Identifying appropriate attachment factors for isolated adult rat cardiomyocyte culture and experimentation

Lumkwana, Dumisile (2014-04)

Thesis (MScMedSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Introduction: Primary culture of isolated adult rat cardiomyocytes (ARCMs) is an important model for cardiovascular research, but successful maintenance of these cells in culture for their use in experiments remains challenging (Xu et al, 2009; Louch et al, 2011). Most studies are done on acutely isolated cardiomyocytes immediately after isolation, which is due to low survival of these cells in culture. Obstacles in culture are due to the type of medium and attachment factors (tissue culture adhesives) used to culture and grow these cells. Although we previously identified an optimum medium and adhesive for culture, an adhesive that permits cells to remain attached to the culture surface until after an ischemia/reperfusion insult was elusive. Aims: We therefore aimed to identify the best attachment factor and concentration that will allow adult rat cardiomyocytes to remain attached to the culture surfaces after ischemia/reperfusion experiments. Methods: Cardiomyocytes were isolated from adult Wistar rat hearts and cultured overnight on different concentrations (25 -200 μg/ml) of collagen 1, collagen 4, extracellular matrix (ECM), laminin/entactin (L/E) and laminin. Following overnight cultures, experiments were done in PBS and in PBS versus MMXCB to compare ARCM attachment and viability. Cardiomyocytes cultured on ECM, L/E and L (25−200μg/ml) were subjected to 1 hour of simulated ischemia using MMXCB that contained 3mM SDT and 10mM 2DG, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view of 1.17μm/mm2 were captured using fluorescence microscopy. The cells were analysed according to morphology and fluorescence intensity. Results: Total and rod-shaped ARCMs attachment was improved when MMXCB was used as an experimental buffer instead of PBS. Regardless of the buffer used, morphological viability was poor on substrates of Col 1 and Col 4. In contrast to collagens, ARCMs attached efficiently and morphological viability was high on substrates of ECM, L/E and L in MMXCB, but this was greatly reduced in PBS. Mitochondrial viability was high in MMXCB compared to PBS on Col 1 and Col 4 at 75−175μg/ml and on ECM, L/E and L at all concentrations, except at 50 and 150μg/ml ECM, 175μg/ml L/E and 25μg/ml L. When cardiomyocytes cultured on ECM, L/E and L were subjected to simulated ischemia, total ARCMs, rod-shaped and R/G fluorescence (mitochondrial viability) was reduced at all concentrations compared to the control group. Hypercontracted cells were higher in the ischemic treated cells compared to the controls on ECM at 75−150μg/ml and 200μg/ml, L/E at 50,100μg/ml and 175μg/ml and on L at 125μg/ml. Total numbers of ARCMs attached on ECM, L/E and L in the ischemic group consisted of similar numbers of non-viable hypercontracted and viable rod-shaped cells. Conclusion: Cardiomyocytes should be cultured on ECM or L/E or L at concentrations from 25−200μg/ml in MMXCB. PBS is harmful to cultured ARCMs and should thus not be used as an experimental buffer. Ischemia/reperfusion can be simulated on ARCMs cultured on ECM, L/E or L from 25−200μg/ml, provided that a modified culture buffer is used as experimental buffer.

AFRIKAANSE OPSOMMING: Inleiding: Primêre selkulture van geïsoleerde volwasse rot kardiomiosiete (VRKMe) is ‘n belangrike model vir kardiovaskulêre navorsing, maar om hierdie selle suksesvol in kultuur te onderhou is ‘n groot uitdaging (Xu et al, 2009; Louch et al, 2011). Die meeste navorsingstudies maak gebruik van akuut geïsoleerde kardiomiosiete onmiddelik na isolasie omdat oorlewing van hierdie selle in kultuur baie laag is. Die struikelblokke in kultuur is as gevolg van die tipe medium en weefselkultuurgom wat gebruik word. Ons het voorheen 'n optimale medium en weefselkultuurgom geïdentifiseer vir VRKM kultuur oorlewing, maar die weefselkultuurgom was nie effektief genoeg om die selle aan die kultuuroppervlak te laat bly vaskleef, tot na die einde van 'n isgemie/herperfusie eksperiment nie. Doel: Die doel was dus om die beste weefselkultuurgom en konsentrasie te identifiseer, wat sal toelaat dat VRKMe verbonde bly aan die kultuuroppervlaktes tot na die einde van isgemie/herperfusie eksperimente. Metodes: Kardiomiosiete was geïsoleer vanaf volwasse Wistar rotharte en oornag in kultuur op verskillende konsentrasies (25 -200 μg/ml) van kollageen 1, kollageen 4, ekstrasellulêre matriks (ESM), laminin/entactin (L/E) en laminin onderhou. Die volgende dag was die VRKMe vir eksperimentasie in PBS en in PBS teenoor MMXCB gebruik, om selbehoud en oorlewing te vergelyk. Kardiomiosiete op ESM, L/E en L (25−200μg/ml) was aan 1 uur van gesimuleerde isgemie blootgestel, in MMXCB wat 3mM SDT en 10mM 2DG bevat het, gevolg deur 15 minute herperfusie. Sel oorlewing was bepaal deur selle te kleur met JC-1 en daarna was fluoressensiebeelde van die selle in ‘n veldgebied van 1.17μm/mm2 geneem. Die selle was volgens selmorfologie en fluoressensie intensiteit ontleed. Resultate: Met die gebruik van MMXCB as eksperimentele buffer in plaas van PBS, het die aantal totale en staafvormige VRKMe verbinding verbeter. Morfologiese onderhoud was sleg op kollageen 1 en 4, ongeag van watter buffer gebruik was. In kontras met die kollagene was die VRKM verbinding en morfologiese onderhoud op ESM, L/E en L in MMXCB effektief verbeter, maar in PBS aansienlik verminder. Mitochondriale lewensvatbaarheid in MMXCB teenoor PBS op kollageen 1 en 4 by 75−175μg/ml, sowel as op ECM, L/E en L by alle konsentrasies, was hoog, behalwe by 50 en 150μg/ml ESM, 175μg/ml L/E en 25μg/ml L. Isgemie blootstelling van kardiomiosiete gekultuur op alle konsentrasies van ESM, L/E en L, het ‘n afname in die totale, staafvormige en R/G fluoressensie (mitochondriale lewensvatbaarheid) teweeggebring. Meer hiperkontrakteerde kardiomiosiete was in die isgemie behandelde groepe as in die kontrole groepe teenwoordig, spesifiek op ESM by 75−150μg/ml en 200μg/ml, op L/E by 50,100μg/ml en 175μg/ml asook op L by 125μg/ml. In die isgemie groepe het die totale aantal VRKMe op ESM, L/E en L meestal uit ‘n gelyke hoeveelheid hiperkontrakteerde en staafvormige selle bestaan. Gevolgtrekking: Kardiomiosiete moet op ESM of L/E of L by konsentrasises van 25−200μg/ml in MMXCB gekultuur word. PBS is nadelig vir VRKMe in kultuur en moet dus nie gebruik word as eksperimentele buffer nie. Isgemie/herperfusie eksperimente kan gesimuleer word op VRKMe wat op 25−200μg/ml ESM, L/E of L gekultuur is, mits ‘n gemodifiseerde kultuur buffer gebruik word as eksperimentele buffer.

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