Materials and Reagents

Falcon tubes (50 ml) (Sarsdtedt)
Sterile pipets (Sarsdtedt)
100 mm cell culture dishes (Sarsdtedt)
Aluminum foil
Adult mouse hearts from freshly euthanized mice (C57Bl6/J)
NaCl (Carl Roth, catalog number: 3957.2 )
KCl (Carl Roth, catalog number: HNO2.3 )
Na₂HPO₄ (Carl Roth, catalog number: 4984.1 )
KH₂PO₄ (AppliChem, catalog number: A2946 )
CaCl₂ (AppliChem, catalog number: A3652 )
MgSO_4^.7H₂O (AppliChem, catalog number: A1037 )
NaHCO₃ (AppliChem, catalog number: A1940 )
Collagenase II (Worthington, catalog number: LS004177 )
2.5% Trypsin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15090-046 )
DMEM/F12 (Thermo Fisher Scientific, Gibco^TM, catalog number: 31331-028 )
PBS (without CaCl₂, MgCl₂) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190-94 )
FBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
PenStrep (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
L-glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-024 )
Ascorbic acid (AppliChem, catalog number: A1052 )
PBS (see Recipes)
Hanks balanced salt solution (HBSS) (see Recipes)
Collagenase II stock (see Recipes)
Digestion buffer (see Recipes)
Fibroblast medium (see Recipes)

Equipment

Scalpel, forceps and scissors (Fine Science Tools)
Autoclaved glass beaker (100 ml) with magnetic stirrer
Sterile cell culture hood
Cell culture incubator (Labotect Labor-Technik Göttingen)
Magnetic stirrer with thermostat (IKA^®-Werke GmbH & CO. KG)
Cell culture centrifuge (Eppendorf, model: 5417R )
Autoclave

Procedure

Remove 3 adult mouse hearts from freshly euthanized mice and place them on a Petri dish containing ice cold PBS.
Place the hearts under a sterile cell culture hood and perform the following steps under sterile conditions.
Pump the blood out of the hearts by the use of forceps.
Mince the 3 hearts on a Petri dish on ice. By the use of scissors cut the heart into 10 pieces and by the use of a scalpel reduce them into a size of 1 mm.
Transfer the minced tissue (using the scalpel as a spatula) in an autoclaved glass beaker with a magnetic stirrer and cover the beaker with aluminum foil.
Add 25 ml digestion buffer and digest tissue under constant stirring at 37 °C for 5 min.
Note: The stirring speed should be adjusted so that all the pieces of tissue just flow and do not sit at the bottom but should not be too strong to destroy the cells.
Leave the mixture for 1 min for the tissue to sit down and discard the first supernatant that contains debris and blood cells.
Add 25 ml digestion buffer and digest tissue under constant stirring at 37 °C for 10 min.
Leave the mixture 1 min for the tissue to sit down and collect the supernatant with a 25 ml pipette (around 22 ml). Do not collect the tissue pieces that tend to float.
Place the supernatants in plastic 50 ml falcon tubes on ice containing 2 ml fibroblast medium. If possible supernatants can be combined in one falcon but for each supernatant 2 ml of fibroblast medium is required.
Repeat steps 6-10 until all the tissue is dissolved (typically 7-10 times).
Spin the cells down for 5 min at 300 x g at 4 °C.
Resuspend cells in 20 ml fibroblast medium.
Plate the cell suspension into two 100 mm cell culture dishes (10 ml cell suspension/dish) and incubate them at 37 °C in a cell culture incubator with 5% CO₂ for 2 h (“Pre-plating”).
2 h upon plating alive and healthy fibroblasts should have adhered to the dish. Check the cell confluence under the microscope (it should be around 50%). At this point fibroblasts resemble little round dots.
Collect and discard supernatant. Wash 3x with 2.5 ml warm PBS and replenish with 10 ml fresh fibroblast medium. Cultivate at 37 °C and 5% CO₂ in a cell culture incubator.
Primary adult mouse fibroblasts divide less than embryonic ones and under our culture conditions have a doubling time of 36-48 h. Typically, 2-3 days upon plating the fibroblasts reach 90% confluence.
Note: Caution! Do not split the fibroblasts until they reached a minimum confluence of 80% because the fibroblasts growth rate will decrease and they will differentiate.
Once confluence is reached fibroblasts are trypsinized as follows.
Wash two times with 2 ml pre-warmed PBS and then add 2 ml warm trypsin. Incubate for 5 min at 37 °C, in a cell culture incubator. Control if fibroblasts detached from plate. If not prolong incubation until the cells detach.
Neutralize trypsin by adding 7 ml pre-warmed fibroblast medium.
To achieve a re-plating confluence of 30%, split 1:3 into 3 new 100 mm plates. Add 3 ml of fibroblast suspension in a plate containing 7 ml fresh and warm fibroblast medium (final volume/plate 10 ml)

Representative data

For representative photos and results please refer to Zafiriou et al. (2014).

