Isolation of Human Fibroblasts

Materials and Reagents

1.5 ml syringes (BD Plastipak, catalog number: 307731)

2.Pasteur pipette (Ucallm)

3.50 ml centrifugetube (Ucallm)

4.15 ml centrifugetube (Ucallm)

5.Sterile scalpel #21 blade (Swann-Morton, catalog number: 0507)

6.Polystyrene cell culture dish (Ucallm)

7.Syringe filtration2 µm (Ucallm)

8.Scissors

9.Collagenase P from Clostridium histolyticum (Merck, Roche, catalog number:11213857001). Reconstitute in PBS to 150 U/ml, store 100 μl aliquots at -20 ℃

10.TrypLE Express Enzyme (no phenol red; Thermo Fisher, catalog number:12604013). Store at room temperature protected from light for up to 2 years

11.Fetal bovine serum (Biosera, catalog number: FB-1001/500). Store at -20 ℃ for up to 60 months

12.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 ℃

13.Dulbecco’s Modified Eagle Medium (Merck, Sigma-Aldrich, catalog number: D5671-500ML). Store at 4 ℃

14.L-glutamine (Merck, Sigma-Aldrich, catalog number: G7513-100ML). Store at -20 ℃ for up to 2 years

15.Penicillin-streptomycin (Merck, Sigma-Aldrich, catalog number: P4333-100ML). Store at -20 ℃ for up to 2 years

16.Phosphate-buffered saline (PBS)

17.Amphotericin B (250 μg/ml; Gibco, catalog number: 15290-018). Store at -20 ℃ for 1 year

18.Sterile double-distilled H₂O

19.Red blood cell lysis buffer (10x; BioLegend, catalog number: 420301). Store at 4 ℃

20.DNase stock solutions

21.“Complete”DMEM (see Recipes)

22.“Empty”DMEM (see Recipes)

Recipes

1.“Complete” DMEM

Dulbecco’s Modified Eagle Medium (Sigma-Aldrich)

10% (v/v) fetal bovine 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

Equipment

1.Orbital shaker-incubator (e.g., Grant-bio Orbital Shaker-Incubator ES-20)

2.Cell Strainer 40 μm (Ucallm)

3.Centrifuge

Procedure

1.Sample collection

Transport the tissue sample in“empty”DMEM (Recipe 2) on ice.

2.Prepare working solutions

1)In a 50 ml centrifugetube, add 100 μl Collagenase P and 40 μl DNase stock solutions to 5 ml“complete”DMEM (Recipe 1).

2)Make PBS-A: add 1 μlAmphotericin to 10 ml PBS in a 15 ml centrifuge tube.

3)Dilute the 10X RBC Lysis Buffer to 1X working concentration with Sterile double-distilled H₂O.

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 pipettebulb 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 centrifugetube containing the Collagenase P/DNase  Solution.

3)Transfer the 50 ml centrifugetube to the orbital shaker. 

4)Incubate at 37 ℃with agitation (200 rpm) for 15 min.

5)After 15 min, remove from the orbital shaker and sequentially pipette with 50ml, 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.TrypLEtreatment

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 ℃for 10 min.

7.Removing non-digested tissue fragments

1)Add tissue strainer to a new 50 ml centrifugetube 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 ℃.

2)Aspirate the medium.

3)Re-suspend the pellet in 1 ml 1Xred blood cell lysis solution.

4)Incubate at 4 ℃for 10 min.

5)Centrifuge at 450 x g for 5 min at 4 ℃.

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.

2.To establish primary cell cultures, plate 1 x 10⁵cells/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 [Erezet 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).

References

1.Waise, S., Parker, R., Rose-Zerilli, M. J. J., Layfield, D. M., Wood, O., West, J., Ottensmeier, C.H., Thomas, G. J. and Hanley, C. J. (2019). An Optimized Method to Isolate Human Fibroblasts from Tissue for Ex Vivo Analysis. Bio-protocol 9(23): e3440.

2.Sugimoto, H., Mundel, T. M., Kieran, M. W. and Kalluri, R. (2006). Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol Ther 5(12): 1640-1646.

3.Waise, S., Parker, R., Rose-Zerilli, M. J. J., Layfield, D. M., Wood, O., West, J., Ottensmeier, C. H.,Thomas, G. J. and Hanley, C. J. (2019). An optimised tissue disaggregation and data processing pipeline for characterising fibroblast phenotypes using single-cell RNA sequencing. Sci Rep 9(1): 9580.

4.Erez, N., Truitt, M., Olson, P., Arron, S. T. and Hanahan, D. (2010). Cancer-associated fibroblasts are activated in incipient neoplasia to orchestrate tumor-promoting inflammation in an NF-kappaB-Dependent manner. Cancer Cell 17(2): 135-147.

5.Gray, D. H., Chidgey, A. P. and Boyd, R. L. (2002). Analysis of thymic stromal cell populations using flow cytometry. J Immunol Methods 260(1-2): 15-28.

6.Grange, C., Letourneau, J., Forget, M. A., Godin-Ethier, J., Martin, J., Liberman, M., Latour, M.,Widmer, H., Lattouf, J. B., Piccirillo, C. A., Cailhier, J. F. and Lapointe, R. (2011). Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation. J Immunol Methods 372(1-2): 119-126.

7.Quatromoni, J. G., Singhal, S., Bhojnagarwala, P., Hancock, W. W., Albelda, S. M. and Eruslanov, E.An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells. J Leukoc Biol 97(1): 201-209.

8.Lambrechts, D., Wauters, E., Boeckx, B., Aibar, S., Nittner, D., Burton, O., Bassez, A., Decaluwe, H.,Pircher, A., Van den Eynde, K., Weynand, B., Verbeken, E., De Leyn, P., Liston, A., Vansteenkiste, J., Carmeliet, P., Aerts, S. and Thienpont, B. (2018). Phenotype molding of stromal cells in the lung tumor microenvironment. Nat Med 24(8): 1277-1289.

Manufacturer’s Link

Becton Dickinson: https://www.bd.com

Swann-Morton: https://www.swann-morton.com

Merck.com: https://www.merck.com>news>roche

Thermo Fisher Scientific: https://www.thermofisher.com

Biosera: https://www.biosera.com

Sigma-Aldrich: https://www.sigmaaldrich.com

Thermo Fisher Scientific: https://www.thermofishercom >home > brands >gibco

BioLegend: https://www.biolegend.com

Grant Instruments: https://www.grantinstruments.com

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