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Cited by 2163D Printing in Biomedical ResearchMicrofluidic and Capillary Electrophoresis ApplicationsHydrogels: synthesis, properties, applications

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Engineering a Brain Cancer Chip for High-throughput Drug Screening

Open access
Fan, Yantao, Nguyen, Duong Thanh, Akay, Yasemin, Xu, Feng, Akay, Metin·2016·Scientific Reports
DOI 10.1038/srep25062PMC PMC4858657
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Abstract

Glioblastoma multiforme (GBM) is the most common and malignant of all human primary brain cancers, in which drug treatment is still one of the most effective treatments. However, existing drug discovery and development methods rely on the use of conventional two-dimensional (2D) cell cultures, which have been proven to be poor representatives of native physiology. Here, we developed a novel three-dimensional (3D) brain cancer chip composed of photo-polymerizable poly(ethylene) glycol diacrylate (PEGDA) hydrogel for drug screening. This chip can be produced after a few seconds of photolithography and requires no silicon wafer, replica molding, and plasma bonding like microfluidic devices made of poly(dimethylsiloxane) (PDMS). We then cultured glioblastoma cells (U87), which formed 3D brain cancer tissues on the chip, and used the GBM chip to perform combinatorial treatment of Pitavastatin and Irinotecan. The results indicate that this chip is capable of high-throughput GBM cancer spheroids formation, multiple-simultaneous drug administration, and a massive parallel testing of drug response. Our approach is easily reproducible, and this chip has the potential to be a powerful platform in cases such as high-throughput drug screening and prolonged drug release. The chip is also commercially promising for other clinical applications, including 3D cell culture and micro-scale tissue engineering.

