GMPグレードの溶解性多孔質マイクロキャリアに基づく大規模セル生産

要約

ここでは、GMPグレードの溶解性マイクロキャリアに基づく完全クローズドシステムによる接着細胞の大規模製造を実現するためのプロトコルを紹介します。ヒト間葉系幹細胞、HEK293T細胞、およびVero細胞の培養は検証され、細胞および遺伝子治療業界の量的要求と品質基準の両方を満たしました。

要約

細胞・遺伝子治療(CGT)業界の研究者は、細胞を効率的かつ大規模に増殖させるという手ごわい課題に長い間直面してきました。2次元(2D)平面培養システムの主な欠点に対処するために、ヒト間葉系幹細胞/間質細胞(hMSC)、HEK293T細胞、Vero細胞などの接着細胞の3D培養用に、GMPグレードの溶解性多孔質マイクロキャリアに基づく自動クローズド工業スケール細胞生産(ACISCP)プラットフォームを革新的に開発しました。大規模な拡張を達成するために、5 Lおよび15 Lの攪拌タンクバイオリアクターで2段階の拡張を行い、1.1 x 10^{10 hMSC} を9日以内に全体で128倍に拡大しました。細胞は、マイクロキャリアを完全に溶解して回収し、濃縮、洗浄し、連続フロー遠心分離機ベースの細胞処理システムで調製した後、細胞充填システムで分注しました。2D平面培養と比較して、3D培養から採取したhMSCの品質に有意差はありません。また、これらの溶解性多孔質マイクロキャリアをCGT分野で人気のある他の細胞タイプにも適用しています。具体的には、HEK293T細胞とVero細胞をそれぞれ1.68 x 10⁷ cells/mLおよび1.08 x 10⁷ cells/mLのピーク細胞密度まで培養しました。本研究では、GMPグレードの溶解性マイクロキャリアと高度なクローズド機器の特性を利用して、接着細胞の工業規模の製造を実現するためのバイオプロセスエンジニアリングプラットフォームを使用するためのプロトコルを提供します。

概要

CGT業界は、過去20年間で指数関数的な拡大を目の当たりにしてきました。次世代医薬品の進化により、多くの難治性疾患の治療・治癒が期待されています¹。2017年に食品医薬品局(FDA)がCGT製品「キムリア」を初めて承認して以来、世界のCGT関連の研究開発は急速に成長を続けており、FDAはCGTの積極的な治験薬申請が2018年に500件に増加したことを確認し^{ています2。}2030年までに米国でのCGT製品の承認数は54〜74件になると予測^{されていました2。}

CGTの研究とイノベーションの急速な成長は喜ばしいことですが、これらの有望な医薬品を手頃な価格で必要なだけ多くの患者に届けることができるラボ研究と工業規模の製造の間には、まだ大きな技術的ギャップがあります。これらの臨床試験に採用されている現在のプロセスは、小規模な実験のために実験室で確立されたものであり、CGT製造の改善と革新には多大な努力が必要です³。CGT製品には多くの種類があり、そのほとんどは生細胞に基づいており、同種、自家、人工、または天然のものがあります。これらの生きた医薬品は、低分子の実体や生物学的製剤よりもはるかに複雑であるため、大規模な製造は大きな課題となっています

プロトコル

ヒトのへその緒は北京清華長源病院から入手した。ヒト臍帯間葉系幹細胞(UCMSC)の取得、単離、培養に関するすべての手順とプロトコルは、インフォームドコンセントと北京清華長源病院の倫理委員会(出願番号22035-4-02)の承認を得て実施され、手順とプロトコルは1964年のヘルシンキ宣言およびその後の改正または同等の倫理基準に準拠していました。

1. hMSC、HEK293T細胞、Vero細胞の単層培養

1. 報告された方法²¹に従って、ウォートンゼリーから初代ヒトUCMSCを分離します。初代UCMSCの単離および増殖には無血清(SF)hMSC培地を使用し、継代2で細胞を回収し、マスターセルバンク(MCB)として凍結保存します。
2. 15 mLの完全なSF hMSC培地で単層培養のために、1つのT75フラスコ(すなわち、2D平面血管上の播種密度は8,000細胞/cm 2)に6 x 10⁵ 個のヒトUCMSC(できれば継代²または継代3細胞)を接種します。フラスコを8の字に動かして均一に播種します。5%_CO2 細胞培養インキュベーターで37°Cで80%〜90%のコンフルエントまで培養します。
3. 回収するには、細胞培養培地をデカントし、3〜5 mLのリン酸緩衝生理食塩水(PBS)で細胞を2....

ディスカッション

免疫療法と幹細胞療法はどちらも、生細胞を薬剤として利用します。ただし、それらの最終生成物は、低分子やウイルスと同じ方法で精製または滅菌されるべきではありません。したがって、Quality by Design(QbD)の原則を常に念頭に置き、セル製造中の化学品製造および制御(CMC)プロセスに実際に適用する必要があります²³。完全閉鎖細胞培養システム、ならびに処理システム?.......

資料

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.