Notes

Note that primary isolated cells as fibroblasts are differentiated when cultured for a long time period (more than 20 days) cease to divide (Bayreuther et al.,1988). Moreover, using cells from the same passage leads to better reproducibility. Therefore, we recommend using passage 4 fibroblasts for all co-culture experiments.

Recipes

PBS, 1 L
Dissolve the following in 800 ml distilled H₂O (dH₂O)
8 g of NaCl
0.2 g of KCl
1.44 g of Na₂HPO₄
0.24 g of KH₂PO₄
Adjust pH to 7.4 with HCl, add dH₂O to 1 L, sterilized by autoclaving
Hanks balanced salt solution (HBSS) (100 ml)

Stock solution 1: 8.0 g NaCl, 0.4 g KCl in 100 ml dH₂O
Stock solution 2: 0.358 g Na₂HPO₄, 0.6 g KH₂PO₄ in 100 ml dH₂O
Stock solution 3: 0.72 g CaCl₂ in 50 ml dH₂O
Stock solution 4: 1.23 g MgSO₄^.7H₂O in 50 ml dH₂O
Stock solution 5: 0.35 g NaHCO₃ in 10 ml dH₂O
HBSS buffer: 10 ml Solution 1, 1 ml Solution 2, 1 ml Solution 3, 86 ml dH₂O, 1 ml Solution 4, 1 ml Solution 5 (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 0.44 KH₂PO₄, 1.3 mM CaCl₂, 1 mM MgSO₄, 4.2 mM NaHCO₃)

Collagenase II stock (1 ml)
Weight the appropriate amount of collagenase (varies from lot to lot) to obtain 10,000 U and dissolve in 1 ml sterile water
Digestion buffer (100 ml)
95 ml HBSS buffer
1 ml collagenase II (final conc. 100 U/ml)
4 ml 2.5% Trypsin (final conc. 0.1%)
Filter sterilize
For optimal enzymatic activity always prepare fresh.
Fibroblast medium
DMEM/F12
10% FBS
100 U/ml PenStrep
1x L-glutamine
100 µM ascorbic acid
Filter sterilize the whole medium and keep at 4 °C

Acknowledgments

This protocol was adapted by methods described by Lafontant et al. (2006) and Ohkubo et al. (1997).
The HBSS recipe was retrieved from the internet site labrat.com (link: http://www.thelabrat.com/protocols/Hanks.shtml).
This work was supported by the Deutsche Forschungsgemeinschaft (ZE900/1-1; ZI708/7-1, 8-1, 10-1; SFB1002; exFLIII) and the German Center for Cardiovascular Research (DZHK).

References

Bayreuther, K., Rodemann, H. P., Hommel, R., Dittmann, K., Albiez, M. and Francz, P. I. (1988). Human skin fibroblasts in vitro differentiate along a terminal cell lineage. Proc Natl Acad Sci U S A 85(14): 5112-5116.
Lafontant, P. J., Burns, A. R., Donnachie, E., Haudek, S. B., Smith, C. W. and Entman, M. L. (2006). Oncostatin M differentially regulates CXC chemokines in mouse cardiac fibroblasts. Am J Physiol Cell Physiol 291(1): C18-26.
Ohkubo, N., Matsubara, H., Nozawa, Y., Mori, Y., Murasawa, S., Kijima, K., Maruyama, K., Masaki, H., Tsutumi, Y., Shibazaki, Y., Iwasaka, T. and Inada, M. (1997). Angiotensin type 2 receptors are reexpressed by cardiac fibroblasts from failing myopathic hamster hearts and inhibit cell growth and fibrillar collagen metabolism. Circulation 96(11): 3954-3962.
Zafiriou, M. P., Noack, C., Unsold, B., Didie, M., Pavlova, E., Fischer, H. J., Reichardt, H. M., Bergmann, M. W., El-Armouche, A., Zimmermann, W. H. and Zelarayan, L. C. (2014). Erythropoietin responsive cardiomyogenic cells contribute to heart repair post myocardial infarction. Stem Cells 32(9): 2480-2491.