Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

The cell and gene therapy industry has witnessed an exponential expansion over the past two decades. However, a large technological gap exists between lab research and industrial skill manufacturing. Compliant with regulatory policies, and to meet the green demands of the CGT industry, automated closed systems have been adopted to produce cell therapeutics.

For adherent cells, cultivation using microcarriers in scalable stirred-tank bioreactors is a promising method. Conventionally, commercialized microcarriers are non-desirable. Cell harvesting using trypsinization and the sieving out microcarriers by size is not only inefficient and challenging, but also impacts cell quality and causes safety concerns.

In this study, we have successfully achieved large-scale cell production based on GMP-grade 3D TableTrix dissolvable microcarriers in an automated and closed system. Over 11 billion human mesenchymal stem cells were yield in a two-stage expansion within nine days. We also demonstrated high density culture of HEK293T cells and Vero cells, in which both densities reached above 10 million cells per milliliter.

This advanced biomanufacturing platform allows adherence cells to be effectively expanded and completely harvested in an automated and closed manner. Our protocol offers an opportunity to enhance production efficiency and refine quality control at industrial-scale manufacturing of CGT products. To begin, perform two point calibration on the pH probe with a bioreactor controller.

Then to assemble a bioreactor vessel, install parts such as stainless tubes and impellers onto the head plate according to the manufacturer's instructions. Attach a ready-to-use culture disposable consumable kit to the appropriate stainless steel parts, tubes, and ports on the head plate, following the manufacturer's guidance. Upon assembling the vessel, perform an air tightness check of the fully assembled vessel as specified by the manufacturer's instructions.

Autoclave the vessel with the consumable kit at 15 PSI and 121 degrees Celsius for 60 minutes. After cooling the vessel to room temperature, insert the temperature probe into the thermo well tube and connect the cables for the monitor, pH probe, and DO probe from the controller to the respective parts on the vessel. Wrap the heat mat around the vessel tightly.

Log into the system of the bioreactor controller and enter the parameters into the system according to the manufacturer's instructions. Weld the outlet tube on the medium bottle to the inlet C-flex tube on the bottle containing 10 grams of pre-sterilized W01 micro carriers for MSC. Pump 500 milliliters of culture medium from the storage bag into the bottle of micro carriers W01.

From the stirred tank bioreactor vessel, stop the temperature, pH, and DO control, and the bioreactor controller temporarily and fully dispense all the PBS from the vessel into the waste bottle. Weld the 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 th PUMP1 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 DO again as per the manufacturer's instructions. Seal and disconnect the tube connecting the feed bag 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 resuspend 2.5 times 10 to the 8 hMSCs 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 a 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 resuspension 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 2 times 10 to the 6 cells per milliliter using the complete serum-free medium. For the cell formulation process in a 15 liter bioreactor, use formulation buffer to resuspend the harvested cells.

Weld 250 milliliters of resuspended 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 resuspended. 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, cumulative 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 cryopreserved cells showed good adhesion, normal expansion behavior, and a typical spindle shape with spiral growth after thawing and replating to 2D flasks. Furthermore, the cells also maintain 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 bead to bead transfer process in the 15 liter bioreactor, the HEK293T cells reached a peak cell concentration of 1.68 times 10 to the 7 cells per milliliter. While the Vero cells reached a concentration of 1.08 times 10 to the 7 cells. Both cell types maintained high viability.