Cryopreservation of Cells and Tissues: Approaches and Applications

JSPS Science Dialogue, 2009

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 stud ies? 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 techhlnologies •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. Thermal 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. Struct ure of 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 steps

Removal of CPAs from the thawed samples

Clinical applications

Necessary steps for thawing of biomaterials and preparation of clinical use 37oC Water Bath 37oC 恒恒水浴温水浴

http://www.agr.kuleuven.ac.be/dtp/tro/cost871/Home.htm http://blog.bioethics.net/cryopreservation.jpg http://gb.cri.cn/15884/2007/04/26/[email protected] JSPS Science Dialogue, 2009

CPA Loading Process JSPS Science Dialogue,Loading 2009 of an impermeant CPA JSPS Science Dialogue,Loading 2009 of a permeant CPA JSPSCell Science volume Dialogue, change 2009 during loading/unloading of CPA

Cell volume tolerance limits during CPA addition and removal JSPS Science Dialogue, 2009

Freezing process JSPS Science Dialogue,Cell 2009freezing in CPA solution JSPS Science Dialogue, 2009Inverted “U” shape

赤血球幹細胞精子生存率

、残存

率、活着酵母細胞

率

冷却速度

http://www.benbest.com/cryonics/cooling.html JSPS ScienceWhy Dialogue, there 2009 is an optimal freezing rate?

You hurt me. Optimal

If the freezing process is too slow, the cell will lose too much water and its volume will become too small , it will suffer from severe “Solution Injjyury”

http://www.scq.ubc.ca/a-cold-greeting-an-introduction-to-cryobiology/ The first picture was selected from Prof. DY Gao’s ppt file JSPS Science Dialogue, 2009

Why Optimal it’s me?

If the freezing is too fast, the cell will lose too little water and the water kept in the cell will become ice, its volume may not change or even become bigger, it will suffer from severe “ Intracellular Ice Injury”

http://www.scq.ubc.ca/a-cold-greeting-an-introduction-to-cryobiology/ The first picture was selected from Prof. DY Gao’s ppt file JSPS Science Dialogue, 2009

I am

Optimal Lucky.

If the freezing is not too slow and too fast, it will lose enough water while still keep some water, its volume become smaller but not too small, it will suffer from minor “Solution Injury” and “ Intracellular Ice Injury” .

http://www.scq.ubc.ca/a-cold-greeting-an-introduction-to-cryobiology/ The first picture was selected from Prof. DY Gao’s ppt file JSPS Science Dialogue,Cell specified 2009 optimal freezing rate

http://upload.wikimedia.org/wikipedia/commons/9/93/Cryopreservation.jpg http://www.scq.ubc.ca/a-cold-greeting-an-introduction-to-cryobiology/ JSPS ScienceTwo typical Dialogue, events 2009 during cell cryopreservation

Movie 1: Freezing of a mouse oocyte

The movie is selected from Prof. DY Gao’s ppt JSPS ScienceTwo typical Dialogue, events 2009 during cell cryopreservation

freezing CPA Loadi ng

Control the water transport speed

Control Best these two cryopreservation things outcomes

The effective approach to optimize cryopreservation

Two i mportant thi ngs: (1) Ice formati on, (2 ) C ell volume change JSPS Science Dialogue, 2009

Different cells have different optimal cooling rates

Different membranes have different ability for water transport

Different cells have different membranes

Conclusion: To study the ability of cell membrane for water transport is very important https://eapbiofield.wikispaces.com/file/view/plasma_membrane.gif http://www.benbest.com/cryonics/cooling.html http://en.wikipedia.org/wiki/Cell_(biology) JSPS Science Dialogue, 2009

1 Lp

Volume 090.9 0.8 d Cell 0.7

malize 0.6 rr

No Weight Height 0.5 体重身長 0 50 100 150 200 Time (s)

Question: how to measure the cell membrane permeability (the ability of the membrane for water transport) ? Fortunately, the cryobiologists found that each cell has its own hydraulic condtiitductivity Lp va lue (m3/(m2.s.Pa)) (like you, you have your we ig ht and hei ght)

http://www.diseaseproof.com/Weight.jpg JSPSThe Science Perfusion Dialogue, Microscope 2009 Developed by Prof. Takamatsu ---- a Powerful Tool for the Study of Osmotic Response of Cells

Takamatsu et al. Biomech. Eng. 2004, 126(4): 402-409 JSPSThe Science Perfusion Dialogue, Microscope 2009 Developed by Prof. Takamatsu ---- a Powerful Tool for the Study of Osmotic Response of Cells

Depth of the chamber is only 50um (0.05mm)

Takamatsu et al. Biomech. Eng. 2004, 126(4): 402-409 JSPS Science Dialogue, 2009

Movie 2: HH cell volume change (from 015M0.15M NaCl to 005M.5M NaCl solution)

0.15 mol/L 塩水 Æ 0.5 mol/L 塩水 JSPS Science Dialogue, 2009

t3 Photo of a cell at time t PjProjec tdted area o fthf the ce ll

Area: A=4πr2 Volume: V=(4/3)πr3 A=A0 Î V=?? JSPS ScienceLp Dialogue, value 2009 of a typical HH cell at 23oC

