The role of bloom index of gelatin on the interaction with retinal pigment epithelial cells

Abstract

Biocompatible materials are of considerable interest in the development of cell/drug delivery carriers for therapeutic applications. This paper investigates the effects of the Bloom index of gelatin on its interaction with retinal pigment epithelial (RPE) cells. Following two days of culture of ARPE-19 cells with gelatin samples G75-100, G175, and G300, the in vitro biocompatibility was determined by cell proliferation and viability assays, and glutamate uptake measurements, as well as cytokine expression analyses. The mitochondrial dehydrogenase activity in the G300 groups was significantly lower than that of G75-100 and G175 groups. The Live/Dead assays also showed that the gelatin samples G300 induced mild cytotoxicity. In comparison with the treatment of gelatins with low Bloom index, the exposure to high Bloom strength gelatins markedly reduced the glutamate uptake capacity of ARPE-19 cells. One possible explanation for these observations is that the presence of gelatin samples G300 with high viscosity in the medium may affect the nutrient availability to cultured cells. The analyses of pro-inflammatory cytokine IL-6 expression at both mRNA and protein levels showed that the gelatins with low Bloom index caused less cellular inflammatory reaction and had more acceptable biocompatibility than their high Bloom strength counterparts. These findings suggest that the Bloom index gives influence on cellular responses to gelatin materials.

Keywords: Bloom index; gelatin; in vitro biocompatibility; retinal pigment epithelial cells.

Figure 1.

Shear stress versus shear rate curves for various gelatin samples in BSS at 37 °C.

Figure 2.

Cell proliferation assay of ARPE-19 cell cultures incubated in the presence of various dissolved gelatin materials for two days at 37 °C. Results are expressed as percentage of controls (MTS activity of cells cultured in the absence of materials). An asterisk indicates statistically significant differences (*p<0.05; n = 4) as compared to controls.

Figure 3.

Cell viability of ARPE-19 cultures was determined by staining with Live/Dead Viability/Cytotoxicity Kit in which the live cells fluoresce green and the dead cells fluoresce red. Green (A, C, E, G) and red (B, D, F, H) fluorescence images of cells in (A, B) controls (without materials) after exposure to various dissolved gelatin materials (C, D) G75-100, (E, F) G175, and (G, H) G300 for 2 days at 37 °C. Scale bars indicate 100 μm.

Figure 4.

Mean percentage of live cells in the ARPE-19 cultures exposed to various dissolved gelatin materials as measured by the Live/Dead assay. An asterisk indicates statistically significant differences (*p<0.05; n = 6) as compared to controls (without materials).

Figure 5.

Glutamate uptake in the ARPE-19 cultures exposed to various dissolved gelatin materials for 2 days. Data in the experimental groups are percentages relative to that of control groups (without materials). An asterisk indicates statistically significant differences (*p<0.05; n = 3) as compared to controls.

Figure 6.

Gene expression of IL-6 in ARPE-19 cells incubated with various dissolved gelatin materials for two days by real-time RT-PCR. Normalization was done by using GAPDH. Data in the experimental groups are percentages relative to that of control groups (without materials). An asterisk indicates statistically significant differences (*p<0.05; n = 3) as compared to controls.

Figure 7.

Level of IL-6 released from ARPE-19 cell cultures after incubation with various dissolved gelatin materials for two days. An asterisk indicates statistically significant differences (*p<0.05; n = 4) as compared to controls (without materials).