Establishment and validation of a terminally differentiated adult rat ventricular cardiomyocyte model

Hanser, Sidney (2014-04)

Thesis (MScMedSc)--Stellenbosch University, 2014.

Thesis

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.

AFRIKAANSE OPSOMMING: Verskeie eksperimentele modelle word gebruik in kardiovaskulêre navorsing wat insluit die heel dier model, heel hart model en die hartsel model waar elkeen voor-en-nadele het. Alhoewel die primêre volwasse rot kardiomiosiet (VRKe) model vir talle jare al gebruik was, word dit nie algemeen gebruik nie as gevolg van die probleme wat betrokke is by die opstel van hierdie model. Doelwitte: Hierdie studie het gepoog om optimale toestande vir die isolasie van hoë opbrengs lewensvatbare VRKe te ontwikkel, en kweek toestande te optimaliseer om die % lewensvatbaarheid van geïsoleerde VRKe te verbeter tydens oornag kultuur. Metode: VRKe was geïsoleer uit harte van manlike wistar rotte deur ensiematiese vertering by lae Ca2+ konsentrasies, wat later verhoog is tot fisiologiese Ca2+ vlakke. Sel tellings was ingesamel om die totale getal en die % lewensvatbaarheid van VRKe te bepaal. Die hoof kondisies wat getoets was gedurende isolasie sluit in: (1) die effek van die verhoging van Ca2+ konsentrasie aan die einde, by 1.2mM teenoor 1.8mM, (2) die teenwoordigheid van insulien gedurende isolasie en die verhoging van Ca2+ en (3) die effek van vinnige in vergelyking met stadig kalsium verhoging. VRKe was oornag gekweek in 96-put plate en die % lewensvatbaarheid was getoets met die JC-1 of TMRM. Laminin was geondersoek as kultuurgom, en M199 wat Hank se soute (M199 (H)) bevat was in vergelyking met M199 wat Earle se soute (M199 (E)) bevat as ‘n kweekmedium. Drie aanvullings groepe was gemaak met elke M199 en vergelyk, insluitend (1) M199 met energie substrate, (2) M199 met energie substrate en blebbistatin, en (3) M199 met energie substrate, blebbistatin en ‘n finale modifikasie (patent hangende). Resultate: Die vermindering van die finale Ca2+ konsentrasie vanaf 1.8mM tot 1.2mM het verbetering in sel oorlewing getoon. Insulien het nie die % lewensvatbaarheid en die totale aantal VRKe verbeter tydens die vertering fase, stadige of vinnige Ca2+ verhogings metodes. Hoë laminin konsentrasies (100μg/ml) is nodig om 'n hoë sel getal te behou op die kultuur oppervlak gedurende eksperimentele wasse. Die energie substraat aangevulde M199 (E) en M199 (H) het die sel lewensvatbaarheid van die VRKe vernietig . ‘n Bykomende blebbistatin aanvulling het die oorlewing van VRKe na oornag kultuur en sel vlekke verbeter. M199 (H) met die finale modifikasie (patent hangende) het nog hoër oorlewing getoon in vergelyking met M199 (E), maar dit was net duidelik met die JC-1 en nie TMRM vlekke nie. Ons is van mening dat JC-1 'n meer akkurate meting van mitokondriale funksie gee as TMRM. Dis omdat JC-1 'n rasiometriese kleurstof is, terwyl TMRM ‘n enkele kleur vertoon. Gevolgtrekking: Die finale Ca2+ konsentrasie van 1.2mM is meer voordelig as 1.8mM. Insulien toediening is nie nodig vir die isolasie proses nie. Nie vinnig of stadige Ca2+ verhoging was meer doeltreffend nie. Die basiese energie aanvullings wat algemeen gebruik word in die literatuur is nie voldoende vir M199 (E) of M199 (H) medium vir die oorlewing van VRKe in kultuur nie. In plaas daarvan, moet blebbistatin teenwoordig wees met die basiese aanvullings om die lewensvatbaarheid te verbeter tydens kultuur. ‘n Nuut geformuleerde kultuur medium met M199 (H) het die hoogste oorlewing na oornag kultuur getoon. Die isolasie en kultuur van lewensvatbare VRKe model is dus suksesvol gestig.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/86762
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