Patient‐derived in vitro skin models for investigation of small fiber pathology

Abstract

Objective To establish individually expandable primary fibroblast and keratinocyte cultures from 3‐mm skin punch biopsies for patient‐derived in vitro skin models to investigate of small fiber pathology. Methods We obtained 6‐mm skin punch biopsies from the calf of two patients with small fiber neuropathy (SFN) and two healthy controls. One half (3 mm) was used for diagnostic intraepidermal nerve fiber density (IENFD). From the second half, we isolated and cultured fibroblasts and keratinocytes. Cells were used to generate patient‐derived full‐thickness three‐dimensional (3D) skin models containing a dermal and epidermal component. Cells and skin models were characterized morphologically, immunocyto‐ and ‐histochemically (vimentin, cytokeratin (CK)‐10, CK 14, ki67, collagen1, and procollagen), and by electrical impedance. Results Distal IENFD was reduced in the SFN patients (2 fibers/mm each), while IENFD was normal in the controls (8 fibers/mm, 7 fibers/mm). Two‐dimensional (2D) cultured skin cells showed normal morphology, adequate viability, and proliferation, and expressed cell‐specific markers without relevant difference between SFN patient and healthy control. Using 2D cultured fibroblasts and keratinocytes, we obtained subject‐derived 3D skin models. Morphology of the 3D model was analogous to the respective skin biopsy specimens. Both, the dermal and the epidermal layer carried cell‐specific markers and showed a homogenous expression of extracellular matrix proteins. Interpretation Our protocol allows the generation of disease‐specific 2D and 3D skin models, which can be used to investigate the cross‐talk between skin cells and sensory neurons in small fiber pathology.

Long-term small-fiber neuropathy and pain sensitization in survivors of pediatric acute lymphoblastic leukemia after stem cell transplantation

“…Still, pathomechanisms of SFN and pain sensitization are still unknown (Terkelsen et al 2017 ). Previous investigations suggest that elevation of macrophages and mast cells in immune-mediated SFN leads via chemokines and other mediators to microglia cell activation (Marchand et al 2005 ; Karl et al 2019 ). This peripheral activation together with altered sensory stimuli processing in the CNS may contribute to pain sensitization (Hoeijmakers et al 2012 ).…”

Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

“…Neurons were resuspended in 70 µl of filtered conditioned neuronal medium and seeded into one chamber compartment and acclimated for one week. Healthy control- derived primary keratinocytes were acquired and cultured via routine methods (Karl et al ., 2019) and seeded into the corresponding compartment. The chamber insert barrier separating the associated chambers was removed after 24 h and medium exchanged either with fresh keratinocyte medium, comprising of EpiLife Medium supplemented with 1% EpiLife defined growth supplement, and 1% pen/strep (all Thermo Fisher Scientific, Waltham, USA) or stored conditioned neuronal medium.…”

“…For srAT, a 2-mm skin punch biopsy was taken from the back at th10 level (device by Stiefel GmbH, Offenbach, Germany) under local anesthesia following a standard procedure (Üçeyler et al ., 2010). Tissue sections for ExM and cell cultures were acquired from 6- mm skin biopsy samples taken from the upper thigh according to a previously published protocol (Karl et al ., 2019). Our study was approved by the Würzburg Medical School Ethics committee (#135/15).…”

Fibroblasts: the neglected cell type in peripheral sensitisation and chronic pain? A review based on a systematic search of the literature

“…Considering the intimate relationship between fibroblasts and the immune system, it is therefore unsurprising that, already two decades ago, they were included on a list of cells thought to be capable of inducing peripheral neuron sensitisation 20. Since then, however, they seem to have engendered little interest, with the exception, perhaps, of synovial fibroblasts in the knee21 and recent pioneering work on fibroblasts taken from patients with small fibre neuropathy and fibromyalgia 22–24. However, fibroblasts are a key component of our body’s inflammatory response,25–27 and abnormal fibroblast function has been implicated in painful immune-mediated diseases like arthritis 28–30.…”

“…20 Since then, however, they seem to have engendered little interest, with the exception, perhaps, of synovial fibroblasts in the knee 21 and recent pioneering work on fibroblasts taken from patients with small fibre neuropathy and fibromyalgia. [22][23][24] However, fibroblasts are a key component of our body's inflammatory response, [25][26][27] and abnormal fibroblast function has been implicated in painful immune-mediated diseases like arthritis. [28][29][30] They therefore seem a sensible cell type to explore in the study of peripheral sensitisation.…”