An Optimized Method to Isolate Human Fibroblasts from Tissue for Ex Vivo Analysis

Materials and Reagents

1. 5 ml syringes (BD Plastipak, catalog number: 307731)
2. 10 ml syringes (BD Plastipak, catalog number: 307736)
3. Pasteur pipette (Scientific Laboratory Supplies, catalog number: PIP4210)
4. 50 ml Falcon tube (Sarstedt, Brand, model/catalog number: 114 x 28 mm, 62.547.004)
5. 15 ml Falcon tube (Sarstedt, Brand, model/catalog number: 120 x 17 mm, 62.554.002)
6. Sterile scalpel #21 blade (Swann-Morton, catalog number: 0507)
7. Polystyrene cell culture dish (Sarstedt, catalog number: 83.3902)
8. Syringe filtration unit Filtropur S 0.2 (Sarstedt, catalog number: 83.1826.001)
9. Scissors
10. Collagenase P from Clostridium histolyticum (Merck, Roche, catalog number: 11213857001). Reconstitute in PBS to 150 U/ml, store 100 μl aliquots at -20 °C
11. TrypLE Express Enzyme (no phenol red; Thermo Fisher, catalog number: 12604013). Store at room temperature protected from light for up to 2 years
12. Fetal bovine serum (Biosera, catalog number: FB-1001/500). Store at -20 °C for up to 60 months
13. Deoxyribonuclease I from bovine pancreas (Merck, Sigma-Aldrich, catalog number: D4263). Reconstitute in 1 ml PBS (2000 U/ml), store 40 μl aliquots at -20 °C
14. Dulbecco’s Modified Eagle Medium (Merck, Sigma-Aldrich, catalog number: D5671-500ML). Store at 4 °C
15. L-glutamine (Merck, Sigma-Aldrich, catalog number: G7513-100ML). Store at -20 °C for up to 2 years
16. Penicillin-streptomycin (Merck, Sigma-Aldrich, catalog number: P4333-100ML). Store at -20 °C for up to 2 years
17. Phosphate-buffered saline (PBS)
18. Amphotericin B (250 μg/ml; Gibco, catalog number: 15290-018). Store at -20 °C for 1 year
19. Sterile double-distilled H₂O
20. Red blood cell lysis buffer (10x; BioLegend, catalog number: 420301). Store at 4 °C
21. DNase stock solutions
22. “Complete” DMEM (see Recipes)
23. “Empty” DMEM (see Recipes)

Equipment

1. Orbital shaker-incubator (e.g., Grant-bio Orbital Shaker-Incubator ES-20)
2. EASYStrainer 40 μm (Grener Bio-One, catalog number: 542040)
3. Centrifuge
   

Procedure

1. Tissue dissociation
   1. Sample collection
      Transport the tissue sample in “empty” DMEM (Recipe 2) on ice
   2. Prepare working solutions
      1. In a 50 ml Falcon tube, add 100 μl Collagenase P and 40 μl DNase stock solutions to 5 ml “complete” DMEM (Recipe 1)
      2. Make PBS-A: add 1 μl Amphotericin to 10 ml PBS in a 15 ml Falcon tube 
   3. Mechanical dissociation
      In a polystyrene Petri dish, incise the tissue 10-12 times to relax the tissue
   4. Wash sample in PBS-A
      1. Add 5 ml PBS-A to incised tissue, leave at room temperature for 5 min
      2. This process may be repeated if the sample is particularly congested
   5. Enzymatic dissociation
      1. Using scissors, cut the Pasteur pipette bulb at a 45-degree angle to create a scoop. Use this to remove the tissue from the PBS-A
      2. Transfer to the 50 ml Falcon tube containing the Collagenase P/DNase solution
      3. Transfer the 50 ml Falcon tube to the orbital shaker 
      4. Incubate at 37 °C with agitation (200 rpm) for 15 min
      5. After 15 min, remove from the orbital shaker and sequentially pipette with 50 ml, 10 ml and 5 ml pipette tips to promote dissociation
      6. Return to the orbital shaker for a further 15 min, then repeat sequential pipetting
      7. Return to the orbital shaker for a further 30 min, then repeat sequential pipetting
   6. TrypLE treatment
      1. Centrifuge the sample at 450 x g for 5 min
      2. Remove the supernatant and re-suspend the resulting pellet in 1 ml of undiluted TrypLE 
      3. Incubate at 37 °C for 10 min
   7. Removing non-digested tissue fragments
      1. Add tissue strainer to a new 50 ml Falcon tube on ice 
      2. Strain tissue/enzyme suspension, pressing through with plunger of 5 ml syringe, simultaneously washing with “empty” DMEM
      3. Keep the sample on ice from this point
   8. Red blood cell lysis
      1. Centrifuge the sample at 450 x g for 5 min at 4 °C
      2. Aspirate the medium
      3. Re-suspend the pellet in 1 ml red blood cell lysis solution
      4. Incubate at 4 °C for 10 min
      5. Centrifuge at 450 x g for 5 min at 4 °C
   9. Sample collection
      
