Journal of Tissue Science and Engineering

Development of a Protocol for Human Adipose Stem Cell Culture in CO₂ Independent Medium and Perfusion Bioreactor

Abstract

Silva ARP, Paula ACC, Zonari A, Martins TMM, A M Goes and M M Pereira1

Advances in research on stem cells derived from human Adipose tissue (hASC) may allow its use for bone tissue engineering. In such context, it is important to standardize a methodology to culture cells in high quantity. Bioreactors, in which cells are cultured in three-dimension, mimic the physiological environment in vitro, allowing the hASC maintenance, proliferation and differentiation. The aim of the study was to assemble a perfusion bioreactor, and to develop a new protocol for hASC cultivation in a CO2 independent medium to be used in the bioreactor. The hASC were isolated from human lipoaspirate, and a two-dimensional cell culture was performed in DMEM, supplemented
with 10% Fetal Bovine Serum (FBS). The follow step was to adapt the cells to Leibovitz’s CO2 Independent Medium (LEI+10% FBS), and compare the MTT and Alkaline Phosphatase Activity (ALP) assays to cells cultured in DMEM. The cellular adaptation from DMEM to LEI chosen was the most gradual, beginning in the first passage with 10% LEI, continuing with 25%, 50%, 75% and 100% LEI in the subsequent passages. Phenotypic characterization was performed and hASC cultivated in DMEM and LEI expressed the following markers: CD29, CD44, CD73, and HLA-ABC. In a second step, hASC were evaluated in relation to MTT, ALP, and collagen synthesis, when cultivated in LEI and osteogenic LEI (LEI O), compared to DMEM and osteogenic DMEM. And finally, analysis of imunofluorescence were made of hASC cultivated in LEI and LEI O, showing that in induction medium, this cells expressed osteopontin, osteocalcin, and type I collagen, characteristic of osteogenic cell. The results suggest that
Leibovitz’s CO2 Independent Medium supplemented with 10% FBS is an adequate model for in vitro cultivation of hASC in perfusion bioreactor.