Name: Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers (Video) | JoVE
Uploaded: 2023-07-07T14:30:00
Description: Watch this Scientific Journal Video about Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers at JoVE.com

この研究は、National Science Foundation for Distinguished Young Scholars(82125018)の財政的支援を受けました。

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?...

Closed System Production of Adherent Cells Based on Dissolvable Microcarriers

<ol>
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Part B Reviews. 19 (6), 485-502 (2013).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMP-grade+microcarrier+and+automated+closed+industrial+scale+cell+production+platform+for+culture+of+MSCs.">GMP-grade microcarrier and automated closed industrial scale cell production platform for culture of MSCs.</a> Journal of Tissue Engineering and Regenerative Medicine. 16 (10), 934-944 (2022).</li><li>NMPA. Pharmaceutical Excipient Registration Database: Microcarrier tablets for cells. Center for Drug Evaluation Available from: <a target="_blank" href="https://www.cde.org.cn/main/xxgk/listpage/ba7aed094c29ae314670a3563a716e">https://www.cde.org.cn/main/xxgk/listpage/ba7aed094c29ae314670a3563a716e</a> (2023)</li><li>List of Drug Master Files (DMFs). US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/drug-mater-files-dmfs/list-drug-master-files-dmfs">https://www.fda.gov/drug-mater-files-dmfs/list-drug-master-files-dmfs</a> (2023)</li><li>Beeravolu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mesenchymal+stromal+cells+from+human+umbilical+cord+and+fetal+placenta.">Isolation and characterization of mesenchymal stromal cells from human umbilical cord and fetal placenta.</a> Journal of Visualized Experiments. (122), e55224 (2017).</li><li>Xie, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quality+evaluation+system+establishment+of+mesenchymal+stromal+cells+for+cell-based+therapy+products.">The quality evaluation system establishment of mesenchymal stromal cells for cell-based therapy products.</a> Stem Cell Research &amp; Therapy. 11 (1), 176 (2020).</li><li>Maillot, C., Sion, C., De Isla, N., Toye, D., Olmos, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+by+design+to+define+critical+process+parameters+for+mesenchymal+stem+cell+expansion.">Quality by design to define critical process parameters for mesenchymal stem cell expansion.</a> Biotechnology Advances. 50, 107765 (2021).</li><li>Silva Couto, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+human+mesenchymal+stem/stromal+cells+(hMSCs)+in+bioreactors+using+microcarriers:+Lessons+learnt+and+what+the+future+holds.">Expansion of human mesenchymal stem/stromal cells (hMSCs) in bioreactors using microcarriers: Lessons learnt and what the future holds.</a> Biotechnology Advances. 45, 107636 (2020).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+bead-to-bead+cell-transfer+method+for+serial+subculture+and+large-scale+expansion+of+human+mesenchymal+stem+cells+in+bioreactors.">Facile bead-to-bead cell-transfer method for serial subculture and large-scale expansion of human mesenchymal stem cells in bioreactors.</a> Stem Cells Translational Medicine. 10 (9), 1329-1342 (2021).</li><li>Tsai, A. C., Pacak, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprocessing+of+human+mesenchymal+stem+cells:+From+planar+culture+to+microcarrier-based+bioreactors.">Bioprocessing of human mesenchymal stem cells: From planar culture to microcarrier-based bioreactors.</a> Bioengineering. 8 (7), 96 (2021).</li><li>Hewitt, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+human+mesenchymal+stem+cells+on+microcarriers.">Expansion of human mesenchymal stem cells on microcarriers.</a> Biotechnology Letters. 33 (11), 2325-2335 (2011).</li><li>Mawji, I., Roberts, E. L., Dang, T., Abraham, B., Kallos, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+opportunities+in+downstream+separation+processes+for+mesenchymal+stromal+cells+cultured+in+microcarrier-based+stirred+suspension+bioreactors.">Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier-based stirred suspension bioreactors.</a> Biotechnology and Bioengineering. 119 (11), 3062-3078 (2022).</li><li>Schirmaier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-up+of+adipose+tissue-derived+mesenchymal+stem+cell+production+in+stirred+single-use+bioreactors+under+low-serum+conditions.">Scale-up of adipose tissue-derived mesenchymal stem cell production in stirred single-use bioreactors under low-serum conditions.</a> Engineering in Life Sciences. 14 (3), 292-303 (2014).</li><li>Lawson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+development+for+expansion+of+human+mesenchymal+stromal+cells+in+a+50L+single-use+stirred+tank+bioreactor.">Process development for expansion of human mesenchymal stromal cells in a 50L single-use stirred tank bioreactor.</a> Biochemical Engineering Journal. 120, 49-62 (2017).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+microcarrier+culture+of+HEK293T+cells+and+Vero+cells+in+single-use+bioreactors.">Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors.</a> AMB Express. 9 (1), 70 (2019).</li><li>Fang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+scalable+vaccinia+virus-based+vector+production+process+using+dissolvable+porous+microcarriers.">Development of scalable vaccinia virus-based vector production process using dissolvable porous microcarriers.</a> 25th Annual Meeting of the American Society of Gene &amp; Cell Therapy. Molecular Therapy. 30, 195-196 (2022).</li></ol>

