Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Summary

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Abstract

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

Protocol

The skin punch biopsy obtained using standard procedure³ (e.g. obtained with 4mm round Visipunch instrument) should be kept in complete DMEM 20% FBS media on ice. Once the sample has arrived in the laboratory, process the biopsy as soon as possible.

1. Preparation of the Skin Punch Biopsy

STEPS 1.1- 1.3 ARE TO BE PERFORMED INSIDE A BIOSAFETY CABINET

1. Prepare in advance: Add 1 ml of 0.1% Gelatin to each well of a 6-well plate. Set the plate aside for 30-60 min. Aspirate the gelatin solution and add 800 μl of complete DMEM/20% FBS media to each well. Ensure that t....

Results

Keratinocytes growing out of the biopsy pieces as soon as 48 hr after dissection. First fibroblasts outgrowth can be observed about one week after processing. Once the wells have reached confluency, fibroblasts are passaged for two more passages to reach three 150 cm flasks (T150) and cells are cryopreserved. We generate with this method 15-20 million cells. The fibroblasts are positive for anti-SERPINH1 (also known as HSP-47) (Figure 1), which is a collagen-specific molecular chaperone localized in the.......

Discussion

With this protocol, relatively pure cultures of skin fibroblasts can be obtained. The fibroblasts have characteristic morphological features of elongated, spindle-like cell bodies, round to oval cell nuclei, and the fibroblasts grow aligned and in bundles when confluent. The media supports the growth of fibroblasts whereas other cell populations e.g. keratinocytes need additional supplements and growth factors, or are mitotically less active, this allows for relatively pure fibroblast cultures.

Materials

Name	Company	Catalog Number	Comments
Name of Equipment	Company	Catalogue Number
Dissecting microscope	Leica	S6E
10 cm Tissue Culture Petri dish	VWR	25382-166
6-well Tissue Culture plate	VWR	73520-906
Stainless steel disposable sterile scalpels (2), blade No#15, Miltex	VWR	21909-660
Sterile pointed-tip forceps
50 mL Conical tubes	VWR	21008-940
2 mL Serological Pipettes	VWR	89130-894
5 mL Serological Pipettes	VWR	89130-896
10 mL Serological Pipettes	VWR	89130-898
Pasteur Pipettes	VWR	14672-380

Name of Reagent	Company	Catalogue Number
1X DMEM: High Glucose	Invitrogen/Gibco	11960-069
20% Fetal Bovine Serum	Invitrogen/Gibco	26140-079
1X L-Glutamine	Invitrogen/Gibco	25030-164
1X MEM Non-essential Amino Acids	Invitrogen/Gibco	11140-076
1X Penicillin/Streptomycin	Invitrogen/Gibco	15140-163