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Figure 1 Brain cancer chip design and preparation. (a) Final hydrogel device with microchannels and microwells after adding inlet and outlet reservoirs on top of the inlet and outlet orifices. (b) Schematic of the layers that are assembled during device fabrication. (c) Time protocol of the brain cancer chip preparation. (d) Photograph of the device from above. (e) Christmas tree-shaped channel system (gradient generator) of the brain cancer chip with channels of gradually decreasing width from 300 μm to 100 μm, an array of 24 individual culture chambers, and three inlet reservoirs and one outlet reservoir. The sub-channels, which link the microwells to the main channel, prevented captured cells from escaping the microwell.
Figure 1 Brain cancer chip design and preparation. (a) Final hydrogel device with microchannels and microwells after adding inlet and outlet reservoirs on top of the inlet and outlet orifices. (b) Schematic of the layers that are assembled during device fabrication. (c) Time protocol of the brain cancer chip preparation. (d) Photograph of the device from above. (e) Christmas tree-shaped channel system (gradient generator) of the brain cancer chip with channels of gradually decreasing width from 300 μm to 100 μm, an array of 24 individual culture chambers, and three inlet reservoirs and one outlet reservoir. The sub-channels, which link the microwells to the main channel, prevented captured cells from escaping the microwell.CC BY · PubMed Central
Figure 2 Characterization of the brain cancer chip. (a) Diagram for the optimization of PEGDA hydrogel layer thickness and UV exposure time for cross-linking PEGDA. Circles indicate exposure times that did not result in fully cross-linked PEGDA hydrogel, triangles indicate over-cross-linking in which the channels were blocked, and squares indicate the optimal time for crosslinking PEGDA hydrogel. (b) Images of photomasks with different channel widths and their resulting hydrogels. Channels were observed from top view. (c) Quantitative results of channel widths produced in the PEGDA hydrogel versus their corresponding photomasks. (d) Combinatorial screening of solutes from inlets via microfluidic networks. Eosin (red) and Chicago blue (blue) were injected into the two outer inlets. Color in the microchannels changed from red (left) to blue (right). (e) Fluorescent imaging of FITC in hydrogel microfluidic channels after simultaneous injection of FITC (left) and PBS (right). The concentrations of FITC solution in different microchannels were calculated by comparing fluorescent intensities between each microchannel and the inlet.
Figure 2 Characterization of the brain cancer chip. (a) Diagram for the optimization of PEGDA hydrogel layer thickness and UV exposure time for cross-linking PEGDA. Circles indicate exposure times that did not result in fully cross-linked PEGDA hydrogel, triangles indicate over-cross-linking in which the channels were blocked, and squares indicate the optimal time for crosslinking PEGDA hydrogel. (b) Images of photomasks with different channel widths and their resulting hydrogels. Channels were observed from top view. (c) Quantitative results of channel widths produced in the PEGDA hydrogel versus their corresponding photomasks. (d) Combinatorial screening of solutes from inlets via microfluidic networks. Eosin (red) and Chicago blue (blue) were injected into the two outer inlets. Color in the microchannels changed from red (left) to blue (right). (e) Fluorescent imaging of FITC in hydrogel microfluidic channels after simultaneous injection of FITC (left) and PBS (right). The concentrations of FITC solution in different microchannels were calculated by comparing fluorescent intensities between each microchannel and the inlet.CC BY · PubMed Central
Figure 3 Diffusion properties of the hydrogel chip. (a) Diffusion of FITC-glucose in the microchannel at the time points: 15 seconds, 1 min, 3 min, 10 min, 20 min and 30 min. Scale bar, 500 μm. Plot of normalized fluorescent intensity of FITC solution for each time point. (b) Graph showing the diffusion of FITC-glucose (MW = 150,000 Da) and DAPI (MW = 277 Da) 30 minutes after injection. (c) Time-lapse images of FITC diffusion in microwell (i) and tunable release of FITC after washing microwell by PBS (ii). (d) Three-dimensional graph showing the increase in fluorescent intensity of the microwell and its surrounding hydrogel diagonally across the microwell over time after injection (i) and the change in fluorescent intensity of the microwell and its surrounding hydrogel after washing with PBS, showing the release of chemicals back into microwell.
Figure 3 Diffusion properties of the hydrogel chip. (a) Diffusion of FITC-glucose in the microchannel at the time points: 15 seconds, 1 min, 3 min, 10 min, 20 min and 30 min. Scale bar, 500 μm. Plot of normalized fluorescent intensity of FITC solution for each time point. (b) Graph showing the diffusion of FITC-glucose (MW = 150,000 Da) and DAPI (MW = 277 Da) 30 minutes after injection. (c) Time-lapse images of FITC diffusion in microwell (i) and tunable release of FITC after washing microwell by PBS (ii). (d) Three-dimensional graph showing the increase in fluorescent intensity of the microwell and its surrounding hydrogel diagonally across the microwell over time after injection (i) and the change in fluorescent intensity of the microwell and its surrounding hydrogel after washing with PBS, showing the release of chemicals back into microwell.CC BY · PubMed Central
Figure 4 GBM cell capture, culture and 3D-spheroid formation. (a) Optimization of initial cell-seeding concentrations. Cells in each microwell were counted 30 minutes after the injection. Error bars represent standard error of the mean. Scale bar, 200 μm. (b) U87 cells were injected into the microfluidic network from the inlets and captured in the microwell. Cells were cultured in DMEM and aggregated to form cancer spheroids. Scale bar, 200 μm. (c) Bright field and fluorescence micrograph showing cancer spheroid. (ii) Staining of live cells with 4 mM Calcein AM (green) and (iii) dead cells with 2 mM ethidium homodimer-1 (red). (iv) Merged image of stained cancer spheroid. (d) GBM spheroids in microwells of brain cancer chip (i) showed a uniform size (361.3 ± 36.2 μm-diameter) (ii). Scale bar, 200 μm.
Figure 4 GBM cell capture, culture and 3D-spheroid formation. (a) Optimization of initial cell-seeding concentrations. Cells in each microwell were counted 30 minutes after the injection. Error bars represent standard error of the mean. Scale bar, 200 μm. (b) U87 cells were injected into the microfluidic network from the inlets and captured in the microwell. Cells were cultured in DMEM and aggregated to form cancer spheroids. Scale bar, 200 μm. (c) Bright field and fluorescence micrograph showing cancer spheroid. (ii) Staining of live cells with 4 mM Calcein AM (green) and (iii) dead cells with 2 mM ethidium homodimer-1 (red). (iv) Merged image of stained cancer spheroid. (d) GBM spheroids in microwells of brain cancer chip (i) showed a uniform size (361.3 ± 36.2 μm-diameter) (ii). Scale bar, 200 μm.CC BY · PubMed Central
Figure 5 Combinatorial drug treatment and drug screening. (a) Pitavastatin (10 μM, 100 μl) and Irinotecan (100 μM, 100 μl) were injected into the two outer inlets on day 7 of cancer spheroid culture. (b) Time-lapse images of cancer spheroid in microwells were taken before drug administration (day 0) and after drug administration (day 1, day 4 and day 7). Scale bar, 200 μm. (c) Images of trypan blue staining of each channel on day 4 after drug administration. Scale bar, 200 μm. (d) Combinatorial drug screening results. Cancer spheroids were cultured for 7 days. Pitavastatin (10 μM, 100 μl) and Irinotecan (100 μM, 100 μl) were then injected into the two outer inlets. The graph shows cell viability in the microwells on day 1, day 4 and day 7 after dual-drugs treatment. (e) Comparison of combinatorial drug screening results (Channel D) with individual drug screening results by Pitavastatin (10 μM, 100 μl) and Irinotecan (100 μM, 100 μl) and untreated GBM cancer spheroids on day 1, day 4 and day 7 after dual-drug and individual drug treatments.
Figure 5 Combinatorial drug treatment and drug screening. (a) Pitavastatin (10 μM, 100 μl) and Irinotecan (100 μM, 100 μl) were injected into the two outer inlets on day 7 of cancer spheroid culture. (b) Time-lapse images of cancer spheroid in microwells were taken before drug administration (day 0) and after drug administration (day 1, day 4 and day 7). Scale bar, 200 μm. (c) Images of trypan blue staining of each channel on day 4 after drug administration. Scale bar, 200 μm. (d) Combinatorial drug screening results. Cancer spheroids were cultured for 7 days. Pitavastatin (10 μM, 100 μl) and Irinotecan (100 μM, 100 μl) were then injected into the two outer inlets. The graph shows cell viability in the microwells on day 1, day 4 and day 7 after dual-drugs treatment. (e) Comparison of combinatorial drug screening results (Channel D) with individual drug screening results by Pitavastatin (10 μM, 100 μl) and Irinotecan (100 μM, 100 μl) and untreated GBM cancer spheroids on day 1, day 4 and day 7 after dual-drug and individual drug treatments.CC BY · PubMed Central
Figure 6 Tunable release of drugs. (a) Time protocol of the formation of cancer spheroids and drugs testing in hydrogel chip. (b) Time-lapse images and cell viability of U87 cells in microwell during the application of a single dose of drugs on day 7. Drugs were continuously released from the hydrogel layer to microwell and affected cancer spheroid.
Figure 6 Tunable release of drugs. (a) Time protocol of the formation of cancer spheroids and drugs testing in hydrogel chip. (b) Time-lapse images and cell viability of U87 cells in microwell during the application of a single dose of drugs on day 7. Drugs were continuously released from the hydrogel layer to microwell and affected cancer spheroid.CC BY · PubMed Central