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?...

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4,5,6。本研究では、CGTに広く適用されている3つの足場依存性細胞の大規模細胞産生プロトコルを実証する。これらには、細胞ベースの治療に使用されているヒト間葉系幹細胞/間質細胞(hMSC)や、最終的な治療用細胞製品の遺伝子操作のためのウイルスの産生に使用されるHEK293T細胞およびVero細胞が含まれます。足場依存性細胞は、通常、手作業による処理を必要とする平面系で培養されます。しかし、手作業による培養法は多大な労力を必要とし、汚染されやすく、最終製品の品質を損なう可能性があります。さらに、インラインプロセス制御がないため、バッチ間の品質に大きなばらつきが生じます7。幹細胞治療を例にとると、200を超える幹細胞治療候補の有望なパイプラインがあり、臨床応用の需要を満たすためには、年間300兆個のhMSCが必要になると推定されています8。したがって、治療用細胞の大規模製造は、このような高い細胞需要でこれらの治療的介入を実施するための前提条件となっています9。
平面システムの挫折を防ぐために、従来の非溶解性マイクロキャリアを備えた攪拌タンクバイオリアクターでの大規模な製造プロセスの開発が努力されてきました10,11,12,13が、これらは複雑な調製手順と低い細胞回収効率に悩まされています14.最近、我々は幹細胞増殖のための溶解性マイクロキャリアを革新し、従来の非溶解性商用マイクロキャリアからの細胞採取の課題を回避することを目指している15。この新規で市販されているGMPグレードの3D溶解性多孔質マイクロキャリアである3D TableTrixは、大規模な細胞生産に大きな可能性を示しています。実際、これらの多孔質マイクロキャリアに基づく3D培養は、細胞の接着、増殖、遊走、および活性化を促進するために、好ましい生体模倣微小環境を再現する可能性があります16。マイクロキャリアの多孔質構造と相互接続された細孔ネットワークは、より大きな細胞接着領域を作り出し、酸素、栄養素、および代謝産物の交換を促進し、したがって、in vitro細胞増殖のための最適な基質を作り出すことができる17。これらのGMPグレードの3D溶解性多孔質マイクロキャリアの高い多孔性は、hMSCの大規模な増殖を可能にし、細胞を完全に溶解する能力により、これらの増殖した細胞の効率的な収穫を可能にする18。また、GMPグレードの製品であり、中国医薬品評価センター(出願番号:F20210000003およびF20200000496)19および米国FDA(FDA、USA;ドラッグマスターファイル番号:35481)20。
本稿では、hMSC、HEK293T細胞、およびVero細胞増殖のために、これらの分散性および溶解性多孔性マイクロキャリアを用いた自動閉鎖型工業スケール細胞生産(ACISCP)システム18を例示する。5 L バイオリアクターから 15 L バイオリアクターへの hMSC の 2 層拡張 (9 日間で 128 倍の累積増殖) に成功し、最終的に 1 つのバッチの生産から最大 1.1 x 1010 hMSC を取得しました。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。さらに、hMSC製品の品質を評価し、適合性を確認しました。また、CGT産業で広く適用されている他の2種類の足場細胞、HEK293T細胞とVero細胞のスケールアップ生産へのこれらの溶解性マイクロキャリアの応用を実証しました。HEK293T細胞のピーク細胞密度は1.68 x 10 7 cells/mLに達し、Vero細胞のピーク密度は1.08 x 107 cells/mLに達しました。ACISCPシステムは、様々な接着細胞の培養に応用でき、CGTの工業化促進に寄与する強力なプラットフォームとなる可能性があります。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.25% trypsin EDTA</td>
			<td >BasalMedia</td>
			<td >S310JV</td>
			<td >Used for 2D cell harvest.</td>
		</tr>
		<tr >
			<td >3D FloTrix Digest</td>
			<td >CytoNiche Biotech</td>
			<td >R001-500</td>
			<td >This is a reagent that specifically dissolves 3D TableTrix microcarriers.</td>
		</tr>
		<tr >
			<td >3D FloTrix MSC Serum Free Medium</td>
			<td >CytoNiche Biotech</td>
			<td >RMZ112</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >3D FloTrix Single-Use Filtration Module</td>
			<td >CytoNiche Biotech</td>
			<td >R020-00-10</td>
			<td >This module contains 0.22 &#956;m capsule filters used for filtration of culture medium and digest solution.</td>
		</tr>
		<tr >
			<td >3D FloTrix Single-Use Storage Bag (10 L)</td>
			<td >CytoNiche Biotech</td>
			<td >R020-00-03</td>
			<td >Used as feed bag for 5 L bioreactor.</td>
		</tr>
		<tr >
			<td >3D FloTrix Single-Use Storage Bag (3 L)</td>
			<td >CytoNiche Biotech</td>
			<td >R020-00-01</td>
			<td >Used as cell seeding or transfer bags.</td>
		</tr>
		<tr >
			<td >3D FloTrix Single-Use Storage Bag (50 L)</td>
			<td >CytoNiche Biotech</td>
			<td >R020-00-04</td>
			<td >Used as feed bag for 15 L bioreactor.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaPACK Disposable Fill&#38;Finish Consumable Kit</td>
			<td >CytoNiche Biotech</td>
