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Materials

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Bioengineering

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

Published: July 7th, 2023

DOI:

10.3791/65469

1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Beijing CytoNiche Biotechnology Co. Ltd.
* These authors contributed equally

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 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.

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.

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The human umbilical cord was obtained from Beijing Tsinghua Changgeng Hospital. All procedures and protocols regarding the acquisition, isolation, and culture of human umbilical cord mesenchymal stem cells (UCMSCs) were conducted with informed consent and with the approval of the Ethics Committee of Beijing Tsinghua Changgeng Hospital (filing number 22035-4-02), and the procedures and protocols complied with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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The ACISCP platform is a fully closed system that employs a series of stirred-tank bioreactors for scale-up expansion, a cell processing system for automated cell harvesting and formulation, and a cell filling system (Figure 1). Adherent cells attach to the porous microcarriers, which can be dispersed into the bioreactor, thus achieving the suspended cultivation of adherent cells.

Following the protocol as described, 2.5 x 108 hMSCs with 10 g of W01 mic.......

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Both immunotherapy and stem cell therapy utilize live cells as drugs; however, their final products should not be purified or sterilized in the same way as small molecules or viruses. Therefore, the principle of Quality by Design (QbD) should always be kept in mind and practically applied to the Chemical Manufacturing and Control (CMC) process during cell production23. A fully closed cell culture system, as well as a processing system and a filling system, are preferentially considered to meet the.......

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This work was financially supported by the National Science Foundation for Distinguished Young Scholars (82125018).

