High-glucose 3D INS-1 cell model combined with a microfluidic circular concentration gradient generator for high throughput screening of drugs against type 2 diabetes

Abstract

In vitro models for screening of drugs against type 2 diabetes are crucial for the pharmaceutical industry. This paper presents a new approach for integration of a three-dimensionally-cultured insulinoma cell line (INS-1 cell) incubated in a high concentration of glucose as a new model. In this model, INS-1 cells tended to aggregate in the 3D gel (basement membrane extractant, BME), in a similar way to 3D in vivo cell culture models. The proliferation of INS-1 cells in BME was initially promoted and then suppressed by the high concentration of glucose, and the function of insulin secretion also was initially enhanced and then inhibited by the high concentration of glucose. These phenomena were similar to hyperglycemia symptoms, proving the validity of the proposed model. This model can help find the drugs that stimulate insulin secretion. Then, we identified the difference between the new model and the traditional two-dimensional model in terms of cell morphology, inhibition rate of cell proliferation, and insulin secretion. Simultaneously, we developed a circular drug concentration gradient generator based on microfluidic technology. We integrated the high-glucose 3D INS-1 cell model and the circular concentration gradient generator in the same microdevice, tested the utility of this microdevice in the field of drug screening with glipizide as a model drug, and found that the microdevice was more sensitive than the traditional device to screen the anti-diabetic drugs.

Fig. 1. Observation of cell growth of INS-1 cells in three-dimensional and two-dimensional culture conditions. (A) Fluorescence image of INS-1 cells in well plate (6 days). Scale bar = 200 μm; (B) fluorescence image of INS-1 cells in BME (6 days). Scale bar = 50 μm.

Fig. 2. Time-resolved fluorescence images of INS-1 cells cultured in the BME. The green and red colors indicated live and dead cells, respectively. Scale bar = 50 μm.

Fig. 3. (A) Time-dependence of inhibition rate of INS-1 cell proliferation in 3D BME and 2D well plate, respectively. The high glucose concentration was 25.5 mmol L⁻¹ and 25.0 mmol L⁻¹ in BME and well plate, respectively. The control was 5.6 mmol L⁻¹. The inhibition rates of control in BME and well plate were same. n = 3; (B) time-dependence of insulin secretion of INS-1 cells cultured in 3D BME and 2D well plate, respectively. The high glucose concentration was 25.5 mmol L⁻¹ and 25.0 mmol L⁻¹ in BME and well plate, respectively. The control was 5.6 mmol L⁻¹. n = 3.

Fig. 4. (A) Illustration of channel design of the circular concentration gradient. The red and white spots in the center were solution inlets, and the spots labelled with numbers were solution outlets. The color gradient was simple illustration to show the concentration distribution within the microdevice; (B) plot of the concentration at each outlet, theoretical value and experimental value; n = 3. (C) Exploded view of the integrated microdevice. The top PDMS layer was concentration gradient generator. The bottom PDMS layer embedded 20 identical chambers (indicated by brown color), in which 3D INS-1 cells were cultured. The light green color indicated porous membrane sandwiched in between; (D) photograph of the real microdevice.

Fig. 5. (A) Variation of inhibition rate of 3D INS-1 cell proliferation with culture time in the presence of high glucose (25.5 mmol L⁻¹). At 96 h, glipizide was added into the culture medium; n = 4. (B) Variation of inhibition rate of 2D INS-1 cell proliferation with culture time in the presence of high glucose (25.5 mmol L⁻¹). At 72 h, glipizide was added into the culture medium; n = 3. (C) Variation of inhibition rate with glipizide concentration in 3D and 2D high-glucose models at 24 h after addition of glipizide; (D) variation of inhibition rate with glipizide concentration in 3D and 2D high-glucose models at 48 h after addition of glipizide; (E) variation of insulin secretion function of 3D INS-1 cells with culture time in the presence of high glucose (25.5 mmol L⁻¹). At 96 h, glipizide was added into the culture medium; n = 4. (F) Variation of insulin secretion function of 2D INS-1 cells with culture time in the presence of high glucose (25.5 mmol L⁻¹). At 72 h, glipizide was added into the culture medium; n = 3. (G) Variation of insulin secretion with glipizide concentration in 3D and 2D high-glucose models at 24 h after addition of glipizide; (H) variation of insulin secretion with glipizide concentration in 3D and 2D high-glucose models at 48 h after addition of glipizide.