Upon the development of cell-processed products and regenerative medical products, scale-up of cell culture process is considered to be essential. However, there is concern that to achieve scale-up could affect the efficiency of cell culture or other processes. Besides, in case of using aoutologous cells, the cells can’t be used for threatment immediately because it takes long time to cultivate after collection. For this reason, allogenic cells (ES cells, iPS cells), cells donated by donors have been attracting attention recently. By collecting and cultivating a lot of cells from donors beforehand, they can be used for treatment sooner. This time we evaluate cryopreservation of human iPS cell-derived cardiomyocytes for treatment volume based on the success of cryopreservation of undifferentiated iPS cells at preliminary experiment. Also, we used CryoMACS Freezing Bag and ThawSTAR CB the same as preliminary experiment. <\/span><\/p>\n<\/div>\n<\/div>\n

Preliminary experiment<\/p>\n<\/div>\n

Conditions<\/p>\n

\u3010Cells\u3011 undifferentiated human iPS cells<\/li>\n
\u3010Volume\u3011 70mL cell suspension<\/li>\n
\u3010Concentration\u3011 5\u00d710\u2076cells\/mL<\/li>\n
\u3010Freezing medium\u3011 STEM-CELLBANKER®<\/span><\/li>\n
\u3010Freezing period\u3011 approx. 1.5 months<\/li>\n

