|Ahead of print publication
Indigenous primary culture protocols for human adult skin fibroblast, pancreatic stellate cells, and peritoneal fibroblasts
Supriya Sharma1, Gaurav Pande2, Mohit Kumar Rai3, Vikas Agarwal3
1 Department of Surgical Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Medical Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Submission||22-Jun-2020|
|Date of Acceptance||23-Jun-2020|
Department of Medical Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
Mohit Kumar Rai,
Department of Clinical Immunology and Rheumatology, SGPGIMS, Lucknow, UP
Source of Support: None, Conflict of Interest: None
Despite availability of commercially available cell lines, primary cultures of cells remain the best available natural resource for in vitro research work. However, establishing primary cultures is challenging and despite availability of many protocols for primary cell cultures reproducibility is lacking either due to cumbersome protocol, need for special reagents, and requirement of training. Herein, we present our simplistic least manipulative protocols for establishing primary human dermal fibroblasts, human pancreatic stellate cells, and human peritoneal fibroblasts that may be followed by other research laboratories for in vitro studies.
Keywords: Culture protocols, dermal fibroblasts, enzymatic digestion, explant culture, pancreatic stellate cells, peritoneal fibroblasts
|How to cite this URL:|
Sharma S, Pande G, Rai MK, Agarwal V. Indigenous primary culture protocols for human adult skin fibroblast, pancreatic stellate cells, and peritoneal fibroblasts. Indian J Rheumatol [Epub ahead of print] [cited 2020 Oct 27]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=298044
| Introduction|| |
Fibrosis is the mechanism of wound healing whereby normal parenchymal tissue is replaced by the extracellular matrix (ECM) proteins. Fibroblasts are the key cells that are implicated in fibrosis. Upon stimulation, these fibroblasts get activated and converted to myofibroblast phenotype that secretes the lots of ECM proteins. Currently, fibrosis is believed to be the end result of inflammatory damage and is irreversible. Despite the availability of various commercial fibroblast cell lines, the biology of fibroblasts is best understood by culturing organ-specific fibroblasts as their biological behavior varies with the site of origin., However, unlike established cell lines that grow indefinitely, primary cells eventually undergo senescence in culture and need to be frequently re-established. Establishing primary cultures is often difficult and challenging. Some of the protocols, well established for primary human cell cultures, are difficult to follow and require special reagents which may require expertise and escalated costs of the cultures. There is a need for exploration of simple, reproducible, and cost-effective culture protocols which may be followed widely and allow researchers in cost-constraint settings to carry out their research work.
In the present study, we present our indigenous simple and cost-effective primary culture protocols for human skin fibroblasts, human pancreatic stellate cells (PSCs), and human peritoneal fibroblasts.
| Methods|| |
The study was approved by the Institutional Ethics Committee, and written informed consent was obtained from all the patients/participants as per the declaration of Helsinki.
Protocol for primary human dermal fibroblast
During skin wound healing, dermal fibroblasts switch from a proliferative and migratory phenotype to a contractile, matrix-remodeling myofibroblasts. Uncontrolled healing leads to fibrosis and formation and may lead to the formation of hypertrophic scars/keloids. For study of mechanism of fibrosis and finding newer cures, primary fibroblasts are the best models for the study of fibroproliferative disorders.
Preparation of culture medium
Prepare complete culture medium by adding the following components to Dulbecco's modified eagle medium (DMEM): 10% fetal calf serum (FCS), 2 mM glutamine, and 1% penicillin-streptomycin solution. Filter media through 0.22 um filters.
Preparation of enzyme solutions
- Always use freshly prepared enzyme solution
- Preparation of dispase solution in 50 ml tube: Dissolve 0.5 g dispase (Sigma, USA) in 50 ml phosphate buffer saline (PBS)
- Preparation of collagenase solution in 15 ml tube:Dissolve 40 mg Type II collagenase in 10 ml PBS
- Filter enzyme solutions with 0.22 um syringe filters.
Collection of skin biopsies
After written informed consent, identify the site of skin biopsy from where the biopsy is to be obtained. Under aseptic conditions and suitable local anesthesia, remove 4 mm × 4 mm skin (up to dermis layer) and immediately transport it in sterile PBS or normal saline to the culture laboratory for processing. Skin biopsies were obtained from ten patients with scleroderma.
