Kubo Kentaro, Kuroyanagi Yoshimitsu
Regenerative Tissue Engineering, Graduate School of Medical Sciences, Kitasato University, Sagamihara, Kanagawa, Japan.
Artif Organs. 2005 Oct;29(10):800-5. doi: 10.1111/j.1525-1594.2005.00132.x.
Allogeneic cultured dermal substitute (CDS) was prepared by cultivating fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). CDS can be cryopreserved and transported to other hospitals in a frozen state. To evaluate cell viability, cell growth, and release of VEGF after long-term cryopreservation, the CDS was cryopreserved at -85 degrees C or -152 degrees C for a given period. We measured cell viability immediately after thawing and cell growth in CDS that was recultured for 1 week after thawing. In addition, the amount of vascular endothelial growth factor (VEGF) released from CDS that was recultured for 1 week after thawing was measured. The cell viability and cell growth of control CDS that was thawed within 3 weeks after freezing was 56.2% and 132.7%, respectively. The cell viability and cell growth of the CDS that was cryopreserved at -85 degrees C for 6 months was 43.4% and 119.7%, respectively. When cryopreserved at -152 degrees C for 1 year, the cell viability and cell growth was 52.0% and 110.8%, respectively. These values were comparable to those of the control. The amount of VEGF released from CDS cryopreserved at -85 degrees C for 6 months (491.0 pg/mL) or at -152 degrees C for 1 year (586.8 pg/mL) was comparable to that of the control CDS (587.3 pg/mL). In contrast, the amounts of VEGF released from CDS cryopreserved at -85 degrees C for 1 year (322.5 pg/mL) or at -152 degrees C for 2 years (210.8 pg/mL) were low, with a marked decrease in cell viability and cell growth. These findings suggest that CDS cryopreserved at -85 degrees C for 6 months or at -152 degrees C for 1 year maintains sufficient cell viability and the ability to proliferate and release a significant amount of VEGF. The release of VEGF from CDS after long-term cryopreservation is a useful therapeutic effect, and is important for clinical use.
异体培养真皮替代物（CDS）是通过在透明质酸（HA）和脱细胞胶原蛋白（Col）的双层海绵基质上培养成纤维细胞制备而成。CDS可以冷冻保存，并以冷冻状态运输到其他医院。为了评估长期冷冻保存后细胞活力、细胞生长以及VEGF的释放情况，将CDS在-85℃或-152℃下冷冻保存一定时间。解冻后立即测量细胞活力，并测量解冻后再培养1周的CDS中的细胞生长情况。此外，还测量了解冻后再培养1周的CDS释放的血管内皮生长因子（VEGF）的量。冷冻后3周内解冻的对照CDS的细胞活力和细胞生长分别为56.2%和132.7%。在-85℃下冷冻保存6个月的CDS的细胞活力和细胞生长分别为43.4%和119.7%。在-152℃下冷冻保存1年时，细胞活力和细胞生长分别为52.0%和110.8%。这些值与对照相当。在-85℃下冷冻保存6个月（491.0 pg/mL）或在-152℃下冷冻保存1年（586.8 pg/mL）的CDS释放的VEGF量与对照CDS（587.3 pg/mL）相当。相比之下，在-85℃下冷冻保存1年（322.5 pg/mL）或在-152℃下冷冻保存2年（210.8 pg/mL）的CDS释放的VEGF量较低，细胞活力和细胞生长明显下降。这些发现表明，在-85℃下冷冻保存6个月或在-152℃下冷冻保存1年的CDS保持了足够的细胞活力、增殖能力以及释放大量VEGF的能力。长期冷冻保存后CDS释放VEGF是一种有益的治疗效果，对临床应用很重要。
