Construction of three-dimensional dermoid tissue based on cell sheets technology <i>in vitro</i>_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XIANG Hua ¹ , CHEN Rui ¹ , WU Shan ¹ , XI Dali ² , LONG Siqi ¹ , SHEN Yuan ¹ , FAN Zengjie ¹ ,  REN Liling ¹

1. School of Stomatology, Lanzhou University, Lanzhou Gansu, 730000, P.R.China;
2. Department of Pathology, the First Affiliated Hospital of Lanzhou University, Lanzhou Gansu, 730000, P.R.China;

Corresponding author：

REN Liling, Email: renlil@lzu.edu.cn

Keywords：

Tissue engineered dermis; cell sheet technology; bone marrow mesenchymal stem cells; endothelial cells; pre-vascularization

DOI：

10.7507/1002-1892.201904039

Video：

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Objective To explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology.Methods Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression.Results HDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a " honeycomb” network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant (P>0.05).Conclusion Three-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.

Citation： XIANG Hua, CHEN Rui, WU Shan, XI Dali, LONG Siqi, SHEN Yuan, FAN Zengjie, REN Liling. Construction of three-dimensional dermoid tissue based on cell sheets technology in vitro. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(1): 116-123. doi: 10.7507/1002-1892.201904039 Copy

1.	Fan ZJ, Liu B, Wang JQ, et al. A novel wound dressing based on Ag/Graphene polymer hydrogel: effectively kill bacteria and accelerate wound healing. Advanced Functional Materials, 2014, 24(25): 3933-3943.
2.	赵跃华, 张永红, 林凯. 人表皮干细胞复合裸鼠脱细胞真皮支架制备的人工皮肤. 中国组织工程研究, 2016, 20(25): 3771-3777.
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4.	Elloumi‐Hannachi I, Yamato M, Okano T. Cell sheet engineering: a unique nanotechnology for scaffold‐free tissue reconstruction with clinical applications in regenerative medicine. J Intern Med, 2010, 267(1): 54-70.
5.	肖斌, 刘毅. 以脂肪源性干细胞膜片构建组织工程皮肤. 中国组织工程研究, 2014, 18(33): 5361-5365.
6.	支振亚, 邢飞, 陈龙, 等. 细胞膜片技术在骨与软骨组织工程领域的研究进展. 中国修复重建外科杂志, 2018, 32(2): 237-241.
7.	Kim K, Utoh R, Ohashi K, et al. Fabrication of functional 3D hepatic tissues with polarized hepatocytes by stacking endothelial cell sheets in vitro. J Tissue Eng Regen Med, 2017, 11(7): 2071-2080.
8.	Sekine H, Shimizu T, Hobo K, et al. Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neo-vascularization and improves cardiac function of ischemic hearts. Circulation, 2008, 118(14 Suppl): S145-S152.
9.	李海艳, 邹柳, 申利贤, 等. 支架、细胞、因子: 组织工程血管化策略的研究与前景. 中国组织工程研究, 2017, 21(14): 2259-2265.
10.	Chen L, Xing Q, Zhai QY, et al. Pre-vascularization enhances therapeutic effects of human mesenchymal stem cell sheets in full thickness skin wound repair. Theranostics, 2017, 7(1): 117-131.
11.	Karoline P, Johanna M, Severin M, et al. Microvascular networks from endothelial cells and mesenchymal stromal cells from adipose tissue and bone marrow: A comparison. Frontiers in Bioengineering and Biotechnology, 2018, 6: 2296-4185.
12.	Kim DK, Kim JI, Hwang TI, et al. Bioengineered osteoinductive broussonetia kazinoki/silk fibroin composite scaffolds for bone tissue regeneration. ACS Appl Mater Interfaces, 2017, 9(2): 1384-1394.
13.	Gurel Pekozer G, Torun Kose G, Hasirci V. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells. Microvasc Res, 2016, 108: 1-9.
14.	Thulabandu V, Chen D, Atit RP. Dermal fibroblast in cutaneous development and healing. Wiley Interdiscip Rev Dev Biol, 2018, 7(2).
15.	Ren L, Ma D, Liu B, et al. Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration. Biomed Res Int, 2014, 2014: 301279.
16.	Ma D, Ren L, Liu Y, et al. Engineering scaffold-free bone tissue using bone marrow stromal cell sheets. J Orthop Res, 2010, 28(5): 697-702.
17.	Sasagawa T, Shimizu T, Sekiya S, et al. Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials, 2010, 31(7): 1646-1654.
18.	Narita T, Shintani Y, Ikebe C, et al. The use of scaffold-free cell sheet technique to refine mesenchymal stromal cell-based therapy for heart failure. Mol Ther, 2013, 21(4): 860-867.
19.	Madathil BK, Kumar PR, Kumary TV. N-isopropylacrylamide-co-glycidylmethacrylate as a thermoresponsive substrate for corneal endothelial cell sheet engineering. Biomed Res Int, 2014, 2014: 450672.
20.	Shudo Y, Miyagawa S, Ohkura H, et al. Addition of mesenchymal stem cells enhances the therapeutic effects of skeletal myoblast cell-sheet transplantation in a rat ischemic cardiomyopathy model. Tissue Eng Part A, 2014, 20(3-4): 728-739.
21.	Kanai N, Yamato M, Okano T. Cell sheets engineering for esophageal regenerative medicine. Ann Transl Med, 2014, 2(3): 28.
22.	Liu H, Zhou W, Ren N, et al. Cell sheets of co-cultured endothelial progenitor cells and mesenchymal stromal cells promote osseointegration in irradiated rat bone. Sci Rep, 2017, 7(1): 3038.
23.	Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A, 2001, 98(18): 10344-10349.
24.	Guan X, Delo DM, Atala A, et al. In vitro cardiomyogenic potential of human amniotic fluid stem cells. J Tissue Eng Regen Med, 2011, 5(3): 220-228.
25.	赵娜, 李宏玲, 李聪, 等. 异种及同种异体间充质干细胞移植免疫排斥及免疫调节作用研究. 广东省职业健康协会学术交流会, 2015.

