Study On In Vitro Culture Of HUCB-derived MSCs And Its Effectson The Proliferation Of Allogeneic Lymphocytes

A lot of experiment reports have confirmed that mesenchymal stem cells (MSCs) can be cultivated. from umbilical cord blood successfully. HUCB - derived MSCs has biology features similar to the bone marrow derived MSCs. While HUCB - derived MSCs still has the following characteristics:1.Due to umbilical cord blood is waste ,It is convenient to be obtained and not cause any damage to donors.2.HUCB - derived MSCs is more naive and has stronger ability of the proliferation and differentiation than BM MSCs.Therefore, HUCB - derived MSCs has an important research and clinical application value. But there are still many unsolved problems, such as separation methods of the effective components of umbilical cord blood are not stable, a long time of culture, low frequency of success etc. This paper wants to study on the preparation ,in vitro culture conditions, growth characteristics and some biological characteristics of HUCB– derived MSCs,, so as to establish a more suitable for the separation and in vitro culture conditions of HUCB - derived MSCs , and a more in-depth understanding.to the biological characteristics of the HUCB - derived MSCsUsing the methods of red blood cell lysis combined with adherence to isolate and purificate HUCB - derived MSCs .Using different culture conditions to screen the method of more suitable for the growth of HUCB - derived MSCs. With optical microscope to observe its morphological characteristics of : HUCB - derived MSCs. Cell population doubling time measured with trypan blue staining and understand its growth curve, Through the method of cell chemical dyeing further understanding of the cell chemical characteristics, By MTT colorimetric detection the effect of HUCB - derived MSCs on the proliferation of allogeneic lymphocyte from human umbilical cord blood.Results show that HUCB - derived MSCs can be isolated and purificated by red blood cell lysis combined with adherence. It can obviously improve the adhesive rate of HUCB - derived MSCs that fetal bovine serum is used to coate the culture plates ,Containing 5% FBS in medium is more advantageous to growth of HUCB- derived MSCs. than that of containing 2% FBS and 10% FBS in medium. When Containing 2% FBS in medium, HUCB- derived MSCs gain rates are :: 0% in not coated group,: 5.6% in coated group When Containing 5% FBS in medium, HUCB- derived MSCs gain rates are :: 16.7% in not coated group,: 27.8% in coated group. When Containing 10% FBS in medium, HUCB- derived MSCs gain rates are :: 16.7% in not coated group,: 22.2% in coated group.: Morphological characteristics of HUCB - derived MSCs is fibroblasts-like cells. In vitro culture for 8 days, the number of cells increase gradually with the extension of time . Cell population doubling time is 56.6 h. The results of cell chemical dyeing show PAS (+), NBE (+). HUCB- derived MSCs has the inhibition effect on the proliferation capacity of allogeneic lymphocytes from human umbilical cord blood. And the inhibition effect is relevant with the proportion of inoculated cell number of lymphocytes from umbilical cord blood and HUCB - derived MSCs. And compared to the control group of no HUCB - derived MSCs , when the proportion of inoculated cell number of lymphocytes from umbilical cord blood with HUCB - derived MSCs is for 100:1, the inhibition rate is 21.3%, while the proportion of inoculated cell number of lymphocytes from umbilical cord blood with HUCB - derived MSCs is for 50:1, the inhibition rate is 53.3%。.This can be concluded : 1. Using the methods of red blood cell lysis combined with adherence to isolate and purificate HUCB - derived MSCs and the method saves time and money., the chance of contamination is greatly reduced .So it is more suitable for separation and purification of HUCB derived - MSCs . 2. It can obviously improve the adhesive rate of HUCB - derived MSCs that fetal bovine serum is used to coate the culture plates .Containing 5% FBS in medium is more advantageous to growth of HUCB- derived MSCs. than that of containing 2% FBS and 10% FBS in medium. 3. through observation of cell morphology and cell growth curve, doubling time and biological characteristics of cells ,it can confirm that the cells are HUCB - derived MSCs. 4 HUCB - derived MSCs has negative immune control function, it can inhibit the proliferation of allogeneic lymphocytes, and at the same time, the inhibition effect is relevant with the proportion of inoculated cell number of lymphocytes from umbilical cord blood and HUCB - derived MSCs.