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大学・研究所にある論文を検索できる 「Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。




Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

Hiramatsu, Satoe Morizane, Asuka Kikuchi, Tetsuhiro Doi, Daisuke Yoshida, Kenji Takahashi, Jun 京都大学 DOI:10.3233/JPD-212934



[Background:] Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. [Objective:] To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. [Methods:] We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. [Results:] The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. [Conclusion:] These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.



1. Parmar M, Torper O, Drouin-Ouellet J (2019) Cell-based therapy for Parkinson’s disease: A journey through decades toward the light side of the Force. Eur J Neurosci 49, 463- 471.

2. Nolbrant S, Heuer A, Parmar M, Kirkeby A (2017) Gener- ation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral trans- plantation. Nat Protoc 12, 1962-1979.

3. Kikuchi T, Morizane A, Doi D, Magotani H, Onoe H, Hayashi T, Mizuma H, Takara S, Takahashi R, Inoue H, Morita S, Yamamoto M, Okita K, Nakagawa M, Parmar M, Takahashi J (2017) Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model. Nature 548, 592-596.

4. Kim TW, Koo SY, Studer L (2020) Pluripotent stem cell therapies for Parkinson disease: present challenges and future opportunities. Front Cell Dev Biol 8, 729.

5. Kim TW, Piao J, Koo SY, Kriks S, Chung SY, Betel D, Socci ND, Choi SJ, Zabierowski S, Dubose BN, Hill EJ, Mosharov E V., Irion S, Tomishima MJ, Tabar V, Studer L (2021) Biphasic activation of WNT signaling facilitates the derivation of midbrain dopamine neurons from hESCs for translational use. Cell Stem Cell 28, 343-355.e5.

6. Morizane A, Takahashi J, Takagi Y, Sasai Y, Hashimoto N (2002) Optimal conditions for in vivo induction of dopamin- ergic neurons from embryonic stem cells through stromal cell-derived inducing activity. J Neurosci Res 69, 934-939.

7. Koyanagi M, Takahashi J, Arakawa Y, Doi D, Fukuda H, Hayashi H, Narumiya S, Hashimoto N (2008) Inhibition of the Rho/ROCK pathway reduces apoptosis during trans- plantation of embryonic stem cell-derived neural precursors. J Neurosci Res 86, 270-280.

8. Frodl EM, Duan WM, Sauer H, Kupsch A, Brundin P (1994) Human embryonic dopamine neurons xenografted to the rat: effects of cryopreservation and varying regional source of donor cells on transplant survival, morphology and function. Brain Res 647, 286-298.

9. Sautter J, Strecker S, Kupsch A, Oertel WH (1996) Methyl- cellulose during cryopreservation of ventral mesencephalic tissue fragments fails to improve survival and function of cell suspension grafts. J Neurosci Methods 64, 173-179.

10. Sautter J, Ho¨glinger GU, Oertel WH, Earl CD (2000) Sys- temic treatment with GM1 ganglioside improves survival and function of cryopreserved embryonic midbrain grafted to the 6-hydroxydopamine-lesioned rat striatum. Exp Neurol 164, 121-129.

11. Chong Y-K, Toh T-B, Zaiden N, Poonepalli A, Leong SH, Ong CEL, Yu Y, Tan PB, See S-J, Ng W-H, Ng I, Hande MP, Kon OL, Ang B-T, Tang C (2009) Cryopreservation of neurospheres derived from human glioblastoma multi- forme. Stem Cells 27, 29-39.

12. Smith GD, Serafini PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, Alegretti JR, Motta EL (2010) Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril 94, 2088-2095.

13. Jang TH, Park SC, Yang JH, Kim JY, Seok JH, Park US, Choi CW, Lee SR, Han J (2017) Cryopreservation and its clinical applications. Integr Med Res 6, 12-18.

14. Fahy GM, Wowk B (2015) Principles of cryopreservation by vitrification, Springer, New York, NY.

15. Nagano M, Atabay EP, Atabay EC, Hishinuma M, Kata- giri S, Takahashi Y (2007) Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vit- rification on in vitro viability of mouse preantral follicles. Biomed Res 28, 153-160.

16. Schwartz PH, Bryant PJ, Fuja TJ, Su H, O’Dowd DK, Klassen H (2003) Isolation and characterization of neural progenitor cells from post-mortem human cortex. JNeurosci Res 74, 838-851.

