Influence of cyclosporine A on contractile function, calcium handling, and energetics in isolated human and rabbit myocardium.

OBJECTIVE: The immunosuppressive drug Cyclosporine A (CsA) is a key substance in pharmacological therapy following solid organ transplantation and has been suggested to prevent cardiac hypertrophy. We investigated the direct effects of CsA on myocardial function, because these are largely unknown. METHODS: In multicellular cardiac muscle preparations from end-stage failing and non-failing human hearts as well as from non-failing rabbit hearts we investigated the effects of CsA on contractile performance, sarcoplasmic reticulum (SR) Ca2+-load, cytosolic calcium transients, calcium sensitivity of the myofilaments, and myocardial oxygen consumption. RESULTS: In failing human muscle preparations there was a concentration dependent decrease in contractile force; the maximal effect amounted to 55.6+/-6.4% of control while EC50 was reached at 1.0+/-0.3 nM (n=6). These concentrations are at and even below the therapeutic plasma levels. CsA decreased the aequorin light signal in human failing trabeculae to 71.5+/-5.9% (n=5), indicating decreased calcium transients. Estimation of the SR calcium load via measurement of rapid cooling contractures revealed a decrease to 84.4+/-6.5% in failing human preparations (n=6). Measurements of both decreased SR calcium load and force development in presence of CsA were also observed in four non-failing human muscle preparations. In rabbit muscle preparations (n=8), developed force decreased to 50.2+/-7.7% (n=8, EC50: 1.9+/-0.4 nM) and rapid cooling contractures to 74.0+/-7.4% of control at 100 nmol/l CsA. No direct effects were observed on myofilament calcium sensitivity nor on maximal force development of permeabilized preparations from the rabbit (n=7). Oxygen consumption measurements showed that CsA decreased the economy of contraction to 76.4+/-7.9% in rabbit preparations (n=8). CONCLUSIONS: CsA causes a direct cardio-depressive effect at clinically relevant concentrations, most likely due to altered handling of Ca2+ by the SR.

Agitation, aeration and perfusion modules for cell culture bioreactors.

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode. Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated. Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 microns) as well as anchorage dependent cells grown on microcarriers (pore size 75 microns) over six weeks to 3 months. Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.