Processing of skin for isolation of fibroblast
Wash skin biopsy tissue with PBS 2–3 times at 1200 rpm for 5 min each. Incubate with 0.5% (W/V) Dispase (Sigma, USA) solution at 37°C water bath for 45 min followed by washing with PBS at 37°C at 1200 rpm for 7 min. Peel keratin layer by forceps and treat tissue with 4 mg/ml Type II collagenase (Sigma, USA) for 4 h. Repeat washings 2–3 times in complete culture media to remove the remaining collagenase and suspended cell debris. Culture cells (lying at the bottom of the tube after centrifugation) in complete DMEM (Sigma, USA) at 37°C and 5% CO2 for 24 h. Changefirst culture medium after 24 h and thereafter every 48–72 h. After 5–7 days, cells adhered to the surface start forming blast such as bunches are visible. Keep changing culture media every 48–72 h and around days 20–25, spindle-shaped fibroblasts [Figure 1] are visible. At 80% or more of confluence, trypsinize the flask with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution and wash thrice with complete media at 1000 RPM for 7 min each. If required, fibroblasts can be subcultured in fresh flasks or stored in the liquid nitrogen for future use.
|Figure 1: Elongated spindle-shaped primary dermal fibroblasts derived from the left mid forearm of a patient with systemic sclerosis (×40)|
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After the second passage, the purity of fibroblasts was >95% as evidenced by prolyly-4-hydroxylase staining [Figure 2].
|Figure 2: Dermal fibroblasts were stained with FITC conjugated prolyl-4-hydroxylase antibody and analyzed by FACS CANTO (Beckton-Dickinson, San Jose, CA). Minimum of 10,000 events were counted with Flow Jo software. Figure on the left show's purity of dermal fibroblasts 70% positivity after the first passage and 95% after the third passage on the right|
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Protocol for the isolation of pancreatic stellate cells from the pancreatic tissue
Pancreatic tissue was obtained from patients undergoing pancreatoduodenectomy for carcinoma of pancreas and lateral pancreatojejunostomy for chronic pancreatitis (CP). Pancreatic tissue of 0.5 cm × 0.5 cm was collected in sterile saline/PBS. Pancreatic tissue was incubated with PBS containing 2% antibiotics for 15 min and then decanted. It was repeated three times. Tissue was chopped into small pieces (<1 mm2) by using scalpel and mixed with 1 ml DMEM containing 5% FCS followed by their spread on culture flask/six well culture plates. Chopped tissues were allowed to adhere to the surface for 10 min. Later, fresh DMEM was added slowly by the sides of wall of culture flask/six well culture plate so as not to detach the adhered tissues. Enough medium was added so that tissues get covered by it. Flask/culture plate was incubated at 37°C and 5% CO2. After 72–96 h of culture, rounded cells [Figure 3] emerged out adjacent to the adhered tissues. After around 120–144 h, these round cells changed into polygonal shapes [Figure 4]. Culture medium was changed every 48–72 h. It was added carefully from the side of wall of culture flask/six well culture plate, so that cells remained adherent. Around 8–10 days after initiation of culture, polygonal or star-shaped cells were visible with numerous vacuoles in it [Figure 5].
|Figure 3: Bunch of rounded cells seen adjacent to the pancreatic tissue after 96 h|
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|Figure 4: Elongated and polygonal cells of early pancreatic stellate cells after 120 h|
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|Figure 5: Polygonal and star-shaped pancreatic stellate cells with multiple vacuoles after 10 days (×200)|
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Pancreatic tissue was obtained from two patients of pancreatic cancer undergoing pancreatoduodenectomy.
Protocol for primary fibroblast from human peritoneum
Peritoneum biopsy tissue of size 1 cm × 1 cm was obtained from the patients undergoing laparotomy after written informed consent and transported in sterile PBS to the laboratory for fibroblasts isolation. Tissue was washed with PBS 2–3 times by centrifuging at 1200 rpm followed by chopping of the tissue into small pieces (<1 mm size) by scalpel. Chopped pieces were treated with Type II collagenase (4 mg/ml) for 4 h at 37°C and 5% CO2. Thereafter, tissue pieces were washed 2–3 times with PBS by centrifuging at 1200 rpm to remove remaining collagenase and cell debris. Now, complete DMEM (Sigma, USA) containing 10% heat-inactivated FCS (GIBCO, NZ) was added, and tissue pieces were cultured in the culture flask at 37°C and 10% CO2. First medium was changed after 24 h and thereafter every 48–72 h depending on depletion of medium. After 6–8 days, aggregates of rounded cells were visible which later become elongated. Fibroblasts became confluent after 10–15 days [Figure 6]. Primary cultures were subpassaged with 0.25% trypsin - EDTA solution (Sigma, USA). Purity of peritoneal fibroblasts was 95% after the third passage [Figure 7]. These cells were sub-cultured or stored in liquid nitrogen for future studies.
|Figure 6: Thin elongated spindle-shaped cells after 12 days of peritoneal tissue culture (×40)|
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|Figure 7: Peritoneal fibroblasts were stained with anti-fibroblast FITC antibody and analyzed by FACSCanto™II (Beckton-Dickinson, San Jose, CA). Minimum of 10,000 events were counted with FACSDiva™ software (Beckton-Dickinson Labware, Franklin Lakes, NF). Cell debris and dead cells were excluded from the analysis. Red peak indicates cells after the first passage that represent purity level of 70%, blue peak indicates cells after the second passage that represent purity level of 90%, and yellow peak indicates cells after the third passage that represent the purity level of 95%|
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Peritoneal tissue was obtained from four patients undergoing elective cholecystectomy.
