JSPS Science Dialogue, 2009 Cryopreservation of Cells and Tissues: Approaches and Applications Gang Zhao PhD JSPS Research Fellow, Department of Mechanical Engineering Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Associate Professor, Department of Modern Mechanics, University of Science and Technology of China, Hefei 230027, China. Host Professor: Hiroshi Takamatsu JSPS Science Dialogue: Kumamoto Prefectural Daini High School (熊本県立第二高等学校) 細胞の凍結保存：方法と応用 , Friday, June, 26th, 2009 JSPS Science Dialogue, 2009 Outline Self introduction 1) Who am I? 2) Where I come from? Research works 1) Why I become a natural science researcher? 2) Why I come to Japan for furth er studies? 3) What’s cryobiology? 4) Whyypy we need cryopreservation? 5) Why we can cryopreserve biomaterials? 6) How to cryopreserve biomaterials? 7) Two typical events during cell cryopreservation. 8) Cell specified optimal freezing rate. 9) Measurement of hydraulic permeability by perfusion microscope. 10) Status quo and future of cryopreservation. JSPS Science Dialogue, 2009 Self introduction http://www.kyushu-u.ac.jp/ Kumamoto Prefectural Daini High School, Friday, June 26, 2009 JSPS Science Dialogue,Who 2009 am I? My Institution. Full Name: Gang Zhao PhD 趙剛 (チヨウ ゴウ) PtSttPresent Status: 1. JSPS Research Fellow Kyushu University, Japan 日本会本学術振興会 外国人特究特別研究 員 九州大学, 日本 2. Associate Professor University of Science and Technology of China, China 副教授 中国科学技术大学, 中国 MjMajor: Cryobiology JSPS ScienceWhere Dialogue, I come 2009 from? My County: China. http://ditu.google.cn/ JSPS Science Dialogue, 2009 Research Works http://www.kyushu-u.ac.jp/ Kumamoto Prefectural Daini High School, Friday, June 26, 2009 JSPS ScienceWhy Dialogue,I become 2009 a natural science researcher? •Shown on TV in 1980s in China • A whole generation was deeply moved by this TV play (ドラ マ ) • All the people love the two actors (山口百恵、三浦友和) very much • The leading actress died from Leukemia (白血病, a cancer of the blood or bone marrow)->all the spectators were very sorrowful. Natural Science 赤い疑惑（あかいぎわく） は、1975年10月3日から •Save our lives 年 月 日まで で EiEngineer ing Biology 1976 4 16 TBS •Improve our 放送されたテレビドラマ。 (工学) (生物学) 赤いシリーズ第2弾。平均 quality of life 視聴率23. 4%、最高30. 9% •Keep us healthy （関東地区）を記録した。 and happy 主な出演者は山口百恵、 Clinical medicine 三浦友和、宇津井健。 (臨床医学) http://imgsrc.baidu.com/baike/pic/item/79b1e93635a830220a55a9a4.jpg JSPSWhy Science I come Dialogue, to Japan 2009 for research and further studies? •The most advanced techlhnologies •The highest life expectancy SONY •The beautiful landscapes •The world famous universities ASIMO and professors 高松洋 教授 九州大学 工 学院 http://en.wikipedia.org/wiki/Japan http://sanyo.com/ http://panasonic.cn/ http://www.toshiba.com/tai/ JSPS ScienceWhat Dialogue, is cryobiology? 2009 From Star Strek. Interstellar travel (星間旅行) 地球(ちきゅう) 火星(かせい) Planets and dwarf ppylanets of the Solar System http://en.wikipedia.org/wiki/File:Planets2008.jpg JSPS ScienceWhat Dialogue, is cryobiology? 2009 A typical example. Spacecraft (宇宙船 ) 地球 火星 Journey: 6~9 months Or even about 2 years 50 million miles Earth is the third planet from Mars is the fourth planet from the Sun in the Solar System. the Sun in the Solar System. What to do for the astronauts during the travel: months or even years? Internet: none; TV: none; Restaurant: none; Hospital: none …… http://en.wikipedia.org/wiki/Star_travel http://en.wikipedia.org/wiki/File:Planets2008.jpg http://en.wikipedia.org/wiki/Earth http://en.wikipedia.org/wiki/Mars JSPS ScienceWhat Dialogue, is cryobiology? 2009 A typical example. Travelling To http://www.rsc.org/chemistryworld/Issues/2006/May/MissionMarzzz.asp The Mars With “Slowed down Time” : Hibernating Like Hibernation A Brown Bear (冬眠) http://www.china.com.cn/news/zhuanti/xzk/txt/2006-11/16/content_7367944_3.htm http://www.appcrash.net/uploaded_images/hibernate_bear-751535.jpg http://img1.qq.com/luxury/pics/3737/3737672.jpg JSPS ScienceWhat Dialogue, is cryobiology? 2009 A typical example. http://news.xinhuanet.com/photo/2009-03/31/xin_5020306310904390105526.jpg Travelling To The Mars With Cryopreservation “Stopped Time” : (低温保存 ) Cryopreservation Of The Astronauts http://www.china.com.cn/news/zhuanti/xzk/txt/2006-11/16/content_7367944_3.htm http://www.appcrash.net/uploaded_images/hibernate_bear-751535.jpg http://img1.qq.com/luxury/pics/3737/3737672.jpg JSPS ScienceWhat Dialogue,is cryobiology? 