Skin punch biopsy explant culture for derivation of primary human fibroblasts.

摘要: Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used the derivation of induced pluripotent stem cells model disease1. Early passages these can also be for cell-based functional assays specific pathways, mechanisms2 subsequent drug screening approaches. The advantage presented over enzymatic procedures 1) reproducibility technique small amounts tissue older patients, e.g. patients affected Parkinson's disease, 2) technically simple approach more challenging methodologies using treatments, 3) time consideration: takes 15-20 min performed immediately after biopsy arrival. Enzymatic treatments take up 4 hr problems overdigestion, reduction viability attachment when not handled properly. This describes dissection preparation a 4-mm human skin fibroblast culture has very high success rate which is important dealing patient-derived samples. In culture, keratinocytes migrate out within first week preparation. Fibroblasts appear 7-10 days outgrowth keratinocytes. DMEM glucose media supplemented 20% FBS favors growth will overgrow After 2 diluted resulting relatively homogenous cultures expresses marker SERPINH1 (HSP-47). Using approach, million 4-8 weeks banking. about min, then monitored once day under microscope, changed every 2-3 cells.