Ultrasonic-augmented Primary Adult Fibroblast Isolation

Summary

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Abstract

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method’s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

Introduction

Fibroblasts are flat, spindle-shaped cells with multiple stellate processes and an extensive rough endoplasmic reticulum^1,2. An average fibroblast measures 30 - 100 µm and has a life span of 57 ± 3 days^1,3. The average cell cycle duration of human fibroblasts ranges from 16 - 48 h depending on the culture conditions⁴. There is evidence that the replicative capacity and functional quality of cultured primary fibroblasts negatively correlates with the donor age, suggesting that younger donors (animals or patients) should be preferred if possible^5,6.

Fibroblasts constitute a predominant cell type of most mammalian body tissues. Despite their ubiquitous presence, the molecular identification of fibroblasts is still a challenge⁷. Fibroblasts migrate to developing tissues and organs from different sources during embryonic development⁸. For this reason, there is a plethora of marker proteins that can be found in fibroblasts whereas unique marker proteins, which are present in every fibroblast population and exclusive for fibroblasts, are still missing. Thus, expression patterns of several recognized markers are usually used to identify fibroblasts. Among the most recognized markers are vimentin, human fibroblast surface protein (hFSP), discoidin domain receptor 2 (DDR2) and alpha smooth muscle actin (αSMA).

Fibroblasts are the major extracellular matrix (ECM)-producing cell type. Thereby, fibroblasts maintain an orderly tissue architecture and provide mechanical support for neighboring cells¹. The balance between ECM synthesis and degradation is a well-regulated process. Shifts towards synthesis mark the beginning of excessive ECM deposition which, if not terminated, leads to fibrosis. Fibrosis is mediated by myofibroblasts, which originate from activated fibroblasts undergoing molecular and phenotypical changes. One hallmark of myofibroblasts is enhanced secretion of ECM and cytokines and the expression of orderly arranged αSMA microfilaments⁹.

Primary fibroblasts have been in the spotlight of recent research focusing on fibrosis, tissue inflammation and fibroblast-cancer-cell interactions^10,11. However, to effectively study fibroblast properties in health and disease, it is necessary to isolate viable primary adult fibroblasts on a regular basis. There are several methods available to isolate fibroblasts^12,13,14. The three major methods of fibroblast isolation are outgrowth from tissue chunks¹², enzymatic tissue digestion¹⁵, and enzymatic perfusion of hollow organs^9,13,16. The advantage of outgrowth is a gentle isolation process without enzymatic cell degradation. On the other hand, outgrowth cultures usually require prolonged culture periods until cells can be used for experiments. Common enzymatic digestion is fast but bears a risk of contamination with other cell types (e.g., endothelial cells) or bacteria in the agitation process, which is necessary to mechanically dissolve the tissue. Furthermore, these methods are often elaborate and require time and skill to learn.

Regarding the importance of primary fibroblasts in research, there is still a need to optimize existing cell isolation approaches in terms of quickness, simplicity and reliability. Here, a novel ultrasonic-based enzymatic fibroblast isolation method delivering high quality cells is provided.

Protocol

The following protocol follows the institutional animal care guidelines of Technische Universität Dresden, Germany (File number: T 2014/4) as well as internationally accepted animal care guidelines (FELASA)¹⁷. Figure 1 visualizes the cell isolation process.

1. Preparing the setup, material and media

1. Prepare cell culture medium, PBS solution, collagenase blend stock solution (reconstitute 50 mg of lyophilized collagenase blend in 12 mL of sterile ultrapure water), and 0.25% trypsin solution.
2. Warm up the medium, the PBS and the trypsin solution to 37 °C.
3. Preheat the ultrasonic water bath to 37 °C.
4. Disinfect forceps, a stainless-steel spatula, scalpels (2x scalpels per organ) and 2 glass beakers with 70% ethanol and place these materials under the cell culture hood.
5. Fill one beaker with 70% ethanol and the other with sterile water or PBS solution. These beakers are required to disinfect and wash the instrument after each organ procession.
6. Place sterile 15 mL plastic tubes containing cold PBS on wet ice. The number of tubes depends on the number of organs you want to isolate fibroblasts from.