      1. Aspirate the red cell lysis buffer
      2. Re-suspend the pellet in 1 ml of “complete” DMEM
      3. The cells are now ready for quantification (if necessary) and use
         

Notes

1. The single-cell suspension may be used for a number of downstream applications, including single-cell RNA sequencing, establishing primary fibroblast cultures, and isolating fibroblast subpopulations. For single-cell RNA sequencing with the Drop-seq platform, re-suspend the cells in double-distilled H₂O supplemented with 9% Optiprep (Sigma-Aldrich), 1% PBS and 0.1% BSA, and perform as per Macosko et al. (2015) with the following modifications: 500 ng cDNA for PCR, 15 PCR cycles. The number of cells it is feasible to use will depend on the yield from the microfluidic step.
2. To establish primary cell cultures, plate 1 x 10⁵ cells/cm² to tissue culture plates in ‘Complete’ DMEM. Incubate in a humidified incubator at 37 °C and 5% CO₂ for 2 h to allow cells to adhere, before washing three times with PBS to remove non-adherent cells. This typically yields a 99.1% pure fibroblast (CD45-EpCAM-CD31-CD90⁺) culture, as determined by flow cytometry analysis. However, users should note that these culture conditions will not maintain in vivo fibroblast phenotypes (Waise et al., 2019).
3. Antibodies directed to specific fibroblast surface markers (e.g., PDGFR-α [Erez et al., 2010]), in combination with fluorescence-activated or magnetic cell sorting, can be used for isolation of fibroblast subpopulations. It is of note when employing these methods that enzymatic disaggregation can alter surface marker expression (Gray et al., 2002; Grange et al., 2011; Quatromoni et al., 2015), and that no single surface marker will reliably identify or differentiate all fibroblast populations (Sugimoto et al., 2006; Lambrechts et al., 2018).

Recipes

1. “Complete” DMEM
   Dulbecco’s Modified Eagle Medium (Sigma-Aldrich)
   10% (v/v) fetal calf serum (Biosera)
   1% (v/v) L-glutamine (Sigma-Aldrich)
   1% (v/v) penicillin-streptomycin (Sigma-Aldrich)
2. “Empty” DMEM
   Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) only
   

Acknowledgments

This protocol was derived from previously-published data ²⁸. This work was supported by Cancer Research UK and Medical Research Council Clinical Research Training Fellowships and a Pathological Society Trainee’s Small Grant to SW. Implementation of Drop-seq was supported by a Medical Research Council Discovery award (MC_PC_15078) and a Southampton Cancer Research UK Centre Development Fund Award to MJJRZ, CHO, JW, CJH & GJT. RP was supported by a John Goldman Fellowship for Future Science (2016/JGF/0003; Leuka Charity) awarded to MJJRZ. The authors thank Evan Macosko, Melissa Goldman and Steve McCarroll for their helpful advice, Dr. Serena Chee (University Hospital Southampton), Benjamin Johnson, Carine Fixmer and Maria Lane (TargetLung Clinical Trials Associates) for enabling access to clinical samples, and the patients involved in this study.

Competing interests

The authors declare no competing interests.

Ethics

Lung samples were received fresh from patients undergoing surgery at Southampton General Hospital (TargetLung study; approved by NRES Committee South Central: Hampshire A, REC number 14/SC/0186). All research was performed in accordance with the appropriate regulations. Informed consent was obtained from patients or their legal guardians.

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