THREE-DIMENSIONAL CULTURED ADIPOSE-DERIVED STEM CELLS BASED ON MICROBIAL TRANSGLUTAMINASE ENZYME CROSSLINKED GELATIN HYDROGEL_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

RENXiaomei ¹ , QIANHong ² , XIAOZhenghua ² , LONGHaiyan ³ , GUOYingqiang ² ,  YANGGang ¹

1. Department of Medical Information Engineering, College of Electrical and Information Engineering, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
2. Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
3. Engineering Training Center, Chengdu Aeronautic Polytechnic, Chengdu Sichuan, 610041, P. R. China;

Corresponding author：

YANGGang, Email: yang_gang@scu.edu.cn

Keywords：

Adipose-derived stem cells; Three-dimensional culture; Hydrogel; Gelatin; Microbial transglutaminase

DOI：

10.7507/1002-1892.20160316

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To study the growth of adipose-derived stem cells (ADSCs) planted in three-dimensional (3D) materials, a 3D cultured ADSCs system based on microbial transglutaminase (mTG) enzyme crosslinked gelatin hydrogel was constructed. Methods ADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated in situ (2D environment) and cultured in the 3D materials (3D environment). The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment. Results The result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant (P < 0.05). But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly. Conclusion ADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.

Citation： RENXiaomei, QIANHong, XIAOZhenghua, LONGHaiyan, GUOYingqiang, YANGGang. THREE-DIMENSIONAL CULTURED ADIPOSE-DERIVED STEM CELLS BASED ON MICROBIAL TRANSGLUTAMINASE ENZYME CROSSLINKED GELATIN HYDROGEL. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(12): 1532-1537. doi: 10.7507/1002-1892.20160316 Copy

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2.	Rasmussen JG, Frøbert O, Holst-Hansen C, et al. Comparison of human adipose-derived stem cells and bone marrow-derived stem cells in a myocardial infarction model. Cell Transplant, 2014, 23(2):195-206.
3.	Heydarkhan-Hagvall S, Schenke-Layland K, Yang JQ, et al. Human adipose stem cells:a potential cell source for cardiovascular tissue engineering. Cells Tissues Organs, 2008, 4(187):263-274.
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5.	Sugii S, Kida Y, Berggren WT, et al. Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells. Nat Protoc, 2011, 6(3):346-358.
6.	李志园, 杨刚, 黄华, 等.双相脉冲电刺激可促进脂肪干细胞分化为心肌细胞样细胞.细胞与分子免疫学杂志, 2015, 31(9):1200-1204.
7.	Lin L, Marchant RE, Zhu J, et al. Extracellular matrix-mimetic poly (ethylene glycol) hydrogels engineered to regulate smooth muscle cell proliferation in 3-D. Acta Biomater, 2014, 10(12):5106-5115.
8.	Seliktar D. Designing cell-compatible hydrogels for biomedical applications. Science, 2012, 336(6085):1124-1128.
9.	Ghezzi C, Marelli B, Donelli I, et al. The role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen-silk fibroin construct. Biomaterials, 2014, 35(24):6236-6247.
10.	Skopinska-Wisniewska J, Olszewski K, Bajek A, et al. Dialysis as a method of obtaining neutral collagen gels. Mater Sci Eng C Mater Biol Appl, 2014, 40:65-70.
11.	Chuang CH, Lin RZ, Tien HW, et al. Enzymatic regulation of functional vascular networks using gelatin hydrogels. Acta Biomaterialia, 2015, 19:85-99.
12.	Sell SA, McClure MJ, Garg K, et al. Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering. Adv Drug Deliv Rev, 2009, 61(12):1007-1019.
13.	员海超, 蒲春晓, 魏强, 等.组织工程细胞外基质材料研究进展.中国修复重建外科杂志, 2012, 26(10):1251-1254.
14.	王觅格, 夏亚一, 王栓科, 等.骨髓间充质干细胞复合消旋聚乳酸/明胶修复兔关节软骨缺损的实验研究.中国修复重建外科杂志, 2007, 21(7):753-758.
15.	Marelli B, Achilli M, Alessandrino A, et al. Collagen-reinforced electrospun silk fibroin tubular construct as small calibre vascular graft. Macromol Biosci, 2012, 12(11):1566-1574.
16.	Lin CC. Recent advances in crosslinking chemistry of biomimetic poly (ethylene glycol) hydrogels. RSC Adv, 2015, 5(50):39844-39853.
17.	Ito A, Mase A, Takizawa Y, et al. Transglutaminase-mediated gelatin matrices incorporating cell adhesion factors as a biomaterial for tissue engineering, J Biosci Bioeng, 2003, 95(2):196-199.
18.	Chen RN, Ho HO, Sheu MT. Characterization of collagen matrices crosslinked using microbial transglutaminase. Biomaterials, 2005, 26(20):4229-4235.
19.	丁克毅.转谷氨酰胺酶改性明胶高强度薄膜的制备.食品与生物技术学报, 2007, 26(1):25-28.
20.	Mizuno A, Mitsuiki M, Motoki M. Effect of transglutaminase treatment on the glass transition of soy protein. J Agric Food Chem, 2000, 48(8):3286-3291.
21.	Kieliszek M, Misiewicz A. Microbial transglutaminase and its application in the food industry. A review. Folia Microbiol (Praha), 2014, 59(3):241-250.
22.	徐幸莲, 陈巧芬, 周光宏.转谷氨酰胺酶对蛋白质凝胶性能的影响.食品科学, 2003, 24(10):38-43.
23.	Hiroyasu A, Masae A, Koichi U, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms. Agriculture and Biological Chemistry, 1989, 53(10):2613-2617.

