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Summary
Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
Materials
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Automatic Translation
Bioengineering

Storskala celleproduktion baseret på GMP-grade opløselige porøse mikrobærere

Published: July 07, 2023
doi:
10.3791/65469
, , , , , , , ,
1Department of Biomedical Engineering, School of Medicine,Tsinghua University, 2Beijing CytoNiche Biotechnology Co. Ltd.

Summary

Her præsenterer vi en protokol for at opnå storskala fremstilling af klæbende celler gennem et helt lukket system baseret på opløselige mikrobærere af GMP-kvalitet. Dyrkningen af humane mesenkymale stamceller, HEK293T celler og Vero-celler blev valideret og opfyldte både kvantitetskrav og kvalitetskriterier for celle- og genterapiindustrien.

Abstract

Forskere i celle- og genterapiindustrien (CGT) har længe stået over for en formidabel udfordring i effektiv og storstilet udvidelse af celler. For at løse de primære mangler ved det todimensionelle (2D) plane dyrkningssystem udviklede vi innovativt en automatiseret ACISCP-platform (Closed Industrial Scale Cell Production) baseret på en GMP-kvalitet, opløselig og porøs mikrobærer til 3D-kulturen af klæbende celler, herunder humane mesenkymale stamceller / stromale celler (hMSC’er), HEK293T celler og Vero-celler. For at opnå storstilet ekspansion blev der udført en to-trins udvidelse med 5 L og 15 L omrørte tankbioreaktorer for at give 1,1 x 1010 hMSC’er med en samlet 128 gange udvidelse inden for 9 dage. Cellerne blev høstet ved fuldstændig opløsning af mikrobærerne, koncentreret, vasket og formuleret med et kontinuerligt flow centrifugebaseret cellebehandlingssystem og derefter aliquoteret med et cellepåfyldningssystem. Sammenlignet med 2D plan kultur er der ingen signifikante forskelle i kvaliteten af hMSC’er høstet fra 3D-kultur. Vi har også anvendt disse opløselige porøse mikrobærere på andre populære celletyper i CGT-sektoren; specifikt er HEK293T celler og Vero-celler blevet dyrket til maksimale celletætheder på henholdsvis 1,68 x 10 7 celler / ml og 1,08 x 107 celler / ml. Denne undersøgelse giver en protokol til anvendelse af en bioprocesteknisk platform, der udnytter egenskaberne ved opløselige mikrobærere af GMP-kvalitet og avanceret lukket udstyr til at opnå industriel fremstilling af klæbende celler.

Introduction

CGT-industrien har været vidne til en eksponentiel ekspansion i løbet af de sidste to årtier. Udviklingen af næste generation af lægemidler forventes at behandle og helbrede talrige ildfaste sygdomme1. Siden den første Food and Drug Administration (FDA) godkendelse af et CGT-produkt, Kymriah, i 2017 er CGT-relateret forskning og udvikling i verden fortsat med at vokse hurtigt, hvor FDA ser aktive undersøgelsesansøgninger om nye lægemidler til CGT steget til 500 i 20182. Det var blevet forudsagt, at antallet af godkendelser af CGT-produkter sandsynligvis vil være 54-74 i USA i 20302.

Mens den hurtige vækst i CGT-forskning og innovation er spændende, er der stadig en stor teknologisk kløft mellem laboratorieforskning og industriel produktion, der kunne levere disse lovende lægemidler til at nå så mange patienter som nødvendigt til overkommelige omkostninger. De nuværende processer, der blev vedtaget for disse kliniske forsøg, blev etableret i laboratorier til mindre eksperimenter, og der er behov for en betydelig indsats for at forbedre og innovere CGT-fremstilling3. Der er mange typer CGT-produkter, de fleste af dem baseret på levende celler, som kan være allogene, autologe, konstruerede eller naturlige. Disse levende lægemidler er meget mere komplekse end små molekylære enheder eller biologiske lægemidler, hvilket gør storskala fremstilling til en betydelig udfordring 4,5,6. I dette arbejde demonstrerer vi en storskala celleproduktionsprotokol for tre forankringsafhængige celler, der anvendes bredt i CGT’er. Disse omfatter humane mesenkymale stamceller / stromale celler (hMSC’er), som er blevet brugt til cellebaseret terapi, og HEK293T celler og Vero-celler, som begge bruges til at producere vira til gensplejsning af det endelige terapeutiske celleprodukt. Forankringsafhængige celler dyrkes almindeligvis på plane systemer, som kræver manuel behandling. Imidlertid kræver manuelle kulturmetoder en betydelig mængde arbejdskraft og er tilbøjelige til forurening, hvilket kan kompromittere slutproduktets kvalitet. Desuden er der ingen in-line proceskontrol, hvilket fører til betydelige variationer i kvaliteten mellem batcher7. Hvis man tager stamcelleterapi som et eksempel, med en lovende pipeline på over 200 stamcelleterapikandidater, anslås det, at der vil være behov for 300 billioner hMSC’er om året for at opfylde kravene til kliniske anvendelser8. Derfor er storskala fremstilling af terapeutiske celler blevet en forudsætning for at udføre disse terapeutiske indgreb med et så højt cellebehov9.

