Separation, Culture And Cryopreservation Of Cells From Source Tissues Of Placenta And Umbilical Cord

Background: In recent years the adult stem cells has been more and more used as the seed cells of cellular therapy, gene therapy and tissue engineering. In this study, we established the separation method of the purposed cells, including human placental derived adherent cells (HPDACs) and human umbilical artery smooth muscle cells (HUA-SMCs), from the placenta and umbilical cord, cultured and identified the separated cells, and employed low-temperature cryopreservation for the preservation of the placental tissue, so as to provide plentiful cells and tissue resource for the realization of the individualized gene therapy and cell therapy in the future. Part 1Objective: We employed and compared the different methods of human placental tissue cryopreservation, analyzed the influences of the cryopreservation on the separation efficiency of HPDAC from the placental tissue and biological characteristics of HPDAC.Method: The placental tissue was divided into five groups. Each group, weighing up to 20g, is broken into tissue masses with 3mm x 3 mm size by the mixer and divided into five tubes. The PVS2, preservation and vitrification solution, with different concentrations (5%,10%,20%,40%,60%) were added to five groups respectively as an anti-creaming agent to bring down the temperature quickly, which were then preserved in the liquid nitrogen. After they had been cryopreserved for three months, the cryopreservation tissues were revived. The placental tissue in the former and later cryopreservation were respectively analized by sodium and potassium-ATP enzyme, vitality of catalase and separation efficiency of HPDAC tests Meanwhile, the above separated HPDACs were made a morphological observation, immune phenotype identification, test of curve of growth, as well as the inductive differentiation towards osteogenesis and adipogenesis.Result: It's found out that the activity of 10% enzyme in PVS2 group is the highest, the second is 20% in PVS2, and the lowest is 60% in PVS2. The average separation efficiency in the HPDACs number of fresh placental tissue is (0.575±0.30)×10⁵/g. After cryopreservation, the HPDACs could be separated from only 10% of PVS₂ group of placental tissue. Among them, the average separation efficiency of HPDACs' number is (0.158±0.05)×10⁵/g; the HPDAC morphology of placental tissue from cryopreservation revival, immune phenotype, curve of growing and HPDACs of fresh placental tissue source are in a basic agreement.Part 2Objective: In this part of study, we separated the HUA-SMCs from human umbilical artery and explored the influences of low-temperature preservation on the biological characteristics.Method: The original generation of HUA-SMCs were separated from the membrane tissue drawn from human umbilical artery through an adherent method and the HUA-SMCs could be preserved in the low temperature through a two-step method. After 3-month cryopreservation, we compared the cell morphology of HUA-SMCs before cryopreservation with those after cryopreservation, utilized anti-α-smooth muscle actin antibody to have an identification of immune cytochemistry and analized the biological characteristics through curve of growth.Result: It's found out that the tissue adherent method could get a large number of HUA-SMCs with fusiform shape. It proves positive by the identification ofα-smooth muscle actin. Moreover, comparing HUA-SMC under the low-temperature cryopreservation with fresh HUA-SMC, we found that there is no obvious difference between the cell morphology and biological characteristics.conclusion(1) In this study the HPDACs have been separated from the placental tissue successfully, which has the characteristics of stem cells and the potentials towards osteogenesis and adipogenesis.(2) It is indicated that after the revival of placental tissue cryopreservation, the separation effiency of HPDAC is very low, and the influence of low-temperature cryopreservation on the survival of placental tissue is significant.(3) The HUA-SMCs have been separated from the smooth muscle of human umbilicus artery vessels successfully. After a long-term of cryopreservation, the HUA-SMCs remains the basic biological characteristics of the smooth muscle cells of human umbilicus artery vessels. After the revival, its original proliferation capacity can be recovered, resulting in a kind of new cell source for the seed cells of tissue engineering in the future.