IN VITRO INVESTIGATION OF THE EFFECT OF STROMAL VASCULAR FRACTION ISOLATED BY MECHANICAL AND ENZYMATIC METHODS ON WOUND HEALING

Öz

Objective: The role of Stromal Vascular Fraction (SVF) cell suspension obtained from adipose tissue in wound healing is gaining importance day by day. In this study, the role of SVF cell population, isolated by enzymatic and mechanical methods in wound healing, will be examined in vitro. Material and Method: An SVF cell cocktail was isolated by mechanical and enzymatic methods after adipose tissue was obtained from 10 different patients by the liposuction method (the adipose tissue was divided into two equal groups for enzymatic and mechanical methods). After the isolation process is done, cell viability and cell number were counted using flow cytometry, and cell proliferation rates were measured by WST-1 assay. The Wound Healing Scratch Assay test, which is used as an in vitro model for wound healing, was performed with cell cocktails obtained from both groups. After isolation, the level of expression of the collagen type 1 gene, known to be involved in wound healing, was measured in both groups. Results: While the SVF (E-SVF) cell cocktail obtained bythe enzymatic method was 1,52x106/ml (±3,63, n=10, p=0,015), the number of SVF (M-SVF) cell cocktails obtained by mechanical method was 0,67x106/ml (±1,69, n=10, p=0,015). No significant difference was observed between cell viability of SVF cell cocktails isolated by two different methods. The M-SVF cell cocktail had a 10% higher cell proliferation rate compared to E-SVF. Furthermore, M-SVF cell migration rates and Col 1 gene levels were found to be higher than E-SVF cells. Conclusion: Although the cell number of the M-SVF cell cocktail was less than the cell number of the E-SVF cell cocktail, the collagen type 1 gene level of the M-SVF cell cocktail was found to be higher, so the M-SVF cell cocktail had higher wound healing properties.

Anahtar Kelimeler

• Stromal vascular fraction
• wound healing
• stem cell

MEKANİK VE ENZİMATİK YÖNTEM İLE İZOLE EDİLEN STROMAL VASKÜLER FRAKSİYON’UN YARA İYİLEŞMESİ ÜZERİNDEKİ ETKİSİNİN IN VİTRO İNCELENMESİ

Öz

Amaç: Yağ dokusundan elde edilen Stromal Vasküler Fraksiyon (SVF) hücre süspansiyonun yara iyileşmesindeki rolü gün geçtikçe önem kazanmaktadır. Bu çalışmada enzimatik ve mekanik yöntemler ile izole edilen SVF hücre popülasyonun yara iyileşmesindeki rolü incelenecektir. Gereç ve Yöntem: 10 farklı hastadan yağ alma yöntemi ile edilen yağ dokusu (eşit miktarda iki farklı gruba bölündükten sonra) mekanik ve enzimatik yöntemler kullanılarak SVF hücre kokteyli izole edilmiştir. İzolasyon sonrasında hücre canlılığı ve hücre sayısı akış sitometrisi kullanılarak sayılmış, hücre proliferasyon hızları WST-1 testi ile ölçümlenmiştir. Yara iyileşmesinde in-vitro model olarak kullanılan Scratch Assay testi her iki grup ile elde edilen hücre kokteylleri ile için gerçekleştirilmiştir. İzolasyon sonrasında, yara iyileşmesinde rol aldığı bilinen kolajen tip 1 geninin ifade edilme seviyesi her iki grup içinde ölçümlenmiştir. Bulgular: Enzimatik yöntem ile elde edilen SVF (E-SVF) hücre kokteyli 1,52x106/ml (±3,63, n=10, p=0,015) iken, mekanik yöntem ile elde edilen SVF (M-SVF) hücre kokteyli sayısı 0,67x106/ ml (±1,69, n=10, p=0,015) olarak ölçümlenmiştir. İki farklı yöntem ile izole edilen SVF hücre kokteylinin izolasyon sonrası hücre canlıkları arasında anlamlı bir fark gözlenmemiştir. M-SVF hücre kokteylinin, E-SVF ile karşılaştırıldığında %10 daha fazla hücre proliferasyon hızına sahip olduğu görülmüştür. Ayrıca, M-SVF hücrelerinin migrasyon hızlarının ve Col 1 gen seviyelerinin E-SVF hücrelerine oranla daha fazla olduğu saptanmıştır. Sonuç: M-SVF hücre kokteylinin hücre sayısının E-SVF hücre kokteylinin hücre sayısından az olmasına rağmen, M-SVF hücre kokteylinin kolajen tip 1 gen seviyesinin arttığı görülmüş, böylece M-SVF hücre kokteylinin daha yüksek yara iyileşme özelliklerine sahip olduğunu gözlemlenmiştir.

