Cultured porcine epithelial grafts: an improved method.
Cultured porcine epithelial grafts: an improved method.
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DOI：
10.1111/1523-1747.ep12874554
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发表时间：
1990-02
期刊：
影响因子：
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通讯作者：
S. Regauer;C. Compton
中科院分区：
文献类型：
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作者：
S. Regauer;C. Compton
描述了猪角质形成细胞体外培养的改进方法，通过该方法可以快速产生适合移植的角质形成细胞片。在 3 小时内，用 0.25% 分散酶溶液（37 摄氏度）将猪皮的表皮与真皮酶促分离，并用胰蛋白酶消化成单细胞悬浮液。角质形成细胞在补充有 20 ng/ml 氢化可的松、100 微克/ml 青霉素、100 微克/ml 链霉素和 20%（来自六个月大猪的细胞）或 10% 胎牛血清（来自两个月大猪的细胞）的 Dulbecco-Vogt 改良 Eagle 培养基中生长。新鲜分离的角质形成细胞以 1.25 X 10(6) 细胞/ml 的密度进行铺板，因为它们的铺板效率比在类似条件下生长的人类角质形成细胞低约 15 倍。铺在塑料上的原代角质形成细胞比铺在致命辐射的 3T3-J2 饲养层细胞上的原代角质形成细胞生长汇合更快。在塑料上生长的猪角质形成细胞在第三次传代时达到衰老，但随后在经致死辐射的 3T3-J2 饲养层上培养时，最多可传代七次。然而，第二代猪角质形成细胞的铺板效率仅为5%-7%左右，而人新生儿包皮角质形成细胞的铺板效率为20%-30%。在塑料上生长的汇合分层原代培养物或在饲养层上生长的次生培养物用于嫁接。用分散酶溶液将片材分离并钉在凡士林纱布上以方便处理。通过这种方法产生的猪移植物移植到供体动物的肌肉筋膜切除的全层伤口后，已证实表皮再生。
An improved method of in vitro cultivation of porcine keratinocytes by which keratinocyte sheets suitable for grafting can be generated rapidly is described. Epidermis from split-thickness porcine skin is enzymatically separated from dermis with 0.25% Dispase solution (37 degrees C) within 3 h, and trypsinized to a single cell suspension. Keratinocytes are grown in Dulbecco-Vogt modified Eagle medium supplemented with 20 ng/ml hydrocortisone, 100 micrograms/ml penicillin, 100 micrograms/ml streptomycin, and 20% (cells from six-month-old pigs) or 10% fetal calf serum (cells from two-month-old pigs). Freshly isolated keratinocytes are plated at a density of 1.25 X 10(6) cells/ml since their plating efficiency is about 15 times lower than that of human keratinocytes grown under comparable conditions. Primary keratinocytes plated on plastic grow to confluence faster than those plated on lethally irradiated 3T3-J2 feeder layer cells. Porcine keratinocytes grown on plastic reach senescence in the third passage but, when subsequently cultivated on a lethally irradiated 3T3-J2 feeder layer, can be passaged up to seven times. Nevertheless, plating efficiency of second-passage porcine keratinocytes is only about 5%-7%, whereas that of human newborn foreskin keratinocytes is 20%-30%. Confluent stratified primary cultures grown on plastic, or secondary cultures grown on feeder layers, are used for grafting. The sheets are detached with Dispase solution and stapled to vaseline gauze to facilitate handling. Epidermal regeneration from porcine grafts produced by this method has been demonstrated after transplantation to full-thickness wounds excised to muscle fascia in donor animals.