JSPS Science Dialogue, 2009 Cryopreservation of Cells and Tissues: Cryopreservation of Cells and Tissues: Approaches and Applications Gang Zhao PhD JSPS Research Fellow Department of Mechanical Engineering Science 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 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
Transcript
JSPS Science Dialogue, 2009
Cryopreservation of Cells and Tissues: Cryopreservation of Cells and Tissues: Approaches and Applications
Gang Zhao PhDJSPS Research Fellow Department of Mechanical Engineering Science 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 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
OutlineOutlineSelf introduction
1) Who am I?2) Where I come from?
Research works1) Why I become a natural science researcher?2) Wh f f h d2) Why I come to Japan for further studies?3) What’s cryobiology?4) Why we need cryopreservation?y y p5) Why we can cryopreserve biomaterials?6) How to cryopreserve biomaterials? 7) Two typical events during cell cryopreservation.7) wo typ cal events dur ng cell cryopreservat on.8) Cell specified optimal freezing rate.9) Measurement of hydraulic permeability by perfusion microscope.
10) Status quo and future of cryopreservation10) Status quo and future of cryopreservation.
JSPS Science Dialogue, 2009
Self introductionSelf introduction
http://www.kyushu-u.ac.jp/
Kumamoto Prefectural Daini High School, Friday, June 26, 2009
JSPS Science Dialogue, 2009Who am I? My Institution.
Full Name: Gang Zhao PhD 趙剛 (チヨウ ゴウ)
P t St tPresent Status: 1. JSPS Research Fellow
Kyushu University, Japan本 会 特 究日本学術振興会 外国人特別研究
員九州大学, 日本
2. Associate ProfessorUniversity of Science and
Technology of China, China副教授
中国科学技术大学, 中国
M jMajor:Cryobiology
JSPS Science Dialogue, 2009Where I come from? My County: China.
http://ditu.google.cn/
JSPS Science Dialogue, 2009
Research WorksResearch Works
http://www.kyushu-u.ac.jp/
Kumamoto Prefectural Daini High School, Friday, June 26, 2009
JSPS Science Dialogue, 2009Why I become a natural science researcher?
h TV i 1980 i Chi• 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 • The leading actress died from Leukemia (白血病, a cancer of the blood or bone marrow)->all the spectators were very sorrowful.
Typical Temperature Range for Cryobiology: <37oC~-196oC
JSPS Science Dialogue, 2009What is cryobiology? Definition.
Cryobiology
--the branch of biology --the branch of biology that studies the effects of low temperatures on
Natural Sciencep
living things.
--the Greek words "cryo"
Engineering(工学)
Biology(生物学)
the Greek words cryo = cold, "bios" = life, and "logos" = science. Clinical medicine
(臨床医学)
Briefly, cryobiology is the study of life at low
( )
ytemperatures.
JSPS Science Dialogue, 200946th Annual Meeting of the Society for CryobiologyCRYO 2009 covers a variety of
1 C ti f C ll Ti O
CRYO 2009 covers a variety of topics in low temperature science and technology1. Cryopreservation of Cell, Tissue, Organ
and Genetic Resources2. Cryosurgery and Cryomedicine3. Cell, Tissue and Organ Banking4 H th i P ti d4. Hypothermic Preservation and
Transplantation5. Hypothermia6. Tissue Engineering7 Th l d M h i l P ti7. Thermal and Mechanical Properties8. Medical Devices in Cryobiology9. Cryomicroscope and Cryo EM Techniques
and Imaging10 St t f W t d I
第46回国際低温生物学会7月19日～7月23日札幌 日本10. Structure of Water and Ice
and Vitrification14. Desiccation, Anhydrobiosis, Lyophilization
and Freeze-Drying15. Anti-Ice Nucleation Substances/Antifreeze
Proteins and Glycoproteins
17. Cold Acclimation18. Impact of Global Warming on Cryobiology19. Biological Adaptation to Cold and Freezing20. Ecology at Low and Freezing Temperatures
Proteins and Glycoproteins16. Hibernation
http://www.agr.hokudai.ac.jp/cryo09sapporo/
21. Cryobiology for Agriculture
JSPS Science Dialogue, 2009Major areas 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) 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 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 conditions for transplantation. 3. Preservat on of organs under hypotherm c cond t ons for transplantat on. 4. Lyophilization (freeze-drying) of pharmaceuticals. 5. Cryosurgery, a (minimally) invasive approach for the destruction of
unhealthy tissue using cryogenic gases/fluids. y g y g g6. Physics of supercooling, ice nucleation/growth and mechanical
engineering aspects of heat transfer during cooling and warming.
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
1/6
http://en.wikipedia.org/wiki/Cryobiology
細胞や組織、器官、そして身体全体の凍結保存
JSPS Science Dialogue, 2009Why we need cryopreservation?
d l fbridge arteries myocardial infarction
心筋梗塞 (しんきんこうそく)
bridge arteries
Thrombus ['θrɔmbəs] 栓血栓 (けっせん)
Coronary artery bypass surgery (artery transplantation )
JSPS Science Dialogue, 2009Loading of an impermeant CPA
JSPS Science Dialogue, 2009Loading of a permeant CPA
JSPS Science Dialogue, 2009Cell volume change during loading/unloading of CPA
Cell volume tolerance limits during CPA addition and removal Cell volume tolerance limits during CPA addition and removal
JSPS Science Dialogue, 2009
Freezing processFreezing process
JSPS Science Dialogue, 2009Cell freezing in CPA solution
JSPS Science Dialogue, 2009Inverted “U” shape
精子赤血球幹細胞
生存率
酵
、残存率
酵母細胞率、活着率率
冷却速度
http://www.benbest.com/cryonics/cooling.html
冷却速度
JSPS Science Dialogue, 2009Why there is an optimal freezing rate?
