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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 lacking; therefore, there is an urgent need for further research^4,5.

As there is no ideal animal model for keloid research in vivo^6,7,building an in vitro model by acquiring primary fibroblasts from keloid tissues can offer feasibility and reliability for keloid research^2,6. 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 lines^8,9. 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.

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Protokół

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) with PSA by adding 1% PSA into 1x PBS. Prepare PBS with FBS by adding 1% FBS to 1x PBS.
3. Prepare several sterilized scissors, forceps, and scalpels by autoclaving.

2. Obtaining removed tissues

1. Obtain tissues from keloid patients through surgery. Collect the fresh keloid tissues in a sterile packing bag or sterile centrifuge tube, and transfer them to a biological safety cabinet in the laboratory as soon as possible.
   ​NOTE: In this protocol, the size of the acquired keloid tissue was approximately ~20 x 20 x 10 mm³, and the size may vary depending on the surgery.

3. Isolation

1. Take out the keloid tissue using sterile tweezers, and place it in a 50 mL sterile centrifuge tube containing 10-25 mL of PBS with 1% PSA for 10 min. Then, prepare a 6-well plate, and add 4 mL of PBS supplemented with 1% PSA to each well. Take out the tissue using sterilized tweezers, and wash it twice with PBS supplemented with 1% PSA. Using sterile forceps, transfer the tissue sequentially from one well to the next.
2. Remove the adipose andepidermis layers using surgical scissors or a surgical scalpel, and leave the dermis layer untouched. Trim and dissect the dermis layer into 3-5 mm² pieces with scissors, transfer these pieces using sterile forceps to the next well, and wash them in PBS with 1% PSA solution again.

4. Culture

1. Place the dermis tissue pieces in Petri dishes using sterilized forceps; ensure that the number of pieces is between 10 and 30 and the distance between each piece is >5 mm. Put the Petri dishes upside down in a 5% CO₂ incubator at 37 °C for 30-60 min until the pieces of tissues dry a little and stick to the Petri dish. Then, add DMEM supplemented with 10% FBS and 1% PSA, and carefully place the Petri dishes into a 5% CO₂ incubator at 37 °C.
   NOTE: Fibroblasts are a morphologically and functionally heterogeneous cell population. Considering this complexity, the isolation and culture steps should be performed with care; select the keloid dermal tissue pieces evenly, and mix these pieces well before placing them in the Petri dish.
2. After 3 days, replace half of the supernatant with a complete culture medium. Change the culture medium every 2-3 days. Observe the fibroblasts under a microscope at 40x magnification every day.
   NOTE: All the steps should be performed gently. Do not move the Petri dish for 2 days after isolating, as the tissue pieces take time to adhere to the Petri dishes.
3. When the fibroblasts growing around the tissue pieces reach approximately 90% confluency, remove the tissue pieces and the culture medium. Wash the fibroblasts with sterile 1x PBS, and add 2 mL of sterile 1x trypsin-EDTA solution to the plates.Incubate the cells for approximately ~3-5 min at 37 °C in a humidified 5% CO₂ incubator.Gently tap the culture dish, and observe it under a microscope. When the majority of the cells detach from the plate, add 2 mL of complete medium to end the digestion process.
4. Transfer the cell suspension to a 15 mL sterile centrifuge tube, and centrifuge the tube at 300 × g for 3 min at room temperature. Discard the supernatant carefully, and resuspend the cell pellet in complete medium.Seed the fibroblasts into a 9 cm cell culture dish, and incubate at 37 °C in a humidified 5% CO₂ incubator.

5. Maintenance and preservation

1. Approximately 3-4 days later, when the fibroblasts have grown to 80% confluency, repeat steps 4.2-4.4, and passage the fibroblasts at a ratio of 1:3. Use the passaged fibroblasts for further experiments.
   ​NOTE: The fibroblast culture should be stopped after 10 passages, because the cells may start to show changed characteristics compared to the original cells.
2. Cryopreserve the passaged cells of P1-P3 in liquid nitrogen for further usage.
   1. Repeat steps 4.2-4.3. Transfer the cell suspension to a 15 mL sterile centrifuge tube, and centrifuge the tube for 3 min at 300 × g. Discard the supernatant carefully, resuspend the cell pellet in 1 mL of cell freezing medium containing 90% FBS and 10% DMSO, and transfer the suspension into cell cryotubes.
   2. Move the cells into a frozen box, and then put the frozen box in a −80 °C freezer. After 1 day, transfer the cells to liquid nitrogen for long-term preservation.

6. Identification of fibroblasts by immunofluorescence staining

1. Place round coverslips in a 24-well plate, and culture the passaged fibroblasts at a concentration of 1 × 10⁴ cells/well. When the fibroblasts reach 60% confluency, remove the culture medium. Add 1 mL of 4% paraformaldehyde to fix the fibroblasts for 20 min at room temperature, and wash 3x with PBS for 1 min each time.
   NOTE: Count the number of cells by using a hemocytometer slide under a microscope or an automatic cell counter.
2. Remove the PBS, incubate with 0.5% Triton X-100 for 20 min for the permeabilization of cell membranes, and then wash 3x with PBS for 1 min each time. Add PBS with 0.5% bovine serum albumin, and soak for 30 min. Then, remove it, and add the PDGFR-α/vimentin antibody diluted in antibody diluent at 1:1,000. Incubate overnight at 4 °C.
3. The next day, remove the primary antibody, wash the cells 3x with PBS for 3 min, and add the secondary antibody Alexa Fluor-555 goat anti-rabbit IgG diluted with antibody diluent at 1:200 to soak for 1 h.
4. Remove the secondary antibody, and wash the cells 3x with PBS. Take out the round coverslips using forceps, put them on glass slides, and add 50 µL of 5 µg/mL 4',6-diamidino-2-phenylindole (DAPI) solution to stain the cellular nuclei. Keep the samples in a wet, dark box, and observe them under a laser confocal fluorescence microscope.
   ​NOTE: Add a negative control group without a primary antibody but with a secondary antibody to exclude nonspecific staining.

7. Identification of fibroblasts by flow cytometry

1. Collect the cell pellet into a 1.5 mL sterile centrifuge tube, and resuspend with 50 µL of PBS containing 1% FBS. Incubate with anti-CD90 for 30 min in the dark, and add an anti-IgG isotype control to the control group. Then, add 200 µL of PBS containing 1% FBS, and centrifuge the tube for 10 min at 300 × g at 4 °C.
2. Remove the supernatant, and add 200 µL of PBS containing 1% FBS. Resuspend and filter the suspension through a cell strainer (70 mesh). Add a 5 µg/mL stock solution of DAPI at 1:100 to the filtrate, and then acquire the results by flow cytometry. Set the forward scatter (FSC) as the abscissa and the side scatter (SSC) as the ordinate, and circle the main cell population. Select the cell population, and set the phycoerythrin as the abscissa and the count as the ordinate for analysis.

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Wyniki

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...

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Dyskusje

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

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Materiały

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