Experimental study on cultivation and purification of bone marrow-derived mesenchymal stem cells and its co-culture with chitosan porous scaffolds in vitro

Abstract

Background As commonly used scaffold material in tissue engineering, chitosan has many advantages, such as strong biodegradability, low antigenicity, good biocompatibility and no pyrogen reaction. This study aims to isolate, cultivate and purify Sprague-Dawley (SD) rat bone marrow-derived mesenchymal stem cells (BMSCs), and to observe the growth of BMSCs when co-cultured with self-made chitosan porous scaffold in vitro and to test the biocompatibility of this tissue engineering scaffold, so as to lay the foundation for promoting nerve regeneration of transplant treatment.  Methods Three-week-old healthy male SD rats were used in this study, and BMSCs were isolated and purified through bone marrow adherent culture method. The surface markers of BMSCs at Passage 3 were detected and identified by flow cytometry (FCM) and the BMSCs were three?dimensionally cultured in vitro on chitosan porous scaffolds produced by freeze-drying method. Ethanol alternative method was used to detect the chitosan scaffold porosity. Scanning electron microscope was used to explore the internal structure of the scaffold, measure the size of its aperture, and observe the morphology and development of the cells within the scaffold. Methyl thiazolyl tetrazolium (MTT) method was used to determine the cells' proliferation.  Results The cultured BMSCs were uniform and similiar to fibrous arrangement, and mixed cells reduced obviously. The identification result of FCM showed the CD29 positive rate was 98.49% and CD45RA positive rate was only 0.85%. The chitosan scaffold had an interlinked, uniform similar three-dimensional porous structure and its aperture porosity was 90%. Some cells stretched out pseudopod and infiltrated into the porous structure of scaffold, even fusing with them. The BMSCs were seeded in the scaffold successfully. The chitosan scaffold had no obvious effect on BMSCs' proliferation. Conclusions Chitosan porous scaffolds have good structural character and biocompatibility, and can be used as an alternative cell-carrier for later cell replace treatment of central nervous system diseases.

doi: 10.3969/j.issn.1672-6731.2014.11.015