Heart Chamber |
Myocyte Harvesting Diagram |
Myocyte Harvesting System |
Isolation of viable myocytes from perfused hearts is the initial and most critical step in many optical and/or electrophysiological experiments or for long term culturing.
Also can be used for Constant pressure Langendorff, Constant Flow Langendorff, both recirculating and non recirculating models.
The Radnoti isolated myocyte system is complete including stand assembly, water jacketed heart chamber with a collecting trap, perfusate manifolds and valves, 3 water jacketed reservoirs with aerators, 3 water jacketed bubble traps, water jacketed perfusate lines, peristaltic pump, isolated cell bath with shaker (optional) and thermal circulator pump.
The system can be used for either constant pressure or constant flow Langendorff preparations. If the experimenter decides to use the more common constant flow isolation , a manifold is used to select the appropriate solution that is then pumped through a bubble trap and into the heart. First, crystalloid solutions (Kreb’s or Tyrode’s) are used to remove blood cells and stabilize the preparation, then a solution containing EGTA is used to remove residual calcium. A solution containing collagenase and sometimes other digestive enzymes is then recirculated through the preparation until the heart becomes flaccid, indicating that the intercellular matrix holding the cells together is dissolving.
The partially digested tissue is then cut into small chunks and placed in a shaking bath and subjected to continued dissociation treatment until individual myocytes are released. The protocol may require several changes of solution to select the myocyte fraction that has the best yield and viability, the latter determined through the use of microscopic examination in the presence of trypan blue or other vital dye staining. To increase viability, step changes in the calcium concentration of the incubation media are used to return the myocyte to a normal ionic environment prior to their use.
If the experimenter decides to use a constant pressure isolation, the reservoir(s) are elevated to the appropriate hydrostatic head and a manifold directs the selected solution to the appropriate bubble trap and then to the heart. A valve at the bottom of the heart chamber diverts the perfusate either to waste or to the pump for recirculation back to the source of the solution. |
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