The Experimental Study On The Human Nucleus Pulposus Cells Culture In Vitro And Cryopreservation

Objective:(1) To study the biological characteristics of the nucleus pulposus cells whichcoming from the nucleus pulposus tissues of the anterior cervical operation.(2) To searchfor the best generation of the nucleus pulposus cells suitable as seed cells in tissueengineering.(3) To study the effects of cryopreservation and recovery processing onnucleus pulposus cells of type II collagen synthesis and cell proliferation.Methods:(1) The nucleus pulposus cells of the human nucleus pulposus tissues obtainedfrom anterior cervical operation is extracted by enzymatic digestion method.(2) Thecellular morphology and growth were observed under an inverted phase contrastmicroscope, and the cell climbing piece of nucleus pulposus cells of the cell climbing piecewere identified by type II collagen immunohistochemical staining method, When thenucleus pulposus cells were in vitro.(3) The mean optical density of the first, second, third,fourth and fifth generation of immunohistochemical staining photograph is measured byImage-Pro Plus6.0software, so as to observe the difference between type II collagencontent in cytoplasm.(4) The primary nucleus pulposus cells were divided into A, B twogroups. The cells in group A were frozen and stored at-80℃refrigerator, be thawed inthe2weeks later, and group B cells without the cryopreserved. The OD value of A groupand B group were measured by MTT method to observe the difference of A, B two groupsof nucleus pulposus cells proliferation activity. The mean optical density values ofimmunohistochemical staining photos of A group and B group nucleus pulposus cells weremeasured by Image-Pro Plus6.0software, which reflects in the cryopreservation andrecovery effect on the synthesis of nucleus pulposus cells of type II collagen.Results:(1) The enzyme digestion method was successfully extracted and culturedhuman nucleus pulposus cells from the human nucleus pulposus tissues obtained fromanterior cervical operation. The first and second generation cell is a short fusiform shape,triangle or polygon. After the cells passaged to the third generation, the nucleus pulposuscells became spindle.(2) The mean optical density of the first generation and the second generation of nucleus pulposus cells had no significant difference, in the type II collagenimmunohistochemical staining(P＞0.05). There was statistically significant that the firstand the second generation of nucleus pulposus cells on the mean optical density comparedwith the third generation, fourth generation and fifth generation, in the type II collagenimmunohistochemical staining(P＜0.05).(3) Group A compared with B group was noobvious statistical difference on the mean optical density of the type II collagenimmunohistochemical staining(P＞0.05). The difference of optical density of MTT assay atA group and B group didn’t have statistical significance(P＞0.05).Conclusion:(1) The nucleus pulposus cells of the human nucleus pulposus tissuesobtained from anterior cervical operation could be cultured in vitro and passaged cells.(2)The first and the second generation of nucleus pulposus could act as seed cells of tissueengineering intervertebral disc for their intracellular type two collagen synthesis and cellmorphology in the normal state.(3)The cryopreservation and recovery processing coundnot reduce nucleus cytoplasm of type II collagen synthesis, also cound not reduce cellproliferation.