Benchtop centrifugation is an open and manual process that is challenging to scale. Our protocol shows how to close and automate the dissociation, washing, and harvesting of mesenchymal stem cells, or MSCs, in a single run. The setup is simple.
The entire process can be performed outside the biosafety cabinet in a class cGMP facility. Closed automated cell processing reduces manual handling steps that could be prone to risk and enable cGMP compliance. This method can be applied to other cell types expanded in multi-layered culture systems.
The Counterflow Centrifugation System allows modification of any protocol steps for different applications. Demonstrating this procedure will be Dr.Alan Lam, staff scientist from the stem cell bioprocessing group at Bioprocessing Technology Institute, A star in Singapore. To begin the single-use counterflow centrifugation kit assembly, connect the single-use kit with the single-use PVC transfer bags via tube welding, as outlined in the image.
Then, inside a biosafety cabinet, attach a 10-layer multi-layered flask containing confluent, Wharton's Jelly-derived human mesenchymal stem cells or Wharton's Jelly HMSCs to the custom tubing assembly. Transfer the attached 10-layer culture vessel with the custom tubing assembly to a bench and weld it to line E of the single-use kit.Also. weld a sterile sample port to line G of the high-flow single-use kit.
Next, connect the harvest line H to a sterile lure, fitted with a 50 milliliter syringe. To set up the instrument run, switch the instrument on using the toggle switch at the rear of the instrument and connect the laptop to the USBC port on the instrument using the provided USBC cable. Run the Counterflow Centrifuge Graphical User Interface, or GUI software from the desktop or start menu and sign in.
Then, load the protocol by clicking the Select a protocol button on the main welcome page. Next, press the blue unlock button on the instrument and open the glass door to load the assembled single-use kit onto the Counterflow Centrifugation System. Start by hanging the bags on the hanger hooks in an order that lines them up best with the tube ports on the bubble sensor strips with the 10-layered multi-layered flask placed at an angle.
Line up the kit with the two kit location buttons. Stretch the pump tubing around the peristaltic pump and press the white bulb-shaped connector into place. Attach the centrifuge chamber by lifting the silver lever of the counterflow centrifuge chamber carrier and securing it by returning the lever to its upright position.
Press the tubing from each port on the kit into the tracks along the bubble sensor strips. Close the door by pressing down on the door latch. Hit the Initiate button on the GUI.
A checklist will appear. The first four items are instrument checks, while the last two are user checks. At this point, ensure the bags and connections match the kit image and the manual clamps are open.
Press Confirm to show the Protocol inputs screen. Set the data entry or harvest volume dialogue box value as 45 milliliters and press Confirm. Press the green start button on the instrument to start the protocol run.
Once the initial priming is completed, ensure that the spent medium is completely pumped out into the waste bag. In pause steps as marked in the workflow table, lift the multi-layered flask up and shake it to distribute the buffer equally to all the trays. Once completed, place the 10-layer multi-layered flask back in its original draw position for the next steps.
In steps 20 and 23, ensure that the trypsinized cells are transferred completely into the intermediate bag. Mix the intermediate bag well, manually, in step 25. In step 26, use a two milliliter lure syringe to sample through the sampling port.
In steps 29 and 30, check the stable formation of the fluidized cell bed. It should be like the fluidized cell bed shown here. The run is complete at the ramp to stop step, and all the pinch values on the Counterflow Centrifugation System will close automatically.
After the protocol run is complete, press the blue unlock button on the instrument and open the glass door. Aseptically seal the harvest line using a hand-carry tube sealer and carefully transfer the sealed syringe filled with the concentrated cell harvest into the biological safety cabinet for cell counting and cryopreservation. Detach the chamber again using the lever.
Pull the bulb connector out of its fitting and carefully lift the kit away and dispose of it into a biohazard bag. Clean the instrument using ethanol wipes and close the door. Finally, close the GUI application first before turning off the switch at the rear of the instrument.
The Wharton's Jelly MSCs proliferated well when expanded in both serum-containing and serum-free, xeno-free media. However, those in serum-free, xeno-free medium exhibited slightly longer fibroblast-like spindle-shaped morphology, with a larger average cell size of 17 microns, as compared to 15 microns in serum medium. In both media across the three passages, the Wharton's Jelly HMSCs consistently reached a maximum cell density of about 23, 000 cells per square centimeter and a population-doubling time of 34 hours.
When the Wharton's Jelly HMSCs were expanded in 10-layer culture vessels using the demonstrated counterflow centrifugation, a tenfold volume reduction was achieved, generating cell concentrations as high as 5.3 million cells per milliliter. The protocol consistently achieved a high cell recovery of about 98%and a high cell viability of about 99%for three independent runs. The Wharton's Jelly HMSCs harvested using both methods displayed characteristic surface marker profiles that are CD73, CD90, and CD105 positive, as well as CD34 and CD45 negative.
The cells harvested from counterflow centrifugation displayed a similar colony-forming unit fibroblast assay or CFU potential, compared with those harvested by manual centrifugation. In both methods, the cells retained the ability to differentiate into adipocytes, osteoblasts, and chondrocytes. The cytokine secretion profiles of cells post-counterflow centrifugation was comparable to pre-counterflow centrifugation.
Make sure to assemble the single use kit with reference to the bed setup, displace it in this Counterflow Centrifusion System Protocol Builder. The protocol for washing residual impurities from the final product may be used in clinical applications to support the users in meeting their lot release criteria. To quicken the process of fluid transfer for large-scale processing, external pumps can be used to transfer the trypsinized contents into a transfer bag first, instead of directly harvesting from the multi-layered flask.