			<td >PACK-01-01</td>
			<td >This is a standard kit adapted to 3D vivaPACK fill and finish system.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaPACK fill and finish system for cells</td>
			<td >CytoNiche Biotech</td>
			<td >vivaPACK</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaPREP PLUS cell processing system</td>
			<td >CytoNiche Biotech</td>
			<td >vivaPREP PLUS</td>
			<td >This system is a continuous flow centrifuge-based device.Cells can be concentrated, washed, and resuspended under completely closed procedures.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaPREP PLUS Disposable Cell Processing Kit</td>
			<td >CytoNiche Biotech</td>
			<td >PREP-PLUS-00</td>
			<td >This is a standard kit adapted to 3D vivaPREP PLUS cell processing.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaSPIN&#160; bioreactor 15 L</td>
			<td >CytoNiche Biotech</td>
			<td >FTVS15</td>
			<td >This bioreactor product employs a controller, a 15 L glass stirred-tank vessel, and assessories. A special perfusion tube is available.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaSPIN&#160; bioreactor 5 L</td>
			<td >CytoNiche Biotech</td>
			<td >FTVS05</td>
			<td >This bioreactor product employs a controller, a 5 L glass stirred-tank vessel, and assessories.A special perfusion tube is available.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaSPIN Closed System Consumable Pack (10/15 L)</td>
			<td >CytoNiche Biotech</td>
			<td >R020-10-10</td>
			<td >This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 15 L, containing sampling bags.</td>
		</tr>
		<tr >
			<td >3D FloTrix vivaSPIN Closed System Consumable Pack (2/5 L)</td>
			<td >CytoNiche Biotech</td>
			<td >R020-05-10</td>
			<td >This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 5 L, containing sampling bags.</td>
		</tr>
		<tr >
			<td >3D TableTrix microcarriers G02</td>
			<td >CytoNiche Biotech</td>
			<td >G02-10-10g</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >3D TableTrix microcarriers V01</td>
			<td >CytoNiche Biotech</td>
			<td >V01-100-10g</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >3D TableTrix microcarriers W01</td>
			<td >CytoNiche Biotech</td>
			<td >W01-10-10g (single-use packaging); 
			W01-200 (tablets)</td>
			<td >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]</td>
		</tr>
		<tr >
			<td >APC anti-human CD45 Antibody</td>
			<td >BioLegend</td>
			<td >368512</td>
			<td >Used in flow cytometry for MSC identity assessment</td>
		</tr>
		<tr >
			<td >Calcein-AM/PI Double Staining Kit</td>
			<td >Dojindo</td>
			<td >C542</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >Cap for EZ Top Container Closures for NALGENE-containers (500mL)</td>
			<td >Saint-Gobain</td>
			<td>CAP-38</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >C-Flex Tubing, Formulation 374 (0.25 in x 0.44 in)</td>
			<td >Saint-Gobain</td>
			<td >374-250-3</td>
			<td >Used for tube welding and disconnection.</td>
		</tr>
		<tr >
			<td >CryoMACS Freezing Bag 50</td>
			<td >Miltenyi Biotec&#160;</td>
			<td >200-074-400</td>