2. Mouse dissection and organ removal

1. Wear two pairs of gloves one above the other, so the first pair can be removed as soon as the animal has been dissected.
   NOTE: This procedure prevents bacteria from the animal's fur and skin from spreading over the organs.
2. Euthanize the mouse (e.g., cervical dislocation) and pin the carcass with needles to every limb to a polystyrene pad.
3. Disinfect the mouse carcass using 70% ethanol spray. Make sure the fur is soaked in ethanol so the hair will not swirl up.
4. Cut the fur right above the urogenital tract using surgical forceps and atraumatic scissors. Cut the skin alongside the middle line from the point of the initial incision to the neck (3 - 4 cm) and add relief cuts at the limbs.
   CAUTION: Do not perforate the muscular layer at this step to avoid bacterial contamination!
5. Pin the skin to the polystyrene foam pad to have optimal access to the musculature covering the abdominal cavity.
6. Disinfect the abdominal musculature twice using 70% ethanol. Let the ethanol dry before continuing to the next step.
7. Remove the first pair of gloves. Use a new, sterile set of forceps and scissors.
8. Open the abdominal cavity and the thorax by incising the muscular layer with surgical scissors to gently remove the organs of choice. Therefore, hold the organ gently with surgical forceps (do not pierce the organ, use minimal pressure) and cut the supplying blood vessels near the entry point at the organ with scissors.
9. Put the organs into the sterile tubes containing cold PBS. Close the tubes tightly. Place the tubes on wet ice until continuing with step 3.1.

3. Tissue mincing, digestion and cell extraction

1. Transfer the tubes under the sterile cell culture hood.
   CAUTION: Wear a fresh pair of gloves and disinfect the tubes with 70% ethanol before transferring them under the hood!
2. Take the organ out of the 15 mL tube using sterile forceps. Place the organ onto one half of a sterile 6 cm Petri dish and wash the organ briefly with PBS to remove excess blood. Transfer the organ to the second half of the Petri dish, remove excess PBS.
3. Mince the tissue using two sterile scalpels. The remaining tissue fragments should not be larger than 1 - 2 mm.
4. Transfer the minced tissue into a new sterile 15 mL tube using the sterile spatula and add 2 mL of 0.25% trypsin solution. Place the tube into a cell culture incubator at 37 °C for 5 min.
5. Vortex the tube gently (circa 1400/min) for 10 s.
6. Stop the trypsin reaction under the cell culture hood by adding 4 mL FCS-containing cell culture medium (Dulbecco's Modified Eagle Medium (DMEM), e.g.).
7. Add 250 µL of collagenase blend solution to each tube containing heart or lung tissue and 100 µL for kidney or liver, respectively.
8. Place the tubes into an ultrasonic water bath (37 °C) and activate the ultrasonic sonicator for 10 min.
   NOTE: The ultrasonic water bath used in this protocol has an operating frequency of 35 kHz and a maximal power of 320 W.
9. Vortex the tubes gently (circa 1400/min) for 10 s.
10. Place the tubes again into the ultrasonic water bath for 10 min.
11. Vortex gently (circa 1400/min) for 10 s.
12. Disinfect the tubes with 70% ethanol and transfer them under the sterile cell culture hood.
13. Filter the solution with a 40 µm mesh into a new sterile 15 mL tube.
14. Centrifuge the tube at 500 x g for 5 min.
15. Remove the supernatant and resuspend the pellet in 1 mL of fresh medium.
16. Transfer the cells into a suitable cell culture vessel (e.g., 6-well plate) and place the vessel into the cell culture incubator overnight at 37 °C and 5% CO₂.
17. The next day, remove the medium, wash 3 times with PBS, then add fresh medium (the added volume depends on the cell culture vessel of choice, 2 mL per well of a 6-well plate etc.).
18. Change the medium every other day.
    NOTE: Fibroblasts can be split after reaching optical confluence of 90% (usually after 5-7 days).

Representative Results

The ability of this protocol to isolate adult fibroblasts from solid murine tissue was demonstrated. Viable fibroblasts were obtained that could be used for subsequent experiments such as immunofluorescence staining or proliferation experiments (Figure 2D-F, Figure 5A).

Adult fibroblasts are flat spindle-shaped cells with multiple cellular processes that typically grow in monolayers^12,18. The obtained results reflect these morphological hallmarks and indicate distinct morphological differences in fibroblast populations from different organs (Figure 2). Renal fibroblasts were especially smaller and grew at higher densities than cardiac, pulmonary or hepatic fibroblasts (Figure 2).

The upper panel of Figure 3 depicts the process of cell maturation and proliferation via the example of cardiac fibroblasts following the isolation. The cells displayed high proliferation rates reaching optical confluence of >90% after 6 ± 1 days. At this level of confluence fibroblasts stop to proliferate¹⁸ and the cells can be split, using 0.25% trypsin, and transferred to larger cell culture flasks for subsequent culture or experiments.

