Improvement Of Bone Marrow Stromal Stem Cells In Vitro Culture Method

Bone marrow mesenchymal stem cells (bone mesenchymal stem cells, BMSCs) also called bone marrow stromal source of stem cells, exist in bone marrow hematopoietic stem cells in addition to the outside another kind has the multiplex differentiation potential of stem cells。In certain induction conditions, this kind of cell differentiation can be directed for a variety of hematopoietic outside of the organization,especially mesoderm and neural epiblast progenitors of source organization cells, such as the osteoblast and into cartilage cells, fat cells, tendon cells, muscle cells, nerve cells, etc. Another mesenchymal stem cells (mesenchymal stem cells, MSCs) for easy in vitro, trauma tiny, with an infinite proliferation and multiplex differentiation potential, plus BMSCs has posted the characteristics of the growth wall, the in vitro easy separating and amplification, but also easily of heterologous gene expression and into, so BMSCs by the medical profession’s favour, it becomes a kind of ideal cells to treat tools and gene therapy to target cells. It heralds the regenerative medicine and tissue engineering precedent. However mesenchymal stem cells in the bone marrow content is very low, in need of separation and purification and proliferation. This is the main problem that we are facing at present.ObjectiveThrough the observation of different micro environment on rats serum bone marrow stromal stem cell proliferation the influence of the growth of the state to get a more suitable for BMSCs in vitro growth environment, so as to facilitate the improvement of bone marrow stromal stem cells in vitro methods, to improve the in vitro culture get rate, so that its expand in vitro and purification, better applied to clinical treatment.MethodsSince6-8W age SD male rats femoral, tibia and fibula extraction in bone marrow stromal stem cells. Adopted the bone marrow stick wall of training. In the following three respectively to serum micro environmental culture:Allogeneic serum groups:Extraction are the cells containing10%after substitute rat allogeneic serum culture medium training, later in ChuanDai liquid containing10%in the womb of bovine serum media training。 Tire bovine serum groups:Extraction are the cells containing10%after substitute tire bovine serum culture medium training, later in ChuanDai liquid containing10%when still using the womb bovine serum culture medium training; No serum groups:Extraction cells do not contain the alternative tire of bovine serum DMEM medium training, later in ChuanDai liquid containing10%in the womb of bovine serum media training. In the inverted microscope micro environment different serum bone marrow stromal stem cell growth form, cell growth curve, flow cytometric testing different under the environment of serum micro cell cycle and cell surface CD45and CD90expression. SPSS17.0software application of statistical data analysis.ResultsRats allogeneic serum micro environment culture cells form small, a spindle, mesh growth.10%tire bovine serum micro environment culture cell with a spindle formation fiber appearance, JiLa type growth. No serum micro environment culture cell rare, with only slight JiLa type growth. The rat growth curve allograft serum group multiply fastest, followed by10%tire bovine serum group, and no serum group has not seen the obvious rate doubled. Flow cytometric detection contain serum medium under the culture condition of the third generation of the cells CD90positive expression rate of80%or more, CD45positive expression rate under2%. And in serum-free culture conditions CD90positive expression rate at74.09%, CD45positive rate of only6.09%in expression. This shows that under the environment of serum-free micro cultured cells purity was significantly lower than the serum micro environment with the training of cells.ConclusionsRats allogeneic serum micro environment culture cells form good uniformity, alignment are higher than the other groups, and for the first time ChuanDai significantly longer cells in other serum micro short environment. Allogeneic serum and10%tire bovine serum are suitable for bone marrow stromal stem cell growth, but in allogeneic serum micro environment cell proliferation faster, and in serum-free culture conditions did not see the obvious phenomenon of proliferation. According to these results demonstrate that allogeneic serum micro environment is more suitable bone marrow stromal stem cell growth and purification, is more advantageous to the in vitro culture and improvement of the method.