Improved BALB/c mice granulosa cell functions using purified alginate scaffold

Document Type : Full paper (Original article)


1 Stem Cell Research Laboratory, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

2 Ph.D. Student in Anatomy, Stem Cell Research Laboratory, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran


Alginate, a non-toxic polysaccharide isolated from brown algae, is a widely used 3-dimensional (3D) porous scaffold for the granulosa cell and follicle encapsulation. However, impurities in commercial alginate can lead to alginate biocompatibility reduction. The aim of this study was to evaluate in vitro behavior of the granulosa cells seeded on the purified alginate in varying concentrations compared with matched non-purified ones. We produced a purified alginate using a simple and efficient method. Then, the granulosa cells from mice were isolated and seeded in various concentrations of (0.5%, 1% weight/volume) purified and non-purified alginate. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used on the 3rd, 5th and the 8th days of culture as an index of cell viability and proliferation. Furthermore, the secreted estradiol, progesterone and alkaline phosphatase enzyme (ALP) were measured in the granulosa cells culture media using radioimmunoassay kits. The cells cultured on purified and low concentration alginate showed a higher proliferation rate, sex hormone production and ALP activity. The results confirmed the impact of the alginate hydrogel properties on proliferative rate and function of granulosa cells in a 3D culture system. sa cells in a 3D culture system. 


