Hi, I'm Dr.Jihad, the Center P Lab Director. The goal today of this video is to show an adapted cooled cell processor used in the eyelet isolation facility with no impact in the environment as well in the recovery of the cells. Begin by pre-cooling the light and heavy density gradients and the cell processor kit at four degrees Celsius.
Next, prepare the cell processor by pre-cooling for 45 minutes. Insert the pre-cool cell processor kit into the cell processor for the standard ethanol cooled double jacketed gradient maker. Connect the two glass chambers and place them on a magnetic stir Clamp the tubing between the two chambers.Cool.
The gradient maker with an ethanol circulating chiller at zero degrees celsius after cooling. Set the pump speed to 150 milliliters per minute to fill the chamber with 130 milliliters of heavy density gradient at the bottom of the bag. Once the heavy gradient is in the bag, close the hemostat between two chambers.
Then add 130 milliliters of high density to the front beaker and 140 milliliters of low density gradients to the back beaker.Dec. Clamp the hemostat between the two chambers, then set a pump speed of 50 milliliters per minute to create a continuous gradient between two chambers. Next, prepare human mononuclear buffy coat cells for purification from multiple pooled anonymous Buffy coats collected from whole blood waste products provided by the blood bank.
After separation, add five milliliters of specific gravity resembling low density mononuclear cell suspension into 95 milliliters of high density gradient medium in a transfer bag using a peristaltic pump set at 25 milliliters per minute. Top load the preformed gradient with 30 milliliters of mononuclear cell suspension using a pre calibrated thermo probe.Continuously. Measure the temperature inside the cell processor using a digital thermocouple connected to a sterile thermo probe.
Monitor the temperature of gradients and cells during the process. At the end of gradient loading, set the cell processor at 537 G to minimize the heat generated by the rotor. Turn off the cell processor to allow the hydraulic system to return to pre-run levels and release air.
Then restart the cell processor at 537 G and add 130 milliliters of heavy and 140 milliliters of low density gradients to create a continuous density gradient at a flow rate of 50 milliliters per minute and 4.3 degrees celsius. Next, load 30 milliliters of Buffy coat cell suspension on top of a load density gradient at a flow rate of 25 milliliters per minute and six degrees Celsius. After cell loading, add 50 milliliters of washing solution into the cell processor after five minutes.
Collect the spun gradient in 12 bottles at a flow rate of 100 milliliters per minute and five degrees Celsius fill tube, one with 100 milliliters of wash media and 150 milliliters of cells. Then fill tubes two to 12 with 225 milliliters of wash media and 25 milliliters of cells. The airborne particle assessment in clean room environment, grade C and grade B showed that there was no excess of 0.5 and five microns airborne particulate as per clean room, GMP grade C, grade B and BSL three standards.
No significant difference in gradient temperatures was observed during the first four steps with both processors maintaining approximately 4.5 degrees Celsius. However, after centrifugation and during collection steps, the temperature rose at the beginning and the end of the collection steps for both processors. Further, the cooled cell processors impact on human cell purification.
Efficiency and viability was determined after centrifugation. The temperature of the collected cells rose slightly to 8.5 degrees Celsius. Active air sampling and sterility screening showed no growth.
