Tülay İREZ, Mustafa TAŞYÜREKLİ, Özlem Tuğçe ÇİLİNGİR, Sema Bilgiç GAZİOĞLU

Kordon kanı hücrelerinin saklanmasında vitrifikasyon metodu

Amaç: Çeşitli hastalıkların tedavisinde uygulayabilmek amacı ile kök hücrelerin saklanarak sürekli bir kök hücre bankasında toplanması fikri giderek yaygın hale gelmektedir. Saklanma sırasında hücre kaybının en aza indirilmesi için kullanılan metotların geliştirilmesi gerekmekledir. Çalışmamızda, kordon kanlarından ayrılan mononükleer hücrelere farklı saklama yöntemleri uygulayarak, yapılan karşılaştırmalar sonucunda hangi yöntemin daha verimli olduğunu belirlemek amaçlanmıştır. Yöntem: Gönüllü vericilerden alınan kordon kanlarından mononükleer hücreler, yoğunluk derecelendirme yöntemi kullanılarak ayrıldı. Biri tek aşamalı diğeri iki aşamalı olarak 1 ayrı vitrifikasyon metodu uygulandı. Vitrifikasvon öncesi ve sonrasında İri pan mavisi boyama yöntemi ile luic.re canlılığındaki, akım silomelri tekniği kullanılarak CD34+,CD45+ ve CD34+45+ hücre oranlarındaki, ışık mikroskobu kullanarak morfolojide meydana gelen farklılıklar belirlendi. Bulgular: Elde ettiğimiz bulgular sonucunda, dondurma öncesi ve sonrasında CD45+,CD34+,CD34+/45+ hücre oranlarında anlamlı düşüşler gözlendi. İstatistiksel değerlendirmelerde, uygulanan iki farklı vitrifikasyon metodu arasında anlamlı bir fark görülmedi. Yapılan ışık mikroskobi değerlendirmelerinde ise hücrelerin dondurma sonrasında membran yapılarının bozulduğu, hücrelerde şişmeler oluştuğu ve hücre kümeleri meydana geldiği görüldü. Sonuç: Çalışmamızda vitrifikasyon sonrası hücre kayıpları saptansa da kordon kanı hücrelerinin saklanmasında zamandan ve maddi açıdan tasarruf sağlayacak vitrifikasyon tekniklerinin kullanılabilirliğinin sağlanmasının, kordon kanı bankaları ve diğer kök hücre depolama merkezleri açısından da kullanışlı olacağı açıktır.

Vitrification method in banking of cord blood mononuclear cells

Objectives: The idea of protection of stem cells in a cord blood bank for treatment of various diseases, becomes more widespread.The methods when used during the protection, must be developed for minimizing the risk of cell lost. According to our purpose, we made a comparison by using different protection methods for the cord blood stem cells, during the experiments. Methods: "Density-gradient" method was used for mononuclear cell separation from cord blood, which is taken from voluntary donors. We applied 2 different vitrification method. One of them consist of two steps. Before and after the freezing process, cell viabilities by trypan blue dye method, differences in CD34+,CD45+ and CD34+45+ cells ratio by flow cytometry method and, differences in morphology by light microscope had been determinated. Results: According to our experiment results. CD45+,CD34+ and CD34+/45+ cell ratios significantly decreased alter vitrification processes. But there is no significant differences between 2 different vitrification method, we used. In the light microscopy analysis, cell membran structures have damaged, cells have seen like swell and cells come together and have seen attaching. Conclusion: Vitrification methods are usefull for cell banking because of their short, cheap and easy process. We are thinking that our study is helpful for improving new and suitable methods.