Amorim, CA; Van Langendonckt, A; David, A; Dolmans, MM and Donnez, J (2008). Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix. Human Reprod., 24: 92-99.
Andersen, T; Auk-Emblem, P and Dornish, M (2015). 3D cell culture in alginate hydrogels. Microarrays. 4: 133-161.
Belani, M; Purohit, N; Pillai, P; Gupta, S and Gupta, S (2014). Modulation of steroidogenic pathway in rat granulosa cells with subclinical Cd exposure and insulin resistance: an impact on female fertility. BioMed. Res. Int., 2014, Article ID 46025.
Berkholtz, CB; Shea, LD and Woodruff, TK (2006). Extracellular matrix functions in follicle maturation. Semin. Reprod. Med., 24: 262-269.
Campbell, KL (1979). Ovarian granulosa cells isolated with EGTA and hypertonic sucrose: cellular integrity and function. Biol. Reprod., 21: 773-786.
Deka, S; Kalita, D; Sarma, S and Dutta, D (2014). Some biochemical constituents in follicular fluid of indigenous cows of Assam. Vet. World. 7: 976-979.
Desai, N; Alex, A; AbdelHafez, F; Calabro, A; Goldfarb, J; Fleischman, A and Falcone, T (2010). Three-dimensional in vitro follicle growth: overview of culture models, biomaterial, design, parametersand futures directions. Reprod. Biol. Endocrinol., 8: 119.
Dorati, R; Genta, I; Ferrari, M; Vigone, G; Merico, V; Garagna, S; Zuccotti, M and Conti, B (2016). Formulation and stability evaluation of 3D alginate beads potentially useful for cumulus-oocyte complexes culture. J. Microencapsul., 33: 137-145.
Dusseault, J; Tam, SK; Ménard, M; Polizu, S; Jourdan, G; Yahia, LH and Hallé, JP (2006). Evaluation of alginate purification methods: effect on polyphenol, endotoxin, and protein contamination. J. Biomed. Materials Res. Part A. 76: 243-251.
Dzafic, E; Stimpfel, M and Virant-Klun, I (2013). Plasticity of granulosa cells: on the crossroad of stemness and transdifferentiation potential. J. Assist. Reprod. Genet., 30: 1255-1261.
Ganguly, S (2013). Role of biochemical factors and mineral supplementation in livestock ration for maintenance of their fertility and healthy reproductive status: a review. Res. J. Chem. Sci., 3: 102-106.
Heise, M; Koepsel, R; Russell, AJ and McGee, EA (2005). Calcium alginate microencapsulation of ovarian follicles impacts FSH delivery and follicle morphology. Reprod. Biol. Endocrinol., 3: 47.
Hornick, J; Duncan, F; Shea, L and Woodruff, T (2012). Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro. Human Reprod., 27: 1801-1810.
Hummitzsch, K; Ricken, AM; Kloß, D; Erdmann, S; Nowicki, MS; Rothermel, A; Robitzki, AA and Spanel-Borowski, K (2009). Spheroids of granulosa cells provide an in vitro model for programmed cell death coupled to steroidogenesis. Differentiation. 77: 60-69.
Jeong, SM; Kim, EY; Hwang, JH; Lee, GY; Cho, SJ; Bae, JY; Song, JE; Yoon, KH; Joo, CK and Lee, D (2011). A study on proliferation and behavior of retinal pigment epithelial cells on purified alginate films. Int. J. Stem Cells. 4: 105.
Joo, S; Oh, SH; Sittadjody, S; Opara, EC; Jackson, JD; Lee, SJ; Yoo, JJ and Atala, A (2016). The effect of collagen hydrogel on 3D culture of ovarian follicles. Biomed. Materials. 1: 065009.
Kim, AR; Hwang, JH; Kim, HM; Kim, HN; Song, JE; Yang, YI; Yoon, KH; Lee, D and Khang, G (2013). Reduction of inflammatory reaction in the use of purified alginate microcapsules. J. Biomater. Sci. Polym. Ed., 24: 1084-1098.
King, SM; Quartuccio, S; Hilliard, TS; Inoue, K and Burdette, JE (2011). Alginate hydrogels for three-dimensional organ culture of ovaries and oviducts. J. Vis. Exp., 52: pii: 2804. doi: 10.3791/2804.
Kreeger, PK; Deck, JW; Woodruff, TK and Shea, LD (2006). The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials. 27: 714-723.
Kreeger, PK; Fernandes, NN; Woodruff, TK and Shea, LD (2005). Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose. Biol. Reprod., 73: 942-950.
Langlois, G; Dusseault, J; Bilodeau, S; Tam, SK; Magassouba, D and Hallé, JP (2009). Direct effect of alginate purification on the survival of islets immobilized in alginate-based microcapsules. Acta Biomaterialia. 5: 3433-3440.
Lin, SCY; Wang, Y; Wertheim, DF and Coombes, AG (2017). Production and in vitro evaluation of macroporous, cell-encapsulating alginate fibres for nerve repair. Mater. Sci. Eng. C. Mater. Biol. Appl., 73: 653-664.
Mainigi, MA; Ord, T and Schultz, RM (2011). Meiotic and developmental competence in mice are compromised following follicle development in vitro using an alginate-based culture system. Biol. Reprod., 85: 269-276.
Ménard, M; Dusseault, J; Langlois, G; Baille, WE; Tam, SK; Yahia, L; Zhu, XX and Hallé, JP (2010). Role of protein contaminants in the immunogenicity of alginates. J. Biomed. Mater. Res. B. Appl. Biomater., 93: 333-340.
Mohanty, S; Wu, Y; Chakraborty, N; Mohanty, P and Ghosh, G (2016). Impact of alginate concentration on the viability, cryostorage, and angiogenic activity of encapsulated fibroblasts. Mater. Sci. Eng. C. Mater. Biol. Appl., 65: 269-277.
Pangas, SA; Saudye, H; Shea, LD and Woodruff, TK (2003). Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes. Tissue Eng., 9: 1013-1021.
Pravdyuk, AI; Petrenko, YA; Fuller, BJ and Petrenko, AY (2013). Cryopreservation of alginate encapsulated mesenchymal stromal cells. Cryobiology. 66: 215-222.
Qi, Y; Lu, L; Zhou, C and Luo, B (2009). Purification of alginate for tissue engineering. In Bioinformatics and Biomedical Engineering, 2009. ICBBE 2009. 3rd International Conference on. IEEE. PP: 1-4.
Sèdes, L; Leclerc, A; Moindjie, H; Cate, RL; Picard, JY; Di Clemente, N and Jamin, SP (2013). Anti-Müllerian hormone recruits BMPR-IA in immature granulosa cells. PLoS One. 8: e81551.
Selimoglu, SM; Ayyildiz-Tamis, D; Gurhan, ID and Elibol, M (2012). Purification of alginate and feasible production of alginate-immobilized hybridoma monoclonal antibodies by the cells. J. Biosci. Bioeng., 113: 233-238.
Singh, D; Zo, SM; Kumar, A and Han, SS (2013). Engineering three-dimensional macroporous hydroxyethyl methacrylate-alginate-gelatin cryogel for growth and proliferation of lung epithelial cells. J. Biomater. Sci. Polym. Ed., 24: 1343-1359.
Sondermeijer, HP; Witkowski, P; Woodland, D; Seki, T; Aangenendt, FJ; van der Laarse, A; Itescu, S and Hardy, MA (2016). Optimization of alginate purification using polyvinylidene difluoride membrane filtration: effects on immunogenecity and biocompatibility of three-dimensional alginate scaffolds. J. Biomater. Appl., 31:510-520.
Song, JE; Kim, AR; Lee, CJ; Tripathy, N; Yoon, KH; Lee, D and Khang, G (2015). Effects of purified alginate sponge on the regeneration of chondrocytes: in vitro and in vivo. J. Biomater. Sci. Polym. Ed., 26: 181-195.
Vigo, D; Villani, S; Faustini, M; Accorsi, P; Galeati, G; Spinaci, M; Munari, E; Russo, V; Asti, A and Conte, U (2005). Follicle-like model by granulosa cell encapsulation in a barium alginate-protamine membrane. Tissue Eng., 11: 709-714.
West, ER; Xu, M; Woodruff, TK and Shea, LD (2007). Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials. 28: 4439-4448.
Woodruff, TK and Shea, LD (2007). The role of the extracellular matrix in ovarian follicle development. Reprod. Sci., (8_Suppl), 14: 6-10.
Xu, M; Kreeger, PK; Shea, LD and Woodruff, TK (2006a). Tissue-engineered follicles produce live, fertile offspring. Tissue Eng., 12: 2739-2746.
Xu, M; West, E; Shea, LD and Woodruff, TK (2006b). Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development. Biol. Reprod., 75: 916-923.
Xu, M; West-Farrell, ER; Stouffer, RL; Shea, LD; Woodruff, TK and Zelinski, MB (2009). Encapsulated three-dimensional culture supports development of nonhuman primate secondary follicles. Biol. Reprod., 81: 587-594.
Zhao, Y; Gao, S; Zhao, S; Li, Y; Cheng, L; Li, J and Yin, Y (2012). Synthesis and characterization of disulfide-crosslinked alginate hydrogel scaffolds. Mater. Sci. Eng: C., 32: 2153-2162.