The release of single cells from embryoid bodies in a capillary flow device

The recovery of intact cells from embryoid bodies (EBs) is an essential stage in the development of many cell-based therapies. The use of acapillary device is explored where by a suspension of EBs is exposed to controlled flow for the purpose of their break up and the release of single cells. The device was designed to avoid exposure of the released cells to shear stresses greater than 50 N m(-2) at the wall or entrance; this shear stress level is a reported value above which loss of cell integrity may occur. The disaggregation of the EBs leads to amixed population of intermediate bodies and single cells. The break-up process was described using a first-order relationship with both the rate and final extent of loss of EBs being a function of the flow rate and the total time of exposure to shear stress as determined by capillary length and number of passes. The release of single cells is shown to be related to the loss of EBs and hence again a first-order relationship may be used. The overall relationship developed is used to predict successfully the release of single cells in a capillary device configuration suited to cell preparation for therapy, i.e. for defined sterile operation with the use of long capillaries with a low number of passes. The break up of the EBs was achieved without the use of reagents such as trypsin to degrade the extracellular matrix that links the cells together and where there may be concern of the loss of key components at the cell surface. 60% of the cells released by capillary shear retained their cell wall integrity. Analysis of the phases of break up identified the release of cells from the EBs as being the point of loss of integrity. Exposure of already released cells to shear stress within the capillary led to no further loss of integrity. The potential of this method for releasing cells for therapy is discussed. (c) 2010 Elsevier Ltd. All rights reserved.