A novel in vitro angiogenesis model based on a microfluidic device - PubMed (original) (raw)
A novel in vitro angiogenesis model based on a microfluidic device
Dai Xiaozhen et al. Chin Sci Bull. 2011.
Abstract
Angiogenesis is very important for many physiological and pathological processes. However, the molecular mechanisms of angiogenesis are unclear. To elucidate the molecular mechanisms of angiogenesis and to develop treatments for "angiogenesis- dependent" diseases, it is essential to establish a suitable in vitro angiogenesis model. In this study, we created a novel in vitro angiogenesis model based on a microfluidic device. Our model provides an in vivo-like microenvironment for endothelial cells (ECs) cultures and monitors the response of ECs to changes in their microenvironment in real time. To evaluate the potential of this microfluidic device for researching angiogenesis, the effects of pro-angiogenic factors on ECs proliferation, migration and tube-like structure formation were investigated. Our results showed the proliferation rate of ECs in 3D matrix was significantly promoted by the pro-angiogenic factors (with an increase of 59.12%). With the stimulation of pro-angiogenic factors gradients, ECs directionally migrated into the Matrigel from low concentrations to high concentrations and consequently formed multi-cell chords and tube-like structures. These results suggest that the device can provide a suitable platform for elucidating the mechanisms of angiogenesis and for screening pro-angiogenic or anti-angiogenic drugs for "angiogenesis-dependent" diseases.
Figures
Figure 1
Design and structure of a microfluidic device for establishing an in vitro angiogenesis model. (a) Configuration of the device. The microfluidic device is composed of three main parallel channels connected by a series of smaller horizontal microchannels. (b) The fabricated device. The middle channel is injected with Matrigel and red dye, while the side channels are filled with medium containing either blue or yellow dye. (c) The schematic diagram of the microfluidic device for 3D culture. ECs suspended in Matrigel were injected into the middle channel, and both side channels were filled with medium to supply nutrition for the ECs in the 3D gel. (d) The schematic diagram of the microfluidic device for the migration assay of ECs. After Matrigel was filled into the middle channels and polymerized, ECs were seeded into one of the side channels. The other side channel was filled with medium with or without pro-angiogenic factors to induce ECs to migrate into the gel.
Figure 2
The gradient of fluorescence across the gel channel was quantified by measuring the fluorescence intensity. (a) After gel polymerization, the source channel (up) and the sink channel (down) were loaded with a FITC-dextran solution and PBS, respectively. The length of the Matrigel region was 1000 µm. The dashed line indicates the location of the fluorescence measurements. (b) The plot of the fluorescence intensity profile across the Matrigel region. A linear steady-state gradient was established after 90 min which was maintained for about 10 h.
Figure 3
The viability of ECs in the microfluidic device was investigated by Calcein-AM and PI staining. (a) HUVECs were seeded into one side channel for 3 d; most cells were viable (green). (b) HUVECs in the 3D matrix formed viable multi-cellular aggregates after 72 h in culture and exhibited a rounded morphology distinct from that seen in 2D culture. The viability of HUVECs in 3D culture was up to 95% (the green label is viable cells, while the red label depicts dead cells). The scale bar is 100 µm.
Figure 4
The proliferation of HUVECs in 3D culture induced by pro-angiogenic factors. (a) The proliferation of HUVECs was investigated using the Cell-Light EdU DNA Cell Proliferation Kit. HUVECs suspended in 3D matrigel were injected into the middle channel to culture for 8 h with or without pro-angiogenic factors and then incubated with EdU for 2 h. After incubation with EdU, dividing cells incorporated EdU (red). Cells were counterstained with Hoechst 33324 (blue). Only a few cells were labeled with EdU in the control group, while most cells in the experimental group were labeled with EdU after induction with pro-angiogenic factors. The scale bar is 100 µm. (b) This graph depicts the percentage of dividing cells incorporated with EdU. The results are given as mean ± SD (_n_=6, **, P < 0.01).
Figure 5
HUVECs migrated into the 3D matrigel to form tube-like structures under the stimulation of the gradient of pro-angiogenic factors (A, B). A sequence of micrographs recorded the migration of HUVECs into the 3D Matrigel over a 4 d period: (A) The control group without pro-angiogenic factors; (B) the migration of HUVECs because of the gradient of pro-angiogenic factors. (B(e)) During the invasion of HUVECs into the 3D Matrigel, the “lead-cells” extended filopodial projections (indicated by arrows) into the gel. The scale bar is 200 µm. (C) At the end of the experiments, cells were fixed and stained for actin. The results showed that tube-like structures were formed after cells invaded into the 3D Matrigel (indicated by arrows). The scale bar is 200 µm. (D) Quantitative migration assays of HUVECs into the 3D Matrigel. The migration distance (a) and area (b) of HUVECs into the 3D Matrigel significantly increased with pro-angiogenic factors, compared with those of the control without pro-angiogenic factors. Data are given as mean ± SD (_n_=6, **, P < 0.01).
Figure 6
The tube formation of HUVECs suspended in the 3D matrigel. Fixed samples of HUVECs encapsulated in Matrigel were cultured for 5 d with or without pro-angiogenic factors. At the end of the experiments, samples were fixed and stained for the actin cytoskeleton (green) and nuclei (blue). (a) Cells cultured in DMEM medium (control); (b) cells cultured in DMEM supplemented with pro-angiogenic factors (VEGF, bFGF, EGF, each at 10 ng/mL final concentration). The results showed that 3D encapsulated HUVECs formed many multi-cellular chords and complex tube-like structures in the presence of pro-angiogenic factors, while most cells are isolated and present a round shape in the control sample. White arrows indicate the tube like structures. The scale bar is 50 µm.
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