Almqvist K F, Wang L, Broddelez C, Veys E M, Verbruggen G
Department of Rheumatology, Ghent, University Hospital, University of Ghent, Belgium.
Osteoarthritis Cartilage. 2001 May;9(4):341-50. doi: 10.1053/joca.2000.0394.
To preserve viable, metabolically active chondrocytes cultured in alginate beads at -196 degrees C for further use in in vitro and in vivo studies.
Human articular chondrocytes were isolated from femoral condyles within 24 h post mortem. To optimize the biological freezing procedure, the chondrocytes were control-rate frozen in different concentrations of dimethyl sulfoxide (DMSO) in Dulbecco's MEM supplemented with 10% FCS before being thawed and the cell viability was determined by Trypan Blue exclusion test. To investigate the effect of control-rate freezing on chondrocyte metabolism, control-rate frozen chondrocytes in 5% DMSO were thawed and cultured in gelled agarose for 2 weeks. Non-frozen chondrocytes cultured in agarose served as controls. Furthermore, human articular chondrocytes were cultured in 2% alginate beads for 2 weeks after which the beads were incubated with 5% DMSO for 0 h, 2.5 h, 5 h and 10 h and frozen at -196 degrees C. Non-frozen alginate beads containing chondrocytes and incubated with 5% DMSO served as a control. After 2 weeks in culture, chondrocytes in agarose or in alginate were sulfated with 10 microCi(35)SO(4)/ml for 48 h. The total production of aggrecans, and the aggrecan subtypes, were subsequently determined.
Five percent DMSO in the culture medium was the optimal condition to control-rate freeze and recover viable and functional isolated chondrocytes. Total aggrecan synthesis of control-rate frozen chondrocytes cultured in gelled agarose was not significantly reduced when compared with control cells. The proportion of aggrecan in the aggregate form of control-rate frozen chondrocytes kept in agarose remained unaltered. Chondrocytes, control-rate frozen in the alginate matrix, showed a 0-30% decrease in total aggrecan synthesis rates in culture when compared with the non-frozen chondrocytes. The optimal pre-incubation time of the alginate beads with 5% DMSO was 5 h, without any change in aggrecan synthesis rates when compared with the control situation. Shorter pre-incubation times resulted in an insufficient diffusion of DMSO into the beads and in cell death. There was no difference in the synthesis of the different aggrecan subtypes between frozen and non-frozen chondrocytes in alginate.
Human articular chondrocytes can be stored at -196 degrees C for 24 h without important decreases in their aggrecan synthesis rates when control-rate frozen as a cell suspension in 5% DMSO. Proportions of the aggrecan subtypes (monomers, aggregates) synthesized by chondrocytes cultured in agarose remained unchanged. The control-rate freezing procedure in the alginate beads pre-incubated with 5% DMSO for 5 h produced no decrease in total aggrecan synthesis rates and no change in the synthesized aggrecan subtypes. Further experiments have to confirm the suitability of this freezing method for long-term storage of chondrocytes allowing us to set up a 'chondrocyte' bank for further use in in vitro and in vivo manipulations.
将培养在藻酸盐珠中的活的、具有代谢活性的软骨细胞保存在-196℃，以供进一步用于体外和体内研究。
在死后24小时内从股骨髁分离出人关节软骨细胞。为优化生物冷冻程序，在补充有10%胎牛血清的杜氏改良 Eagle 培养基中，将软骨细胞在不同浓度的二甲基亚砜（DMSO）中进行程序降温冷冻，然后解冻，并通过台盼蓝排斥试验测定细胞活力。为研究程序降温冷冻对软骨细胞代谢的影响，将在5%DMSO中程序降温冷冻的软骨细胞解冻，并在凝胶化的琼脂糖中培养2周。在琼脂糖中培养的未冷冻软骨细胞作为对照。此外，将人关节软骨细胞在2%藻酸盐珠中培养2周，之后将珠子与5%DMSO分别孵育0小时、2.5小时、5小时和10小时，然后在-196℃冷冻。含有软骨细胞且与5%DMSO孵育的未冷冻藻酸盐珠作为对照。培养2周后，用10微居里(35)SO(4)/毫升对琼脂糖或藻酸盐中的软骨细胞进行硫酸化处理48小时。随后测定聚集蛋白聚糖的总产量以及聚集蛋白聚糖亚型。
培养基中5%DMSO是程序降温冷冻并复苏活的且功能正常的分离软骨细胞的最佳条件。与对照细胞相比，在凝胶化琼脂糖中培养的程序降温冷冻软骨细胞的聚集蛋白聚糖总合成量没有显著降低。保存在琼脂糖中的程序降温冷冻软骨细胞中聚集形式的聚集蛋白聚糖比例保持不变。与未冷冻的软骨细胞相比，在藻酸盐基质中程序降温冷冻的软骨细胞在培养中的聚集蛋白聚糖总合成率降低了0 - 3%。藻酸盐珠与5%DMSO的最佳预孵育时间为5小时，与对照情况相比，聚集蛋白聚糖合成率没有变化。预孵育时间较短会导致DMSO向珠子内扩散不足并导致细胞死亡。藻酸盐中冷冻和未冷冻软骨细胞之间不同聚集蛋白聚糖亚型的合成没有差异。
当作为细胞悬液在5%DMSO中进行程序降温冷冻时，人关节软骨细胞可以在-196℃保存24小时，其聚集蛋白聚糖合成率不会显著降低。在琼脂糖中培养的软骨细胞合成的聚集蛋白聚糖亚型（单体、聚集体）比例保持不变。用5%DMSO预孵育5小时的藻酸盐珠中的程序降温冷冻程序不会导致聚集蛋白聚糖总合成率降低，也不会使合成的聚集蛋白聚糖亚型发生变化。进一步的实验必须证实这种冷冻方法是否适合软骨细胞的长期保存，以便我们建立一个“软骨细胞”库，供进一步用于体外和体内操作。
