Establishment of A Three-Dimensional Culture Condition for The U266 Cell Line Based on Peripheral Blood Plasma-Derived Fibrin Gels

Abstract

Objective: The study of pathophysiology as well as cellular and molecular aspects of diseases, especially cancer, requires appropriate disease models. In vitro three-dimensional (3D) structures attracted more attention to recapitulate diseases rather than in vitro two-dimensional (2D) cell culture conditions because they generated more similar physiological and structural properties. Accordingly, in the case of multiple myeloma (MM), the generation of 3D structures has attracted a lot of attention. However, the availability and cost of most of these structures can restrict their use. Therefore, in this study, we aimed to generate an affordable and suitable 3D culture condition for the U266 MM cell line.

Materials and methods: In this experimental study, peripheral blood-derived plasma was used to generate fibrin gels for the culture of U266 cells. Moreover, different factors affecting the formation and stability of gels were evaluated. Furthermore, the proliferation rate and cell distribution of cultured U266 cells in fibrin gels were assessed.

Results: The optimal calcium chloride and tranexamic acid concentrations were 1 mg/ml and 5 mg/ml for gel formation and stability, respectively. Moreover, the usage of frozen plasma samples did not significantly affect gel formation and stability, which makes it possible to generate reproducible and available culture conditions. Furthermore, U266 cells could distribute and proliferate inside the gel.

Conclusion: This available and simple fibrin gel-based 3D structure can be used for the culture of U266 MM cells in a condition similar to the disease microenvironment.

Keywords: Blood plasma; Fibrin; Multiple Myeloma; Three-Dimensional Culture.

Fig.1

The schematic of our study design. A simple and cost-beneficial 3D culture condition was developed using peripheral blood-derived plasma, 1 mg/ ml calcium chloride and 5 mg/ml tranexamic acid for the culture of U266 multiple myeloma cells in order to recapitulate a condition closer to the disease microenvironment.

Fig.2

The effect of different concentrations of tranexamic acidon the stability of fibrin gels formed by fresh plasma and 1 or 2 mg/ml calcium chloride.A. Fibrin gels formed by 1 mg/ml calcium chloride and different concentrations of tranexamic acid at day five. B. The percentages of weight changes as a measure of gel degradation in the formed gels using different concentrations of tranexamic acid and 1 mg/ml calcium chloride during seven subsequent days. C. Fibrin gels formed by 2 mg/ml calcium chloride and different concentrations of tranexamic acid at day five. D. The percentages of weight changes as a measure of gel degradation in the formed gels using different concentrations of tranexamic acid and 2 mg/ml calcium chloride during seven subsequent days. The mean ± standard deviation is used to present all data. The statistically significant differences compared to the group without tranexamic acid (TX0) at day one and compared to the group with 1 mg/ml tranexamic acid (TX1) at day five are displayed with * and #, respectively. *, #; P<0.05 and **; P<0.01. The exact numbers of P values are presented in the text. Statistical analysis was performed by ANOVA (Tukey post hoc) method with three experimental replicates. TX stands for tranexamic acid, and D stands for day. Numbers displayed alongside TX indicate different concentrations (mg/ml) of tranexamic acid.

Fig.3

The effect of different concentrations of tranexamic acid on the stability of fibrin gels formed by frozen plasma and 1 or 2 mg/ml calcium chloride. A. Fibrin gels formed by 1 mg/ml calcium chloride and different concentrations of tranexamic acid at day five. B. The percentages of weight changes as a measure of gel degradation in the formed gels using different concentrations of tranexamic acid and 1 mg/ml calcium chloride during seven subsequent days. C. Fibrin gels formed by 2 mg/ml calcium chloride and different concentrations of tranexamic acid at day five. D. The percentages of weight changes as a measure of gel degradation in the formed gels using different concentrations of tranexamic acid and 2 mg/ml calcium chloride during seven subsequent days. The mean ± standard deviation is used to present all data. The statistically significant differences between groups with different concentrations of tranexamic acid compared to the gels without tranexamic acid (TX0) at days one, two, three and five are displayed with *, #, $ and &, respectively. *, #, $, &; P<0.05 and **, ##, $$; P<0.01. The exact numbers of P values are presented in the text. Statistical analysis was performed by ANOVA (Tukey post hoc) method with three experimental replicates. TX stands for tranexamic acid, and D stands for day. Numbers displayed alongside TX indicate different concentrations (mg/ml) of tranexamic acid.

Fig.4

The degradation rates for the gels formed with fresh or frozen plasma samples in the presence of 1 mg/ml calcium chlorideand 5 mg/ml tranexamic acid over the course of seven days. The data is presented in the form of mean ± standard deviation. Statistical analysis was performed by ANOVA (Tukey post hoc) method with three experimental replicates. D stands for day.

Fig.5

Gel-free culture of U266 cells. A. The morphology of U266 cells at day three of culture (×20). B. Flow cytometry analysis of CD138 marker expression on U266 cells. C. The effect of different seeding densities on the U266 cell fold after three days of culture versus day zero. The data is presented in the form of mean ± standard deviation. The statistically significant groups compared to the 3×10⁴ and to 4×10⁴ seeding density groups are presented by * and #, respectively, at the same corresponding days post-culture. *, #; P<0.05 and **, ##; P<0.01. The exact numbers of P values are presented in the text. Statistical analysis was performed by ANOVA (Tukey post hoc) method with three experimental replicates. D stands for day.

Fig.6

Culture of U266 cells within the fibrin gels. A. The droplets generated by the mixture of U266 cells and fibrin gel components, after gelation. B. Expansion fold of U266 cells at day three post-culture compared to day zero in two different culture conditions: in culture plates and in fibrin gels. Statistical analysis was performed by the t test method with three experimental replicates. C. Phase-contrast image of U266 cells in fibrin gels (×20). D. Distribution of U266 cells in fibrin gels using H&E staining (×20).