Spleen extracellular matrix provides a supportive microenvironment for β-cell function

Document Type : Original Article


1 Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Department of Anatomical Sciences, School of Medicine, Dezful University of Medical Sciences, Dezful, Iran


Objective(s): Type 1 diabetes mellitus is a common autoimmune and multifactorial disorder. Researchers have been interested in making a favorable islet-like tissue model for the treatment of diabetes. The main objective of this study was to determine the effects of the spleen extracellular matrix (S-ECM) on the function of the MIN6 cell line (a β-cell model). 
Materials and Methods: In this experimental research, Wistar rat spleens were decellularized by sodium dodecyl sulfate (SDS) and Triton X-100. S-ECM was characterized by histological assessments, scanning electron microscopy, determination of residua DNA, and examination of the mechanical tensile property. Then, MIN6 cells were seeded on S-ECM scaffold. Glucose-stimulated insulin secretion and mRNA expression of insulin-related genes were examined to confirm the function of the cells. 
Results: The main components of S-ECM such as collagen and glycosaminoglycan remained after decellularization. Furthermore, very low residual DNA and appropriate mechanical behavior of S-ECM provided an ideal extracellular microenvironment for the MIN6 cells. GSIS results showed that the seeded cells in S-ECM secreted more insulin than the traditional two-dimensional (2D) culture. The expression of specific insulin-related genes such as PDX-1, insulin, Maf-A, and Glut-2 in the recellularized scaffold was more significant than in the 2D traditional cultured cells. Also, MTT assay results showed that S-ECM were no cytotoxic effects on the MIN6 cells. 
Conclusion: These results collectively have evidenced that S-ECM is a suitable scaffold for stabilizing artificial pancreatic islands. 


1. Aghazadeh Y, Nostro MC. Cell therapy for Type 1 diabetes: Current and future strategies. Curr Diab Rep 2017; 17: 37-45.
2.    Salg GA, Giese NA, Schenk M, Hüttner FJ, Felix K, Probst P, et al. The emerging field of pancreatic tissue engineering: A systematic review and evidence map of scaffold materials and scaffolding techniques for insulin-secreting cells. J Tissue Eng 2019; 30: 10-35.
3.    Mirmalek-Sani SH, Orlando G, McQuilling JP, Pareta R, Mack DL, Salvatori M, et al. Porcine pancreas extracellular matrix as a platform for endocrine pancreas bioengineering. Biomaterials 2013; 34: 5488-5495.
4.    Ludwig B, Reichel A, Steffen A, Zimerman B, Schally AV, Block NL, et al. Transplantation of human islets without immunosuppression. Proc Natl Acad Sci U S A 2013; 110: 19054-19058.
5.    Niknamasl A, Ostad SN, Soleimani M, Azami M, Salmani MK, Lotfibakhshaiesh N, et al. A new approach for pancreatic tissue engineering: human endometrial stem cells encapsulated in fibrin gel can differentiate to pancreatic islet beta-cell. Cell Biol Int 2014; 38: 1174-1182.
6.    Yang W, Xia R, Zhang Y, Zhang H, Bai L. Decellularized liver scaffold for liver regeneration. Methods Mol Biol 2018; 1577: 11-23.
7.    Wüthrich T, Lese I, Haberthür D, Zubler C, Hlushchuk R, Hewer E, et al. Development of vascularized nerve scaffold using perfusion-decellularization and recellularization. Mater Sci Eng C Mater Biol Appl 2020; 117: 111311.
8.    Simões IN, Vale P, Soker S, Atala A, Keller D, Noiva R, et al. Acellular urethra bioscaffold: decellularization of whole urethras for tissue engineering applications. Sci Rep 2017; 7: 41934.
9.    Roth SP, Erbe I, Burk J. Decellularization of large tendon specimens: combination of manually performed freeze-thaw cycles and detergent treatment. Methods Mol Biol 2018; 1577: 227-237.
10.    Mendibil U, Ruiz-Hernandez R, Retegi-Carrion S, Garcia-Urquia N, Olalde-Graells B, Abarrategi A. Tissue-specific decellularization methods: rationale and strategies to achieve regenerative compounds. Int J Mol Sci 2020; 21: 5447-5476.