\u3010Bag\u3011 CryoMACS Freezing Bag 250mL<\/li>\n<\/ol>\n<\/div>\n

Before freezing<\/p>\n<\/figure>\n
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CryoMACS Freezing Bag after thawing with ThawSTAR CB<\/p>\n<\/figure>\n<\/div>\n<\/div>\n\n
Results<\/p>\n
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\u3010Thawing time\u3011 30 min. (47 \u00d7 2 tubes: 90min)<\/li>\n
\u3010Viability after thawing\u3011 95.5%<\/li>\n
\u3010Proliferration rate after thawing\u3011
Seeded cells: 0.1\u00d710\u2076cells, Culture period: 4 days<\/li>\n<\/ol>\n
250 mL bag: 2.55 times<\/li>\n
Control 1 tube: 2.49 times<\/li>\n
47\u00d72mL tubes: 1.85 times<\/li>\n<\/div>\n
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47 tubes<\/p>\n<\/figure>\n
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250 mL bag<\/p>\n<\/figure>\n
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Control (1 tube)<\/p>\n<\/figure>\n<\/div>\n<\/div>\n\n
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Conclusion<\/p>\n<\/div>\n
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In large scale of freeezng and thawing, using bag allows us to increase work efficiency and reduce damage to cells.<\/span><\/p>\n<\/div>\n<\/div>\n<\/div>\n\n
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Experiment<\/p>\n<\/div> \n
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In this experiment, cardiomyocytes derivrd from human iPS cells were used, and we froze and thawed the cells twice.<\/p>\n<\/div>\n<\/div>\n
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\u3010Cells\u3011 Cardiomyocytes derived from research iPS cells
Cells were collected in 15-17 days after starting differentiated.<\/li>\n
\u3010Volume\u3011 20mL cell suspension<\/li>\n
\u3010Concentration\u3011 5\u00d710\u2076cells\/mL<\/li>\n
\u3010Freezing period\u3011 1-2 weeks at -150C<\/li>\n
\u3010Bag\u3011 CryoMACS Freezing Bag 50mL
50 mL bag is equivalent to 14 \u00d7 2 mL tubes (total liquid volume: 21mL)<\/li>\n<\/ul>\n<\/div>\n
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1<\/p>\n<\/div>\n
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Create and paste a label<\/p>\n<\/div>\n<\/div>\n
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2<\/p>\n<\/div>\n
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Freezing<\/p>\n<\/div>\n<\/div>\n
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3<\/p>\n<\/div>\n
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Cryopreservation<\/p>\n<\/div>\n<\/div>\n
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4<\/p>\n<\/div>\n
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Thawing<\/p>\n<\/div>\n<\/div>\n
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5<\/p>\n<\/div>\n
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Cell culture<\/p>\n<\/div>\n<\/div>\n<\/div>\n
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Working time and post-thawing viability<\/li>\n<\/ol>\n
* 14 tubes (total liquid volume: 21mL)<\/p>\n
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1<\/p>\n<\/div>\n
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7.5 min.<\/p>\n<\/div>\n
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2<\/p>\n<\/div>\n
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6 min.<\/p>\n<\/div>\n
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4<\/p>\n<\/div>\n
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60 min.<\/p>\n<\/div>\n
▶Total: 1 hour or more Viability: 94%<\/p>\n<\/div>\n
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* 50mL bag (Liquid volume: 20mL) 1st experiment<\/p>\n
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1<\/p>\n<\/div>\n
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3.6 min.<\/p>\n<\/div>\n
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2<\/p>\n<\/div>\n
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3 min.<\/p>\n<\/div>\n
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4<\/p>\n<\/div>\n
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10 min.<\/p>\n<\/div>\n
▶Total: 20 minutes or less Viability Viability: 91%<\/p>\n<\/div>\n
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*50mL bag (Liquid volume: 20mL) 2nd experiment<\/p>\n
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1<\/p>\n<\/div>\n
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3.6 min.<\/p>\n<\/div>\n
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2<\/p>\n<\/div>\n
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3 min.<\/p>\n<\/div>\n
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4<\/p>\n<\/div>\n
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10 min.<\/p>\n<\/div>\n
▶Total: 20 minutes or less Viability: 91.5%<\/p>\n<\/div>\n
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5<\/span> Eveluation after culture<\/span><\/p>\n<\/div>\n
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▶Post-thawing adhesive culture<\/p>\n<\/div>\n\n
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<\/td>\n 14 tubes<\/td>\n 50mL bag (1st)<\/td>\n 50mL bag (2nd)<\/td>\n<\/tr>\n
2 days<\/td>\n <\/td>\n <\/td>\n <\/td>\n<\/tr>\n
6 days<\/td>\n <\/td>\n <\/td>\n <\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n\n
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Adhesiveness of 50 mL bag cells was equivalent to that of 14 tubes. There was no diffrence.<\/span><\/p>\n<\/div>\n
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▶Motion analysis (SONY SI8000): After 6 days of post-thawing adhesive culture<\/p>\n<\/div>\n
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14vial<\/td>\n 50mL bag<\/td>\n<\/tr>\n
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Pulse rate: 32.3\/min<\/td>\n Pulse rate: 31.4\/min<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n<\/div>\n
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Pulse rate of 50mL bag cells was equivalent to that of 14 tubes.. There was no diffrence.<\/span><\/span><\/p>\n<\/div>\n\n
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▶Ability to generate cardiac spheroids<\/p>\n<\/div>\n
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Before freezing<\/td>\n 14 tubes<\/td>\n 50mL bag (1st)<\/td>\n 50mL bag (2nd)<\/td>\n<\/tr>\n
<\/td>\n <\/td>\n <\/td>\n <\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n
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Ability to generate cardiiac spheroids using 50mL bag cells was equivalent to that of 14 tubes.There was no diffrence.<\/span><\/p>\n<\/div>\n
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DATA: proveded by the research fgroup of Dr. Shugo Toyama.
Department of Cardiovascular Medicine. Keio University School of Medicine<\/p>\n<\/div>\n\n
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ThawSTAR CB<\/a><\/p>\n\n<\/div>\n
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Please contact us through the following button if you have any questions on this blog.<\/p>\n
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Contact<\/a><\/p>\n
PDF<\/a><\/p>\n<\/div>\n","protected":false},"featured_media":2174,"parent":0,"template":"","categories":[51],"_links":{"self":[{"href":"https:\/\/www.wakenbtech.co.jp\/eng\/wp-json\/wp\/v2\/_blog\/3467"}],"collection":[{"href":"https:\/\/www.wakenbtech.co.jp\/eng\/wp-json\/wp\/v2\/_blog"}],"about":[{"href":"https:\/\/www.wakenbtech.co.jp\/eng\/wp-json\/wp\/v2\/types\/_blog"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.wakenbtech.co.jp\/eng\/wp-json\/wp\/v2\/media\/2174"}],"wp:attachment":[{"href":"https:\/\/www.wakenbtech.co.jp\/eng\/wp-json\/wp\/v2\/media?parent=3467"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.wakenbtech.co.jp\/eng\/wp-json\/wp\/v2\/categories?post=3467"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}