Requirements for the primary human cell cultures
Dedicated culture room, uninterrupted CO2 supply (cylinder or line supply), laminar flow cabinet, incubator, centrifuge, refrigerator, freezer, microscope, hemocytometer, pipette boy, and micropipette.
| Discussion|| |
Commercial cell lines are easy to culture and predictable behavior has inherent drawbacks; they are genetically modified/transformed which can alter their physiological properties and not represent thein vivo state. Moreover, their genotypic and phenotypic expressions may change over time with repeated passaging. Therefore, primary cell cultures remain the gold standard to understand the biological behavior of particular cells as it would have behaved invivo. In addition, primary cell cultures avoid ethical issues related to animal experiments and are the closest to human tissues. Establishing primary cultures from the animal tissues is relatively easy as there is no dearth of tissues; however, it is generally not the case when primary cultures from the human tissues are required. Some of the available cell culture protocols are difficult to follow and require special training and specialized reagents; therefore, simple easy and reproducible cell culture protocols are needed with an attempt to maintain the natural genotype and phenotype of the cultured cells.
Fibroblasts depict a vibrant population of cells showing functional and phenotypical diversity. All the fibroblasts have their exclusive origin, activation status, and functional capacity, and therefore, all fibroblasts do not express a similar marker profile at the same moment. Furthermore, fibroblasts have heterogeneous nature and tissue-specific properties. Therefore, studies and results obtained using an individual fibroblast of organs and tissues cannot be generalized for others organs and tissues fibroblasts. Alpha smooth muscle actin is of great importance among the various markers associated with different fibroblast phenotypes as it reflects conversion of fibroblast to active myofibroblast phenotype that secrete ECM proteins. Myofibroblasts represent a cell with intermediate features between a classic spindle-shaped elongated fibroblast and a smooth muscle cell (smooth muscle-like fibroblast).
Human skin fibroblasts are located in the dermal layer of skin where they produce extracellular matrix rich in Type I and/or Type III collagen. Dermal fibroblasts have spindle-shaped morphology and express markers such as fibronectin, vimentin, and TH-1 (CD90). However, fibroblasts from different organs, anatomical sites, and locations express different markers and have different functional characteristics.,, During wound healing, dermal fibroblasts change their proliferative and migratory behavior to ECM secreting matrix-remodeling myofibroblast., Our protocol of enzymatic digestion (dispase and collagenase) is simple to follow and good yield of dermal fibroblasts.
Various methods for isolating PSCs have been published, either utilizing enzymatic digestion and density gradient centrifugation, or the explant tissue culture from the pancreatic tissue., These methods require large amounts of tissue to obtain a sufficient number of cells. Although immortalized PSCs are available, they may not be ideal for all experimental research works., Therefore, the successful isolation of primary culture remains essential to the study of PSCs. The main method of isolating quiescent PSCs is through density gradient centrifugation with nycodenz percoll, iohexol, and oriodixanol. Reasonable digestion is the key step for the isolation of PSCs. Alterations in the concentration of digestive enzymes, shaking speed in water bath, and digestion time have led to better yield of PSCs.
In normal physiological states, PSCs are quiescent and are laden with Vitamin A lipid droplets. On the exposure to tissue injury or inflammation PSCs transform into activated myofibroblast-like cells with the loss of Vitamin A droplets, enhanced proliferation and migration ability, production of abundant amounts of ECM proteins. Explant techniques are mainly utilized for the isolation of activated state PSCs from CP or pancreatic tumors. However, our method of explant technique yielded quiescent PSCs in majority. Our technique was simple, least manipulative of the pancreatic tissue, without any density centrifugation and high yield of typical star shaped stellate cells filled with lipid-laden vacuoles.
Myofibroblasts have been reported to be easily detected in chronic ambulatory peritoneal dialysis (CAPD) patients on long-term peritoneal dialysis (PD)., In a specific study, myofibroblasts have been observed in very early stages of CAPD patients on long-term PD within their peritoneum and with greater density in the submesothelial compact zone. Not all fibroblasts have the ability of getting transformed into myofibroblasts and that this capacity is limited to certain subtypes and correlated to their tissue origin. CD34 is an antigen characteristic of bone marrow hematopoietic stem cells, which is also expressed in several resident fibroblast cells. Further, their expression reduces in normal peritoneal fibroblasts upon progression towards peritoneal fibrosis. There are now evidences that myofibroblasts might originate from the various sources. Primarily, they may differentiate from resident tissue stem cells or residing fibroblasts, or through epithelial to mesenchymal transition (EMT) process. Process of EMT in peritoneum has been demonstrated earlier. Peritoneal myofibroblasts, which originated from bone marrow, are reported to appear in granulation tissue secondary to the peritoneal implantation of a foreign body., The purity of peritoneal fibroblasts was evaluated by Fluorescein isothiocyanate (FITC) labeled anti-fibroblast antibody [Figure 7]. Our protocol of isolation of peritoneal fibroblasts is simple and least manipulative with a high yield of fibroblasts.
| Conclusion|| |
We have been able to establish simple and cost-effective primary fibroblast cultures from the skin, pancreas, and peritoneum that may be followed easily at various laboratories.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]