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Name Company Catalog Number Comments
0.25% trypsin EDTA BasalMedia S310JV Used for 2D cell harvest.
3D FloTrix Digest CytoNiche Biotech R001-500 This is a reagent that specifically dissolves 3D TableTrix microcarriers.
3D FloTrix MSC Serum Free Medium CytoNiche Biotech RMZ112 This is a serum-free,animal-free medium for mesenchymal stem cell expansion and maintenance in 2D planar culture as well as 3D culture on 3D TableTrix microcarriers.
3D FloTrix Single-Use Filtration Module CytoNiche Biotech R020-00-10 This module contains 0.22 μm capsule filters used for filtration of culture medium and digest solution.
3D FloTrix Single-Use Storage Bag (10 L) CytoNiche Biotech R020-00-03 Used as feed bag for 5 L bioreactor.
3D FloTrix Single-Use Storage Bag (3 L) CytoNiche Biotech R020-00-01 Used as cell seeding or transfer bags.
3D FloTrix Single-Use Storage Bag (50 L) CytoNiche Biotech R020-00-04 Used as feed bag for 15 L bioreactor.
3D FloTrix vivaPACK Disposable Fill&Finish Consumable Kit CytoNiche Biotech PACK-01-01 This is a standard kit adapted to 3D vivaPACK fill and finish system.
3D FloTrix vivaPACK fill and finish system for cells CytoNiche Biotech vivaPACK This system is a closed liquid handling device, with automated mixing and gas exhausting functions. Cells resuspended in cryopreservation buffer can be rapidly and evenly aliquoted into 20 bags per batch.
3D FloTrix vivaPREP PLUS cell processing system CytoNiche Biotech vivaPREP PLUS This system is a continuous flow centrifuge-based device.Cells can be concentrated, washed, and resuspended under completely closed procedures.
3D FloTrix vivaPREP PLUS Disposable Cell Processing Kit CytoNiche Biotech PREP-PLUS-00 This is a standard kit adapted to 3D vivaPREP PLUS cell processing.
3D FloTrix vivaSPIN  bioreactor 15 L CytoNiche Biotech FTVS15 This bioreactor product employs a controller, a 15 L glass stirred-tank vessel, and assessories. A special perfusion tube is available.
3D FloTrix vivaSPIN  bioreactor 5 L CytoNiche Biotech FTVS05 This bioreactor product employs a controller, a 5 L glass stirred-tank vessel, and assessories.A special perfusion tube is available.
3D FloTrix vivaSPIN Closed System Consumable Pack (10/15 L) CytoNiche Biotech R020-10-10 This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 15 L, containing sampling bags.
3D FloTrix vivaSPIN Closed System Consumable Pack (2/5 L) CytoNiche Biotech R020-05-10 This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 5 L, containing sampling bags.
3D TableTrix microcarriers G02 CytoNiche Biotech G02-10-10g These porous and degradable microcarriers are suitable for HEK293T cell culture. They come pre-sterilized in 10g/bottle with C-Flex tubings for welding to tubes on bioreactors.
3D TableTrix microcarriers V01 CytoNiche Biotech V01-100-10g These porous and degradable microcarriers are suitable for adherent cell culture, they come as non-sterilized microcarriers that need to be autoclaved in PBS before use. They are especially suitable for vaccine production.
3D TableTrix microcarriers W01 CytoNiche Biotech W01-10-10g (single-use packaging);
W01-200 (tablets)
These porous and degradable microcarriers are suitable for adherent cell culture, especially for cells that need to be harvested as end products. They come pre-sterilized in 10g/bottle with C-Flex tubings for welding to tubes on bioreactors.The product has obtained 2 qualifications for pharmaceutical excipients from CDE, with the registration numbers of [F20200000496; F20210000003]. It has also received DMF qualification for pharmaceutical excipients from FDA, with the registration number of [DMF:35481]
APC anti-human CD45 Antibody BioLegend 368512 Used in flow cytometry for MSC identity assessment
Calcein-AM/PI Double Staining Kit Dojindo C542 Calcein-AM/PI Double Staining Kit is utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stain viable and dead cells, respectively.
Cap for EZ Top Container Closures for NALGENE-containers (500mL) Saint-Gobain CAP-38 Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.
C-Flex Tubing, Formulation 374 (0.25 in x 0.44 in) Saint-Gobain 374-250-3 Used for tube welding and disconnection.
CryoMACS Freezing Bag 50 Miltenyi Biotec  200-074-400 Used for expanding the 3D FloTrix vivaPACK Disposable Fill&Finish Consumable Kit.
Dimethyl Sulfate (DMSO)  Sigma D2650-100mL Used for preparation of cryopreservation solution.
Dulbecco's Modified Eagle Medium (DMEM) BasalMedia L120KJ Used for cultivation of HEK293T and Vero cells.
DURAN Original GL 45 Laboratory bottle (2 L) DWK life sciences 218016357 Used for waste collection from the 5 L bioreactor.
DURAN Original GL 45 Laboratory bottle (5 L) DWK life sciences 218017353 Used for waste collection from the 15 L bioreactor.
DURAN Original GL 45 Laboratory bottle (500 mL) DWK life sciences 218014459 Used for supplementary bottle of 0.1 M NaOH.
EZ Top Container Closures for NALGENE-containers (500mL) Saint-Gobain EZ500 ML-38-2 Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.
Fetal bovine serum (FBS) superior quality Wisent 086-150 Used for cultivation of HEK293T cells.
FITC anti-human CD14 Antibody BioLegend 301804 Used in flow cytometry for MSC identity assessment.
FITC anti-human CD34 Antibody BioLegend 343504 Used in flow cytometry for MSC identity assessment.
FITC anti-human CD90 (Thy1) Antibody BioLegend 328108 Used in flow cytometry for MSC identity assessment.
Flow cytometry Beckman Coulter CytoFLEX Used for cell identity assessment.
Fluorescence Cell Analyzer Alit life science Countstar Rigel S2 Used for cell counting. Cell viability can be calculated by staining with AO/PI dyes.
GL 45 Multiport Connector Screw Cap with 2 ports  DWK life sciences 292632806 Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.
Glucose Meter Sinocare 6243578 Used for detecting glucose concentration in cell culture medium and supernatant.
Hank's Balanced Salt Solution (HBSS), with calcium and magnesium Gibco 14025092 Used for preparation of digest solution.
Human Albumin 20% Behring (HSA) CSL Behring N/A Used for preparation of wash buffer.
Inverted fluorescent microscope OLYMBUS CKX53SF Used for brifgt field and fluorescent observation and imaging.
Nalgene Measuring Cylinder (500 mL) Thermo Scientific 3662-0500PK Used for calibrating the liquid handling volume speed of peristaltic pumps.
Newborn calf serum (NBS) superfine MINHAI BIO SC101.02 Used for cultivation of Vero cells.
OriCell human mesenchymal stem cell adipogenic differentiation and characterization kit Cyagen HUXUC-90031 Used for tri-lineage differentiation of hUCMSCs.
OriCell human mesenchymal stem cell chondrogenic differentiation and characterization kit Cyagen HUXUC-90041 Used for tri-lineage differentiation of hUCMSCs.
OriCell human mesenchymal stem cell osteogenic differentiation and characterization kit Cyagen HUXUC-90021 Used for tri-lineage differentiation of hUCMSCs.
PE anti-human CD105 Antibody BioLegend 800504 Used in flow cytometry for MSC identity assessment.
PE anti-human CD19 Antibody BioLegend 302208 Used in flow cytometry for MSC identity assessment.
PE anti-human CD73 (Ecto-5'-nucleotidase) Antibody BioLegend 344004 Used in flow cytometry for MSC identity assessment.
PE anti-human HLA-DR Antibody BioLegend 307605 Used in flow cytometry for MSC identity assessment.
Phosphate Buffered Saline (PBS) Wisent 311-010-CL Used in autoclaving of glass vessel and V01 microcarriers, and replacement of culture medium.
Sani-Tech Platinum Cured Sanitary Silicone Tubing (0.13 in x 0.25 in) Saint-Gobain ULTRA-C-125-2F Used for solution transfering driven by peristaltic pumps.
Sterile Saline Hopebiol HBPP008-500 Used for preparation of wash buffer.
Trypzyme Recombinant Trypsin BasalMedia S342JV This reagent is used for bead-to-bead transfer of HEK293T and Vero cells.
Tube Sealer Yingqi Biotech Tube Sealer I This sealer is compatible with both C-Flex tubing and PVC tubing.
Tube Welder for PVC tubing Chu Biotech Tube Welder Micro I Used for welding of PVC tubing.
Tube Welder for TPE tubing Yingqi Biotech Tube Welder I-V2 Used for welding of TPE tubing.
ViaStain AO / PI Viability Stains Nexcelom CS2-0106-25mL Dual-Fluorescence Viability, using acridine orange (AO) and propidium iodide (PI), is the recommended method for accurate viability analysis of primary cells, such as PBMCs, and stem cells in samples containing debris.

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