Boram Kim and Sanghoon Lee Department of Obstetrics and Gynecology, Korea University College of Medicine, Seoul, Korea
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1 Comparison of slow freezing versus vitrification for human ovarian tissue cryopreservation and xenotransplantation Boram Kim and Sanghoon Lee Department of Obstetrics and Gynecology, Korea University College of Medicine, Seoul, Korea
2 CONTENTS Introduction Slow freezing SLIDES 5 Process SLIDES 6 Vitrification SLIDES 8 Process SLIDES 10 Results SLIDES 14 Xenotransplantation Purpose SLIDES 19 Method SLIDES 20 Results SLIDES 24 Conclusion SLIDES 30
3 Introduction 1 Cancer patients have been increasing steadily. 2 Chemotherapy and radiotherapy for the cancer patients can be induced ovarian deficiency, premature ovarian failure and infertility. 3 Young women who had cancer treatment are not only can preserve their fertility but also their ovary can be restored endocrine function.
4 Purpose 1 To demonstrate superior method btw slow freezing and vitrification. 2 To establish the safety and effectiveness of hot cryopreservation and transplantation using xenotransplantation model.
5 Slow freezing medium * Reagent Medium 199 Sigma Aldrich, catalog# M4530, 500ml DMSO Sigma Aldrich, catalog# D2650, 100ml SSS(Serum substitute Supplement) Irvine Scientific, catalog# 99193, 100ml * Method Solution 1 : 5% HSA(or SSS) supplemented M199(medium) Solution 2 : 10% DMSO + 5% HSA(or SSS) supplemented M199 Solution 3 : 12.5% DMSO + 5% HSA (or SSS) supplemented M199
6 Slow freezing process Method 1 1. Tissue selection 2 2. Tissue cutting 3 3. Add sol. 1, 2, 3
7 4 4. Slow freezing setting 5 5. Seeding process 6 6. Store in the LN tank
8 Vitrification medium * Reagent HEPES(1M) Gibco, , 100ml Ethylene Glycol DMSO(dimethyl sulphoxide) Sigma Aldrich, catalog# D2650, 100ml Sucrose Sigma Aldrich, catalog# S1888, 500g SSS(Serum substitute Supplement) IrvineScientific, catalog# 99193, 100ml
9 Vitrification medium * Equilibration Solution(ES) 65ml HEPES supplemented 7.5ml Ethylene Glycol 7.5ml DMSO 20ml SSS(or SPS) (Total concentration: 7.5% EG, 7.5% DMSO, 20% SSS in solution) * Vitrification Solution(VS) 27.5ml HEPES 17.12g Sucrose 20ml Ethylene glycol 20ml DMSO 20ml SSS(or SPS) (Total concentration: 20% EG, 20% DMSO, 20% SSS, 0.5M sucrose in solution)
10 Vitrification process Method 1 1. ES treatment 2 2. Remove solution 3 3. VS treatment
11 4 4. Remove solution 5 5. Put tissues on strainer 6 6. Plunge into LN
12 Process Slow-thawing Vitri-thawing SCID mouse Frozen-thawed tissue
13 Process Human ovarian tissue Punctured with Sectioning Final tissue diameter 4mm (2µm)
14 Hematoxylin & Eosin stain Results Fresh Slow-thawed Vitri-thawed Follicle growth with Hematoxylin and eosin staining after thawing process.
15 γ-h2ax Results Fresh Slow-thawed Vitri-thawed Histological featues of double-strand breaks in the deoxyribonucleic acid.
16 Transmission Electron Microscope Results Slow-thawed primordial follicle Vitri-thawed primordial follicle
17 Western blot Results β -actin Caspase3 Cleaved caspase3 Fresh Slow Vitri
18 Process Slow-thawed Vitri-thawed Human ovarian tissue SCID mouse Frozen-thawed tissue
19 Xenotransplantation * Objective The purpose of this study was to compare the cryopreservation methods and establish safety and effectiveness of human ovarian tissue transplantation using xenotransplantation model. * Material Ovarian tissues were obtained from 15 patients who underwent benign ovarian surgery with informed consent and IRB approval (IRB No.: ED11138) Human ovarian tissues were equally divided and prepared for the slow freezing and vitrification. Frozen thawed human ovarian tissues were transplanted into back muscle of about 100 SCID mice (1 st 40+2 nd 51) 4 weeks after cryopreservation, which were ovariectomized prior to the xenograft.
20 Xenotransplantation Method 1 1. SCID mouse 2 2. Respiratory anesthesia 3 3. Human ovarian tissue
21 4 4. Ovariectomy 5 5. Xenotransplantation 6 6. Tissue retreival
22 Tissue retrieval Slow freezing vitrification
23 Tissue retrieval The Follicle growth and angiogenesis after transplantation
24 Hematoxylin & Eosin stain Results A1 A2 Follicle growth with Hematoxylin and eosin staining after transplantation. B1 B2 A- Slow-thawed and transplanted B- Vitri-thawed and transplanted
25 Ovarian follicle count Results 20 Number of primordial follicle Fresh Slow freezing Vitrification Transplantation after slow freezing Transplantation after vitrification
26 Ki67 Results A1 A2 Histological featues of cell proliferation B1 B2 A- Slow-thawed and transplanted B- Vitri-thawed and transplanted
27 CD31 Results A1 A2 Histological featues of Angiogenesis B1 B2 A- Slow-thawed and transplanted B- Vitri-thawed and transplanted
28 Merge TUNEL DAPI TUNEL assay Results Control Treated A1 B1 C1 D1 E1 A2 B2 C2 D2 E2 A3 B3 C3 D3 E3 A- Fresh tissue B- Slow-thawed C- Vitri-thawed D- Slow-thawed and transplanted E- Vitri-thawed and transplanted
29 Western blot Results β -actin AMH Transplantation and vitri Transplantation and slow Vitrification Slow freezing Fresh
30 Conclusion Slow freezing for ovarian tissue cryopreservation was superior to vitrification * in terms of the follicle growth and histologic feature of ovarian tissues after xenotransplantation. * More research is needed to improve vitrification results in the future. * We believe that this study will provide useful information for reproductive women with cancer who need cryopreservation for fertility preservation.
31 ! Thank you for your attention Supported by the National Research Foundation of Korea Grant funded by the Korean Government. (NRF-2016R1C1B )
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