Under basal cell culture conditions, fibroblast cultures consist of fibroblasts and a smaller proportion of myofibroblasts^19,20. There are several accepted markers to identify fibroblasts. DDR2 and vimentin expression are common fibroblast markers and the expression of organized αSMA microfilaments indicates myofibroblast differentiation^7,9. A representative image of a differentiated myofibroblast with distinctive αSMA microfilaments and representative overview images for αSMA filament abundance in heart, kidney, liver and lung are presented in Figure 4. While only about 20 - 30% of the isolated and subsequently cultured cells expressed orderly arranged αSMA microfilaments (indicating myofibroblast differentiation), all of the cells (≈ 99%) were positive for vimentin and DDR2 (Figure 3, lower panel).

Figure 1. Overview of the fibroblast isolation procedure. After the organs of interest have been removed from the mouse, they are minced under a cell culture hood using sterile scalpels. Subsequently, the minced tissue is transferred to 0.25% trypsin solution for 5 min. After the first digestion, collagenase blend solution is added and the tubes are transferred to an ultrasonic bath for 20 min at 37 °C. After filtration and centrifugation, the cell pellet can be transferred into a suitable cell culture dish. After at least 8 h to attach, the cultures are washed three times with PBS to remove erythrocytes and debris. Finally, fresh medium (DMEM, 15% FCS, 1% Penicillin/Streptomycin) is added and the cells can be cultured. Images of Servier smart medical art have been used to create the figure (https://creativecommons.org/licenses/by/3.0/). Please click here to view a larger version of this figure.

Figure 2. Primary murine fibroblasts isolated from solid organs after 7 days of primary culture. A) Cardiac fibroblasts. B) Pulmonary fibroblasts. C) Renal fibroblasts. D) Hepatic fibroblasts. The cells were cultured in DMEM (15% FCS, 1% Penicillin/Streptomycin (PS)) at 37 °C and 5% CO₂. 10x magnification and 5.6x optical camera zoom were used to obtain these images. Please click here to view a larger version of this figure.

Figure 3. Identification of fibroblasts in culture. A - C) Attachment and growth of cardiac fibroblasts for 4 days. After washing and medium change the attached fibroblasts develop their distinctive shape and proliferate. The cells were cultured in DMEM (15% FCS, 1% PS). D - F) Immunofluorescence staining images of primary fibroblasts after 7 days of culture for fibroblast markers αSMA (D), DDR2 (E) and Vimentin (F). The nuclei were stained with DAPI (blue). Primary antibodies (Sigma-Aldrich) were diluted 1:200 in 1% BSA solution (αSMA: A5228; DDR2: HPA070112; Vimentin: V5255). Alexa-Fluor 488 was used as secondary antibody. The scale bars equal the indicated lengths above. Please click here to view a larger version of this figure.

Figure 4. Representative immunocytochemical images for αSMA microfilament abundance. A) A typical myofibroblast is displayed (magnification: 20x). B – E) Representative αSMA overview images for heart (B), kidney (C), liver (D) and lung (E) (magnification: 10x). The white circles indicate representative cells which were considered myofibroblasts. The nuclei were stained with DAPI (blue). The primary antibody was diluted 1:200 in 1% BSA solution (αSMA: A5228). Alexa-Fluor 488 was used as secondary antibody. The scale bars equal the indicated lengths above. Please click here to view a larger version of this figure.

Figure 5. Proliferation rates of murine fibroblasts and in vitro senescence development. A) Proliferation rate of murine fibroblasts determined after 7 and 14 days of culture. Cells were harvested and counted with a Buerker chamber at the respective time points. The results represent the cell count in 1 mL of medium. B) Senescence is determined by the presence of β-galactosidase (stained green). Senescent cells are indicated with white circles. The scale bar equals 50 µm. Results are presented as mean ± SEM. Please click here to view a larger version of this figure.

Discussion

Compared to immortalized fibroblast cell lines, primary fibroblasts offer several advantages. They can be isolated cost effectively in high quality and quantity. Furthermore, primary cultures offer the possibility to study cells from multiple individuals, which increases the reliability of the obtained results and decreases the likelihood of merely studying cell culture artifacts. Continuous generation of new primary cultures prevents genetic alterations which commonly occur after repeated passaging²¹. Additionally, cellular senescence^22,23 or increased myofibroblast differentiation occur more frequently in cultures at high passages²⁰. At low passages (2-3), the basal myofibroblast count was approximately 20 - 30% (data not shown) and only 2.89% of the cells were considered senescent based on β-galactosidase expression (Figure 5B). For this reason, it is recommended to passage the cultures up to maximal 5 times and to replace them hereafter. This guarantees reliable and replicable results throughout the experiments. Optimal fibroblast growth and proliferation were obtained with cell culture medium containing 15% FCS instead of 10%. This ensured a robust fibroblast yield and good cell viability after the first passage.

