Race L. Kao, Ph.D.
East Tennessee State University
Johnson City, TN 37614
Isolated cardiac myocytes from adult mammals have been used for metabolic, electrophysiologic, receptor and ligand binding, pharmacologic, and recently cellular cardiomyoplasty studies. Isolated ventricular myocytes have the advantages by providing a uniform population of single cell type free of neural and humoral influences in a controllable environment. Freshly isolated cells have been used for most of the published studies. However, at the present time the intact cells cannot be effectively separated from the damaged ones. Most studies are affected by the damaged cells with the exception of electrophysiologic or biophysical experiments.
This technique relies on the ability of perfusing the heart with different buffers and enzyme solution to degrade and loosen the intracellular and collagen matrixes that hold the myocytes in the myocardium. Being able to perfuse the heart without damage and not mincing the myocardium during cell isolation are key factors to reduce injured myocytes. A high yield of quiescent cardiac myocytes that are tolerant to physiologic levels of extracellular calcium with normal morphology and metabolic activity are desired. Cultured cardiac myocytes that maintained their differentiated morphology and normal physiology may allow the recovery of the cells from traumatic injury during the isolation procedure. However, long-term primary culture of isolated cardiac myocytes from adult animal has not been a common practice.
Making the Buffer:
Glass distilled water should be used for making the buffer. Krebs-Henseleit bicarbonate buffer (in mM: NaCl 119; KCl 4.7; CaCl2 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25) has pH 7.4 at 37o C when equilibrated with 95% O2 : 5% CO2 containing heparin (1000 units/L) and glucose (11 mM) can be used for preliminary perfusion. The buffer should be equilibrated with 95% O2:5%CO2 before adding NaHCO3. The calcium free Krebs-Henseleit bicarbonate buffer containing the same compounds but without CaCl2 and heparin. Part of the calcium free Krebs-Henseleit bicarbonate buffer will be used to make up the enzyme solution by adding 0.1% (wt/vol) of collagenase and 0.1% (wt/vol) of hyaluronidase and 200 µM CaCl2. All buffer and solution that will be used to perfuse the heart must be filtered (0.2µ) to avoid coronary spasm and embolization. Insulin, amino acids, adenosine (1mM), 2,3-butanedione monoxime (BDM, 7.5 mM), pyruvate, fatty acids (most of them need to be conjugated with protein) or taurine by them-self or in combination can be added to the buffer or enzyme solution to improve the morphology and viability of isolated cells. However, their physiologic or metabolic effects should be carefully considered for the study designed using isolated cardiac myocytes. Medium 199 (Earle's salts with NaHCO3 and 0.1 g/L L-glutamine) with 10% serum can be used to wash the isolated cells after enzyme treatment before equilibrating the cells with desired buffer for experiment.
Note: Adenosine or BDM should not be added into the Krebs-Henseleit bicarbonate buffer. The 200 µM CaCl2 (from a stock solution) added into the enzyme solution is to minimize injury from calcium paradox and to allow full activation of collagenase. Although step wise increasing of calcium to physiologic concentrations can be beneficial to the isolated cells, we have been using medium 199 (1.8 mM calcium) with serum to wash the isolated cardiac myocytes immediately after isolation.
Selecting the Enzymes:
Collagenase, Type 2 (LS004176 or LS004177) from Worthington Biochemical Corporation (Lakewood, NJ) or collagenase Type IA (C 9891) from Sigma (St Louis, MO) can be selected. Each lot of collagenase has a different amount of acid residue in it, and additional NaHCO3 should be added to maintain the enzyme solution at proper pH. This can be done by solubilizing an adequate amount of the enzyme and finding out the NaHCO3 needed. From the same lot, the amount of NaHCO3 needed for a known amount of collagenase is quite consistent. Collagen fibers in the heart are coated with a layer of glycoprotein, and pure collagenase will not do well for cell isolation. Hyaluronidase from Sigma ( Type I-S, H 3506) or Worthington (LS002594 or LS002592) will work well for the procedure.
Perfusion of the Heart:
Male Sprague-Dawley rats weighing 250~300 g are commonly used. Female rats and other strains of rats can also be used for isolation of cardiac myocytes. After overnight fasting, the rats will be treated with heparin (50 units/100 g body weight, intraperitoneal injection) for 10 minutes before obtaining their hearts for perfusion. After proper anesthesia, the heart is rapidly excised and dropped into a beaker of cold (on crushed ice ~2o C) Krebs-Henseleit bicarbonate buffer containing heparin and glucose before being perfused with a modified Langendorff preparation. If heart isolation and initiation of perfusion cannot be completed within one to two minutes, in vivo cannulation with or without arresting the heart with cold cardioplegic solution should be considered. After five to ten minutes of preliminary perfusion with buffer (to washout the blood and stabilize the heart), the heart will be perfused with calcium free Krebs-Henseleit bicarbonate buffer for five minutes. The buffers are equilibrated with 95%O2:5%CO2 and passed through the heart a single time without recirculation.
Note: If the heart does not dilate and stop contraction within a few beats after switching to calcium free buffer, calcium content is too high in the water or chemicals. If better water or chemicals cannot be obtained, EGTA or EDTA of titrated amounts can be added to the buffer. However, EGTA and EDTA are known to cause membrane damage of isolated cells.
Isolation of Cardiac Myocytes:
Following another five minutes of perfusion with enzyme solution (can be recirculated), the ventricles are cut into an Erlenmeyer flask containing enzyme solution. The heart is incubated in the enzyme solution gassed with 95%O 2:5%CO2 with gentle agitation (circular motion with the heart swimming clockwise and counter clockwise) at 37o C (Erlenmeyer flask immersed in the water of a water bath). The dispersed cells are harvested after each five minutes of incubation by decantation. Enzyme solution is added to the Erlenmeyer flask to continue the cell isolation. Normally, after three successive periods of incubation (five minutes each), the dispersion of ventricular muscle cells should be completed. The cells harvested from first incubation are usually low in number and contain a high percentage of damaged cells that are not desirable for any study. The isolated heart cells from 2nd and 3rd incubation are washed with Medium 199 containing 10% calf serum by low speed centrifugation (24 G for ½ minutes) before they are used for experimental study. A high yield of isolated heart cells (>80%) with excellent viability (>90% exclude 0.3% trypan blue) and morphology (>80% rod shape cells) retaining normal physiologic and metabolic functions can be achieved with this method.
Note: If active contraction is restarted when enzyme solution is used to perfuse the heart, the calcium free buffer perfusion is not adequate to wash out the calcium from the myocardium. Either the calcium content is too high in the calcium free buffer or longer perfusion using the calcium free buffer should be considered. Slicing or mincing of the myocardium before enzyme incubation is not recommended. This will significantly increase the percent of damaged cells.
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