Dissociation Solution for human ES/iPS Cells (CTK溶液)

製品分類 : ヒトES/iPSC-培養試薬研究テーマ : ES/iPS細胞

Dissociation Solution for human ES/iPS Cells (CTK溶液)

ヒトES細胞、ヒトiPS細胞剥離液 (CTK溶液)

【特徴】

  • 剥離液を加えピペッティング操作するだけで継代に適した大きさのコロニーに分割できるため、カッター等でコロニーを裁断して傷つけることはありません。
  • 高い生存率で継代できます。
  • オンフィーダー、フィーダーレスの両方に使用できます。
  • 全ロットの品質試験を行っておりますので、安心してお使いいただけます。

※保存温度:-20℃以下

特許 4317337号

概要

リプロセルの剥離液は、京都大学 中辻憲夫教授 (iCeMS)・末盛博文准教授 (再生医科学研究所) により開発されたものであり一般的にCTK溶液と呼ばれています。このCTK溶液の特許 (権利化済み) はリプロセルが保有しており、「Dissociation Solution for human ES/iPS Cells」として販売しております。また、CTK溶液はヒトiPS細胞の剥離液のゴールドスタンダードとしても広く使われています。

ヒトES細胞やヒトiPS細胞を継代するときには、single cell suspensionにせずコロニーを維持したままで行うことが重要です。例えば、一般の細胞株を剥離する際に使用されるトリプシン-EDTAなどを使用してヒトES細胞およびサルES細胞を継代した場合、生存率は数パーセントと著しく低下します。

これに対して、当社の剥離液は、添加するだけで図に示すように継代に適した大きさのコロニーを調製することが可能です。このことにより、継代時の細胞の生存率がTrypsin-EDTA溶液に比べ約10倍と高いのが特徴です。また、カニクイザルES細胞やヒトES細胞で、3年以上の染色体異常のない培養実績があります。

【動画】細胞剥離液 (RCHETP001) での細胞の剥離

プロトコル

データシート

製品安全データシート

操作動画

1.ヒトiPS細胞の形態

iPS細胞の形態について解説します。細胞の状態の判断にご活用ください。良い状態のiPS細胞は、コロニーの輪郭が明瞭で内部の細胞密度が高くなっています。

2.フィーダー細胞の播種方法

iPS細胞の培養に使用するフィーダー細胞は、その播種密度が重要になります。3×104 細胞/cm2が目安になります。

3.ヒトiPS細胞の継代方法

Dissociation Solution for human ES/iPS Cells (CTK溶液) を用いることで、高生存率かつ簡便な継代が可能になります。ピペッティング操作だけで、適度な大きさのコロニーにほぐれ、そのまま継代できます。通常3-4日に1回、継代を行います。

4.細胞の凍結保存方法と融解方法

Freezing Medium for human ES/iPS Cellsを使用し、ガラス化法で凍結することで、高い生存率での凍結保存が可能になります。ガラス化法とは、細胞を液体窒素で急速凍結する方法で、細胞へのダメージを最小限に抑えます。また、融解時も、温めておいた培養液を凍結細胞に直接加えることで急速解凍を行います。凍結・融解の作業に時間がかかると、生存率が低下するのでご注意ください。