			<td >Used for expanding the 3D FloTrix vivaPACK Disposable Fill&#38;Finish Consumable Kit.</td>
		</tr>
		<tr >
			<td >Dimethyl Sulfate (DMSO)&#160;</td>
			<td >Sigma</td>
			<td >D2650-100mL</td>
			<td >Used for preparation of cryopreservation solution.</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td >BasalMedia</td>
			<td >L120KJ</td>
			<td >Used for cultivation of HEK293T and Vero cells.</td>
		</tr>
		<tr >
			<td >DURAN Original GL 45 Laboratory bottle (2 L)</td>
			<td >DWK life sciences</td>
			<td >218016357</td>
			<td >Used for waste collection from the 5 L bioreactor.</td>
		</tr>
		<tr >
			<td >DURAN Original GL 45 Laboratory bottle (5 L)</td>
			<td >DWK life sciences</td>
			<td >218017353</td>
			<td >Used for waste collection from the 15 L bioreactor.</td>
		</tr>
		<tr >
			<td >DURAN Original GL 45 Laboratory bottle (500 mL)</td>
			<td >DWK life sciences</td>
			<td >218014459</td>
			<td >Used for supplementary bottle of 0.1 M NaOH.</td>
		</tr>
		<tr >
			<td >EZ Top Container Closures for NALGENE-containers (500mL)</td>
			<td >Saint-Gobain</td>
			<td>EZ500 ML-38-2</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >Fetal bovine serum (FBS) superior quality</td>
			<td >Wisent</td>
			<td >086-150</td>
			<td >Used for cultivation of HEK293T cells.</td>
		</tr>
		<tr >
			<td >FITC anti-human CD14 Antibody</td>
			<td >BioLegend</td>
			<td >301804</td>
			<td >Used in flow cytometry for MSC identity assessment.</td>
		</tr>
		<tr >
			<td >FITC anti-human CD34 Antibody</td>
			<td >BioLegend</td>
			<td >343504</td>
			<td >Used in flow cytometry for MSC identity assessment.</td>
		</tr>
		<tr >
			<td >FITC anti-human CD90 (Thy1) Antibody</td>
			<td >BioLegend</td>
			<td >328108</td>
			<td >Used in flow cytometry for MSC identity assessment.</td>
		</tr>
		<tr >
			<td >Flow cytometry</td>
			<td >Beckman Coulter</td>
			<td >CytoFLEX</td>
			<td >Used for cell identity assessment.</td>
		</tr>
		<tr >
			<td >Fluorescence Cell Analyzer</td>
			<td >Alit life science</td>
			<td >Countstar Rigel S2</td>
			<td >Used for cell counting. Cell viability can be calculated by staining with AO/PI dyes.</td>
		</tr>
		<tr >
			<td >GL 45 Multiport Connector Screw Cap with 2 ports&#160;</td>
			<td >DWK life sciences</td>
			<td >292632806</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >Glucose Meter</td>
			<td >Sinocare</td>
			<td >6243578</td>
			<td >Used for detecting glucose concentration in cell culture medium and supernatant.</td>
		</tr>
		<tr >
			<td >Hank&#39;s Balanced Salt Solution (HBSS), with calcium and magnesium</td>
			<td >Gibco</td>
			<td >14025092</td>
			<td >Used for preparation of digest solution.</td>
		</tr>
		<tr >
			<td >Human Albumin 20% Behring (HSA)</td>
			<td >CSL Behring</td>
			<td >N/A</td>
			<td >Used for preparation of wash buffer.</td>
		</tr>
		<tr >
			<td >Inverted fluorescent microscope</td>
			<td >OLYMBUS</td>
			<td >CKX53SF</td>
			<td >Used for brifgt field and fluorescent observation and imaging.</td>
		</tr>
		<tr >
			<td >Nalgene Measuring Cylinder (500 mL)</td>
