Browsing by Author "Hanser, Sidney"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Establishment and validation of a terminally differentiated adult rat ventricular cardiomyocyte model
    (Stellenbosch : Stellenbosch University, 2014-04) Hanser, Sidney; Lopes, John; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Biomedical Sciences: Medical Physiology.
    ENGLISH ABSTRACT: Various experimental models are used in cardiovascular research which includes the whole animal model, whole heart model and heart cell model, with each one having its advantages and disadvantages. Although primary adult rat cardiomyocytes (ARCMs) have been in used for many years, it is not commonly practiced because of the difficulties involved in setting up this model. Aims: This study aimed to develop optimal conditions for the isolation of high yield viable ARCMs, and to optimize culture conditions to improve the % viability of isolated ARCMs during overnight culture. Method: ARCMs were isolated from the hearts of male wistar rats by enzymatic digestion at low Ca2+ concentrations, which were later raised to physiological levels. Cell counts were collected to determine the total number and the % viability of ARCMs. The main conditions tested during isolation included; (1) the effect of calcium raising to a final concentration of 1.2mM versus 1.8mM, (2) the presence of insulin during isolation and calcium raising, and (3) the effect of fast compared to slow calcium raising. ARCMs were cultured overnight in 96 well plates and the % viability was tested with the JC-1 or TMRM. Laminin was assessed as culture adhesive, and M199 containing Hank’s salts (M199 (H)) was compared with M199 containing Earle’s salts (M199 (E)) as culture buffer. Three groups of supplementations were made with each M199 and compared, including (1) M199 with energy substrates, (2) M199 with energy substrates and blebbistatin, and (3) M199 with energy substrates, blebbistatin and a final modification (patent pending). Results: The reduction of the final Ca2+ concentration from 1.8mM to 1.2mM showed improvement in cell survival. Insulin did not improve the % viability and the total number of ARCMs during digestion phase, slow or fast Ca2+ methods. High laminin concentrations (100μg/ml) were needed to retain high cell numbers to the culture surface during experimental washes. The energy substrate supplemented M199 (E) and M199 (H) destroyed the cell viability of the ARCMs. An additional blebbistatin supplementation dramatically improved ARCMs survival after overnight culture and cell staining. M199 (H) with the final modification (patent pending) provided even higher ARCMs survival compared to M199 (E), but this was only evident with the JC-1 stain and not TMRM stain. We consider JC-1 to be a more accurate measure of mitochondrial function than TMRM, given that JC-1 is a ratiometric dye, while TMRM is a single colour reporter. Conclusion: The final Ca2+ concentration of 1.2mM seemed to be more beneficial. Insulin administration is not necessary for the isolation procedure. Neither slow nor fast Ca2+ re-administration is more efficient. The basic energy supplements that are commonly used in the literature are not sufficient in either M199 (E) or M199 (H) medium for survival of ARCMs in culture. Instead, blebbistatin must be present with the basic supplements to improve viability in culture. A new formulated culture media with M199 (H) showed the highest survival after overnight culture. The isolation and culture model of viable ARCMs was therefore successfully established.