Keratinocyte serum-free medium promotes the growth of rat hair follicle stem cells

Zhiqiang ZHANG; Yujie WANG; Jia LI

ABSTRACT

BACKGROUND:

The hair fol icle stem cells are usual cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum. Recently, keratinocyte serum-free medium has been used to culture hair fol icle stem cells.

OBJECTIVE:

To observe the effects of three different culture media on the proliferation and purity of hair fol icle stem cells.

METHODS:

After sterling, the skin of the rat’s beard was cut off, and the vibrissa fol icles were dissected under a stereocroscope. The fol icles were digested by Dispase Ⅱ firstly and by the mixture of trypsin and EDTA secondly. cellsuspension was divided into three pieces based on the average number of cells and respectively cultured by keratinocyte serum-free medium, Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum and keratinocyte serum-free medium containing 10%fetal bovine serum. Hair fol icle stem cells were selected by rapid adherence on col agen type Ⅳ, and passaged. RESULTS AND

CONCLUSION:

Cultured hair fol icle stem cells spread to the third generation, and trypan blue assay showed there was no difference in cellsurvival rate among the three groups (P>0.05). cellCounting Kit 8 assay showed that the cells grew slowly in the three groups in the first 2 days. At day 4, the hair fol icle stem cells grew into the growth logarithmic phase, and the proliferative activity was ranked as fol owskeratinocyte serum-free medium+10%fetal bovine serum>Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12+10%fetal bovine serum>keratinocyte serum-free medium with a significant difference between the three groups (P<0.05). Flow cytometric examination showed the expression of CD34 in the keratinocyte serum-free medium+10%fetal bovine serum group was lower than that in the keratinocyte serum free medium group (P<0.05). The expressions of CD34, CK15 andβ1-integrin (CD29) in the Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12+10%fetal bovine serum were lower than those in the other two groups (P<0.05). These findings indicate that we can obtain higher purity hair fol icle stem cells in the keratinocyte serum-free medium+10%fetal bovine serum than in the Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12+10%fetal bovine serum.