ObjectiveTo explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology.MethodsRabbit 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.ResultsHDFs 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).ConclusionThree-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.
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.