TISSUE BANKING IN JAPAN CRYOPRESERVED HOMOGRAFT
NEW DEVICES
Noboru MOTOMURA1, Aya SAITO1,
Sumihito TAMURA2,
Toshio OBASE3, Katsuhiko UEDA3
#1 Toho University, Sakura Medical Center,
#2 Univ. of Tokyo Tissue Bank,
#3 Uedaseitai Co. Ltd.
IIR Workshop St Petersburg 2018, Univ. ITMO, Russia 2018/09/13
BACKGROUND Long-term preservation of Cryopreserved heart valve allografts (homografts)
packed in heat-sealed fluorinated ethylene propylene (FEP) film bag
storage under vapor phase of liquid nitrogen.
Shortage of those specific storage bag supply, obtained from limited industries abroad with high cost.
Sealing technique : (present) heat sealer using the specific bags
(alternative) CO2 laser
#1 secure closure using its high temperature (over 500 degree) beam
#2 flexibility in sealing line design (straight line, curved or circled line) #3 domestic supply of FEP bag for tissue cell preservation with reasonable cost
Heart valve tissue Storage(-196℃)
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Aortic Valve Allograft
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Artificial Valve & Graft
2012/06/23 Lecture in Dushanbe 4
Mechanical Valve
Bioprosthetic Valve
Artificial Graft
Mortality in Aortic Valve Replacement
With Severe* Infection
→→ 25%!
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Without Infection →→ 2%
*Severe: Aortic valve ring is destructed with infection
IIR Workshop St. Petersburg, Univ ITMO, Russia 2018/09/13
Replacement of Aortic Root
Aortic root structure (with coronary arteries)
Aortic valve allograft
Aortic valve
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IIR Workshop St. Petersburg, Univ ITMO, Russia 2018/09/13
Aortic Root Surgery for Infective Endocarditis PVE (Prosthetic Valve Endocarditis)
•Replacing severely destroyed aortic root with 3-D reconstruction •Anti-infection property of the allografts
Patient’s coronary arteries need to be reimplanted to the allograft
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Reinforcement of allograft muscle skirt with autopericardial strip
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Russia 2018/09/13 8
Drawing by Dr. Ehson KARIMOV
Operation views
Autopericardial strip attached to the allograft
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Russia 2018/09/13 9
After Surgery with Allograft
The gauze tightly pressed around proximal anastomosis
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Drawing by Dr. Ehson KARIMOV
Cryopreserved allograft aortic root
Aortic Allograft
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Replacement with Aortic Allograft
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Vein Allograft
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Vein Allografts in Surgery Liver Transplant (Living Donor)
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On the back table: Vein allografts, sutured with
donor liver veins
After recirculation
University of Tokyo Tissue Bank (UTTB)
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Russia 2018/09/13 15
PA (pulmonary artery) Aortic Valve
Thoracic Aorta SVC (superior vena cava)
Cardiac Tissue Allografts
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Disinfection with Antibiotic Solution
• RPMI1640 + Antibiotics (4℃、24~48H) • Cefmetazon 240μg/ml
• Lincomycin 120μg/ml
• Vancomycin 50μg/ml
• Polymixin 1000u/ml
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Russia 2018/09/13 17
Sampling for Bacteria Contamination
• Sampling for Bacteria Examination
Antibiotic Solution(24~48hr)
Sample ① on Procurement
Sample ③ from Antibiotic Solution Sample ② on Triming the
Tissue
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Cryopreservation
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Program Freezer Dr. KARIMOV !!
Program Freezing
Liquid Nitrogen Tank - 170 degree
Triming
10% DMSO RPMI 1640
Taking Out from Dry Shipper
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Thawing with Warm Sterile Saline
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Warm Betadine
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Rinsing with AlloFlo
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Checking Valve Function
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Special Bag for Cryopreservation and Sealing System with Laser Beam
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Laser welding vs heat sealing
Sealing line Innovative laser
welding method
Conventional
heat sealing
method
Welding
quality reliable less reliable
Thermal
damage no yes
Shrinkage no yes
Flexibility in
drawing line
straight, curved,
or circled line
limited to
straight line
Heat sink
sheet 1 sheet 2
laser
*Indirect heating by laser
*immediate surface cooling
by heat sink
Innovative laser welding
Conventional heat sealing
*Direct heating with hot plate
sheet 1 sheet 2
Hot plate
cooling
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OBJECTIVE To assess the quality of newly designed tissue(e.g. homograft)/cell (e.g. iPS cell)
storage bag for cryopreservation as domestic products by CO2 laser welding
method, which enables drawing any types of sealing line (straight, curved) under
any ambient temperature.
METHODS Laser welding system :
consisting of ‘CO2 laser oscillating unit (Firestar V30, Synrad
Co. Ltd, USA)’, ‘tabletop-type robot (w/ 2 axial operation +
welding jigs & tools)’, and ‘heat sink(Si)’
maximum A4 size for drawing design
Storage bag preparation : FEP films (0.1 mm thick)
Welding conditions : sealing width, laser intensity,
under fixed operating speed (5mm/s).
Assessment : quality of welding line, changes in neighboring film condition. 2018/09/13 IIR Workshop St. Petersburg, Univ ITMO, Russia 2018/09/13 27
controller
CO2 laser oscillating system
XY stage
Heat sink(Si)
Outlook of the whole system
(original)
Laser welding system
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<Preliminary assessment>
Make sure that laser welding is
applicable to FEP film
<Welding condition>
・heat sink:silicon(Si, 0.8mm thick)
・irradiation power:30 Watt (W) ×duty ratio
・spot diameter of laser beam: 2mm
・operating speed:5mm/s
・duty ratio:15~50%(4.5W~15W)
・bag sample(150×100mm) with straight / curved drawing
FEP film
CO2 laser
heat sink (Si)
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Russia 2018/09/13 29
RESULTS (pressure test)
laser intensity>10.5 (35%)
stability in sealing line against 20 Newton(N)
pressure system
Pressure
transuducer
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RESULTS (quality assessment)
10.5W (35%)
○
15W (50%)
△
4.5W (15%)
×
Stability and quality of welding line;
best achieved at 10.5W, acceptable at 13.5 W
Higher intensity ↓
whitening of sealing line
Lower intensity ↓
*Insufficient sealing
*
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RESULTS (structural assessment)
higher laser intensity : thermal damage with bubbling
at sealing line and neighboring film by polarizing microscope
Higher laser intensity
Overview
Sectional
view
Proper laser intensity
Whitening of sealing line
Thermal damage bubbling
Bag Component
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Further Innovation for Open to Public
・commercialize the technique
・assess heat-resisting property
(low ~ high temperature)
・achieve stable domestic supply
of the storage bag
・achieve more compact design
of the system
Updated laser welding system
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CONCLUSIONS
Tissue Bank in Japan is doing an important role in clinical field.
Cryopreservation technique and its quality are critical to
maintain the high level medical quality in Japan.
CO2 laser welding method is feasible in stability and strength of
the sealing line, and quality of the line was achieved when
performed by appropriate laser intensity.
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