Comparison of characteristics of different generations of rabbit nucleus pulposus cells after cryopreservation

English Keywords:Nucleus pulposus cells  Cryopreseryation  Different generation  Cell viability  Cell culture  Rabbit

English Abstract:

  【Abstract】 Objective：To compare the characteristics of different generations of rabbit nucleus pulposus cells（NPC） after cryopreservate and search for the best generation suitable for cryopreseryation as seed cells.Method：10 New Zealand white rabbits（3-4 months old） were used，and the thoracic and lumbar（T1/2-L7/S1）NPC were separated immediately after general anesthesia under strictly aseptic conditions and cultured in vitro：control group（group A，n=10），the original generational cells；group B，C，D，E and F respectively representing the first，second，third，forth and fifth generation cells（each group n=10 and cell concentration 1×105/ml） were cryopreserved for 2-3 months and then continued for two weeks after recovery.The Trypan Blue and MTT were used for detecting cell survival rates and cell growth activity respectively；the proteoglycan（PG） synthesis and glycosaminoglycans（GAG） accumulation were tested；and the expression of type Ⅱ collagen gene were evaluated by RT-PCR.All the results were analyzed statistically using the SPSS 13．0 software package.Result：Cell survival rate of group B and C was close to that of group A on the 2nd day（P>0.05） and group D，E and F had obviously lower cell survival rate than that of group A，B and C（P<0.05）.Cells of each group proliferated in different degrees.The growth tendency of group A，B，C and D was closer，and group A，B and C showed no time-related difference（P>0.05），while group D had worse cell survival rate than group A（P<0.05）.The proliferation of group E on the 10th and 14th days as well as each time point of group F showed no significant difference（P>0.05）.PG synthesis and GAG accumulation of group A，B and C at each time point were higher than that of group D，E and F at the corresponding time point（P<0.05）.The collagen Ⅱgene expression of group A，B and C was higher than that of group D and E（P<0.05），and group F had lowest expression（P<0.01）.Conclusion：After cryopreservation，different generations of cells present varied characteristics.The first and second generations are suitable as seed cells after cryopreservation due to their better maintenance of NPC characteristics.