Home › Cell Biology › Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting
Cell Biology JoVE (Open Access) Citable · DOI

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

DOI: 10.3791/59372-v
What you'll learn
  • ✓ Isolate papillary and reticular fibroblast subsets from human skin using FACS without in vitro culture
  • ✓ Perform enzymatic digestion and single-cell suspension preparation from dermal tissue
  • ✓ Distinguish fibroblast populations by surface markers (FAP, CD90) and functional properties
Protocol

Biopharma Insights This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Difficulty
advanced
Total time
~4–6 hours per skin sample
Model organism
Human skin (ex vivo)
Biosafety
BSL-1

Steps

1
Section human skin dermis into papillary and reticular layers

Carefully dissect and separate the upper papillary and lower reticular dermal layers from human skin biopsy using anatomical landmarks and scalpel technique.

▶ 01:00
2
Separate epidermis and dermis with enzymatic digestion

Treat skin sections with protease to digest and separate the epidermal layer from the underlying dermis, then process isolated dermal tissue further.

▶ 02:38
3
Prepare single-cell suspension and perform erythrocyte lysis

Enzymatically digest dermal tissue to generate single-cell suspension, then remove red blood cells using lysis buffer to enrich for fibroblasts.

▶ 03:52
4
Culture fibroblasts and perform adipogenesis differentiation assay

Expand isolated fibroblast populations in culture and differentiate subsets using adipogenic induction protocols to assess functional capacity.

▶ 05:58
5
Characterize FAP and CD90 fibroblast subsets by gene expression

Analyze dermal localization, gene expression profiles, and functional differences among FAP+CD90−, FAP+CD90+, and FAP−CD90+ fibroblast populations.

▶ 07:04
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