This protocol provides a solution for closed automated buffer exchange and concentration for small-scale cell manufacturing. Importantly, the buffer exchange is able to be performed with a high recovery and a delivery of a very small volume of concentrated product. New users may struggle to follow all the automation steps.
Practicing programming these steps and performing test runs with medium are good ways to become familiar with the device. Before beginning the buffer exchange, in a class two laminar flow head, use a syringe and needle assembly to remove 50 milliliters of saline solution from a 500 milliliter saline bag. Replace this volume with 50 milliliters of 20%human serum albumin, and harvest the cells of interest from their culture vessels.
After counting, use the syringe to load the cells into a transfer bag, and use the Luer lock to connect the transfer bag to a single-use processing kit. Then connect the previously prepared bag of wash buffer to the single-use processing kit. To set up an automated buffer exchange, open the graphic user interface of the device and click the start button.
Click new to create a new protocol, and click control to select the valves to be opened and closed, the centrifugal and pump speeds, the pump direction, and the action triggers. When all of the parameters have been set, click save. Hang the connected appropriate bags on the device, and place the single-use processing kit on the machine.
Then click the connect button and download the saved program. When the play button lights up, the protocol is ready to be started. To perform the automated buffer exchange, open the manual clamps for the transfer bag tubing, and click start to initiate the buffer exchange program.
At the end of automation step six, the process will pause due to triggering of the bubble sensor by the air in the now empty bag. If the bag is indeed empty, press next to move the process to the next step. When the automated process is finished, close all the tubing clamps and open the door of the device.
Then remove the single-use kit from the device and disconnect the syringe to allow the cells to be collected for subsequent analysis. Typically, the fluidized cell bed will appear as represented in this image with the cells accumulating in the middle and toward the front of the cone, a small space at the tip of the chamber in which the cells do not accumulate, and a visible opening within the cell loading inlet. The fluidized cell bed may be compressed when introducing a new buffer that is at a different viscosity or density.
A high flow rate may be applied to select for live cells as dead cells are smaller and lighter and can be forced out of the chamber by increasing the flow rate. The automated process of buffer exchange processing time is shorter than that of the manual buffer exchange process. In this analysis, the recovery rates between the manual and the automated processes were similar for both types of cells tested, and the cell viability was not affected by either process.
The recovered cells demonstrated similar proliferation rates, abilities to produce cytokines, and ideo-activities, regardless of the type of processing. It's important to remember that this protocol serves only as a guideline, and therefore users need to program the flow rate and centrifugation speed according to their cell type and buffer. Buffer exchange is an intermediate step in cell manufacturing that may follow cell culture expansion or cryopreservation and formulation.
The goal is to develop ways to enclose the entire process. Counterflow centrifugation is a very versatile platform. Future studies will explore applications in cell selection, such as dead cell removal and mononucleus cell isolation from a chorisis product.