			<td >Thermo Scientific</td>
			<td >3662-0500PK</td>
			<td >Used for calibrating the liquid handling volume speed of peristaltic pumps.</td>
		</tr>
		<tr >
			<td >Newborn calf serum (NBS) superfine</td>
			<td >MINHAI BIO</td>
			<td >SC101.02</td>
			<td >Used for cultivation of Vero cells.</td>
		</tr>
		<tr >
			<td >OriCell human mesenchymal stem cell adipogenic differentiation and characterization kit</td>
			<td >Cyagen</td>
			<td >HUXUC-90031</td>
			<td >Used for tri-lineage differentiation of hUCMSCs.</td>
		</tr>
		<tr >
			<td >OriCell human mesenchymal stem cell chondrogenic differentiation and characterization kit</td>
			<td >Cyagen</td>
			<td >HUXUC-90041</td>
			<td >Used for tri-lineage differentiation of hUCMSCs.</td>
		</tr>
		<tr >
			<td >OriCell human mesenchymal stem cell osteogenic differentiation and characterization kit</td>
			<td >Cyagen</td>
			<td >HUXUC-90021</td>
			<td >Used for tri-lineage differentiation of hUCMSCs.</td>
		</tr>
		<tr >
			<td >PE anti-human CD105 Antibody</td>
			<td >BioLegend</td>
			<td >800504</td>
			<td >Used in flow cytometry for MSC identity assessment.</td>
		</tr>
		<tr >
			<td >PE anti-human CD19 Antibody</td>
			<td >BioLegend</td>
			<td >302208</td>
			<td >Used in flow cytometry for MSC identity assessment.</td>
		</tr>
		<tr >
			<td >PE anti-human CD73 (Ecto-5&#39;-nucleotidase) Antibody</td>
			<td >BioLegend</td>
			<td >344004</td>
			<td >Used in flow cytometry for MSC identity assessment.</td>
		</tr>
		<tr >
			<td >PE anti-human HLA-DR Antibody</td>
			<td >BioLegend</td>
			<td >307605</td>
			<td >Used in flow cytometry for MSC identity assessment.</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS)</td>
			<td >Wisent</td>
			<td >311-010-CL</td>
			<td >Used in autoclaving of glass vessel and V01 microcarriers, and replacement of culture medium.</td>
		</tr>
		<tr >
			<td >Sani-Tech Platinum Cured Sanitary Silicone Tubing (0.13 in x 0.25 in)</td>
			<td >Saint-Gobain</td>
			<td >ULTRA-C-125-2F</td>
			<td >Used for solution transfering driven by peristaltic pumps.</td>
		</tr>
		<tr >
			<td >Sterile Saline</td>
			<td >Hopebiol</td>
			<td >HBPP008-500</td>
			<td >Used for preparation of wash buffer.</td>
		</tr>
		<tr >
			<td >Trypzyme Recombinant Trypsin</td>
			<td >BasalMedia</td>
			<td >S342JV</td>
			<td >This reagent is used for bead-to-bead transfer of HEK293T and Vero cells.</td>
		</tr>
		<tr >
			<td >Tube Sealer</td>
			<td >Yingqi Biotech</td>
			<td >Tube Sealer I</td>
			<td >This sealer is compatible with both C-Flex tubing and PVC tubing.</td>
		</tr>
		<tr >
			<td >Tube Welder for PVC tubing</td>
			<td >Chu Biotech</td>
			<td >Tube Welder Micro I</td>
			<td >Used for welding of PVC tubing.</td>
		</tr>
		<tr >
			<td >Tube Welder for TPE tubing</td>
			<td >Yingqi Biotech</td>
			<td >Tube Welder I-V2</td>
			<td >Used for welding of TPE tubing.</td>
		</tr>
		<tr >
			<td >ViaStain AO / PI Viability Stains</td>
			<td >Nexcelom</td>
			<td >CS2-0106-25mL</td>
			<td >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.</td>
		</tr>
	</tbody>
</table>