1. Fan ZJ, Liu B, Wang JQ, et al. A novel wound dressing based on Ag/Graphene polymer hydrogel: effectively kill bacteria and accelerate wound healing. Advanced Functional Materials, 2014, 24(25): 3933-3943.
2. 赵跃华, 张永红, 林凯. 人表皮干细胞复合裸鼠脱细胞真皮支架制备的人工皮肤. 中国组织工程研究, 2016, 20(25): 3771-3777.
3. Supp DM. Skin substitutes for burn wound healing: current and future approaches. Expert Review of Dermatology, 2014, 6(2): 217-227.
4. Elloumi‐Hannachi I, Yamato M, Okano T. Cell sheet engineering: a unique nanotechnology for scaffold‐free tissue reconstruction with clinical applications in regenerative medicine. J Intern Med, 2010, 267(1): 54-70.
5. 肖斌, 刘毅. 以脂肪源性干细胞膜片构建组织工程皮肤. 中国组织工程研究, 2014, 18(33): 5361-5365.
6. 支振亚, 邢飞, 陈龙, 等. 细胞膜片技术在骨与软骨组织工程领域的研究进展. 中国修复重建外科杂志, 2018, 32(2): 237-241.
7. Kim K, Utoh R, Ohashi K, et al. Fabrication of functional 3D hepatic tissues with polarized hepatocytes by stacking endothelial cell sheets in vitro. J Tissue Eng Regen Med, 2017, 11(7): 2071-2080.
8. Sekine H, Shimizu T, Hobo K, et al. Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neo-vascularization and improves cardiac function of ischemic hearts. Circulation, 2008, 118(14 Suppl): S145-S152.
9. 李海艳, 邹柳, 申利贤, 等. 支架、细胞、因子: 组织工程血管化策略的研究与前景. 中国组织工程研究, 2017, 21(14): 2259-2265.
10. Chen L, Xing Q, Zhai QY, et al. Pre-vascularization enhances therapeutic effects of human mesenchymal stem cell sheets in full thickness skin wound repair. Theranostics, 2017, 7(1): 117-131.
11. Karoline P, Johanna M, Severin M, et al. Microvascular networks from endothelial cells and mesenchymal stromal cells from adipose tissue and bone marrow: A comparison. Frontiers in Bioengineering and Biotechnology, 2018, 6: 2296-4185.
12. Kim DK, Kim JI, Hwang TI, et al. Bioengineered osteoinductive broussonetia kazinoki/silk fibroin composite scaffolds for bone tissue regeneration. ACS Appl Mater Interfaces, 2017, 9(2): 1384-1394.
13. Gurel Pekozer G, Torun Kose G, Hasirci V. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells. Microvasc Res, 2016, 108: 1-9.
14. Thulabandu V, Chen D, Atit RP. Dermal fibroblast in cutaneous development and healing. Wiley Interdiscip Rev Dev Biol, 2018, 7(2).
15. Ren L, Ma D, Liu B, et al. Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration. Biomed Res Int, 2014, 2014: 301279.
16. Ma D, Ren L, Liu Y, et al. Engineering scaffold-free bone tissue using bone marrow stromal cell sheets. J Orthop Res, 2010, 28(5): 697-702.
17. Sasagawa T, Shimizu T, Sekiya S, et al. Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials, 2010, 31(7): 1646-1654.
18. Narita T, Shintani Y, Ikebe C, et al. The use of scaffold-free cell sheet technique to refine mesenchymal stromal cell-based therapy for heart failure. Mol Ther, 2013, 21(4): 860-867.
19. Madathil BK, Kumar PR, Kumary TV. N-isopropylacrylamide-co-glycidylmethacrylate as a thermoresponsive substrate for corneal endothelial cell sheet engineering. Biomed Res Int, 2014, 2014: 450672.
20. Shudo Y, Miyagawa S, Ohkura H, et al. Addition of mesenchymal stem cells enhances the therapeutic effects of skeletal myoblast cell-sheet transplantation in a rat ischemic cardiomyopathy model. Tissue Eng Part A, 2014, 20(3-4): 728-739.
21. Kanai N, Yamato M, Okano T. Cell sheets engineering for esophageal regenerative medicine. Ann Transl Med, 2014, 2(3): 28.
22. Liu H, Zhou W, Ren N, et al. Cell sheets of co-cultured endothelial progenitor cells and mesenchymal stromal cells promote osseointegration in irradiated rat bone. Sci Rep, 2017, 7(1): 3038.
23. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A, 2001, 98(18): 10344-10349.
24. Guan X, Delo DM, Atala A, et al. In vitro cardiomyogenic potential of human amniotic fluid stem cells. J Tissue Eng Regen Med, 2011, 5(3): 220-228.
25. 赵娜, 李宏玲, 李聪, 等. 异种及同种异体间充质干细胞移植免疫排斥及免疫调节作用研究. 广东省职业健康协会学术交流会, 2015.

Chinese Journal of Reparative and Reconstructive Surgery

Construction of three-dimensional dermoid tissue based on cell sheets technology in vitro

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