Anahtar Kelimeler

• Stromal vasküler fraksiyon
• kök hücre
• yara iyileşmesi

Kaynakça

1. 1. Balekar N, Katkam NG, Nakpheng T, Jehtae K, Srichana T. Evaluation of the wound healing potential of Wedelia trilobata (L.) leaves. J Ethnopharmacol 2012;141(3):817-24.
2. 2. Yates CC, Hebda P, Wells A. Skin wound healing and scarring: fetal wounds and regenerative restitution. Birth Defects Res C Embryo Today 2012;96(4):325-33.
3. 3. Aronowitz JA, Ellenhorn JD. Adipose stromal vascular fraction isolation: a head-to-head comparison of four commercial cell separation systems. Plast Reconstr Surg 2013;132:932e-939e.
4. 4. Condé-Green A1, Baptista LS, de Amorin NF, de Oliveira ED, da Silva KR, Pedrosa Cda S, et al. Effects of centrifugation on cell composition and viability of aspirated adipose tissue processed for transplantation. Aesthet Surg J 2010;30:24955.
5. 5. Condé-Green A, Kotamarti VS, Sherman LS, Keith JD, Lee ES, Granick MS. Shift toward Mechanical Isolation of Adipose-derived Stromal Vascular Fraction: Review of Upcoming Techniques. Plast Reconstr Surg Glob Open 2016;4:e1017.
6. 6. Tiryaki T. A novel three-step mechanical digestion method for ADSC. Oral presentation at the 14th Annual International Federation of Adipose Therapeutics and Science IFATS meeting. 2016 Nov 17-20, San Diego, California.
7. 7. Tonnard P, Verpaele A, Peeters G, Hamdi M, Cornelissen M, Declercq H. Nanofat grafting: basic research and clinical applications. Plast Reconstr Surg 2013;132:1017-26.
8. 8. Banyard DA, Sarantopoulos CN, Borovikova AA, Qiu X, Wirth GA, Paydar KZ, et al. Phenotypic analysis of stromal vascular fraction after mechanical shear reveals stressinduced progenitor populations. Plast Reconstr Surg 2016;138:237e-247e.

9. 9. Koçak P, Canikyan S, Batukan M, Attar R, Şahin F, Telci D. Comparison of enzymatic and nonenzymatic isolation methods for endometrial stem cells. Turk J Biol 2016;40(5):1081-9.
10. 10. Lauffenburger, DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell J 1996;84(3):359-69.
11. 11. Martin P, Martin P. Wound healing aiming for perfect skin Regeneration. Science 1997;276(5309):75-81.
12. 12. Demirci S, Doğan A, Demirci Y, Şahin F. In vitro wound healing activity of methanol extract of Verbascum speciosum. Int J Appl Res Nat Prod 2014;7(3):37-44.
13. 13. Şahin F, Koçak P, Yıldırım GM, Özkan İ, Yıldırım E, Kala E. In Vitro Wound Healing Activity of Wheat-Derived Nanovesicles. Appl Biochem Biotechnol 2018;188(2):38194.
14. 14. Lockhart RA. Tissue Dissociation Enzymes for Adipose Stromal Vascular Fraction Cell Isolation: A Review, J Stem Cell Res Ther 2015;5:12.
15. 15. Aronowitz JA, Lockhart RA, Hakakian CS. Mechanical versus enzymatic isolation of Stromal vascular fraction cells from adipose tissue. Springerplus 2015;4:713.
16. 16. Tonnard P, Verpaele A, Peeters G, Hamdi M, Cornelissen M, Declercq H. Nanofat Grafting: Basic Research and Clinical Applications. Plast Reconstr Surg 2013.
17. 17. Bora P, Majumdar AS. Adipose tissue-derived Stromal vascular fraction in regenerative medicine: a brief review on biology and translation. Stem Cell Res Ther 2017;8:145.