Cellular cardiomyoplasty: development of a technique to culture human myoblasts for clinical transplantation.

Some recent studies have demonstrated that epicardial injection of autologous myoblasts, obtained from satellite cells of skeletal muscle, in association to coronary artery bypass graft surgery (CABG) in patients with decreased left ventricular function secondary to ischaemic disease could be of some utility to get a better recovery of ventricular function due to the ability of these cells to grow and generate new muscle fibers over the previous fibrotic scar. The aims are the setting up of a process for the collection of the cellular cardiomyoplasty in samples of multiorganic donations and to carry out this technique in the same surgical moment as the revascularisation is performed in two patients. For this purpose we obtained muscle through biopsy of 15 human multiorgan donors and of two patients. Separation of fatty tissue, minced, and further digestion with collagenase type I (1.5 mgr/ml/2 gr by weight) and trypsin 1 x. Filtration of the cellular suspension, centrifugation and sowing of this suspension in culture medium, with 20% of human serum. Culture for three weeks until obtainment of between 200-300 million cells. Inmunohistochemistry and flow cytometry for the identification of the myoblasts was carried out. The results were obtained through flow cytometry, using CD56 as an indicator of the presence of myoblasts, between 70 and 80% of these types of cells were obtained after three weeks of culture. By inmunohistochemistry analyses, different markers were analyzed: desmin and myogenin. The results indicated the presence of a great number of positive cells with these markers, possibly myoblasts. Skeletal myoblast implant was not associated with adverse effects. The culture of autologous myoblasts is a rapid and simple technique where after three weeks of culture a great number of cells for implantation are obtained. In patients with old myocardial infarction, treatment with skeletal myoblast in conjunction with coronary artery bypass is safe and feasible. and it is easy to obtain myoblasts from muscle tissue for transplant into patients.

Viability and histologic structure of porcine valves after cryopreservation.

BACKGROUND: Increased awareness of the limitations of current cardiac valve substitutes has generated a renewed interest in the use of allograft valves. The effects of currently used preservation techniques on the viability of the valve leaflets and the longevity of the implantation however remain controversial. The objective of this study is to analyze the influence of ischemic time, sterilization methods with or without fungicides, and storage procedures on the viability of the valve leaflets and on the histologic structure of the arterial wall, valve leaflet, and myocardium. METHODS: The tissue sources were hearts from 40 pigs with 1 hour of warm ischemic time. The aortic and pulmonary valves were dissected after 2 or 24 hours of cold ischemic time. They were stored in antibiotic solution for 20 hours at 4 degrees C with or without an antifungal agent. The samples were cryopreserved using a programmed temperature decrease method. After 1 week of storage in a liquid nitrogen tank, either in a gas or a liquid phase, the cardiac valves were slowly thawed and examined. RESULTS: Pulmonary valves showed greater viability than aortic valves. Decreased cellular viability was observed independent of cold ischemic time, treatment with amphotericin B, or the storage method used. Treatment with or without amphotericin B had no influence on cellular viability. Conversely it was observed that there was greater cellular viability among those valves stored in a liquid phase. As far as the histologic structure of the valve is concerned we did not observe any influence either in the treatment with amphotericin B or the storage method used although it was observed that reduction of the cold ischemic time minimized histologic injury. CONCLUSIONS: Optimization of preservation methods may decrease the negative effects of cryopreservation on cell viability and histologic structure of the valve.