For at undgå tilbageslag i plane systemer er der gjort en indsats for at udvikle storstilede fremstillingsprocesser i bioreaktorer med omrøring af tanke med konventionelle ikke-opløselige mikrobærere 10,11,12,13, men disse lider under komplicerede forberedelsesprocedurer og lav cellehøstningseffektivitet 14. For nylig har vi innoveret en opløselig mikrobærer til stamcelleekspansion med det formål at omgå udfordringerne ved cellehøstning fra konventionelle ikke-opløselige kommercielle mikrobærere15. Denne nye, kommercielt tilgængelige GMP-grade 3D opløselige porøse mikrocarrier, 3D TableTrix, har vist stort potentiale for storskala celleproduktion. Faktisk kunne 3D-kultur baseret på disse porøse mikrobærere potentielt genskabe gunstige biomimetiske mikromiljøer for at fremme celleadhæsion, spredning, migration og aktivering16. De porøse strukturer og sammenkoblede porenetværk af mikrobærere kunne skabe et større celleadhæsionsområde og fremme udvekslingen af ilt, næringsstoffer og metabolitter og dermed skabe et optimalt substrat til in vitro-celleekspansion17. Den høje porøsitet af disse GMP-grade 3D opløselige porøse mikrobærere muliggør storstilet udvidelse af hMSC’er, og evnen til, at cellerne kan opløses fuldstændigt, muliggør effektiv høst af disse ekspanderede celler18. Det er også et produkt af GMP-kvalitet og er blevet registreret som farmaceutisk hjælpestof hos Chinese Center for Drug Evaluation (arkiveringsnumre: F20210000003 og F20200000496)19 og FDA i USA (FDA, USA; Drug Master Filnummer: 35481)20.

Her illustrerer vi et automatiseret ACISCP-system (Closed Industrial Scale Cell Production)18 ved hjælp af disse dispergerbare og opløselige porøse mikrobærere til hMSC-, HEK293T- og Vero-celleudvidelse. Vi opnåede en vellykket todelt udvidelse af hMSC’er (128 kumulativ foldudvidelse på 9 dage) fra en 5 L bioreaktor til en 15 L bioreaktor og opnåede endelig op til 1,1 x 1010 hMSC’er fra en enkelt produktionsbatch. Cellerne blev høstet ved fuldstændig opløsning af mikrobærerne, koncentreret, vasket og formuleret med et kontinuerligt flow centrifugebaseret cellebehandlingssystem og derefter aliquoteret med et cellefyldningssystem. Desuden vurderede vi kvaliteten af hMSC-produkter for at bekræfte overholdelsen. Vi demonstrerede også anvendelsen af disse opløselige mikrobærere til opskaleret produktion af to andre typer forankringsceller, HEK293T celler og Vero-celler, der anvendes i vid udstrækning i CGT-industrien. Den maksimale celletæthed af HEK293T celler nåede 1,68 x 10 7 celler / ml, mens topdensiteten af Vero-celler nåede 1,08 x 107 celler / ml. ACISCP-systemet kunne tilpasses til dyrkning af en række vedhængende celler, og det kunne potentielt blive en stærk platform, der bidrager til at fremskynde industrialiseringen af CGT.

Protocol

Den menneskelige navlestreng blev opnået fra Beijing Tsinghua Changgeng Hospital. Alle procedurer og protokoller vedrørende erhvervelse, isolering og dyrkning af humane navlesnorsmesenkymale stamceller (UCMSC’er) blev udført med informeret samtykke og med godkendelse fra den etiske komité på Beijing Tsinghua Changgeng Hospital (arkiveringsnummer 22035-4-02), og procedurerne og protokollerne var i overensstemmelse med Helsingfors-erklæringen fra 1964 og dens senere ændringer eller sammenlignelige etiske standarder….

Representative Results

ACISCP-platformen er et helt lukket system, der anvender en række bioreaktorer med omrøring til opskaleringsudvidelse, et cellebehandlingssystem til automatiseret cellehøst og formulering og et cellepåfyldningssystem (figur 1). Vedhængende celler binder sig til de porøse mikrobærere, som kan spredes i bioreaktoren og dermed opnå den suspenderede dyrkning af klæbende celler. Efter den beskrevne protokol blev 2,5 x 108 hMSC’er med 10 g W01-mikrob…

Discussion

Både immunterapi og stamcelleterapi bruger levende celler som lægemidler; Imidlertid bør deres slutprodukter ikke renses eller steriliseres på samme måde som små molekyler eller vira. Derfor bør princippet om Quality by Design (QbD) altid holdes for øje og praktisk anvendes på den kemiske fremstillings- og kontrolproces (CMC) under celleproduktion23. Et fuldt lukket cellekultursystem samt et behandlingssystem og et påfyldningssystem anses fortrinsvis for at opfylde kravene. I denne under…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet økonomisk af National Science Foundation for Distinguished Young Scholars (82125018).

Materials

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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Tags

Cell And Gene TherapyLarge-scale Cell ProductionGMP-grade Dissolvable Porous MicrocarriersAutomated Closed SystemStirred-tank BioreactorAdherent CellsCell HarvestingMesenchymal Stem CellsHEK293T CellsVero CellsIndustrial-scale ManufacturingQuality Control
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Chen, Y., Xu, H., Zhang, Y., Guo, L., Lan, M., Yang, Y., Liu, W., Yan, X., Du, Y. Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers. J. Vis. Exp. (197), e65469, doi:10.3791/65469 (2023).
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