Separating and purifying method for ox adrenal medella chromaffin cell

Abstract

The ox adrenal medella chromaffin cell separating and purifying method includes perfusing collagenase solution backwards to adrenal vein; fully hydrolyzing adrenal medella cell and collagen between cortical cell and medella cell under certain temperature; mechanically shearing and gradiently sieving stripped medella to obtain suspension with discrete adrenal chromaffin cell; and purifying the cell by means of the difference in cell density in the suspension. In the Percoll non-continuous gradient method to purifying ox adrenal medella chromaffin cell, every adrenal can be separated to obtain (0.7-3.1)E8 chromaffin cells of purity over 95 % and with excellent shape and activity over 99 % averagely.

Description

technical field [0001] The invention relates to a method for separating and purifying bovine adrenal medulla chromaffin cells. Background technique [0002] Adrenal medulla chromaffin cells are neuronal cells that synthesize, store and secrete catecholamines. When implanted in the central nervous system, it can remain active for a long time and secrete catecholamines on demand. Although a large number of in vivo tests have also shown its significant effect in alleviating pain and treating Parkinson's disease, the technology is still in the treatment stage of individual clinical cases. There are still some problems in its wide application in clinical practice, such as limited cell sources, adrenal chromaffin cells cannot divide and proliferate in vitro, and there is no cell line with good functional expression, and the cells can only be obtained by separating the adrenal medulla; The separation and purification process of chromaffin cells reported in the past is complicated...

AI Technical Summary

Problems solved by technology

[0004] ①Using collagenase digestion as the main cell separation method, the large-scale use of high-concentration collagenase and long digestion time not only lead to a decrease in cell yield, but also greatly increase the process cost;

[0005] ②Because of the heterogeneity of chromaffin cells and the distribution of cell density within a certain range, the continuous density gradient centrifugation method to purify chromaffin cells will not only cause loss when the cells are collected, but also the cell level is not clear enough after centrifugation;

[0006] ③ Continuous density gradient centrifugation requires tens of thousands of centrifugal forces to form a continuous gradient zone of the separation medium. Only laboratories equipped with ultracentrifuges can use this method, and the amount of centrifugal medium is relatively large;

[0007] ④ The differential adhesion method used in the past has a long duration of purification. Although chromaffin cells with high purity can be obtained, it needs to be at the cost of cell number loss, and the cell yield is extremely low.

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