preparation of the stirred-tank bioreactor for large-scale production of human mesenchymal stem cells

Closed System Production of Adherent Cells Based on Dissolvable Microcarriers

The CGT industry has witnessed an exponential expansion over the past two decades. The evolution of next-generation medicines is anticipated to treat and cure numerous refractory diseases1. Since the first Food and Drug Administration (FDA) approval of a CGT product, Kymriah, in 2017, CGT-related research and development in the world has continued to grow at a fast rate, with the FDA seeing active investigational new drug applications for CGT increased to 500 in 20182. It had been predicted that the number of approvals of CGT products will likely be 54-74 in the United States by 20302.
While the rapid growth in CGT research and innovation is exciting, there is still a large technological gap between lab research and industrial-scale manufacturing that could deliver these promising medicines to reach as many patients as needed at affordable costs. The current processes adopted for these clinical trials were established in labs for small-scale experiments, and significant efforts are needed to improve and innovate on CGT manufacturing3. There are many types of CGT products, most of them based on live cells, which can be allogenic, autologous, engineered, or natural. These living drugs are much more complex than small molecular entities or biologics, hence making large-scale manufacturing a significant challenge4,5,6. In this work, we demonstrate a large-scale cell production protocol for three anchorage-dependent cells that are widely applied in CGTs. These include human mesenchymal stem/stromal cells (hMSCs), which have been used for cell-based therapy, and HEK293T cells and Vero cells, both of which are used to produce viruses for the genetic engineering of the final therapeutic cell product. Anchorage-dependent cells are commonly cultured on planar systems, which require manual processing. However, manual culture methods require a significant amount of labor and are prone to contamination, which can compromise the quality of the end product. Furthermore, there is no in-line process control, leading to substantial variability in quality between batches7. Taking stem cell therapy as an example, with a promising pipeline of over 200 stem-cell therapy candidates, it is estimated that 300 trillion hMSCs would be needed per year to meet the demands of clinical applications8. Hence, the large-scale manufacturing of therapeutic cells has become a prerequisite to perform these therapeutic interventions with such a high cell demand9.
To preclude the setbacks of planar systems, efforts have been made in developing large-scale manufacturing processes in stirred-tank bioreactors with conventional non-dissolvable microcarriers10,11,12,13, but these suffer from complicated preparation procedures and low cell-harvesting efficiency14. Recently, we have innovated a dissolvable microcarrier for stem cell expansion, aiming to circumvent the challenges of cell harvesting from conventional non-dissolvable commercial microcarriers15. This novel, commercially available GMP-grade 3D dissolvable porous microcarrier, 3D TableTrix, has shown great potential for large-scale cell production. Indeed, 3D culture based on these porous microcarriers could potentially recreate favorable biomimetic microenvironments to promote cell adhesion, proliferation, migration, and activation16. The porous structures and interconnected pore networks of microcarriers could create a larger cell adhesion area and promote the exchange of oxygen, nutrients, and metabolites, thus creating an optimal substrate for in vitro cell expansion17. The high porosity of these GMP-grade 3D dissolvable porous microcarriers enables large-scale expansion of hMSCs, and the ability for the cells to be fully dissolved allows for the efficient harvesting of these expanded cells18. It is also a GMP-grade product and has been registered as a pharmaceutical excipient with the Chinese Center for Drug Evaluation (filing numbers: F20210000003 and F20200000496)19 and the FDA of the United States (FDA, USA; Drug Master File number: 35481)20.
Here, we illustrate an automated closed industrial scale cell production (ACISCP) system18 using these dispersible and dissolvable porous microcarriers for hMSC, HEK293T cell, and Vero cell expansion. We achieved a successful two-tiered expansion of hMSCs (128 cumulative fold expansion in 9 days) from a 5 L bioreactor to a 15 L bioreactor and finally obtained up to 1.1 x 1010 hMSCs from a single batch of production. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Furthermore, we assessed the quality of hMSC products to confirm compliance. We also demonstrated the application of these dissolvable microcarriers for the scaled-up production of two other types of anchorage cells, HEK293T cells and Vero cells, that are extensively applied in the CGT industry. The peak cell density of HEK293T cells reached 1.68 x 107 cells/mL, whereas the peak density of Vero cells reached 1.08 x 107 cells/mL. The ACISCP system could be adapted to culture a variety of adherent cells, and it could potentially become a powerful platform contributing to expediting the industrialization of CGT.

Author Spotlight: Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers 

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 saving 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.

Watch this Scientific Journal Video about Preparation of the Stirred-Tank Bioreactor for Large-Scale Production of Human Mesenchymal Stem Cells at JoVE.com

Preparation of the Stirred-Tank Bioreactor for Large-Scale Production of Human Mesenchymal Stem Cells

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 thermowell 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 microcarriers for MSC.Pump 500 milliliters of culture medium from the storage bag into the bottle of microcarriers W01.

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Research

JoVE Journal

Bioengineering

346 Aufrufe.  Tsinghua University. Führen Sie zunächst eine Zwei-Punkt-Kalibrierung an der pH-Sonde mit einem Bioreaktor-Controller durch. Um dann einen Bioreaktorbehälter zusammenzubauen, installieren Sie Teile wie rostfreie Rohre und Laufräder gemäß den Anweisungen des Herstellers an der Kopfplatte. Befestigen Sie ein gebrauchsfertiges Einweg-Verbrauchsmaterialset an den entsprechenden Edelstahlteilen, Rohren und Anschlüssen an der Kopfplatte gemäß den Anweisungen des Herstellers.Führen Sie nach dem Zusammen...

Serie: Vorbereitung des Rührkessel-Bioreaktors für die großtechnische Produktion humaner mesenchymaler Stammzellen

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...

Two-Tiered Expansion of Human Mesenchymal Stem Cells from 5-Liter to 15-Liter Bioreactors and Automated Cell Formulation from a 15-Liter Bioreactor

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.