Method optimization of skin biopsy-derived fibroblast culture for reprogramming into induced pluripotent stem cells (iPSCs)#

Authors#

Kathleen Mommaerts, Camille Bellora, Pauline Lambert, Seval Türkmen, Jens C. Schwamborn, Fay Betsou

Abstract#

Background: Fibroblasts can be isolated from skin biopsies using either a chemical dissociation, a physical dissociation or a combination of both techniques. They can be reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of defined sets of key transcription factors. This study aimed to identify the optimal protocol for skin biopsy dissociation, fibroblast culture and fibroblast cryopreservation in the scope of reprogramming into iPSCs and in the context of biobank accreditation.

Methods: First, four dissociation techniques typically used in laboratory (explant-based, enzymatic and/or mechanical) and two cryopreservation media containing 10% dimethyl sulfoxide (DMSO), either commercial or home-made, were evaluated in terms of post-thaw recovery, viability, growth curves and karyotyping analyses of the fibroblasts. Next, the clones reprogrammed from the fibroblasts isolated with the two optimal dissociation methods and cryopreservation media were further assessed by reprogramming quality prior cryopreservation and post-thaw pluripotency comparison.

Results: Fibroblasts isolated from skin biopsies using an explant-based or enzymatic dissociation method showed higher viability, higher proliferative potential and higher genome stability post-thaw compared to the other dissociation techniques. Fibroblasts obtained by the explant-based dissociation technique showed a slightly higher reprogramming quality. The iPSC reprogrammed from explant-based dissociated fibroblasts showed successful recovery of iPSC clones. No difference between the two cryopreservation media was detected for the tested endpoints with the exception of a higher visual count of colonies at the end of the reprogramming for the explant-based dissociation method.

Conclusions: This article presents formal method optimization for biospecimen processing in the context of accreditation in laboratories and biobanks. We validated skin biopsy-derived fibroblast isolation, culture and cryopreservation for downstream mRNA reprogramming into iPSC. The explant-based dissociation technique and home-made medium are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.