Isolation And Effect Estimation Of Bovine Adrenal Chromaffin Cells

The remarkable analgesic effects of adrenal medullary chromaffin cell(AMCC) transplantation for pain have been shown in many studies, but it has notreached the status of a standard clinical treatment. Limits of available donorshighly restrict the widespread clinical use of this approach. The bovine adrenalmedulla most often serves as a source of these cells because of the large amountand relative availability of this tissue. However, chromaffin cells are obtained bycurrent methods with low cell yield, purity and high technical cost, which make itdifficult to meet a great deal of AMCC transplantation on clinic.As a result, current isolation methods of AMCC, research foundations andexperimental conditions of our lab were considered to build an isolation methodfor Bovine Adrenal Chromaffin Cells (BCC). And the biological characteristics ofBCC in vitro and analgesic effects in vivo were studied to estimate their functionalactivity.Collagenase perfusion digestion, mechanical dissociation and discontinuousdensity gradient centrifugation on Percoll have been used to obtain purified BCC.Under the optimized conditions, 0.7~3.1×108 cells could be isolated per adrenalgland with 99% viabilityand more than 95% purity.The cytoplasm blackened, cell membrane crimpled, cell viability andcatecholamine level dropped with time in culture, but the concentration of glucoseand lactic acid had no remarkable change. These results showed that BCC had thefunctional activity of catecholamine-secretion in culture, but with low metabolismrate.The analgesic effects of BCC transplantation into the spinal arachnoid spacewere studied, and the results proved that microencapsulated BCC producedremarkable analgesic effects to SD rat models for both acute and chronic pain, butBCC transplanted did not alleviate the pain because of immunological rejection. The results of cell morphology, cell viability, cell damage and functionalactivity of BCC before and after cryopreservation were compared, and resultsshowed that cryopreservation was an effective method for BCC activitymaintenance, and microcapsules could hugely reduce the cell damage caused bycryopreservation. In conclusion, collagenase perfusion digestion, mechanical dissociation andused Percoll discontinuous density gradient centrifugation were used to isolateand purify BCC in this thesis, and results showed that it could provide enoughhealthy cell sources with high cell yield, cell purity and cell viability towidespread application of BCC transplantation on clinic.