Wright K A, Nadire K B, Busto P, Tubo R, McPherson J M, Wentworth B M
Genzyme Tissue Repair, Cell Biology Group, Framingham, MA 01710-9322, USA.
Burns. 1998 Feb;24(1):7-17. doi: 10.1016/s0305-4179(97)00075-2.
The Epicel ASAProgram service generates autologous keratinocyte grafts used for the closure of full-thickness wounds in moderately and severely burned patients. The manufacturing process used to generate Epicel service autografts (ESA) is based upon the keratinocyte co-culture technique described by Rheinwald and Green which employs murine Swiss 3T3/J2 fibroblasts as feeder cells. Recently, a technique has been described that employs a polyurethane wound dressing, HydroDerm (HD, Innovative Technologies, Ltd), as a delivery vehicle for cultured keratinocytes intended for autologous grafting. We have examined the practical feasibility of this technique and report on testing the ability of HD to support keratinocyte growth and epithelium formation in vitro, at the air-liquid interface (ALI), and in vivo, after grafting to full-thickness wounds created on the backs of athymic (Swiss Nu/Nu) mice. The results demonstrate that keratinocytes grow on the HD dressing in Gibco SFM at a rate that is approximately 15 per cent of that observed when cells are cultivated on tissue culture (TC) plastic using standard techniques, yet the cells retain their proliferative capacity and form an epithelium in vitro when cultivated at the ALI on a dermal substrate. Keratinocyte-seeded HD membranes were also transferred to full-thickness wounds in athymic mice. Animals grafted with cells seeded to HD developed human epithelium, as revealed by species-specific detection of involucrin and evolved a normal attachment to the wound substratum, as demonstrated through the expression of dermally opposed laminin and alpha 6 beta 4 integrin. The ability of keratinocytes to maintain proliferative potential after seeding onto HD and their ability to form a properly oriented epithelium in vitro and in vivo suggests that this wound dressing may be useful as a vehicle for autologous keratinocyte grafting and help to provide earlier epithelial coverage to the burned patient. However, because of the slow proliferation rate of keratinocytes on HydroDerm, timely graft delivery would be best achieved by combining cell expansion via the Rheinwald and Green culture system, followed by the seeding of cells onto HydroDerm in a reduced calcium medium for subsequent autologous grafting.
Epicel ASA程序服务可生成自体角质形成细胞移植物，用于闭合中度和重度烧伤患者的全层伤口。用于生成Epicel服务自体移植物（ESA）的制造工艺基于Rheinwald和Green所描述的角质形成细胞共培养技术，该技术采用小鼠瑞士3T3/J2成纤维细胞作为饲养细胞。最近，有一种技术被描述，该技术采用聚氨酯伤口敷料HydroDerm（HD，创新技术有限公司）作为用于自体移植的培养角质形成细胞的递送载体。我们研究了该技术的实际可行性，并报告了测试HD在体外、气液界面（ALI）以及体内支持角质形成细胞生长和上皮形成的能力，即在移植到无胸腺（瑞士裸鼠/裸鼠）小鼠背部创建的全层伤口后。结果表明，角质形成细胞在Gibco SFM中的HD敷料上生长，其速率约为使用标准技术在组织培养（TC）塑料上培养细胞时观察到的速率的15%，然而，当在ALI条件下在真皮基质上培养时，细胞保留其增殖能力并在体外形成上皮。接种角质形成细胞的HD膜也被转移到无胸腺小鼠的全层伤口。通过特异性检测兜甲蛋白发现，移植接种到HD上的细胞的动物形成了人上皮，并通过真皮相对的层粘连蛋白和α6β4整合素的表达证明，其与伤口基质形成了正常附着。角质形成细胞接种到HD上后维持增殖潜力的能力以及在体外和体内形成正确定向上皮的能力表明，这种伤口敷料可能作为自体角质形成细胞移植的载体有用，并有助于为烧伤患者提供更早的上皮覆盖。然而，由于角质形成细胞在HydroDerm上的增殖速率较慢，通过结合经由Rheinwald和Green培养系统进行细胞扩增，随后在低钙培养基中将细胞接种到HydroDerm上以进行后续自体移植，将最好地实现及时的移植物递送。
