. 2023 Feb 20;13(4):e4616. doi: 10.21769/BioProtoc.4616

Table 4. Troubleshooting for common problems.

Problem	Possible reasons for problems	Solution
Low yield of cardiac fibroblasts Less viability	Pieces of mouse/rat heart tissues are too big for proper digestion The digestion mixture used is insufficient Optimum conditions such as speed (rpm) or temperature not maintained Improper digestion due to expired or substandard digestion reagents Not enough cycles of digestion Too much digestion Excess mechanical stress Optimum conditions not maintained during harvesting and digestion of heart Improper coating of tissue culture plates	Taking large chunks would cause incomplete digestion of heart tissue. One heart should be cut into pieces of ~1 mm³ (4–5 pieces for rat pups and 2–3 pieces for mouse pups), for better yield. For better digestion, sufficient DM should be added (130 μL per four mice or one rat heart). The shaker incubator should be set at 250 rpm and 37 °C for digestion. Ensure that the reagents used are fresh and new and stored appropriately at the recommended temperatures. Digestion should be done for an adequate number of cycles (9–10 cycles). Digestion of the heart tissue should not exceed 10 cycles. Digestion time for one cycle should not be longer than 7 min. Excessive mechanical stress should be avoided such as excessive mincing of the heart tissue and prolonged vortexing. The speed of the shaker incubator should not exceed 280 rpm. The excised heart should be placed immediately in ice-cold PBSG buffer upon harvesting. After each cycle of digestion, cells should be collected in horse serum and the 50 mL tube should be kept inside a CO₂ incubator maintained at 37 °C and a 5% CO₂ saturation level. Proper coating of tissue culture plates with appropriate adhesion reagent should be done.
Contamination of cells by bacteria/fungi	Use of unsterilized dissection tools and/or reagents Contamination in CO₂ incubator or tissue culture hood Use of expired or bad antibiotics	All the steps should be performed under sterile conditions. All tools and reagents should be sterilized including scissors and forceps. Fresh blades and a new sterile 50 mL conical centrifuge should be used. Buffers should be made fresh and autoclaved. DM, horse serum, complete media, and the reagent used for coating the plates should be filter-sterilized. Heat-sterilize the incubator to decontaminate it. Fumigation or UV sterilization of the tissue culture hood should be practiced frequently. Before starting the digestion process, expose the tissue culture hood to UV for a minimum of 30 min. Antibiotics should be added to the media right before usage. Repeated freeze-thaw cycles should be avoided for the media with antibiotics.
Contamination by non-fibroblast cells	Excessive pre-plating Tissues from other organs not removed completely during harvesting of the hearts	Pre-plating should not exceed 1 h as other cells (cardiomyocytes) may also start to get attached to the coated surface of the plate. Non-cardiac tissues such as pieces of lungs, diaphragm, or any other connective tissue stuck to the heart should be completely removed before starting the digestion process.

Problem

Possible reasons for problems

Solution

Low yield of cardiac fibroblasts

Less viability

Pieces of mouse/rat heart tissues are too big for proper digestion

The digestion mixture used is insufficient

Optimum conditions such as speed (rpm) or temperature not maintained

Improper digestion due to expired or substandard digestion reagents

Not enough cycles of digestion

Too much digestion

Excess mechanical stress

Optimum conditions not maintained during harvesting and digestion of heart

Improper coating of tissue culture plates

Taking large chunks would cause incomplete digestion of heart tissue. One heart should be cut into pieces of ~1 mm³ (4–5 pieces for rat pups and 2–3 pieces for mouse pups), for better yield.

For better digestion, sufficient DM should be added (130 μL per four mice or one rat heart).

The shaker incubator should be set at 250 rpm and 37 °C for digestion.

Ensure that the reagents used are fresh and new and stored appropriately at the recommended temperatures.

Digestion should be done for an adequate number of cycles (9–10 cycles).

Digestion of the heart tissue should not exceed 10 cycles. Digestion time for one cycle should not be longer than 7 min.

Excessive mechanical stress should be avoided such as excessive mincing of the heart tissue and prolonged vortexing. The speed of the shaker incubator should not exceed 280 rpm.

The excised heart should be placed immediately in ice-cold PBSG buffer upon harvesting. After each cycle of digestion, cells should be collected in horse serum and the 50 mL tube should be kept inside a CO₂ incubator maintained at 37 °C and a 5% CO₂ saturation level.

Proper coating of tissue culture plates with appropriate adhesion reagent should be done.

Contamination of cells by bacteria/fungi

Use of unsterilized dissection tools and/or reagents

Contamination in CO₂ incubator or tissue culture hood

Use of expired or bad antibiotics

All the steps should be performed under sterile conditions. All tools and reagents should be sterilized including scissors and forceps. Fresh blades and a new sterile 50 mL conical centrifuge should be used. Buffers should be made fresh and autoclaved. DM, horse serum, complete media, and the reagent used for coating the plates should be filter-sterilized.

Heat-sterilize the incubator to decontaminate it. Fumigation or UV sterilization of the tissue culture hood should be practiced frequently.

Before starting the digestion process, expose the tissue culture hood to UV for a minimum of 30 min.

Antibiotics should be added to the media right before usage. Repeated freeze-thaw cycles should be avoided for the media with antibiotics.

Contamination by non-fibroblast cells

Excessive pre-plating

Tissues from other organs not removed completely during harvesting of the hearts

Pre-plating should not exceed 1 h as other cells (cardiomyocytes) may also start to get attached to the coated surface of the plate.

Non-cardiac tissues such as pieces of lungs, diaphragm, or any other connective tissue stuck to the heart should be completely removed before starting the digestion process.