Engineering a High-Throughput 3-D In Vitro Glioblastoma Model

Abstract

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults because of its highly invasive behavior. The existing treatment for GBM, which involves a combination of resection, chemotherapy, and radiotherapy, has a very limited success rate with a median survival rate of <1 year. This is mainly because of the failure of early detection and effective treatment. We designed a novel 3-D GBM cell culture model based on microwells that could mimic in vitro environment and help to bypass the lack of suitable animal models for preclinical toxicity tests. Microwells were fabricated from simple and inexpensive polyethylene glycol material for the control of in vitro 3-D culture. We applied the 3-D micropatterning system to GBM (U-87) cells using the photolithography technique to control the cell spheroids' shape, size, and thickness. Our preliminary results suggested that uniform GBM spheroids can be formed in 3-D, and the size of these GBM spheroids depends on the size of microwells. The viability of the spheroids generated in this manner was quantitatively evaluated using live/dead assay and shown to improve over 21 days. We believe that in vitro 3-D cell culture model could help to reduce the time of the preclinical brain tumor growth studies. The proposed novel platform could be useful and cost-effective for high-throughput screening of cancer drugs and assessment of treatment responses.

Keywords: Glioblastoma; PEG hydrogel; in vitro; microwells.

FIGURE 1.

Schematic of the process for formation of controlled-size GBM cancer spheriods using microwells. The hydrogel of poly(ethylene glycol) dimethyl acrylate (PEGDA) was used as a biomaterial to fabricate 3D microwells due to its biocompatible, hydrophilic, UV-sensitive properties.

FIGURE 2.

Stability of PEGDA hydrogel on treated and untreated cover glasses. (A and B) Percentage of stable microwells prepared by PEGDA-750 and PEGDA-1000 on TMSPMA treated and untreated cover glasses. The results showed that TMSPMA-treated cover glasses leaded to stable hydrogel attachment comparing to untreated cover glasses. (C and D) Different concentration (10% to 80%) of PEGDA-750 and PEGDA-1000 were prepared and applied on the TMSPMA- treated cover glasses.

FIGURE 3.

Quantification of the seeding density of initial U-87 cells into the microwells. (A-C) The seeding concentrations of initial cells were optimized. The cells were seeded and the images were taken before and after wash with PBS. The number of the cells in the microwells was counted. (D) The representative bright field images of cells in the round and square microwells with the size of . Images showed that cells/ml leads to the optimal formation of cancer spheriods over 3 weeks in culture. Scale bars represent . (E) Graphical representation of U87 cell density versus cell concentration in solution. Cells in the PEGDA microwells counted for calculating the cell density. Error bars represent standard error of the mean.

FIGURE 4.

Time course of GBM cells within PEGDA-750 microwells of different sizes. Each row is a specific microwell size ( round and square, round and square and round and square) and each column is a different time point (Day1, 4, 7, 14 and 21) Bright field images of the U87 cancer cells cultured in PEGDA-750 microwells for 21 days were taken. The U87 cells’ aggregation, proliferation and spheroids’ formation from day 0 to day 21 were observed over a period of 21 days. The size of the microwell affected the U87 cancer spheroids’ formation in the microwells. The more the size of the microwell is smaller, the more the cells get in the microwells and connect with each other easily.

FIGURE 5.

Cellular growth of GBM spheroids over a period of 21 days in the different size of microwells. The cell growth was affected by the size and the shape of the microwell. ( denotes denotes .

FIGURE 6.

GBM aggregates in PEGDA-750 microwells. Bright field and fluorescent images after applying Live/dead assay to GBM cells. Side view images of the aggregates in (A-1, 2) m (B-1, 2) and (C-1, 2) microwells on Day 21. Significant aggregations with controlled sizes were observed. Cells formed 3D spheroids with diameters closely corresponding to the respective microwells. Scale bars correspond to . (D) Aggregates grown in different shape microwells on day 21; PEGDA-750 microwells confirmed that the GBM cells are viable for 3 weeks and the shape of the spheroids can be controlled as well as their size.