TANG Jun , TAN Jinnü , YE Zhaoyang , ZHOU Yan , TAN Wensong
  • State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China;
ZHOU Yan, Email: zhouyan@ecust.edu.cn
Tissue engineering; microstructures; pre-vascularization; rotating bottle dynamic culture; three-dimensional co-culture
10.7507/1002-1892.202203028
Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables References Cited by

Objective  To construct three-dimensional (3D) pre-vascularized microstructures and explore the promoting effect of human fibroblasts (HFs) on the sprout and migration of human umbilical vein endothelial cells (HUVECs) in 3D co-culture system. Methods  HUVECs and HFs were cultured and the 3rd to 5th generation cells were selected for subsequent experiments. In 2D co-culture system, HFs were stained with PKH26 and the cell density was fixed, which co-cultured with HUVECs in different ratios (1∶4, 1∶1, 4∶1) and inoculation methods (HUVECs inoculation at 48 hours after HFs, direct mixed inoculation). Then the formation of vascular like structures was observed under fluorescence microscope. In 3D co-culture system, HUVECs and HFs were labeled with green fluorescent protein and red fluorescent protein by lentivirus transfection, respectively. They were inoculated on porous micro-carriers followed by dynamically culturing in rotating bottles to prepare HF, HUVEC, HF-EC, or HF&EC microstructures. The cell growth in microstructures was testing by low permeability crystal violet staining. Subsequently, the microstructures were embedded in fibrin gel and the cell growth and adhesion in HF and HUVEC microstructures were observed by laser confocal microscopy. Laser confocal microscope were also used to observe the sprouts of 4 kinds of microstructures, as well as the cell composition, the number and length of sprouts from HF-EC and HF&EC microstructures. HFs conditioned medium was prepared to observe its effect on sprouts of HUVEC microstructures with DMEM as control group. Results  In 2D co-culture system, HFs pre-culturing was helpful to the formation and stability of vascular like structures, and the best effect was when the ratio of two kinds of cells was 1∶1. In 3D co-culture system, it was found that the cells grew well on micro-carriers and had the ability of pre-vascularization. HUVEC microstructures did not sprout, but HF, HF-EC, and HF&EC microstructures could which indicated a good vascularization ability. The HF-EC microstructures were superior to HF&EC microstructures in terms of sprouts length and number (P<0.05). The tubes sprouting from co-cultured group were composed of HFs and HUVECs, and HF microstructures migration preceded HUVEC microstructures always, and their migration trajectories were the same. HUVEC microstructures could sprout when cultured in HFs conditioned media. Conclusion  HF-HUVEC pre-vascularized microstructures can be prepared by pre-culturing HFs before HUVECs and with the cell ratio at 1∶1 in a rotating bottle. In 3D co-culture system, HFs can promote and guide the sprout of HUVECs.

Citation： TANG Jun, TAN Jinnü, YE Zhaoyang, ZHOU Yan, TAN Wensong. Effect of fibroblasts on promoting the sprout and migration of endothelial cells in three-dimensional pre-vascularized microstructures. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(7): 881-888. doi: 10.7507/1002-1892.202203028 Copy

  • Previous Article

    Epidemiological and clinical characteristics analysis of 681 cases of thoracolumbar osteoporotic vertebral compression fractures

  • Next Article

    Effects and mechanism of morroniside on osteogenic differentiation and proliferation of mouse MC3T3-E1 cells