The Impact On The Cells Activity Of Articular Chondrocytes Stored In Different Cold-Preservations

ObjectiveTo evaluate the effects of three different preservation methods on morphology of Articular Chondrocytes,the survival rate and activity of the cartilage cells.These three different cryogenic methods which act on fresh Articular Chondrocytes are slow gradient cooling cryopreservation,slow constant cooling cryopreservation,vitrification group of fresh Articular Chondrocytes is the collator.To compare the results of those different preservation methods at two weeks and one month,elect the best preservation method, providing a best way for the organizational engineering.MethodsThe fresh Articular Chondrocytes were derived from healthy rabbit knee joint.Under the aseptic conditions.The rabbits were between two and four weeks old.The cells were cultured Under the aseptic conditions in vitro.Deal with the Articular Chondrocytes with the methods of gradient cooling cryopreservation, constant cooling cryopreservation,and vitrification.After two weeks and one month took out some Articular Chondrocytes, resuscitate ceels in 37℃water condition and observe the results.Constant cooling cryopreservation was the method that frosts the admixture of cells and low-temperature protective agent in two steps.Slow Gradient cooling cryopreservation was a method which put the admixture of cells and low-temperature protective agent to 4 temperature gradients before it droped to-40℃.The vitrification group was that the admixture of cells and low-temperature protective agent was deal with the vitrification solution and then put it into liquid nitrogen at the fastest speed to preserve.Articular Chondrocytes in all experime- ntal groups took out in the preservation of 2 weeks and 1 month,and then used inversion microscope to observe the shape of cells,identified by toluidine blue staining and observe the biological characteristics of chondrocytes.And use CCK8 to detect activity of cartilage cells(OD values reflect the activity of cartilage cells).The differences of the type II collagen were tested by Western blot, and so on the mRNA levels were tested by RT-PCR. Make the fresh group as the control group to compare with the experimental group.Results1.The chondrocytes of the prime generation were anchorage-dependent cells,the cells anchored when it was the 24 hours,the bottom of bottle was full of chondrocytes when it was the 36 hours,The shape of cells was polygonal or triangular.preserved for 2 weeks and1 month the experimental groups compared with the control group had statistical difference. Slow gradient cooling cryopreservation group was similitude to the control group.The cytoplasm of vitrification group and slow constant cooling group were fewer.And the amount were decreased.Preserved for 2 weeks was better than preserved for 1 month. Identified by toluidine blue staining and observe the biological characteristics of chondrocytes.Confirmed that it was conformed to the biological characteristics of chondrocytes.2.Activity of cartilage cells:Preserved for 2 weeks,the experimental groups compared with the control group had statistical difference(P<0.05).Compared slow gradient cooling group with vitrification group and slow gradient cooling group with slow constant cooling group,all of them had statistical difference(P<0.05).Preserved for 1 month,the experimental groups compared with the control group had statistical difference(P<0.05). Compared slow gradient cooling group with vitrification group and slow gradient cooling group withm slow constant cooling group,all of them had statistical difference(P<0.05).In different preservations,the slow gradient cooling group had statistical difference.3.The expression of collagen II:Preserved for 2 weeks,the expression of collagen II ,the experimental groups compared with the control group had statistical difference(P<0.05).Compared slow constant cooling group with vitrification group did not have statistical difference.Preserved for 1 month,the experimental groups compared with the control group had statistical difference.Compared slow gradient cooling group with vitrification group and slow gradient cooling group with slow constant cooling group,all of them had statistical difference(P<0.05).In different preservation,the slow gradient cooling group had statistical difference(P<0.05).4.The expression of mRNA:The expected length of specifie strip in type II collagen mRNA PCR products were visible in all three groups'pecimens.The expression of type II collagen mRNA in the experimental groups was lower significantly in comparison with the control group.Compared slow gradient cooling group with slow constant cooling group and vitrification group had statistical difference.Compared slow constant cooling group with verification group had statistical difference.Conclusion1.The methods of slow gradient cooling cryopereservation effected the Articular Chondrocytes was fewer than the methods of slow constant cooling cryopereservation and verification. The methods of slow gradient cooling cryopereservation had some certain clinical application value.2.The preservation longer the effects of slow gradient cooling cryopereservation, verification and slow constant cooling cryopereservation were more distinctness.