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Metadata

Authors
Fan, Y., Nguyen, D. T., Akay, Y., Xu, F., & Akay, M.
Affiliations
  • University of Houston (US)
  • Xi'an Jiaotong University (CN)
Corresponding author
Akay, Metin
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2016-05-06
Volume
6
Issue
1
Pages
25062-25062
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/srep25062
PMC
PMC4858657
OpenAlex
W2346064008
Cited by
216
References
32
Citations by year
2026: 9 · 2025: 17 · 2024: 18 · 2023: 22 · 2022: 29 · 2021: 32 · 2020: 23 · 2019: 23 · 2018: 23 · 2017: 14 · 2016: 6
FWCI
10.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
  • Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
  • Physical Sciences › Materials Science › Biomaterials › Hydrogels: synthesis, properties, applications
Keywords
glioblastoma, high-throughput drug screening, PEGDA hydrogel, U87 glioblastoma cells, tumor spheroids, pitavastatin, irinotecan, combination therapy, photolithography, microfluidics, 3D cell culture, drug release
MeSH terms
Antineoplastic Agents, Brain Neoplasms, Drug Evaluation, Preclinical, Humans, Reproducibility of Results, Spheroids, Cellular, Cell Culture Techniques, Cell Line, Tumor, High-Throughput Screening Assays

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