiated in osteogenesis media for 14 days, ALP activity was analyzed and alizarine red staining was conducted. Results showed that hUCMSCs expressed CD29, CD44, CD90 and CD105, but negative for CD14 and CD45. After 7 days proliferation, cells on macroporous Cultispher-S had the highest density which reached(8.0±0.32)×105 /ml, with Cytodex-3 followed of(5.8±0.23)×105 /ml, plate culture was the lowest with (4.1±0.1)×105 /ml. Cells on Cultispher-S at late phase formed aggregates, Cell Bridge could be observed. Large amounts of extracellular matrix (ECM) are secreted by cells. Metabolic kinetics showed that cells on Cultispher-S are most efficient in utilization of glucose. Cells recovered from three culture systems all remain the osteogenic potential. In addition, Cultispher-S is made of collagen which could degrade in human bodies, thus avoid trypsinization procedure used to harvest cells. In summary, 3D culture system can provide larger amount of cells than 2D culture. Cultispher-S was more suitable than Cytodex-3 for clinically expansion of hUCMSCs.