RESUMEN
Cell culture of cardiac tissue analog is becoming increasingly interesting for regenerative medicine (cell therapy and tissue engineering) and is widely used for high throughput cardiotoxicity. As a cost-effective approach to rapidly discard new compounds with high toxicity risks, cardiotoxicity evaluation is firstly done in vitro requiring cells/tissue with physiological/pathological characteristics (close to in vivo properties). Studying multicellular electrophysiological and contractile properties is needed to assess drug effects. Techniques favoring process automation which could help in simplifying screening drug candidates are thus of central importance. A lot of effort has been made to ameliorate in vitro models including several in vitro platforms for engineering neonatal rat cardiac tissues. However, most of the initial evaluation is done by studying the rate of activity. In this study, we present new approaches that use the videomicroscopy video of monolayer activity to study contractile properties of beating cells in culture. Two new variables are proposed which are linked to the contraction dynamics and are dependent on the rhythm of activity. Methods for evaluation of regional synchronicity within the image field of view are also presented that can rapidly determine regions with abnormal activity or heterogeneity in contraction dynamics.
RESUMEN
In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS) is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Dimetilpolisiloxanos/administración & dosificación , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Actinas/genética , Actinas/metabolismo , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Miocitos Cardíacos/efectos de los fármacos , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Acute or sustained stretch of cardiac tissue is known to play a key role in arrhythmogenesis. Using a fluorescence approach, we designed a system measuring calcium transients and transmembrane potential changes in monolayers of cultured cardiomyocytes under uniaxial elongation and electrical stimulation. Cardiac myocytes are seeded on a rectangular PDMS template held and stretched by a motorized linear guide system. Electrical stimulation is performed with two parallel carbon electrodes supplied by amplified pulses from a digital-to-analog converter. The cells are stained with either voltage- or calcium-sensitive dye (di-4-ANEPPS and Fluo-4 AM respectively). The two available excitation light sources are both current-controlled LED arrays (λ = 523 ± 45 nm for di-4-ANEPPS and λ = 505 ± 15 nm for Fluo-4 AM). The filtered emitted fluorescence (λ > 610 nm for di-4-ANEPPS and λ = 535 ± 25 nm for Fluo-4 AM) is transduced to current with a photodiode, converted to amplified voltage signals and digitized. The design and preliminary validation results are presented.