Kitosan Kaplı Yüzeylerde Kollajen, Vitronektin ve Jelatinin Üç Boyutlu HEK293 Hücre Kültürüne ve Hücresel Kollajen Salınımına Olan Etkileri

Abstract

Amaç: Son zamanlarda in vivo ve klinik çalışmalara uyarlanmasının daha kolay olması nedeniyle üç boyutlu kültür oluşturulması oldukça önem kazanmıştır. Üç boyutlu kültür çalışmaları açısından hem maliyeti hem de farklı hücrelerde uygulanabilmesi nedeniyle kitosan materyali ön plana çıkmaktadır. Projemizde hücreler arası matriks elemanlarının kitosan yüzeylere kaplanmasının, hücresel sferoid oluşumuna ve Hücrelerarası Matriks Elemenları HME salınımına olan etkileri araştırılmıştır.Gereç ve Yöntemler: Bu çalışmada, HEK293 insan embriyonik böbrek hücre hatlarında farklı hücreler arası matriks elemanları ile kaplanmış kitosan yüzeylerde oluşturdukları sferoid yapıları mikroskop altında incelenmiştir. Ayrıca HEK293 hücrelerinin ortama salgıladıkları kollajen miktarları ELISA yöntemi ile tespit edilmiştir.Bulgular: Farklı hücreler arası matriks elemanları ve onların konsantrasyonlarının HEK293 hücrelerinin sferoid oluşumlarını farklı etkilediği tespit edilmiştir. HEK293 hücreleri yaygın olmayan ama büyük sferoid odakları oluştururken, ortamda sadece kollajen tip 1 ve jelatinin varlığında daha yaygın ve bütün yüzeye yayılım gösteren sferoidler oluşturmaktadır. Vitronektin ise sferoid oluşumunu negatif yönde etkilemektedir. Ayrıca hücreler arası matriks elemanlarının farklı dozları ve kombinasyonları ortama salınan kollajen tip 1 ve kollajen tip 4 miktarını etkilediği saptanmıştır.Sonuç: Değişen hücreler arası matriks elemanları ve konsantrasyonu 3 Boyutlu 3B kültür oluşumunu etkilemektedir. Hücrelerin sferoid oluşumunun artışını sağlayan koşulların, hücrelerden ortama salgılanan kollajen miktarını değiştirdiği de gösterilmiştir

Keywords

• 3 Boyutlu Kültür, Kitosan, Kollajen, Vitronektin, Jelatin

Ertosun MG. Kitosan kaplı yüzeylerde kollajen, vitronektin ve jelatinin üç boyutlu HEK293 hücre kültürüne

Abstract

Objective: Recently, it has become very important to create a three-dimensional culture as it is easier to adapt to in vivo and clinical studies. In terms of three-dimensional culture studies, chitosan material comes to the fore because of its cost and its use in different cells. The effects of chitosan surfaces coated with extracellular matrix elements ECM on cellular spheroid formation and cellular ECM release were investigated in this study.Material and Methods: Spheroid structures formed by human embryonic kidney cell lines HEK293 on different ECM-coated chitosan surfaces were examined under a microscope. In addition, the amount of collagen released by HEK293 cells was determined by the ELISA method.Results: It was determined that the different extracellular matrix elements and their concentrations affect the spheroid formation of HEK293 cells on chitosan surfaces. While HEK293 cells form large spheroid structures with fewer foci on chitosan surfaces, they create larger spheroids in the presence of only collagen type 1 or gelatin on chitosan surfaces. Vitronectin negatively affects spheroid formation. In addition, different doses and combinations of extracellular matrix elements have been found to affect the amount of collagen type-1 and collagen type-4 released into the micro-environment.Conclusion: Changing extracellular matrix elements and their concentrations used with a chitosan surface affects 3D culture formation. It has also been shown that the conditions that increase the spheroid formation of cells change the amount of collagen secreted from cells to the micro-environment

Keywords

• 3D Culture, Chitosan, Collagen, Vitronectin, Gelatin

References

1. Simian M, Bissell MJ. Organoids: A historical perspective of thinking in three dimensions. J Cell Biol 2017; 216(1):31-40.
2. Vinci M, Gowan S, Boxall F, Patterson L, Zimmermann M, Court W, Lomas C, Mendiola M, Hardisson D, Eccles SA. Advances in establishment and analysis of three- dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 2012;10:29.
3. Ravi M, Paramesh V, Kaviya SR, Anuradha E, Paul Solomon FD. 3D cell culture systems: Advantages and applications. J Cell Physiol 2015; 230(1):16-26.
4. Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 2000; 21(24):2589-98.
5. Aiedeh K, Gianasi E, Orienti I, Zecchi V. Chitosan microcapsules as controlled release systems for insulin. J Microencapsul 1997; 14(5):567-76.
6. Zhu A, Zhang M, Wu J, Shen J. Covalent immobilization of chitosan/heparin complex with a photosensitive hetero-bifunctional crosslinking reagent on PLA surface. Biomaterials 2002; 23(23):4657-65.
7. Kim BS, Baez CE, Atala A. Biomaterials for tissue engineering. World J Urol 2000; 18(1):2-9.
8. Pachence JM. Collagen-based devices for soft tissue repair. J Biomed Mater Res 1996; 33(1):35-40.

