From the stirred tank bioreactor vessel, stop the temperature pH and DO control on the bioreactor controller temporarily and fully dispense all the PBS from the vessel into the waste bottle. Weld to single use filtration module to a port on the 10 liter single use storage bag. Filter and transfer the complete serum free MSC medium into the storage bag.
Set the pump one speed to 300 RPM. And dispense one liter of medium from the feed bag into the vessel. Stop the pump.
And start the temperature pH and dissolved to DO again, as per the manufacturer's instructions. Seal and disconnect the tube connecting the feedback to the feed tube. Weld the tube from the bottle of dispersed micro carriers to the feed tube.
And pump all the contents from the bottle into the vessel. Next, re-suspend 2.5 times 10 to the eight HMCs in a serum free medium and transfer them to the empty bottle previously containing the micro carrier suspension. Add serum free medium to 500 milliliters in total inside the bottle and mix the cell suspension.
Pump all the contents from the bottle into the vessel. Seal and disconnect the tube connecting the bottle to the feed tube and then weld back the feed bag. Adjust agitation settings on the controller to initiate cell inoculation to the micro carriers.
And set up an automated medium supplement and exchange regime. Weld the disposable sampling bags to the sampling tube. And collect samples at the desired time points.
At the time of cell harvest on the controller, set the agitation speed to zero for the micro carriers to settle. And dispense supernatant into the feed regime, leaving approximately one liter of culture suspension in the vessel. Weld the bag containing 50 milliliters of freshly prepared digest solution to 500 milliliters of HBSS for dilution.
And pump all solution into the vessel, via the feed tube. Set the agitation speed between 40 to 45 RPM to dissolve the micro carriers. After the micro carriers have dissolved within 40 to 60 minutes, weld the disposable three liter storage bag to the harvest tube and completely pump out the cell suspension.
Turn on the cell processing system during cell harvest. And install a disposable cells processing kit on the instrument, according to the manufacturer's instructions. Weld the bags of cell suspension, wash buffer and culture medium to the kit.
Enter the operational parameters and start the automated wash and re-suspension process. Collect the sample of cells from the centrifuge chamber as indicated by the cell processing systems for cell density calculation. After counting, readjust the cell density to two times 10 to the six cells per milliliter, using the complete serum free medium.
For the cell formulation process in a 15 liter bioreactor, use formulation buffer to re-suspend the harvested cells. Weld 250 milliliters of re-suspended cells to the cell filling. Assemble the disposable fill and finish consumable kit onto the cell filling system.
And turn on the pre-cooling system, following the manufacturer's instructions before the cells have been re-suspended. Follow the instructions on the system and set it to fill 30 milliliters per bag with a total of 20 bags per set. Weld the new set of 20 cryo-preservation bags to the disposable fill and finish consumable kit and repeat the fill and finish until all cells are aliquoted.
For hMSCs, accumulative 128 fold expansion was achieved with cell viability above 90%Glucose intake exhibited a negative correlation with cell expansion, showing metabolism associated with healthy cell growth. Freshly harvested MSCs retain classical phenotypic markers with more than 95%expression for positive markers and less than 2%for negative markers. The cryo-preserved cells showed good adhesion, normal expansion behavior and typical spindle shape with spiral growth after thawing and re-plating to 2D flasks.
Furthermore, the cells also maintained their capability to differentiate into osteogenic, adipogenic and chondrogenic lineages. This platform can be adopted to cultivate other anchorage-dependent cells as well, such as HEK293T and Vero cells. After the B2B transfer process in the 15 liter bioreactor, the HEK293T cells reached a peak cell concentration of 1.68 times 10 of the seven cells per milliliter.
While the Vero cells reached a concentration of 1.08 times 10 to the seven cells. Both cell types maintained high viability.