17. Woods EJ, Perry BC, Hockema JJ, Larson L, Zhou D, Goebel WS (2009) Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology 59, 150- 157.

18. Ohara H, Ando S, Kishino A (2018) GMP Manufactur- ing of iPS Cells and Cell Bank for Regenerative Medicine. Sumitomo Kagaku 2018, 13-20.

19. Doi D, Magotani H, Kikuchi T, Ikeda M, Hiramatsu S, Yoshida K, Amano N, Nomura M, Umekage M, Morizane A, Takahashi J (2020) Pre-clinical study of induced pluripo- tent stem cell-derived dopaminergic progenitor cells for Parkinson’s disease. Nat Commun 11, 3369.

20. Doi D, Samata B, Katsukawa M, Kikuchi T, Morizane A, Ono Y, Sekiguchi K, Nakagawa M, Parmar M, Taka- hashi J (2014) Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation. Stem Cell Reports 2, 337-350.

21. Milosevic J, Storch A, Schwarz J (2005) Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells. Stem Cells 23, 681-688.

22. Malpique R, Oso´rio LM, Ferreira DS, Ehrhart F, Brito C, Zimmermann H, Alves PM (2010) Alginate encapsulation as a novel strategy for the cryopreservation of neurospheres. Tissue Eng Part C Methods 16, 965-977.

23. Mitchell PD, Ratcliffe E, Hourd P, Williams DJ, Thomas RJ (2014) A quality-by-design approach to risk reduction and optimization for human embryonic stem cell cryopreserva- tion processes. Tissue Eng Part C Methods 20, 941-950.

24. Baust JM, Campbell LH, Harbell JW (2017) Best practices for cryopreserving, thawing, recovering, and assessing cells. In Vitro Cell Dev Biol Anim 53, 855-871.

25. Drummond NJ, Singh Dolt K, Canham MA, Kilbride P, Morris GJ, Kunath T (2020) Cryopreservation of human midbrain dopaminergic neural progenitor cells poised for neuronal differentiation. Front Cell Dev Biol 8, 578907.

26. Radio NM, Mundy WR (2008) Developmental neurotoxi- city testing in vitro: Models for assessing chemical effects on neurite outgrowth. Neurotoxicology 29, 361-376.

27. Reinhardt P, Schmid B, Burbulla LF, Scho¨ndorf DC, Wagner L, Glatza M, Ho¨ing S, Hargus G, Heck SA, Dhingra A, Wu G, Mu¨ller S, Brockmann K, Kluba T, Maisel M, Kru¨ger R, Berg D, Tsytsyura Y, Thiel CS, Psathaki OE, Klingauf J, Kuhlmann T, Klewin M, Mu¨ller H, Gasser T, Scho¨ler HR, Sterneckert J (2013) Genetic correction of a lrrk2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression. Cell Stem Cell 12, 354-367.

28. Ryan KR, Sirenko O, Parham F, Hsieh JH, Cromwell EF, Tice RR, Behl M (2016) Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high- throughput screen for developmental neurotoxicity or neurotoxicity. Neurotoxicology 53, 271-281.

29. Pegg DE (2010) The relevance of ice crystal formation for the cryopreservation of tissues and organs. Cryobiology 60, S36-S44.

30. Massie I, Selden C, Hodgson H, Fuller B, Gibbons S, Morris GJ (2014) GMP cryopreservation of large volumes of cells for regenerative medicine: Active control of the freezing process. Tissue Eng Part C Methods 20, 693-702.

31. Li R, Yu G, Azarin SM, Hubel A (2018) Freezing responses in DMSO-based cryopreservation of human iPS cells: aggregates versus single cells. Tissue Eng Part C Methods 24, 289-299.

32. John Morris G, Acton E (2013) Controlled ice nucleation in cryopreservation-A review. Cryobiology 66, 85-92.

33. Yan Y, Sart S, Calixto Bejarano F, Muroski ME, Strouse GF, Grant SC, Li Y (2015) Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging. Biotechnol Prog 31, 510-521.

34. Niclis JC, Gantner CW, Alsanie WF, McDougall SJ, Bye CR, Elefanty AG, Stanley EG, Haynes JM, Pouton CW, Thompson LH, Parish CL (2017) Efficiently specified ventral midbrain dopamine neurons from human pluripo- tent stem cells under xeno-free conditions restore motor deficits in parkinsonian rodents. Stem Cells Transl Med 6, 937-948.