This protocol complements existing techniques in terms of speed and degree of difficulty. Outgrowth techniques require between 10 - 21 days, depending on the species and tissue, until sufficient quantities of fibroblasts can be harvested^12,24. Langendorff-perfusion on the other hand is fast (<5 h) but requires more manual skill and training. Compared to outgrowth methods^12,24 or fibroblast isolation from the supernatant of Langendorff-perfusion¹⁶, this protocol offers especially beginners the opportunity to work with primary cells after a very short training period (2 - 3 isolations) without the requirement of high mechanical skill or technical effort. Contamination with non-fibroblast cells (e.g., endothelial cells) was negligible because cultured fibroblasts rapidly overgrow other cell types in cultures due to their higher proliferation rates²⁵. Additionally, this protocol gives clear instructions that help to avoid bacterial contamination, which is one major problem working with primary murine cells. Here, two critical steps to avoid contamination were identified. The first one is the organ removal. Rodent fur bacteria can be easily transferred to the organs if the gloves and surgical instruments are not changed after removing the fur and the skin. The second critical step is the isolation process itself. The cells have to be removed mechanically from their tissue niche. In the context of Langendorff-perfusion this is realized by a continuous current of liquid. Other techniques use magnetic stirring bars or shaking methods. Particularly, the replacement of magnetic stirring bars by ultrasonic waves reduces the risk of bacterial contamination by avoiding physical contact between the stirrer and the tissue lysate. An additional advantage of the ultrasonic water bath is the even distribution of warmth to the tubes, thereby providing optimal working temperatures for the enzymes.

In conclusion, this protocol provides a quick, reliable and replicable method to isolate primary fibroblasts that is ideal for both advanced and early-stage researchers. This method is a useful addition to the existing spectrum of techniques that can contribute to a better understanding of fibroblast function in health and disease.

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Sigma-Aldrich, St. Louis, USA	T4049-100ML
Antibiotics	Gibco-Life Technologies, Carlsbad, USA	Gibco LS15140148	Penicillin/ Streptomycin (10,000 U/mL)
Cell culture hood	Thermo Fisher Scientific, Waltham, USA	51023608	HeraSafe KSP15
Cell culture incubator	Thermo Fisher Scientific, Waltham, USA	50049176	BBD 6220
Cell culture plates	Thermo Fisher Scientific, Waltham, USA	depends on vessel	6-, 12-, 24-wells Nunclon surface
Cell culture suction	VACUUBRAND GMBH + CO KG, Wertheim, Germany	20727400	BVC professional suction
Cell strainer (mesh)	Corning, Tewksbury, USA	431750	40 µm Nylon
Centrifuge	Thermo Fisher Scientific, Waltham, USA	75007213	Megafuge 8R
Cordless pipetting controller	Hirschmann, Eberstadt, Germany	9907200	Pipetus
Disposable pipette tips	Sigma-Aldrich, St. Louis, USA	depends on volume	SafeSeal tips for pipettes (10 µL, 20 µL, 100 µL, 200 µL, 1000 µL)
Disposable plastic pipettes	Sigma-Aldrich, St. Louis, USA	depends on volume	5 mL, 10 mL, 25 mL, 50 mL
Disposable sterile scalpel	Myco Medical, Cary, USA	n.a.	Techno cut
Dulbeccos Modified Eagle Medium (DMEM)	Thermo Fisher Scientific, Waltham, USA	41965-062	High glucose
Eppendorf tubes	Eppendorf, Hamburg, Germany	depends on volume	50 µL, 500 µL, 1.500µL, 2.000 µL
Fetal calf serum (FCS)	Sigma-Aldrich, St. Louis, USA	F2442-50ML
Collagenase blend	Sigma-Aldrich, St. Louis, USA	5401020001	Liberase TL Research Grade
Petri dish 6 cm	Sigma-Aldrich, St. Louis, USA	P5481-500EA
Phosphate Buffered Saline (PBS)	Sigma-Aldrich, St. Louis, USA	D8537-500ML	500 mL
Senescence detection kit	Abcam, Cambridge, UK	ab65351
Shaker/ Vortex	IKA, Staufen im Breisgau, Germany	n.a.	MS2 Minishaker (subsequent model: Ident-Nr.: 0020016017)
Sterile plastic tubes	Thermo Fisher Scientific, Waltham, USA	Falcon 352095	BD Falcon tubes (15 mL, 50 mL)
Ultrasonic water bath	BANDELIN electronic GmbH & Co. KG, Berlin, Germany	312	Sonorex RK100H
Surgical scissors (atraumatic)	Aesculap AG, Tuttlingen, Germany	NR 82
Surgical scissors	Aesculap AG, Tuttlingen, Germany	eq 1060.09
Surgical forceps	Aesculap AG, Tuttlingen, Germany	BD577