| 【Background】 Nowadays, Diabetes has become one of the most common chronic diseases worldwide causinging heavy burdens to people’s health. Based on this, diabetes researches such as islet function have become a hot topic in medicine institutes of the world. Today, the conventional experiment method in vitro in medical research is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have developed to fabricate three-dimensional(3D) islet models in vitro which can mimic the intact islet in body. These artificial islet models have shown better cell function than monolayer islet cells, indicating their great potential as better experimental platforms to study physiological and pathological islet behaviors, such as the molecular mechanisms of diabetes and future islet transplantation.Some basic researches used microengineering method to fabricate 3D islet models in vitro, but few researches focused on comparing the structure and the function among 3D islet models,monolayer islet cells and primary islet. In this research, we would use hanging-drop method to fabricate 3D islet models in vitro and further evaluate the function of it.【Objective】 1. To explore appropriate size of the 3D islet micro-tissue in vitro by hanging-drop method. 2. To compare the structure and the function between the 3D islet micro-tissue in vitro and the 2D monolayer cell. 3. To compare the structure and the function between the 3D islet micro-tissue in vitro and the primary islet.【Methods】 1. Hanging-drop plate was prepared and the β-cells were cultured.Then tried to fabricate appropriate size of the 3D islet micro-tissue in vitro according to different conditions and tested their viability with kits. 2. Inverted microscope and immunofluorescence technique were used to observe the morphology of the 3D islet micro-tissue in vitro and the 2D monolayer cell culture.After that evaluated the function and differences between them through the glucose-stimulated insulin secretion(GSIS) method. 3. Primary rat islets were isolated and the primary islet model was established. Then inverted microscope, immunofluorescence technique and scanning electron microscope were used to observe the similarities and differences of the 3D islet micro-tissue in vitro and the primary islets in morphology.At last, evaluated the functional differences between them through GSIS.【Results】 1. The most suitable condition to fabricate appropriate size of the 3D islet micro-tissue in vitro by hanging-drop method was 30 μl droplets containing 105/ ml β-cells and 5 mm in spreading diameter of droplet. 2. The 3D islet micro-tissue in vitro maintained good viability of β-cells after culture for 7 days.3. Compared with the 2D monolayer cells, the 3D islet micro-tissue in vitro aggregated into a spheroid(e.g, round, oval and irregular shape) with diameter of approximately 100-200μm. The cells in the micro-tissue were packed densely. Immunofluorescence experiments showed the 3D islet micro-tissue in vitro secreted abundant insulin. 4. Compared the function of them, insulin secretion index(SI) of the 3D islet micro-tissue in vitro(2.08±0.14) was higher than that of the 2D monolayer cells(1.49±0.21)(P<0.05). 5. Compared the morphology of them, the primary islet was a spheroid(e.g, round, oval and irregular shape) with diameter of approximately 100-200 μm. The cells in the primary islets were packed densely. The surfaces were smooth and wrapped up by a layer of membrane. The morphology of 3D islet micro-tissue in vitro was similar to the primary islets. The difference between them was the surface of the 3D islet micro-tissue did not have a layer of membrane. Immunofluorescence experiments showed that distributions of β-cells and expression of insulin were both similar. 6. Compared the function of them, insulin SI of the 3D islet micro-tissue in vitro(2.08±0.14) was lower than that of the primary islets(2.72±0.31)(P<0.05).【Conclusions】 1. By hanging-drop method, the 3D islet micro-tissue in vitro can be successfully fabricated and maintain good viability and functionality. 2. Compared with the 2D monolayer cells, the morphology of the 3D islet micro-tissue in vitro is like a spheroid which similar to the primary islet. The 3D islet micro-tissue in vitro can be superior to the 2D monolayer cells on the functionality such as insulin secretion. 3. The 3D islet micro-tissue in vitro and the primary islets are similar in morphology; the function of 3D islet micro-tissue can be inferior to the primary islets on the insulin secretion. |
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