HH Cell #01, 23oC 1.05 Exp Cal 1 ) 0 0.95 (V/V

0.9 ell Volume Volume ell CC 0.85

rmalized rmalized 0.8 oo N

0.75 Lp=4.86x10-14 m/Pa/s

0.7 0 5 10 15 20 25 30 35 40 45 Time (s) JSPS ScienceOptimization Dialogue, 2009 of CPA Addition or Removal 0 M

0 M 1.7 1.6 1.5 2 Steps 1.4 3 Steps olume 1.3 1 Steps 0.5 M VV 1.2 0.75 M 1.1

ed Cell Cell ed zz 101.0 0.9

Normali 0.8 1.0 M 0.7 1.5 M 0.6 0.5 0 50 100 150 200 250 300 Two steps Time (s) 1.5 M Three steps JSPS Science Dialogue,Optimization 2009 of freezing process

0.405 Intracellular

o Ice Volume 0.4 3.6 C/min 4.8oC/min

w0 o

/V 0.395 6 C/min w o VV 10 C/min 0.39 24oC/min o 60 C/min 10 0.385 100oC/min 3.6oC/min C =8mol/L o g o 1000 C/min 8 4.8 C/min 0.38 o 0 -20 -40 -60 -80 -100 -120 6 C/min

Temperature [oC] 8 10oC/min 6 o

)x10 24 C/min cv 60oC/min /V

ice 4 100oC/min (V 1000oC/min 2 Intracellular C =8mol/L g 0 Water Volume 0 -20 -40 -60 -80 -100 -120 Temperature [oC] JSPS ScienceExamples Dialogue, of2009 Successful Cryopreservation

Many cells: RBC, sperm, mouse oocyte, embryo, stem cells, et al.

Some Tissues: blbldood vesse ls, skin, cornea, tdtendon, e t al.

http://health.howstuffworks.com/sunscreen1.htm http://www.thirdeyehealth.com/cornea.html http://www.heartsupplement.com/ http://www.stem-cell-blog.com/ http://commons.wikimedia.org/wiki/File:Embryo,_8_cells.jpg http://www.the-fitness-motivator.com/achilles-tendonitis.html http://encarta.msn.com/media_461516398/two_human_sperm_cells.html http://www.techsupportforum.com/relaxation-room/offline/148518-bigger-game.html JSPS ScienceExamples Dialogue, 2009 of Failed Cryopreservation

Very few cells: Nearly all the bodies: human bodies, human oocyte, et al. animal bodies, et al.

Nearly all the organs: human kidney, human liver, et al. Can only be kept at hypothermic temperatures for several hours

http://www.cnhands.com/html/news/1046/2008-2-2/news_1911.shtml http://health.allrefer.com/pictures-images/bile-produced-in-the-liver.html

http://www.the-scientist.com/blog/display/55612/ http://www.bio.psu.edu/people/faculty/strauss/anatomy/urogen/modelkidney.htm JSPS Science Dialogue, 2009

How about human cryopreservation? JSPS ScienceThe Dialogue, largest 2009 cryonics organization today

As of May 31, 2009, Alcor had 891 members, and 85 patients in cryopreservation.

Alcor Life Ext ensi on Foun da tion

The largest cryonics organization today, in terms of membership, was established by Fred and Linda Chamberlain in 1972 as the Alcor Society for Solid State Hypothermia (ALCOR).

http://www.alcor.org/AboutAlcor/membershipstats.html http://en.wikipedia.org/wiki/Alcor_Life_Extension_Foundation http://www.alcor.org/notablequotes.html http://www.alcor.org/ JSPS ScienceThe Dialogue, oldest 2009 cryonics organization today

American Cryonics Society

Mon thly St at us U pd at e J une 1 , 2009 Total Members: 797 Human Patients in Cryostasis: 93 Human Tissue/DNA Samples in Cryostasis: 157 Pets in Cryostasis: 60 (ペット ) Pet Tissue/DNA Samppyles in Cryostasis: 31

http://www.cryonics.org/ JSPS Science Dialogue, 2009 Human cryopreservation Today's Dream — Tomorrow's Reality

It is difficult to say what is impossible, for the dream of yesterday is the hope of today and the reality of tomorrow. - Robert H. Goddard

Robert Hutchings Goddard (1882-1945) : U.S. professor of physics and scientist, was a pioneer of controlled, liquid-fueled rocketry. He launched the world‘s first liquid-fueled rocket (液体燃料ロケット ) on March 16, 1926.

http://en.wikipedia.org/wiki/Robert_Goddard_(scientist) JSPSWhat Science the experts Dialogue, said 2009 impossible in the past is now possible.

"There is no h ope f or th e f ancif ul id ea of reaching the Moon because of insurmountable barriers to escaping the Earth's gravity . " — Dr. Forest Ray Moulton, University of Chicago astronomer, 1932.

"All this writing about space travel is utter bilge." — Sir Richard Woolley , Astronomer Royal of Britain, 1956

"To place a man in a multi-stage rocket and project him into the controlling gravitational field of the moon.... I am bold enough to say that such a man-made voyage will never occur regardless of all future advances." Buzz Aldrin, — Dr. Lee De Forest,,g, famous engineer, 1957 July, 1969 http://en.wikipedia.org/wiki/Buzz_Aldrin http://www.alcor.org/notablequotes.html JSPS Science Dialogue, 2009 Than k you for your attenti on! ご静聴ありがとうございます

Where there is a dream, there is a future.