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Jul 2019
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An Optimized Method to Isolate Human Fibroblasts from Tissue for Ex Vivo Analysis    

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Abstract

Despite their involvement in many physiological and pathological processes, fibroblasts remain a poorly-characterized cell type. Analysis of primary fibroblasts while maintaining their in vivo phenotype is challenging: standard methods for fibroblast isolation require cell culture in vitro, which is known to alter phenotypes. Previously-described protocols for the dissociation of primary tissues fail to extract sufficient numbers of fibroblasts, instead largely yielding immune cells. Here, we describe an optimized method for generating a fibroblast-enriched single-cell suspension from human tissues using combined mechanical and enzymatic dissociation. This allows analysis of ex vivo fibroblasts without the need for culture in vitro.

Keywords: Tissue disaggregation, Stroma, Fibroblasts, Lung

Background

Fibroblasts are almost ubiquitous in human tissues, and the most common cell type in the stroma of a number of solid tumours, where they are referred to as cancer-associated fibroblasts (CAFs) (Ishii et al., 2016; Kalluri and Zeisberg, 2006; Rupp et al., 2014; Servais and Erez, 2013). CAFs are associated with multiple hallmarks of malignancy (Ishii et al., 2016; Tao et al., 2017) and correlate with poor prognosis in multiple solid tumours (Hanley et al., 2018).

Given these tumour-promoting effects, and their genetic stability relative to cancer cells (Ishii et al., 2016), it is unsurprising that fibroblasts are an attractive therapeutic target. However, clinical trials targeting CAFs have so far yielded disappointing results (Hofheinz et al., 2003; Narra et al., 2007). This may, in part, be due to variation within the fibroblast population: these cells are known to be heterogeneous in both normal and disease states (Desmoulière et al., 2004; Sugimoto et al., 2006; Anderberg and Pietras, 2009; Servais and Erez, 2013; Witowski et al., 2015; Kalluri, 2016; Mellone et al., 2017). However, this heterogeneity remains poorly-characterised and it is not yet clear how many subtypes are present within a given tissue or tumour type, or the nature of functional differences between groups (Herrera et al., 2013; Servais and Erez, 2013; Ishii et al., 2016).

Characterising heterogeneity of cell populations within human tissues often requires analysis at a single-cell level. Single-cell RNA sequencing is a valuable platform for characterising multicellular ecosystems. However, fibroblasts are embedded within extracellular matrix and are particularly difficult to isolate: this has led to under-representation in, for example, single-cell RNA sequencing datasets (Lambrechts et al., 2018). Unlike many murine models, there is no standardized disaggregation protocol for human solid tissues. A number of different immune cell populations have been successfully isolated and analyzed directly from tissues (Holt et al., 1986; Perrot et al., 2007; Grange et al., 2011; Quatromoni et al., 2015; Ganesan et al., 2017). However, epithelial and non-immune stromal cells are usually cultured in vitro prior to analysis (Lurton et al., 1999; Koumas et al., 2003; Comhair et al., 2012; Barkauskas et al., 2013; Mackay et al., 2013). Culture in vitro has been shown to change fibroblast phenotypes (Öhlund et al., 2017; Waise et al., 2019); thus, how and whether the functional differences described in vitro are maintained in vivo is not yet known (Lurton et al., 1999).

Here, we describe an optimized protocol for the isolation of fibroblasts from primary human tissues, allowing immediate analysis without the need for culture in vitro. In brief, primary samples undergo mechanical and enzymatic dissociation, followed by incubation with TrypLE and red cell lysis buffer (to disrupt intercellular adhesions and remove red blood cells, respectively). Use of this approach yields a single-cell suspension consisting of multiple cell types, with approximately a 4-fold greater proportion of fibroblasts compared to other disaggregation strategies (Waise et al., 2019). We describe use of this protocol for the ex vivo analysis of fibroblasts in both normal and disease states using single-cell RNA sequencing, and highlight alternative downstream applications. In addition, this approach may have applications in the analysis of other cell types (e.g., epithelial cells).

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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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Waise, S., Parker, R., Rose-Zerilli, M. 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. DOI: 10.21769/BioProtoc.3440.
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