This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.
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.
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 collagens2,3. However, the underlying pathogenesis remains unclear, and an effective therapeutic method for keloid is still lacking; therefore, there is an urgent need for further research4,5.
As there is no ideal animal model for keloid research in vivo6,7,building an in vitro model by acquiring primary fibroblasts from keloid tissues can offer feasibility and reliability for keloid research2,6. Primary cells are those derived directly from living tissue, and it is generally recognized that these cells can more closely resemble the physiological state and genetic background of multiple individuals compared with cell lines8,9. Culturing primary cells provides a powerful means to study the growth and metabolism of cells, as well as other cell phenotypes.
At present, there are two methods for acquiring primary fibroblasts: enzyme digestion and explant culture. However, several obstacles have been identified to obtaining primary fibroblasts, such as the risk of contamination by various bacteria or fungi, mixing with other types of cells that are not easily removed, the long period of the culture cycle, the subsequent changes to the cell characteristics compared to the original cells, and so on9. Therefore, developing a feasible and effective process for obtaining primary fibroblasts is the foundation for further studies and applications.This study describes an optimized protocol for extracting primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.
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.
2. Obtaining removed tissues
3. Isolation
4. Culture
5. Maintenance and preservation
6. Identification of fibroblasts by immunofluorescence staining
7. Identification of fibroblasts by flow cytometry
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 under the microscope 5 days after processing. As shown in Figure 3B, the fibroblasts displayed high proliferation rates and reached a high level of confluency after 10 days. These fibroblasts had elongated, spindle-like cell bodies and were aligned in bundles when they reached a high level of confluency. By following this protocol, the fibroblasts were passaged and expanded to the desired quantity within 2-3 weeks.
To verify the identity and purity of the fibroblasts, an immunofluorescence staining assay was employed for detecting the fibroblast-specific markers PDGFRα10 and vimentin. As expected, Figure 4A and Figure 4C show that all the fibroblasts were positively stained by the PDGFRα/vimentin antibody, with red immunofluorescence and blue immunofluorescence in the cellular nucleus. As demonstrated in Figure 4C, the flow cytometry assay also showed CD90 positivity in almost all of the fibroblasts.
Figure 1: Overview of the keloid fibroblast isolation and culture procedure. Please click here to view a larger version of this figure.
Figure 2: Representative images of the isolation and culture of fibroblasts from keloid tissues. (A) Wash the tissue. (B) Remove the epidermis and adipose layers. (C) Dissect the dermis into 3-4 mm3 pieces, and wash with PBS again. (D) Place the tissue fragments in a Petri dish. (E) Put the Petri dishes upside down into the 5% CO2 incubator at 37 °C for 30-60 min to let the pieces of tissue dry a little and stick to the Petri dish. (F) Add complete culture medium into the Petri dish, and culture in the CO2-containing cell incubator. Please click here to view a larger version of this figure.
Figure 3: Representative images of fibroblast outgrowths from the keloid tissue pieces. (A) Fibroblast outgrowths from the tissue pieces in ~5 days. (B) The fibroblasts reached a high level of confluency in ~10 days. Scale bars = 200 µm. Please click here to view a larger version of this figure.
Figure 4: Identification of highly purified fibroblasts by immunofluorescence staining and flow cytometry. (A) Immunofluorescencestaining of fibroblasts by anti-PDGFRα. (B) Negative control group without the primary antibody for PDGFRα. (C) Immunofluorescencestaining of the fibroblasts by anti-vimentin. (D) Negative control group without the primary antibody for vimentin. (E) Flow cytometry analysis of the fibroblasts. Please click here to view a larger version of this figure.
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 culture11,12,13,14. 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 success15,16. This study has described an optimized method and provided clear instructions to solve these existing challenges and increase the chance of success for the isolation and culture of keloid fibroblasts.
The possibility of microbial contamination is one of the major problems in obtaining primary fibroblasts. All the equipment and solutions must be sterilized, and standard aseptic techniques must be implemented during all the steps in the protocol. The protocol contains reminders to remain aseptic to reduce the risk of contamination. The first critical step is the isolation process; the aim of soaking the tissues in PBS supplemented with 1% PSA and washing several times is to remove existing microbes and residual bloodstains. To prevent the possible transfer of contamination, an extra pair of sterilized scissors and forceps should be prepared; these instruments could be replaced by unused ones in the next operation. Furthermore, PSA is added to the culture medium to prevent the outgrowth of microbes. Periodic testing for the mycoplasma during the isolation and subsequent culture of the keloid fibroblasts should be incorporated into the protocol. Through these processes, the possibility of microbial contamination can be significantly reduced.
The other challenge is mixing with other types of cells, such as keratinocytes. This cell type also has an intense proliferative ability, making it difficult to remove. The purity of fibroblasts is highly dependent on the isolation process; the epidermis and adipose layers should be entirely removed to avoid mixing with other cells. PDGFR-α is a fibroblast-specific marker that is expressed in the membrane and cytoplasm of fibroblasts10,17,18. CD90 and vimentin are also specific mesenchymal markers that are specifically expressed in the membranes of fibroblasts19,20,21. The immunofluorescence staining assay in this work showed that all the cells positively expressed PDGFR-α and vimentin. The flow cytometry assay also directly demonstrated CD90 positivity in almost all the fibroblasts and greater CD90 positivity compared with the isotype control, thus confirming that we obtained relatively high-purity fibroblasts using this protocol.
The crucial recommended steps to promote the outgrowth and enhance the proliferative efficiency of fibroblasts are as follows. First, the tissue pieces should be cut into appropriate sizes of 3-5 mm2, as smaller tissue pieces are more likely to float during the movement of the Petri dish, and the cells will not easily outgrow larger pieces.Second, the first medium change should be after the third day, as the tissue pieces take time to adhere to the Petri dishes. Moving the Petri dishes too early tends to disturb the tissues and decrease the possibility of fibroblast outgrowth. Third, all the steps in this protocol should be conducted gently; improper handling will cause unnecessary movement of the tissue pieces and impair the cell outgrowth.
Fibroblasts are a morphologically and functionally heterogeneous cell population that can be divided into several subpopulations. One limitation of this study is that we can obtaingeneral fibroblasts from the entire keloid tissue but not the different subpopulations of fibroblasts. We are trying to find better methods to solve this problem in the future.
As demonstrated in our previous studies by RNA-seq, there are many differences between keloid fibroblasts and normal fibroblasts. Keloid fibroblasts produce more collagen I and collagen III. In addition, keloid fibroblasts express specific markers such as POSTN, among others. After passaging, the fibroblasts still maintain these characteristics, which proves that primary fibroblasts could also exhibit some features found in keloidpatients.
In conclusion, this study provides an optimized method and gives clear instructions to solve the existing difficulties in keloid fibroblast extraction, such as microbial contamination, the mixing with other cell types, and the long culture period, and to increase the chance of success. The current work provides a simple and reproducible method for isolating and culturing keloid primary fibroblasts, which will provide a rich resource for future studies and research or could be frozen in liquid nitrogen for banking.
The authors have nothing to disclose.
This work was supported by grants from the National Natural Science Foundation of China (grant numbers 81903189 and 82073418) and the Science and Technology Foundation of Guangzhou (grant number 202102020025).
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 |