11.    Loh QL, Choong C. Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. Tissue Eng Part B Rev 2013; 19: 485-502.
12.    Zanardo TÉC, Amorim FG, Taufner GH, Pereira RHA, Baiense IM, Destefani AC, et al. Decellularized splenic matrix as a scaffold for spleen bioengineering. Front Bioeng Biotechnol 2020; 8: 573461. 
13.    Lokmic Z, Lämmermann T, Sixt M, Cardell S, Hallmann R, Sorokin L. The extracellular matrix of the spleen as a potential organizer of immune cell compartments. Semin Immunol 2008; 20: 4-13.
14.    Miyazaki S, Tashiro F, Tsuchiya T, Sasaki K, Miyazaki JI. Establishment of a long-term stable β-cell line and its application to analyze the effect of Gcg expression on insulin secretion. Sci Rep. 2021; 12: 11:477-487.
15.    Nakashima K, Kanda Y, Hirokawa Y, Kawasaki F, Matsuki M, Kaku K. MIN6 is not a pure beta cell line but a mixed cell line with other pancreatic endocrine hormones. Endocr J 2009; 56: 45-53.
16.     Nyitray CE, Chavez MG, Desai TA. Compliant 3D microenvironment improves β-cell cluster insulin expression through mechanosensing and β-catenin signaling. Tissue Eng Part A 2014; 20: 1888-1895.
17.    Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials 2011; 32: 3233-3243.
18.    Lehr EJ, Rayat GR, Chiu B, Churchill T, McGann LE, Coe JY, et al. Decellularization reduces immunogenicity of sheep pulmonary artery vascular patches. J Thorac Cardiovasc Surg 2011; 141: 1056-1062.
19.     Baert Y, Stukenborg JB, Landreh M, De Kock J, Jörnvall H, Söder O, et al. Derivation and characterization of a cytocompatible scaffold from human testis. Hum Reprod 2015; 30: 256-267.
20.    Vermeulen M, Del Vento F, de Michele F, Poels J, Wyns C. Development of a cytocompatible scaffold from pig immature testicular tissue allowing human sertoli cell attachment, proliferation and functionality. Int J Mol Sci 2018; 19: 227-234.
21.    Liu WY, Lin SG, Zhuo RY, Xie YY, Pan W, Lin XF, et al. Xenogeneic decellularized scaffold: A novel platform for ovary regeneration. Tissue Eng Part C Methods 2017; 23: 61-71.
22.    Ghasemi A, Akbari E, Imani R. An overview of engineered hydrogel-based biomaterials for improved β-cell survival and insulin secretion. Front Bioeng Biotechnol 2021; 9: 662084.
23.    Wu D, Wan J, Huang Y, Guo Y, Xu T, Zhu M, et al. 3D Culture of MIN-6 cells on decellularized pancreatic scaffold: In vitro and in vivo study. Biomed Res Int 2015; 2015: 432645.
24.    Drzazga A, Cichońska E, Koziołkiewicz M, Gendaszewska-Darmach E. Formation of βTC3 and MIN6 Pseudoislets Changes the Expression Pattern of Gpr40, Gpr55, and Gpr119 receptors and improves lysophosphatidylcholines-potentiated glucose-stimulated insulin secretion. Cells 2020; 9: 2062-2081.
25.    Zhang M, Yan S, Xu X, Yu T, Guo Z, Ma M, et al. Three-dimensional cell-culture platform based on hydrogel with tunable microenvironmental properties to improve insulin-secreting function of MIN6 cells, Biomaterials 2021; 270: 120687.
26.    Chaimov D, Baruch L, Krishtul S, Meivar-Levy I, Ferber S, Machluf M. Innovative encapsulation platform based on pancreatic extracellular matrix achieve substantial insulin delivery. J Control Release 2017; 257: 91-101.
27.    Marghani B, Ateya A, Saleh R, Eltaysh R. Assessing of antidiabetic and ameliorative effect of Lupin seed aqueous extract on hyperglycemia, hyperlipidemia and effect on pdx1, Nkx6.1, Insulin-1, Glut-2 and Glucokinase genes expression in streptozotocin-induced diabetic rats, J. Food Nutr. Res 2019; 7: 333-341.
28.    McKinnon CM, Docherty K. Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function. Diabetologia 2001; 44: 1203-1214.
29.    Brissova M, Shiota M, Nicholson WE, Gannon M, Knobel SM, Piston DW, et al. Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J Biol Chem 2002; 277: 11225-11232.