2009 The Temperature Range. EliEvolution: 進化 /i/,i:və’lu:ʃən/ Microorganisms: 微生物 Adapted: 適応 Himalayan midge: 微生物の一種 Standard Physiological Temperatures for a Variety of Organisms (about -20~110oC) Typical Temperature Range for Cryobiology: <37oC~-196oC http://www.scq.ubc.ca/a-cold-greeting-an-introduction-to-cryobiology/ JSPS Science Dialogue,What 2009is cryobiology? Definition. Cryobiology --the branch of biology Natural Science that studies the effects of low temperatures on Engineering Biology living things. (工学) (生物学) --the Greek words "cryo" = cold, "bios" = life, and Clinical medicine (臨床医学) "logos" = science. Briefly, cryobiology is the study of life at low temperatures. JSPS46th Science Annual Dialogue, Meeting 2009 of the Society for Cryobiology CRYO 2009 covers a variety of topics in low temperature science and technology 1. CtiCryopreservation ofCf Ce llTill, Tissue, Organ and Genetic Resources 2. Cryosurgery and Cryomedicine 3. Cell, Tissue and Organ Banking 4. HthiPHypothermic Preserva tidtion and Transplantation 5. Hypothermia 6. Tissue Engineering 7. Therma l an d Mec han ica l Proper ties 8. Medical Devices in Cryobiology 第46回国際低温生物学会 9. Cryomicroscope and Cryo EM Techniques and Imaging 7月19日～7月23日 10. Struc ture o f W at er and I ce 札幌，日本 11. Cryoprotective Agents 12. Ice-Cell Interactions 13. Freezing, Supercooling, Glass Transition and Vitr ifica tion 17. Cold Acclimation 14. Desiccation, Anhydrobiosis, Lyophilization 18. Impact of Global Warming on Cryobiology and Freeze-Drying 19. Biological Adaptation to Cold and Freezing 15. Anti-Ice Nucleation Substances/Antifreeze 20. Ecology at Low and Freezing Temperatures Proteins and Glycoproteins 21. Cryobiology for Agriculture 16. Hibernation http://www.agr.hokudai.ac.jp/cryo09sapporo/ JSPS Science Dialogue,Major areas 2009 of study in cryobiology At least 6 major areas of study in cryobiology can be identified: 1. Study of cold-adaptation of microorganisms, plants (= cold hardiness), invertebrates, and animals (= hibernation). 2. Cryopreservation of cells, tissues, gametes, and embryos of animal and human origin for (medical) purposes of long-term storage. This usually requires the addition of substances which protect the cells during freezing and thawing (cryoprotectants). 3. Preservation of organs under hypothermic condit ions for transplantation. 4. Lyophilization (freeze-drying) of pharmaceuticals. 5. Cryosurgery, a (minimally) invasive approach for the destruction of unhealthyygygg tissue using cryogenic gases/fluids. 6. Physics of supercooling, ice nucleation/growth and mechanical engineering aspects of heat transfer during cooling and warming. 1/6 Cryopreservation of cells, tissues, organs and even whole bodies. 細胞や組織、器官、そして身体全体の凍結保存 http://en.wikipedia.org/wiki/Cryobiology JSPS Science Dialogue,Why we 2009 need cryopreservation? bridge arteries myocardldial infarction 心筋梗塞 (しんきんこうそく) Thrombus ['θrɔmbəs] 血栓 (けっせん) Coronary artery bypass surgery (artery transplantation ) http://upload.wikimedia.org/wikipedia/commons/2/2e/Liver_1.jpg JSPS Science Dialogue,Why we 2009 need cryopreservation? Organ transplantation (臓器移植[ぞうきいしょく]) Kidney and liver transplantation http://upload.wikimedia.org/wikipedia/commons/2/2e/Liver_1.jpg JSPS Science Dialogue,Why we 2009 need cryopreservation? The Urgent Demand for Cryopreservation of Organs and Tissues. PERSONAL RESEARCH STATEMENT 2007, John C. Bischof, Ph.D. JSPS Science Dialogue,Why we 2009 need cryopreservation? Tissue bank Organ bank Cryopreservation Human Cell, Tissue and Organ resources: Some people are willing to donate (寄付 (きふ) ) their tissues and organs after they die. http://www.transplantation.org.cn/zyienizhonghe/2009-04/3595.htm http://www.inmagine.com/pts0607/we060606-photo http://gb.cri.cn/15884/2007/04/26/[email protected] JSPS Science Dialogue,Why we 2009 need cryopreservation? Cryypopreservation: source of life http://jxgymy.cuepa.cn/show_more.php?doc_id=73356 JSPS ScienceWhy Dialogue, we can 2009 cryopreserve biomaterials? 20℃ 4oC ——> 2 hours n 化学 oo o 反 -40 C ——> several days eacti , k 応速度rr ee -80oC ——> several months rat ℃ 定 0 mical mical 数 ee -20℃ -150℃ o Ch -196 C ——>several centuries Temperature / (℃) What is the scientific theory 温度 behind cryopreservation? Conclusion: low temperature has the ability to “slow down time” or even “stopppgy time” from the viewpoint of biology. JSPS Science Dialogue,How to 2009cryopreserve biomaterials? Obtain and prune the resources Cryoprotective agent (CPA,凍結防止剤、耐凍剤) Loading of cryoprotective --a substance that is used to agents (CPAs) protect biological tissue from Freeze the samples, typically, freezing damage (damage due to -80 or -120oC to ice formation). Permeable: glycerol(グリセリン), DMSO…… Transfer the frozen samples to Impermeable: sugar(糖), PVP…… liqqg(uid nitrogen (液氮, LN2))( (- 196oC), and preserve for long term. Typical steps for cryypopreservation of biom aterials http://www.agr.kuleuven.ac.be/dtp/tro/cost871/Home.htm http://blog.bioethics.net/cryopreservation.jpg JSPS Science Dialogue,How to 2009cryopreserve biomaterials? Take the frozen sample out from LN2 container Rewarming of the frozen samples by several