An allogeneic cultured dermal substitute (CDS) was prepared by cultivating fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). The ability of fibroblasts to secrete cytokines is dependent on the conditions of freezing and thawing. The first experiment was designed to investigate the effects of supplements in a cryoprotective medium, that is, dimethylsulfoxide (DMSO), glycerol, and fetal bovine serum (FBS). In each experiment we measured the cell viability after thawing and the cell growth in CDS recultured after thawing. In addition, the amount of vascular endothelial growth factor (VEGF) released from the CDS recultured for one week after thawing was measured. The highest values of cell viability, cell growth, and the amount of VEGF released were obtained when CDS was frozen in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% DMSO and 40% FBS, and then thawed quickly in a water bath at 37 degrees C. However, due to the high cost of FBS, in clinical applications CDS is usually frozen in DMEM supplemented with 10% DMSO and 20% FBS. In practice, however, physicians often cannot use CDS immediately after thawing, depending on clinical conditions. Therefore, in the second experiment we investigated cell viability at different time points after thawing. In addition, we investigated cell growth and the amount of VEGF released from fibroblasts in CDS at different time points after thawing under different conditions, and after further reculturing for one week. We recommend that CDS be rinsed with lactated Ringer's solution immediately after thawing, and that it be used within 4 h after thawing.

Allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a two-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)-beta1, keratinocytes growth factor (KGF), interleukin (IL)-6 and IL-8 were contained in the culture medium which was used in preparing CDS over a cultivation period of one week (fresh CDS culture medium sample). After thawing a cryopreserved CDS, the CDS was recultured in a culture medium for one week. VEGF, bFGF, HGF, TGF-beta1 and IL-8 were contained in the culture medium which was used in reculturing CDS for one week (cryopreserved CDS culture medium sample), although some cytokines were detected at a 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 with the support of the Millennium Project of the Ministry of Health, Labor and Welfare. It was demonstrated that the allogeneic CDS functions as an excellent cell therapy for intractable skin ulcers as well as burn injuries. The spongy matrix itself, as well as the cytokines released from the allogeneic CDS, seemed to be beneficial for the treatment of intractable skin defect.

Allogeneic cultured dermal substitute (CDS) was prepared by culturing 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. The present study was designed to analyze amounts of cytokines released from fibroblasts in fresh or cryopreserved CDS. The culture medium used in preparing CDS over a cultivation period of 1 week (fresh CDS culture medium sample) contained vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF)-AA, transforming growth factor (TGF)-beta1, keratinocyte growth factor (KGF), interleukin (IL)-6 and IL-8. After thawing of cryopreserved CDS, the CDS was re-cultured in medium for 1 week. The culture medium used in re-culturing CDS for 1 week (cryopreserved CDS culture medium sample) contained VEGF, bFGF, and HGF in the same concentration as before freezing, and TGF-beta1 and IL-8 at half the concentration before freezing. Levels of PDGF-AA, KGF, and IL-6 were significantly less than before freezing. This finding suggests that the cryopreserved CDS retains its ability to release VEGF, bFGF, and HGF that are essential for wound healing.

We have developed an allogeneic cultured dermal substitute (CDS) by cultivating fibroblasts on a 2-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). The HA sponge was designed to have a honeycomb structure with many holes (0.5 mm diameter) separated by a distance of 4 mm. Part of the Col sponge was able to penetrate into these holes, and the resulting anchoring structure allows binding of a HA spongy layer with a Col spongy layer. The preparation of the CDS consists of two steps: (i) attachment of cells to the Col surface of the hydrated 2-layered spongy matrix and (ii) proliferation of cells on this sponge immersed in culture medium. The aim of the present study was to assess properties of fresh and cryopreserved CDS. Fibroblasts seeded on the Col surface of the 2-layered spongy matrix attached, proliferated and released vascular endothelial growth factor (VEGF) and fibronectin. The amount of VEGF released from cryopreserved CDS after thawing slowly in an incubator at 37 degrees C and re-cultivation for 1 week was about 300 pg/ml. After thawing quickly in a water bath at 37 degrees C and re-cultivation for 1 week, the amount of VEGF released was about 600 pg/ml. These findings indicate that the cryopreserved CDS maintained its ability to release a significant amount of VEGF. Retention of the therapeutic properties of CDS after cryopreservation is important for clinical use.

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