You
Optimalhurt me.
If the freezing process is too slow the If the freezing process is too slow, the cell will lose too much water and its
l ill b t ll it ill volume will become too small, it will suffer from severe “Solution Injury”j y
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 Why it’s me?Optimal
If the freezing is too fast the cell will lose 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 cell will become ice, its volume may not change or even become bigger, it will suffer from severe “Intracellular Ice Injury”from severe Intracellular Ice Injury
The first picture was selected from Prof. DY Gao’s ppt filehttp://www.scq.ubc.ca/a-cold-greeting-an-introduction-to-cryobiology/
JSPS Science Dialogue, 2009
I am kLucky.
Optimal
If the freezing is not too slow and too fast, it gwill 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”.Injury and Intracellular Ice Injury .
The first picture was selected from Prof. DY Gao’s ppt filehttp://www.scq.ubc.ca/a-cold-greeting-an-introduction-to-cryobiology/
Question: how to measure the cell membrane permeability (the ability of the 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 d ti it L l ( 3/( 2 P )) (lik h i ht d h i ht)conductivity Lp value (m3/(m2.s.Pa)) (like you, you have your weight and height)
http://www.diseaseproof.com/Weight.jpg
JSPS Science Dialogue, 2009The Perfusion Microscope 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
JSPS Science Dialogue, 2009The Perfusion Microscope Developed by Prof. Takamatsu---- a Powerful Tool for the Study of Osmotic Response of Cells
Depth of the chamber pis 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 0 15M NaCl to 0 5M NaCl solution)0.15M NaCl to 0.5M NaCl solution)
0.15 mol/L 塩水 0.5 mol/L 塩水
JSPS Science Dialogue, 2009
t1t1
t2
P j t d f th llt3 Photo of a cell at time t Projected area of the cellt3
Area: A=4πr2
Volume: V=(4/3)πr3Volume: V=(4/3)πr3
A=A0 V=??
JSPS Science Dialogue, 2009
HH Cell #01 23oC
Lp value of a typical HH cell at 23oC
1
1.05HH Cell #01, 23 C
ExpCal
0.95
(V/V
0)
0.9
Cel
l Vol
ume
0.8
0.85
orm
aliz
ed C
0.75
No
Lp=4.86x10-14 m/Pa/s
0 5 10 15 20 25 30 35 40 450.7
Time (s)
JSPS Science Dialogue, 2009
0 MOptimization of CPA Addition or Removal
1.61.7
0 M
0 M
1.31.41.5
2 Steps 3 Steps1 StepsV
olum
e
0.5 M
1 01.11.21.3
1 Steps
zed
Cel
l V
0.75 M
0.80.91.0
Nor
mal
iz
1.0 M
0 50 100 150 200 250 3000.50.60.71.5 M
0 50 100 150 200 250 300
Time (s) 1.5 MTh t
Two steps
Three steps
JSPS Science Dialogue, 2009Optimization of freezing process
0.405
Intracellular Ice Volume
0.395
0.4
Vw
/Vw
0
3.6oC/min4.8oC/min6oC/min10oC/min
Ice Volume
0.385
0.39
V
Cg=8mol/L
10 C/min24oC/min60oC/min100oC/min
10
3.6oC/mino
-120-100-80-60-40-2000.38
Temperature [oC]
g
1000oC/min
6
8
)x10
8
4.8oC/min6oC/min10oC/min24oC/min
4
(Vic
e/Vcv
60oC/min100oC/min1000oC/min
-120-100-80-60-40-2000
2Cg=8mol/L
Intracellular Water Volume
Temperature [oC]
JSPS Science Dialogue, 2009Examples of Successful Cryopreservation
Many cells: RBC, sperm, mouse oocyte, embryo, stem cells, et al.
S Ti bl d l ki t d t lSome Tissues: blood vessels, skin, cornea, tendon, et al.
American Cryonics SocietyAmerican Cryonics Society
M thl St t U d t J 1 2009Monthly Status Update June 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 Samples in Cryostasis: 31
http://www.cryonics.org/
p y
JSPS Science Dialogue, 2009
H iHuman cryopreservationToday's Dream — Tomorrow's Reality
It is difficult to say what is It is difficult to say what is impossible, for the dream of
yesterday is the hope of yesterday is the hope of today and the reality of
tomorrowtomorrow.- 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 1926liquid fueled rocket (液体燃料 ロケ ト) on March 16, 1926.
What the experts said impossible in the past is now possible.
"There is no hope for the fanciful idea of reaching the Moon because of insurmountable barriers to escaping the Earth's gravity "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— 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
Buzz Aldrin, July 1969
field of the moon.... I am bold enough to say that such a man-made voyage will never occur regardless of all future advances."— Dr. Lee De Forest, famous engineer, 1957 July, 1969 , g ,
