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

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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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Drawing by Dr. Ehson KARIMOV

Operation views

Autopericardial strip attached to the allograft

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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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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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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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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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