Cultivating technique for human keratinocyte under laboratory condition
- VernacularTitle:实验室条件下人角质形成细胞的培养技术
- Author:
Ning LU
;
Ping ZHU
;
Yufeng LIU
;
Gang WANG
;
Hailong ZHANG
;
Xiaodong ZHAO
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(1):180-182
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Along with the establishment and development of in vitro culture technique for human keratinocyte, skin would no longer be considered only as the physiological barrier, it's of important significance in immunity and endocrinology and so on.OBJECTIVE: To explore the experimental cultivating technique for human keratinocytes to provide reliable cell resource forthe appliance of keratinocytes in many ways.DESIGN: An opening study with keratinocytes as the subjects of the experiment.SETTING: Department of Immunology and Department of Dermatology,Xijing Hospital, Fourth Military Medical University of Chinese PLA MATERIALS: This experiment was carried out in the clinical laboratory of Department of Immunology of Xijing Hospital of the Fourth Military Medical University of Chinese PLA between March 2003 and March 2005.Prepuce sample was postoperatively obtained from a 6-year-old boy who was admitted to the department of urology surgery of Xijing hospital in April 2003, prepuce was used as the source for keratinocytes.METHODS: ①Two step digestion technique was used for the isolation of epidermal cells: Firstly the skin flap was digested with dispase of mass concentration of 2.5 g/L at 4 ℃ for overnight, followed by separating epidermis the next day; Secondly it was dipped in trypsin and EDTA mixture for subsequent digestion. ② Improved serum-free cultivating technology was applied to the culture of human keratinocytes. The culture medium is composed of human keratinocyte serum free culture medium +2.5 mg/L bovine pituitary lixivium +5 μg/L epidermal growth factor+438 mg/L glutamine, glutamine can promote the growth of the keratinocyte. ③Human keratinocyte in exponential phase were cryopreserved and rewarmed, then made into cell suspension with additional serum free medium, cells were incubated in culture bottle of 75 cm2 by density of 5×105/mL for subculture. Cell morphological changes were observed under microscope and transmission electron microscope.MAIN OUTCOME MEASURES: ① The growth of primary cells and passage cells. ② The cryopreservation and resuscitation of human keratinocyte from different passages. ③ Morphological observation of Keratinocytes RESULTS: ①The growing state of the primary cells and passage cells:cells suspended primarily and gradually adhered to bottom of the culture dish at about 6-24 hours, most of cells were round and gradually stretched to ellipse shape, at about day 3, some keratinocyte clones and clusters can be observed with satellite-like proliferating epidermal cells scattered around, most of them were polygon and cell homogeneity and transparency became strengthened. Cell fusion amount to 70% at around 5 days, reaching to 90% at day 9 and even completely fused into cell diaphragm at day 1 1. Keratinocyte cultured in vitro can survive 2-3 months without obvious changes in cell morphology and growth speed. ② The cryopreservation and resuscitation of human keratinocyte in different passages: Different passage keratinocytes were separately cryopreserved in liquid nitrogen pot and resuscitated 3 months later, the morphology and growth speed of keratinocytes did not change obviously. ③Morphological observation on Keratinocytes: Cells possess typical epidermal cell characteristics under microscope, displaying higher nucleus/plasma ratio, cells arranged closely with clear boundary and good refraction. Transmission electron microscope revealed that cultured epidermal keratinocyte possessed multiple keratinocyte characteristics, such as massive bundles of tonofibrilla in the plasma, clearly observed mitochondria and rough endoplasmic reticule, cytoplasm sticking out short prominence and cells were found connected by desmosome.CONCLUSION: Keratinocyte can keep its normal morphological characeristics during in vitro culture process by using improved cultivating technique, which can be taken as reliable and abundant cell resource for the experimental and clinical study of human keratinocytes.
