Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60 percent of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Thermolysin in human cultured keratinocyte isolation

BACKGROUND: When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. PURPOSE: To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. METHODS: This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. RESULTS: Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. CONCLUSION: The number of colonies in the thermolysin group was significantly greater than in the trypsin group.

INTRODUÇÃO: No tratamento do paciente grande queimado, onde se usa lâminas de epiderme cultivadas, o principal problema é o tempo necessário para sua produção. O isolamento tradicional de queratinócitos utiliza normalmente tripsina. No presente estudo, foi utilizada uma modificação do método de isolamento tradicional, que poderia produzir uma maior pureza e um maior número de colônias formadas a partir da população de células epidérmicas isoladas. OBJETIVO: Comparar a ação da tripsina e da termolisina no isolamento de queratinócitos usando pele de prepúcio de récem-nascidos. MÉTODOS: Essa metodologia utilizou a termolisina, que realiza a separação seletiva entre a epiderme e a derme. Após essa separação, a epiderme foi submetida à ação da tripsina para a obtenção da suspensão celular. RESULTADOS: Comparado ao método convencional, os experimentos mostraram que no grupo da termolisina mostrou facilidade para a separação entre a epiderme e derme, não houve contaminação por fibroblastos e produziu um maior número de colônias formadas, com diferença estatística significante. CONCLUSÃO: O número de colônias no grupo termolisina foi significantemente maior que no grupo tripsina.