9. Preissner KT. Structure and biological role of vitronectin. Annu Rev Cell Biol 1991; 7:275-310.
10. Burgos-Panadero R, Noguera I, Canete A, Navarro S, Noguera R. Vitronectin as a molecular player of the tumor microenvironment in neuroblastoma. BMC Cancer 2019; 19(1):479.
11. Mao JS, Cui YL, Wang XH, Sun Y, Yin YJ, Zhao HM, Yao KD. A preliminary study on chitosan and gelatin polyelectrolyte complex cytocompatibility by cell cycle and apoptosis analysis. Biomaterials 2004; 25(18):3973- 81.
12. Moscato S, Mattii L, D’Alessandro D, Cascone MG, Lazzeri L, Serino LP, Dolfi A, Bernardini N. Interaction of human gingival fibroblasts with PVA/gelatine sponges. Micron 2008; 39(5):569-79.
13. Gohi B, Liu XY, Zeng HY, Xu S, Ake KMH, Cao XJ, Zou KM, Namulondo S. Enhanced efficiency in isolation and expansion of hAMSCs via dual enzyme digestion and micro-carrier. Cell Biosci 2020; 10:2.
14. Ranganathan S, Balagangadharan K. Selvamurugan N. Chitosan and gelatin-based electrospun fibers for bone tissue engineering. Int J Biol Macromol 2019; 133: 354-64.
15. Oryan A, Alidadi S, Bigham-Sadegh A, Moshiri A, Kamali A. Effectiveness of tissue engineered chitosan- gelatin composite scaffold loaded with human platelet gel in regeneration of critical sized radial bone defect in rat. J Control Release 2017; 254:65-74.
16. Fu JH, Zhao M, Lin YR, Tian XD, Wang YD, Wang ZX, Wang LX. Degradable chitosan-collagen composites seeded with cells as tissue engineered heart valves. Heart Lung Circ 2017; 26(1):94-100.
17. Li M, Han M, Sun Y, Hua Y, Chen G, Zhang L. Oligoarginine mediated collagen/chitosan gel composite for cutaneous wound healing. Int J Biol Macromol 2019; 122: 1120-7.
18. Lin YC, Boone M, Meuris L, Lemmens I, Van Roy N, Soete A, Reumers J, Moisse M, Plaisance S, Drmanac R, Chen J, Speleman F, Lambrechts D, Van de Peer Y, Tavernier J, Callewaert N. Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat Commun 2014; 5:4767.
19. Shenvi SV, Dixon BM, Petersen Shay K, Hagen TM. A rat primary hepatocyte culture model for aging studies. Curr Protoc Toxicol 2008; Chapter 14:14-7.
20. Yap LY, Li J, Phang IY, Ong LT, Ow JZ, Goh JC, Nurcombe V, Hobley J, Choo AB, Oh SK, Cool SM, Birch WR. Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells. Tissue Eng Part C Methods 2011; 17(2):193- 207.
21. Li J, Bardy J, Yap LY, Chen A, Nurcombe V, Cool SM, Oh SK, Birch WR. Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells. Biointerphases 2010; 5(3):132-42.
22. Lin, YM, Lee J, Lim JFY, Choolani M, Chan JKY, Reuveny S, Oh SKW. Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation. Stem Cell Res Ther 2017; 8(1):93.
23. Kapalczynska M, Kolenda T, Przybyla W, Zajaczkowska M, Teresiak A, Filas V, Ibbs M, Blizniak R, Luczewski L, Lamperska K. 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch Med Sci 2018; 14(4):910-9.
24. Duval K, Grover H, Han LH, Mou Y, Pegoraro AF, Fredberg J, Chen Z. Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda) 2017; 32(4): 266-77.
25. Antoni D, Burckel H, Josset E, Noel G. Three-dimensional cell culture: A breakthrough in vivo. Int J Mol Sci 2015; 16(3):5517-27.
26. Chaicharoenaudomrung N, Kunhorm P, Noisa P. Three- dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J Stem Cells 2019; 11(12):1065-83.
27. Mousavi S, Khoshfetrat AB, Khatami N, Ahmadian M, Rahbarghazi R. Comparative study of collagen and gelatin in chitosan-based hydrogels for effective wound dressing: Physical properties and fibroblastic cell behavior. Biochem Biophys Res Commun 2019; 518(4):625-31.
28. Corsi K, Chellat F, Yahia L, Fernandes JC. Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles. Biomaterials 2003; 24(7): 1255-64.
29. Ramezani MR, Naderi-Manesh H, Rafieepour HA. Cytotoxicity assessment of a gold nanoparticle- chitosan nanocomposite as an efficient support for cell immobilization: Comparison with chitosan hydrogel and chitosan-gelatin. Biocell 2014; 38(1-3):11-6.
30. Dhiman HK, Ray AR, Panda AK. Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen. Biomaterials 2005; 26(9):979-86.