30.    Matsuoka TA, Artner I, Henderson E, Means A, Sander M, Stein R. The MafA transcription factor appears to be responsible for tissue-specific expression of insulin, Proc. Natl. Acad. Sci. U S A 2004; 101: 2930-2933.
31.    Zhang C, Moriguchi T, Kajihara M, Esaki R, Harada A, Shimohata H, et al. MafA is a key regulator of glucose-stimulated insulin secretion. Mol Cell Biol 2005; 25: 4969-4976.
32.    Park S, Hong SM, Ahn IS. Can splenocytes enhance pancreatic beta-cell function and mass in 90% pancreatectomized rats fed a high fat diet? Life Sci 2009; 84: 358-363. 
33.    Tremmel DM, Odorico JS. Rebuilding a better home for transplanted islets. Organogenesis 2018; 14: 163-168.
34.    Xiang JX, Zheng XL, Gao R, Wu WQ, Zhu XL, Li JH, et al. Liver regeneration using decellularized splenic scaffold: A novel approach in tissue engineering. Hepatobiliary Pancreat Dis Int 2015; 14: 502-508.
35.    Xiang J, Zheng X, Liu P, Yang L, Dong D, Wu W, et al. Decellularized spleen matrix for reengineering functional hepatic-like tissue based on bone marrow mesenchymal stem cells. Organogenesis 2016; 12: 128-142.
36.     Goh SK, Bertera S, Olsen P, Candiello JE, Halfter W, Uechi G, et al. Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering. Biomaterials 2013; 34: 6760-6772.
37.     Huang G, Greenspan DS. ECM roles in the function of metabolic tissues. Trends Endocrinol Metab 2012; 23: 16-22.
38.    Leite AR, Corrêa-Giannella ML, Dagli ML, Fortes MA, Vegas VM, Giannella-Neto D. Fibronectin and laminin induce expression of islet cell markers in hepatic oval cells in culture. Cell Tissue Res 2007; 327: 529-537.
39.    Hughes SJ, Clark A, McShane P, Contractor HH, Gray DW, Johnson PR. Characterisation of collagen VI within the islet-exocrine interface of the human pancreas: implications for clinical islet isolation? Transplantation 2006; 81: 423-426.
40.    Stendahl JC, Kaufman DB, Stupp SI. Extracellular matrix in pancreatic islets: relevance to scaffold design and transplantation. Cell Transplant 2009; 18: 1-12.
41.    Hulinsky I, Harrington J, Cooney S, Silink M. Insulin secretion and DNA synthesis of cultured islets of Langerhans are influenced by the matrix. Pancreas1995; 11: 309-314.
42.    Lin HY, Tsai CC, Chen LL, Chiou SH, Wang YJ, Hung SC. Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK. J Biomed Sci 2010; 17: 56-75. 
43.    Weber LM, Hayda KN, Anseth KS. Cell-matrix interactions improve beta-cell survival and insulin secretion in three-dimensional culture. Tissue Eng Part A 2008; 14: 1959-1968.
44.    Beenken-Rothkopf LN, Karfeld-Sulzer LS, Davis NE, Forster R, Barron AE, Fontaine MJ. The incorporation of extracellular matrix proteins in protein polymer hydrogels to improve encapsulated beta-cell function. Ann Clin Lab Sci 2013; 43: 111-121.
45.    Llacua LA, Hoek A, de Haan BJ, de Vos P. Collagen type VI interaction improves human islet survival in immunoisolating microcapsules for treatment of diabetes. Islets 2018; 10: 60-68.
46.    Zhu Y, Liu Q, Zhou Z, Ikeda Y. PDX1, Neurogenin-3, and MAFA: Critical transcription regulators for beta cell development and regeneration. Stem Cell Res Ther 2017; 8: 240-247. 
47.    Holland AM, Góñez LJ, Naselli G, Macdonald RJ, Harrison LC. Conditional expression demonstrates the role of the homeodomain transcription factor Pdx1 in maintenance and regeneration of beta-cells in the adult pancreas. Diabetes 2005; 54: 2586-2595.
48.    Sharma A, Zangen DH, Reitz P, Taneja M, Lissauer ME, Miller CP, et al. The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 1999; 48: 507-513.
49.    Liu P, Tian B, Yang L, Zheng X, Zhang X, Li J, et al. Hemocompatibility improvement of decellularized spleen matrix for constructing transplantable bioartificial liver. Biomed Mater 2019; 14: 025003.