1. Yeh DC, Chan TM, Harn HJ, et al. Adipose tissue-derived stem cells in neural regenerative medicine. Cell Transplant, 2015, 24(3):487-492.
2. Rasmussen JG, Frøbert O, Holst-Hansen C, et al. Comparison of human adipose-derived stem cells and bone marrow-derived stem cells in a myocardial infarction model. Cell Transplant, 2014, 23(2):195-206.
3. Heydarkhan-Hagvall S, Schenke-Layland K, Yang JQ, et al. Human adipose stem cells:a potential cell source for cardiovascular tissue engineering. Cells Tissues Organs, 2008, 4(187):263-274.
4. Vallières K, Laterreur V, Tondreau MY, et al. Human adipose-derived stromal cells for the production of completely autologous self-assembled tissue-engineered vascular substitutes. Acta Biomater, 2015, 24:209-219.
5. Sugii S, Kida Y, Berggren WT, et al. Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells. Nat Protoc, 2011, 6(3):346-358.
6. 李志园, 杨刚, 黄华, 等.双相脉冲电刺激可促进脂肪干细胞分化为心肌细胞样细胞.细胞与分子免疫学杂志, 2015, 31(9):1200-1204.
7. Lin L, Marchant RE, Zhu J, et al. Extracellular matrix-mimetic poly (ethylene glycol) hydrogels engineered to regulate smooth muscle cell proliferation in 3-D. Acta Biomater, 2014, 10(12):5106-5115.
8. Seliktar D. Designing cell-compatible hydrogels for biomedical applications. Science, 2012, 336(6085):1124-1128.
9. Ghezzi C, Marelli B, Donelli I, et al. The role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen-silk fibroin construct. Biomaterials, 2014, 35(24):6236-6247.
10. Skopinska-Wisniewska J, Olszewski K, Bajek A, et al. Dialysis as a method of obtaining neutral collagen gels. Mater Sci Eng C Mater Biol Appl, 2014, 40:65-70.
11. Chuang CH, Lin RZ, Tien HW, et al. Enzymatic regulation of functional vascular networks using gelatin hydrogels. Acta Biomaterialia, 2015, 19:85-99.
12. Sell SA, McClure MJ, Garg K, et al. Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering. Adv Drug Deliv Rev, 2009, 61(12):1007-1019.
13. 员海超, 蒲春晓, 魏强, 等.组织工程细胞外基质材料研究进展.中国修复重建外科杂志, 2012, 26(10):1251-1254.
14. 王觅格, 夏亚一, 王栓科, 等.骨髓间充质干细胞复合消旋聚乳酸/明胶修复兔关节软骨缺损的实验研究.中国修复重建外科杂志, 2007, 21(7):753-758.
15. Marelli B, Achilli M, Alessandrino A, et al. Collagen-reinforced electrospun silk fibroin tubular construct as small calibre vascular graft. Macromol Biosci, 2012, 12(11):1566-1574.
16. Lin CC. Recent advances in crosslinking chemistry of biomimetic poly (ethylene glycol) hydrogels. RSC Adv, 2015, 5(50):39844-39853.
17. Ito A, Mase A, Takizawa Y, et al. Transglutaminase-mediated gelatin matrices incorporating cell adhesion factors as a biomaterial for tissue engineering, J Biosci Bioeng, 2003, 95(2):196-199.
18. Chen RN, Ho HO, Sheu MT. Characterization of collagen matrices crosslinked using microbial transglutaminase. Biomaterials, 2005, 26(20):4229-4235.
19. 丁克毅.转谷氨酰胺酶改性明胶高强度薄膜的制备.食品与生物技术学报, 2007, 26(1):25-28.
20. Mizuno A, Mitsuiki M, Motoki M. Effect of transglutaminase treatment on the glass transition of soy protein. J Agric Food Chem, 2000, 48(8):3286-3291.
21. Kieliszek M, Misiewicz A. Microbial transglutaminase and its application in the food industry. A review. Folia Microbiol (Praha), 2014, 59(3):241-250.
22. 徐幸莲, 陈巧芬, 周光宏.转谷氨酰胺酶对蛋白质凝胶性能的影响.食品科学, 2003, 24(10):38-43.
23. Hiroyasu A, Masae A, Koichi U, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms. Agriculture and Biological Chemistry, 1989, 53(10):2613-2617.

Chinese Journal of Reparative and Reconstructive Surgery

THREE-DIMENSIONAL CULTURED ADIPOSE-DERIVED STEM CELLS BASED ON MICROBIAL TRANSGLUTAMINASE ENZYME CROSSLINKED GELATIN HYDROGEL

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