Application Of An In Vitro Organ Culture Model Of Mouse Vibrissa Follicles And Investigation In Artificial Skin With Generated Hair Follicles In Vitro

Objectivewe expect to establish an in vitro organ culture model of mouse follicles and investigate the biology properties of hair follicle induced by FK506 in this model, we also expect to establish an in vitro model of hair follicles damaged by anti-cancer drugs .By utilizing this model, we investigate the protective effects of minocycline and FK506 agaist Ara-c, and also investigate the remolding effects of FK506 agaist Ara-c. We observed the skin and hair follicles regeneration after the implantation of hair follicle bulb cells into collagen/chitosan porous scaffolds in vitro.Material and MethodC57BL/6J mice 4 d old or from 4 w to 6 w old offered by experimentation animal centre of Zhejiang University. Mouse vibrissa follicles were isolated by microdissection and cultured in Wi 11 iams' E serum free medium, and the hair growth length and hair growth period keeping in vitro organ culture were observed compared to the hair follicles cultured in DMEM medium. We also observed effects of FK506 at different concentrations changed hair growth properties and 'H-TdR incorporation in the follicles in vitro. Organ cultured mouse vibrissa follicles were also used to investigate the protective effects of minocycline and FK506 on the hair follicle total growth length, growth speed and hair growth period keeping which were inhibited by Ara-c. 3H-TdR and MTT were used to investgate protective effects of minocycline and FK506 on proliferation of hair follicle cells inhibited by Ara-c.In hair follicle regeneration investigation, the cultured dorsal hair follicle bulb cells of 4 days old C57BL/6 mice at different density and different passages were implanted into collagen/chitosan porous scaffolds or on collagen/chitosan porous scaffolds surface in vitro. And cells proliferating and assembling on surface of collagen/chitosan porous scaffold were observed by upside-down microscope. Histological characteristics were investigated hy HE, Keratin-H, Kreatin-L and vimentin were ditected by immunohistochemistry. Cells alive and hair follicle formation were also investigated by cofocal laser scaning microscopy.ResultAn in vitro organ culture model of mouse vibrissa follicles was established by isolated hair follicles, which isolated by microdissection and cultured in Williams'E medium supply with insulin 10mg/L, hydrocortisone 10 g/L, HEPES 10mmol/U sodium selenite 10 g/L,transferin 10mg/L, penicillin 100U/L, streptomycin 100 g/L etc. Mouse vibrissa follicles grow mainly within 3-4 d after culture, hereafter hair follicles growth slow down gradually. Compared to DMEM medium, Will iams' E medium is more appropriately to organ cultured hair fol licles. From 0. 003mg/L to 0. 3mg/L, FK506 improved mouse vibrissa follicles total growth length, growth speed, hair growth period keeping, and also improved 'H-TdR incorporation in the moue vibrissa follicles in vitro. FK506 at these concentrations also prolonged anagen. Whereas the hair growth was significantly inhibited in vitro when FK506 is over Img/L. Ara-c (lOmg/L and 50mg/L) inhibited mouse vibrissa follicles total growth length, growth speed, growth period keeping and 3H-TdR incorporation, and also inhibited proliferation of hair follicle bulb cells in vitro. FK506 at 0. 01-0. 3 mg/L improved the hair follicles total growth length, growth speed, hair growth period keeping, 3H-TdR incorporation in vitro which were all inhibited by Ara-c. Minocycline at 0. 3 10-6~-10-5mol/L improved mouse vibrissa follicles total growth length, growth speed, growth period keeping in vitro organ culture and proliferation of hair follicle bulb cells in vitro , which are inhibied by Ara-c.It was interesting that we discovered that the skin-like structure formed on the collagen/chitosan porous scaffolds by HE dye, which were implanted by cultured hair follicle bulb cells before 4th passages. Cells cladding formed was thicker than others on collagen/chitosan porous scaffolds 4 weeks later after implanting, which bulb cells were before 3th passages and implanted density were over 5.