論文・発表

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  • Morizane R, et al. “Nephron organoids derived from human pluripotent stem cells model kidney development and injury.” Nat Biotechnol. 2015 Oct 12. doi: 10.1038/nbt.3392.
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  • Kanemura, Hoshimi, et al. “Tumorigenicity Studies of Induced Pluripotent Stem Cell (iPSC)-Derived Retinal Pigment Epithelium (RPE) for the Treatment of Age-Related Macular Degeneration.” PloS one 9.1 (2014): e85336.
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  • Tanabe, Koji, et al. “Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts.” Proceedings of the National Academy of Sciences 110.30 (2013): 12172-12179.
  • Tanaka, Akihito, et al. “Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro.” PloS one 8.4 (2013): e61540.
  • Fujikura, J., et al. “Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation.” Diabetologia 55.6 (2012): 1689-1698.
  • Jung, Dongju, et al. “Incorporation of functionalized gold nanoparticles into nanofibers for enhanced attachment and differentiation of mammalian cells.” Journal of nanobiotechnology 10.1 (2012): 1-10.
  • Kajiwara, Masatoshi, et al. “Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.” Proceedings of the National Academy of Sciences 109.31 (2012): 12538-12543.
  • Kunisada, Yuya, et al. “Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells.” Stem cell research 8.2 (2012): 274-284.
  • Kuroda, Takuya, et al. “Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.” PloS one 7.5 (2012): e37342.
  • Matsuura, Katsuhisa, et al. “Creation of human cardiac cell sheets using pluripotent stem cells.” Biochemical and biophysical research communications 425.2 (2012): 321-327.
  • Nakahata, Tatsutoshi, et al. “Method for producing dendritic cells from pluripotent stem cells.” U.S. Patent Application 14/001,004.
  • Nakamura, Naoko, et al. “Feeder-free and serum-free production of hepatocytes, cholangiocytes, and their proliferating progenitors from human pluripotent stem cells: application to liver-specific functional and cytotoxic assays.” Cellular Reprogramming (Formerly” Cloning and Stem Cells”) 14.2 (2012): 171-185.
  • Okahara-Narita, Junko, et al. “Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors.” Primates 53.2 (2012): 205-213.
  • Shinozawa, Tadahiro, et al. “A novel purification method of murine embryonic stem cell?and human-induced pluripotent stem cell?derived cardiomyocytes by simple manual dissociation.” Journal of biomolecular screening 17.5 (2012): 683-691.
  • Shinozawa, Tadahiro, et al. “Determination of Appropriate Stage of Human-Induced Pluripotent Stem Cell?Derived Cardiomyocytes for Drug Screening and Pharmacological Evaluation In Vitro.” Journal of biomolecular screening 17.9 (2012): 1192-1203.
  • Sonoyama, Takuhiro, et al. “Differentiation of human embryonic stem cells and human induced pluripotent stem cells into steroid-producing cells.” Endocrinology 153.9 (2012): 4336-4345.
  • Tanaka, Takayuki, et al. “Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery.” Blood 120.6 (2012): 1299-1308.
  • Wada, Tamaki, et al. “Amyotrophic lateral sclerosis model derived from human embryonic stem cells overexpressing mutant superoxide dismutase 1.” Stem cells translational medicine 1.5 (2012): 396-402.
  • Wakao, Shohei, et al. “Morphologic and gene expression criteria for identifying human induced pluripotent stem cells.” PloS one 7.12 (2012): e48677.
  • Wutz, Anton. “Epigenetic alterations in human pluripotent stem cells: a tale of two cultures.” Cell stem cell 11.1 (2012): 9-15.
  • Iwabuchi, Kumiko A., et al. “ECAT11/L1td1 Is Enriched in ESCs and Rapidly Activated During iPSCGeneration, but It Is Dispensable for the Maintenance and Induction of Pluripotency.” PloS one 6.5 (2011): e20461.
  • Kobayashi, Hideyuki. “Pluripotent Stem Cells Induced from Testicular Tissue of a Man with Klinefelter Syndrome (47, XXY) by Four Transcription Factors (OCT4, SOX2, KLF4, and C-MYC).” (2011).
  • Maekawa, Momoko, et al. “Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1.” Nature 474.7350 (2011): 225-229.
  • Ogawa, Shin-ichiro, et al. “Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells.” In Vitro Cellular & Developmental Biology-Animal 47.7 (2011): 464-469.
  • Okita, Keisuke, et al. “A more efficient method to generate integration-free human iPS cells.” Nature methods 8.5 (2011): 409-412.
  • Takata, Akemi, et al. “Direct differentiation of hepatic cells from human induced pluripotent stem cells using a limited number of cytokines.” Hepatology international 5.4 (2011): 890-898.
  • Tatsumi, Rie, et al. “Simple and highly efficient method for production of endothelial cells from human embryonic stem cells.” Cell transplantation 20.9 (2011): 1423-1430.
  • Yahata, Naoki, et al. “Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer’s disease.” PLoS One 6.9 (2011): e25788.
  • Fujioka, Tsuyoshi, et al. “Establishment of induced pluripotent stem cells from human neonatal tissues.” Human cell 23.3 (2010): 113-118.
  • Kazuki, Yasuhiro, et al. “Complete genetic correction of ips cells from Duchenne muscular dystrophy.” Molecular Therapy 18.2 (2010): 386-393.
  • Lee, Tae-Hee, et al. “Functional recapitulation of smooth muscle cells via induced pluripotent stem cells from human aortic smooth muscle cells.” Circulation research 106.1 (2010): 120-128.
  • Miyoshi, Keiko, et al. “Generation of human induced pluripotent stem cells from oral mucosa.” Journal of bioscience and bioengineering 110.3 (2010): 345-350.
  • Otsuji, Tomomi G., et al. “Progressive maturation in contracting cardiomyocytes derived from human embryonic stem cells: Qualitative effects on electrophysiological responses to drugs.” Stem cell research 4.3 (2010): 201-213.
  • Sakurai, Kenji, et al. “Efficient integration of transgenes into a defined locus in human embryonic stem cells.” Nucleic acids research 38.7 (2010): e96-e96.
  • Tamaoki, N, et al. “Dental pulp cells for induced pluripotent stem cell banking.” Journal of dental research 89.8 (2010): 773-778.
  • Oka, Y., et al. “293FT cells transduced with four transcription factors (OCT4, SOX2, NANOG, and LIN28) generate aberrant ES-like cells.” J Stem cell Regenerative Med 3 (2010): 149-56.
  • Tamaoki, N., et al. “Dental pulp cells for induced pluripotent stem cell banking.”Journal of dental research 89.8 (2010): 773-778.
  • Chen, Hsin-Fu, et al. “A reduced oxygen tension (5%) is not beneficial for maintaining human embryonic stem cells in the undifferentiated state with short splitting intervals.” Human Reproduction 24.1 (2009): 71-80.
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  • Fusaki, Noemi, et al. “Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.” Proceedings of the Japan Academy. Series B, Physical and biological sciences 85.8 (2008): 348-362.
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製品リスト・価格