35. Nishiyama Y, Iwanami A, Kohyama J, Itakura G, Kawa- bata S, Sugai K, Nishimura S, Kashiwagi R, Yasutake K, Isoda M, Matsumoto M, Nakamura M, Okano H (2016) Safe and efficient method for cryopreservation of human induced pluripotent stem cell-derived neural stem and pro- genitor cells by a programmed freezer with a magnetic field. Neurosci Res 107, 20-29.

36. Rodr´ıguez-Mart´ınez D, Mart´ınez-Losa MM, Alvarez- Dolado M (2017) Cryopreservation of GABAergic neuronal precursors for cell-based therapy. PLoS One 12, e0170776.

37. Paynter SJ (2008) Principles and practical issues for cryop- reservation of nerve cells. Brain Res Bull 75, 1-14.

38. Wakeman DR, Hiller BM, Marmion DJ, McMahon CW, Corbett GT, Mangan KP, Ma J, Little LE, Xie Z, Perez- Rosello T, Guzman JN, Surmeier DJ, Kordower JH (2017) Cryopreservation maintains functionality of human iPSC dopamine neurons and rescues parkinsonian phenotypes in vivo. Stem Cell Rep 9, 149-161.

39. Ma XH, Shi Y, Hou Y, Liu Y, Zhang L, Fan WX, Ge D, Liu TQ, Cui ZF (2010) Slow-freezing cryopreservation of neural stem cell spheres with different diameters. Cryobiology 60, 184-191.

40. Miyamoto Y, Noguchi H, Yukawa H, Oishi K, Matsushita K, Iwata H, Hayashi S (2012) Cryopreservation of induced pluripotent stem cells. Cell Med 3, 89-95.

41. Terry C, Dhawan A, Mitry RR, Lehec SC, Hughes RD (2010) Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplanta- tion. Liver Transplant 16, 229-237.

42. Baert Y, Van Saen D, Haentjens P, In’t Veld P, Tournaye H, Goossens E (2013) What is the best cryopreservation protocol for human testicular tissue banking? Hum Reprod

28, 1816-1826.

43. Fujisaki Y, Amano M. Core unit for freerigeration device and refrigeration device using the same. Japan patent 4424693.

44. Dalvi-Isfahan M, Hamdami N, Xanthakis E, Le-Bail A (2017) Review on the control of ice nucleation by ultra- sound waves, electric and magnetic fields. J Food Eng 195, 222-234.

45. Cheng L, Sun DW, Zhu Z, Zhang Z (2017) Emerging techniques for assisting and accelerating food freezing pro- cesses: A review of recent research progresses. Crit Rev Food Sci Nutr 57, 769-781.

46. Kang T, You Y, Jun S (2020) Supercooling preserva- tion technology in food and biological samples: a review focused on electric and magnetic field applications. Food Sci Biotechnol 29, 303-321.

47. Zhao H, Zhang F, Hu H, Liu S, Han J (2017) Experimen- tal study on freezing of liquids under static magnetic field. Chinese J Chem Eng 25, 1288-1293.

48. Acharya P V., Bahadur V (2018) Fundamental interfacial mechanisms underlying electrofreezing. Adv Colloid Inter- face Sci 251, 26-43.

49. Sun W, Xu X, Zhang H, Sun W, Xu C (2006) The mecha- nism analysis of NaCl solution ice formation suppressed by electric field. Proc IEEE Int Conf Prop Appl Dielectr Mater 770-773.

50. Mok JH, Choi W, Park SH, Lee SH, Jun S (2015) Emerging pulsed electric field (PEF) and static magnetic field (SMF) combination technology for food freezing. Int J Refrig 50, 137-145.

51. Hafezparast-Moadab N, Hamdami N, Dalvi-Isfahan M, Farahnaky A (2018) Effects of radiofrequency-assisted freezing on microstructure and quality of rainbow trout (Oncorhynchus mykiss) fillet. Innov Food Sci Emerg Tech- nol 47, 81-87.

52. Li QY, Zou T, Gong Y, Chen SY, Zeng YX, Gao LX, Weng CH, Xu HW, Yin ZQ (2021) Functional assessment of cry- opreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases. Exp Eye Res 202, 108305.