large-scale cell production based on gmp-grade dissolvable porous microcarriers

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 1010 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。

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

ACISCPプラットフォームは、スケールアップ拡張のための一連の攪拌タンクバイオリアクター、自動細胞回収および製剤のための細胞処理システム、および細胞充填システムを採用した完全閉鎖システムです(図1)。接着細胞は多孔質マイクロキャリアに付着し、バイオリアクター内に分散させることで、接着細胞の懸濁培養を実現します。
記載のプ?...

Watch this Scientific Journal Video about Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers at JoVE.com

Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers (Video) | JoVE

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。

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

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 ...

細胞・遺伝子治療業界は、過去20年間で急激な拡大を遂げてきました。しかし、ラボでの研究と産業技術の製造の間には、大きな技術的ギャップが存在します。規制政策に準拠し、CGT業界のグリーン需要を満たすために、細胞治療薬を製造するために自動化されたクローズドシステムが採用されています。接着細胞の場合、スケーラブルな攪拌タンクバイオリアクターでマイクロキャリアを使用して培養することが有望な方法です。従来、製品化されたマイクロキャリアは望ましくない。トリプシン処理による細胞回収や、サイズによるマイクロキャリアの節約は、非効率的で困難であるだけでなく、細胞の品質に影響を与え、安全性の懸念を引き起こします。本研究では、GMPグレードの3D TableTrix溶解性マイクロキャリアを自動閉鎖系で大規模に生産することに成功しました。110億個以上のヒト間葉系幹細胞が、9日以内に2段階拡大して産出されました。また、HEK293T細胞とVero細胞の高密度培養を実証し、両方の密度が1,000万細胞/ミリリットル以上に達しました。この高度なバイオマニュファクチャリングプラットフォームにより、接着細胞を効果的に増殖させ、自動化された閉鎖的な方法で完全に回収することができます。当社のプロトコルは、CGT製品の工業規模の製造において、生産効率を高め、品質管理を改善する機会を提供します。

Research

JoVE Journal

Bioengineering

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

ヒト間葉系幹細胞の大量生産のための攪拌タンク式バイオリアクターの作製

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

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