Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Summary

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Abstract

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFRα and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

Introduction

Keloid, a fibroproliferative disease, manifests as the continuous growth of plaques that often invade the surrounding normal skin without self-limitation and cause various degrees of itching, pain, and cosmetic and psychological burdens for patients¹. Fibroblasts, the primary cells involved in keloids, play an essential role in the formation and development of this disease through excessive proliferation, redundant extracellular matrix production, and disorganized collagens^2,3. However, the underlying pathogenesis remains unclear, and an effective therapeutic method for keloid is still ....

Protocol

This study was approved by the institutional review board of the Dermatology Hospital, Southern Medical University (2020081). Informed patient consent was obtained before tissues were collectedfrom the individuals.

1. Preparation

NOTE: The following procedures should be performed in a sterile environment under a biological safety cabinet.

1. Prepare complete culture medium by adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphotericin B solution (PSA) to high-glucose Dulbecco's modified Eagle medium (DMEM).
2. Prepare phosphate-buffered saline solution (PBS....

Results

The timeline of the protocol is summarized in Figure 1A. Some representative images of the isolation process are shown in Figure 2; the epidermis and adipose layers were carefully removed, and the dermis layer was separated into small fragments of 3-4 mm², which were inoculated into the Petri dishes.

As shown in Figure 3A, several fibroblast outgrowths of the tissue pieces were observed und.......

Discussion

Obtaining primary fibroblasts from keloid tissues is a critical basis for further research. Up until now, there have been two methods for acquiring primary fibroblasts: enzyme digestion and explant culture^11,12,13,14. However, both traditional methods have limitations, such as susceptibility to contamination, mixing with other types of cells, a long culture period, and a low rate of success

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL sterile centrifuge tube	JETBIOFIL	CFT002015
15 mL sterile centrifuge tube	JETBIOFIL	8076
4% polyformaldehyde	Beyotime Biotechnology	P0099	Cell fixation
50 mL sterile centrifuge tube	JETBIOFIL	8081	Put keloid tissue
Alexa Fluor-555 goat anti-rabbit IgG	Abcam	Alexa Fluor 555	second antibody for immunofluorescence staining assay
Anti human CD90	BioLegend	B301002	Identify the purity of fibroblasts
Antibody diluent	Beyotime Biotechnology	P0262
Biological safety cabinet	Thermo Scientific	1300 series A2	Isolation and culture cells
Bovine serum albumin	aladdin	B265993	Blocking for immunofluorescence staining assay
Carbon dioxide incubator	ESCO	CCL-170B-8	Using for culturing cells
Cell cryotubes	Corning	43513	Store the cells in low temperature
centrifugal machine	Thermo Fisher	ST 16R	Discard supernatant
DAPI	Beyotime Biotechnology	C1006	Stain the cellular nucleus
DMSO	MP Biomedicals	196055	Using for preserving cells
Dulbecco's modified eagle medium	Gibco	C11995500BT	Culture medium solution
Fetal bovine serum	BI	04-001-1A
Flow cytometer	BD	BD FACSCelesta	Observing the identity of cells
frozen box	Thermo Scientific	5100-0050
Inverted microscope	Nikon	ECLIPSE Ts2
Laser confocal microscope	Nikon	AIR-HD25	Observing the immunofluorescence staining assay
PDGFR-α antibody	CST	3174T	First antibody for immunofluorescence staining assay
Penicillin-streptomycin-Am solution	Solarbio	P1410	Add in culture medium solution to avoid contamination
petri dish	JETBIOFIL	7556	Culture fibroblasts
Phosphate buffered saline solution	Gibco	C10010500BT	Culture medium solution
Rabbit (DAIE) mAB IgG XR (R) Isotuge Control (PE)	Cell Signaling Technology	5742S	As a control for flow cytometry
Round coverslip	Biosharp	801007	Cell culture
Triton X 100	Solarbio	T8200	Punch holes in the cell membrane
Trypsin-EDTA	Gibco	25200072	Used for passaging cells
Vimentin antibody	Abcam	ab8978	First antibody for immunofluorescence staining assay