Development of bio-artficial skin composed of autologous keratinocytes layered on cryopreserved allogeneic cultured dermal substitute and its clinical application

将成纤维细胞培养在透明质酸（HA）和端胶原（Col）的双层海绵状基质上，制备同种异体培养真皮替代物（CDS）。同种异体CDS可以冷冻保存，并以冷冻状态运送到其他医院。在用于制备CDS的培养基中含有血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、肝细胞生长因子（HGF）、血小板衍生生长因子（PDGF）-AA、转化生长因子（TGF）-β1、角质形成细胞生长因子（KGF）、白细胞介素（IL）-6和IL-8，培养时间为1周（新鲜CDS培养基样品）。将冷冻保存的CDS解冻后，将CDS在培养基中进行黑麦培养1周。在用于黑麦培养CDS 1周的培养基（冻存CDS培养基样品）中含有VEGF、bFGF、HGF、TGF-β1和IL-8，尽管某些细胞因子的检测水平低于冻存前的水平。这一发现表明，冷冻保存的CDS仍保持着释放这些细胞因子的能力。北里大学人工皮肤研究开发中心新开发的同种异体CDS，已在日本各地的医疗中心进行了临床研究。结果表明，同种异体CDS作为一种优秀的细胞治疗顽固性皮肤溃疡以及烧伤。同种异体CDS本身的海绵状基质以及释放的细胞因子似乎对难治性皮肤缺损的治疗有益。为了扩大这一临床应用，我们开发了一种先进的培养皮肤替代品，生物人工皮肤，它是由自体角质形成细胞层在冷冻保存的CDS。Wye通过无胸腺小鼠的动物试验发现，这种生物艺术皮肤可以成功地

Allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a 2-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). Allogeneic CDS can be cryopreserved and transported to other hospitals in a frozen state. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF)-AA, transforming growth factor (TGF)-β1, keratinocytes growth factor (KGF), interleukin (IL)-6, and IL-8 were contained in the culture medium which used in preparing CDS over a cultivation period of 1 week (fresh CDS culture medium sample). After thawing a cryopreserved CDS, the CDS was rye-cultured in a culture medium for 1 week. VEGF, bFGF, HGF, TGF-β1, and IL-8 were contained in the culture medium which used in rye-culturing CDS for 1 week (cryopreserved CDS culture medium sample), although some cytokines were detected in lower level than those before freezing. This finding suggests that the cryopreserved CDS retains its ability to release these cytokines.Clinical research on allogeneic CDS, which was newly developed at the R&D Center for Artificial Skin of Kitasato University, has been carried out in medical centers across Japan. It was demonstrated that the allogeneic CDS functions as an excellent cell therapy for intractable skin ulcers as well as bum injuries. The spongy matrix itself as well as the cytokines released firm the allogeneic CDS seemed to be beneficial for the treatment of intractable skin defect. To expand this clinical application, we developed an advanced cultured skin substitute, bio-artificial skin, which is composed of autologous keratinocytes layered on a cryopreserved CDS. Wye found with animal test using athymic mouse that this bio-artficial skin take successfully