品番 製品名 希望小売価格(税抜)
RCHETP002 Dissociation solution for human ES/iPS cells(30 mL) ¥15,000

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≪オンフィーダー培養用試薬≫

製品名・概要
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京都大学iPS細胞研究所 (CiRA) の山中伸弥教授が発表された、ヒトiPS細胞樹立、培養に関する論文 (Cell 131, 1-12(2007)) でも利用された、リプロセルブランドのヒトiPS細胞、ヒトES細胞の培養に最適な培地です。
ReproStem
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MEF(3x106 cell)
マウス胎児由来線維芽細胞(Mouse embryonic fibroblast)。マウス、サル、ヒトES細胞、ヒトiPS細胞のフィーダー細胞として幅広くお使いいただけます。
SL10(3x106 cell)
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≪フィーダーレス培養用試薬≫

製品名・概要
StemFitR AK02N
高性能・世界最高水準のヒトiPS細胞用フィーダーレス培地。味の素株式会社と京都大学iPS細胞研究所で共同開発した再生医療の臨床研究用の培地です。
NutriStem™ XF/FF
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ReproXF
ヒトES細胞、ヒトiPS細胞用のXeno-Free (ゼノフリー) 培地。コーティング剤、剥離液とともに、動物由来成分のない完全なXeno-Free培養システムが実現できます。
ReproFF
オンフィーダー培養と同じ感覚で、簡単にフィーダレス培養が可能。ロット単位のまとめ買いに最適な大変お得な5本セット (25%OFF) もございます。
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≪グロースファクター≫

製品名・概要
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ヒトES細胞、ヒトiPS細胞の培養に最適なHuman Recombinant bFGF(FGF-2:Basic fibroblast growth factor)です。
bFGF Xeno-Free(FGF-2)
完全なXeno-Free培養が可能なbFGF。従来品と同様の活性配列で、同様にヒトiPS細胞の未分化維持にて品質確認済みです。

≪細胞剥離液・凍結保存液≫

製品名・概要
ReproCryo DMSO Free RM
今までに無い!DMSO不含、緩慢凍結法のヒトiPS細胞凍結保存液。本製品は、京都大学再生医科学研究所が発明したDMSO不含の凍害保護液を、未分化ヒトES/iPS細胞の凍結保存に適用することで、製品化したものです。
CryoStem™
高い生存率を維持できるヒトES/iPS細胞用凍結保存液です。
CRYO-GOLD™
Chemically defined な組織・細胞用 緩慢凍結法 保存液。ヒトiPS/ES細胞をはじめ、多様な細胞の緩慢凍結に対応可能です。
Dissociation solution for human ES/iPS cells
ヒトES細胞、ヒトiPS細胞剥離液 (CTK溶液)です。剥離液を加えピペッティング操作するだけで継代に適した大きさのコロニーに分割できるため、カッター等でコロニーを裁断して傷つけることはありません。
Freezing medium for human ES/iPS cells
ヒトES細胞、ヒトiPS細胞凍結保存液 (DAP213)です。一般的な保存液と比較して著しく高い生存率で凍結保存が可能です (解凍後3~4日後には継代が可能)。

≪未分化マーカー抗体≫

製品名・概要
Anti mouse Nanog antibody
京都大学中辻教授、多田准教授により開発された抗マウスNanog抗体(ラビット)です。マウスES細胞、iPS細胞の未分化マーカーとしてご使用いただけます。
Anti human Nanog antibody
京都大学中辻教授、多田准教授により開発された抗ヒトNanog抗体(ラビット)です。ヒトES細胞、ヒトiPS細胞の未分化マーカーとしてご使用いただけます。

≪その他 関連試薬≫

製品名・概要
hES Cell Cloning & Recovery Supplement
ES/iPS細胞の生存率を上げることができるサプリメントです。シングルES細胞で30倍以上の生存率向上の実績があります。