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1. Okumuş N. Kordon kanı mononükleer hücrelerinin fenotipik karakterizasyonu ve NK aktivitesi. İstanbul Üniversitesi Sağlık Bilimleri Enst. İmmünoloji Yüksek , . Lisans Tezi. İstanbul, 2003.
2. Broxymeyer HE, Smith FO. Cord blood stem cell transplantation. In: Thomas D, Blume KG, Forman SJ, eds. Hematopoietic cell transplantation. 2nd ed. London: Blackwell; 1999. pp. 431-443.
3. Nash RA. Hematopoietic stem cell transplantation. In: Lee GR, Forester J, Lukens J, Parakevas F, Greer JP,Rodgers GM, eds. Wintrobe's Clinical Hematology. 10th ed. Baltimore: Williams and Wilkins; 1999. pp. 875-896
4. Ramsey NKC. Bone marrow transplantation pediatric oncology. Pizzo PA, Poplack DG, eds. In Principle and Practice of Pediatric Oncology. 2nd ed. Philadelphia; Lippincott; 1993. pp. 314-344.
5. Armitage JO. Bone marrow transplantation. N Engl J Med 1994; 330: 827-838.
6. Han P, Hodge G, Story C, Xu X. Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: releveance to umbilical cord blood transplantation. Br J Haematol 1995; 89: 733-740.
7. Almici C. Biologic and phenotypic analysis of early hematopoietic progenitor cells in umbilical cord blood. Leukemia 1997; 11: 2143-2149.
8. Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of trans¬plantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86: 3828-3832.
9. Wagner JE, De Fort T, Rubinstein P, et al. Transplantation of unreleated donor umbilical cord blood: outcomes and analysis of risk factors. Blood 1997;90:398-402.
10. Laughlin MJ, Baeker Jl, Bambach B, et al. Hematopoietic engrafment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001; 344: 1815-1822.
11. Barker JN, Wagner JE. Umbilical cord blood transplantation: current state of the art. Curr Opin Oncol 2002; 14:160-164.
12. Gluckman E, Rocha V, Boyer Chammard A, et al. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. Outcome of cord- blood transplantation from related and unrelated donors. N Engl J Med 1997; 337: 373-381.
13. Mc Guckin CP, Forraz N, Baradoz MO, et al. Production of stem cells with embryonic characteristics from human umblical cord blood. Cell Proliferation 2005; 38: 245.
14. Dooley DC, Oppenlander BK, Xiano M. Analysis of primitive CD34- and CD34+ hematopoietic cells from adults: gain and loss of CD34 antigen by undifferanti- ated cells are closely linked to proliferative status in culture. Stem Cells 2004; 22: 556-569.
15. Goodel M. CD34- or CD34+: does it really matter? Blood 1999; 94: 2545-2547.
16. Chang H, Jensen L, Quesenberry P, et al. Standardizations of hematopoietic stem cell assays: a summary of workshops and working group meeting sponsored by National Heart Lung and Blood Institute and held at National Institutes of Health, Bethesta, MD, on September 8-9, 1998 and July 30, 1999. Exp Hematol 2000; 28: 743-752.
17. Civin CI, Almeida-Porada G, Lee MJ, et al. Sustained, retransplantable multilineage engraftment of highly purified bone marrow adult stem cells in vivo. Blood 1996; 88: 4102-4109.
18. Michallet M, Thiebaut A, Philip I, et al. Transplantation with selected autologous peripheral blood CD34+ Thy1+ hematopoietic stem cells (HSC) in multiple myeloma: impact of HSC dose on engraf- ment, safety and immun reconstitution. Exp Hematol 2000; 28: 858-870.
19. Kreissig K, Krisch A, Serke S. Characterization and measurement of CD34- expressing haemopoietic cells. J of Haemattherapy 1994; 3: 263-289.
20. Janowska-Wieczorek A, Majka M, Ratajczak J, Ratajczak MZ. Autocrine/paracrine mechanisms in human hematopoiesis. Stem Cells 2001; 19: 99-107.
21. Sogo S, Inaba M, Ogata H, et al. Inductiin of c-kit molecules on human CD34+/c-kit low cells: evidence for CD34+/c-kit low cells as primitive hematopoietic stem cells. Stem Cells 1997; 93: 2302-2307.
22. Süzergöz F. Büyüme faktörleriyle uyarılmış kordon kanı mononükleer hücrelerinin in vitro kültürlerde proliferasyonu ve immünfenotipik özelliklerinin incelenmesi. İstanbul Üniversitesi Sağlık Bilimleri Ens., İmmünoloji Doktora Tezi. İstanbul, 2002.
23. Simmons PJ, Torok Storb B. CD34 expression by stromal precursors in normal human adult bone marrow. Blood 1991; 78: 2848-2853.
24. Civin CI, Banquerigo ML, Strauss LC, et al. Antigenic analysis of hematopoiesis flow cytometric characterization of My 10 positive progenitor cells in normal human bone marrow. Exp Hematol 1987; 15: 10-17.
25. Pasino M,Lanza T, Marotta F, et al. Flow cytometric and functional characterization of AC133 cells from human umbilical cord blood. BJ Haematol 2000; 108: 793-800.
26. Dhot Col PS, Sirohi Maj D, Swamy Brig GLN. Collection, separation, enumeration and cryopreservation of umbilical cord blood haematopoietic stem cells- an experimental study. MJAFI 2003; 59: 298-301.
27. Meyer TPH, Hofmann B, Zaisserer J, et al. Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood. Cytotherapy 2006; 8: 265-276.
28. Shreffler WG, Visness CM, Burger M, et al. Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multi- center study. BMC Immunology 2006; 7: 29.
29. Chivu M, Diaconu CC, Bleotu C, Alexiu I, Brasoveanu L, Cerescu C. The comparison of different protocols for expansion of umbilical cord blood hematopoetic stem cells. J Cell Mol Med 2004; 8: 223-231.
30. Todaro AM, Pafumi C, Pernicone G, et al. Haematopoietic progenitors from umbilical cord blood. Blood Purif 2000; 18: 144-147.
31. Kekarainen T, Mannelin S, Laine J, Jaatinen T. Optimization of immunmagnetic separation for cord blood-derived HSCs. BMC Cell Biology 2006; 7: 30.
32. Cairo MS, Wagner JE. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood 1997; 90: 4665-4978.
33. Moezzi L, Pourfathollah AA, Alimoghaddam K, Soleimani M, Ardjmand AR. The effect of cryopreservation on clonogenic capacity and in vitro expansion potential of umbilical cord blood progenitor cells. Transplan Proc 2005; 37: 4500-4503.
34. Ryu KH, Shin HY, Ahn HS, Kim YJ, Woo SY, Seoh JY. Comparison of efficiency of ex vixo expansion of whole blood mononuclear cells and purified CD34+ cellsfrom human umbilical cord blood. Hematologica 2004; 89: 606-607.
35. Abboud M, Xu F, LaVia M, et al. Study of early hematopoietic precursors in human cord blood. Exp Hematol 1992; 20: 1043-1047.
36. Sousa T, Sousa ME, Godinho MI, et al. Umbilical cord blood processing: Volume reduction and recovery of CD34+ cells. Bone Marrow Transplant 1997; 19:311- 313.
37. Yang H, Acker JP, Hannon J, Miszta-Lane H, Akabutu JJ, McGann LE. Damage and protection of UC blood cells during cryopreservation. Cytotherapy 2001; 3: 377-386.