Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates
Summary
This protocol describes a patterned direct contact glioma-astrocyte co-culture utilizing micro-contact printing on polyelectrolyte multilayers (PEMs) to pattern U87 or A172 GBM cells and primary astrocytes.
Abstract
Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in 2020 and 2021. GBM tumors most often arise from astrocytes and are characterized by their invasive nature, often recruiting healthy tissues into tumor tissue. Understanding communication between astrocytes and glioblastoma cells is vital for the molecular understanding of tumor progression. This protocol demonstrates a novel patterned co-culture method to investigate contact-mediated effects of astrocytes on GBM employing layer-by-layer assembly and micro-capillary-force driven patterning. Advantages include a protein-free cell culture environment and precise control of cellular interaction dictated by the pattern dimensions. This technique provides a versatile, economical, reproducible protocol for mimicking cellular interaction between glioma and astrocytes in glioma tumors. This model can further be used to tease apart changes in GBM molecular biology due to physical contact with astrocytes or with non-contact mediated soluble cofactor communication.
Introduction
Glioblastoma Multiforme (GBM) is the most prolific and deadly brain cancer in the United States with a median survival time of around 15 months1. Fewer than 7% of GBM patients survive more than 5 years post diagnosis1,2. By 10 years, that figure drops to less than 1%1,2. Though other cancer types have made marked improvements in survival in recent decades, the success of GBM patients falls short. To develop successful therapeutic interventions, an appropriate in situ model must be utilized to develop a more thorough understanding of GBM tumor biology. This understanding is crucial in improving clinical outcomes for GBM patients.
The brain contains a large variety of cell types each filling specific niches to promote organism function and survival. In addition to neurons, there are a variety of glial cells, including astrocytes, oligodendrocytes, and microglia. Astrocytes, in particular, have been implicated in GBM tumor growth and invasion through the secretion of pro-migratory soluble factors3. Further, there are some reports that physical contact is the driving force of astrocyte-mediated glioma cell migration and invasion4,5. However, the molecular basis driving this change remains largely unknown.
In order to study the contact-mediated effects of astrocytes on tumor growth, this protocol reports on the development of a reproducible, protein-free method for in vitro cell patterning. In this method, polyelectrolyte multilayers (PEMs) are systematically assembled to form a uniform protein-free surface. PEMs are built using a polycation-polyanion system comprising poly(diallylmethylammonium chloride) (PDAC) and sulfonated poly(sterene) (SPS), respectively. These polymers were chosen based on previous studies that reported preferential attachment of cells to SPS over PDAC6,7,8,9,10. On these PEM surfaces, this protocol utilizes microcapillary-force lithography to engineer patterned co-culture models of primary astrocytes and GBM cells.
The techniques presented herein allow for the precise engineering of specific cell-cell interactions through the control of surface patterning, thereby supporting the highly reproducible investigation of cellular communication. Furthermore, the biomimetic surface inherent in this platform facilitates the study of direct cell-cell communication that is crucial for deepening mechanistic understanding of the communication between different cell types. Beyond this, the method is low-cost, providing a significant advantage for conducting in vitro studies to explore cellular communication. Specifically, this protocol exploits differential attachment of GBM on SPS (-) over PDAC (+) to create patterned co-cultures of GBM cell lines and primary astrocytes.
Protocol
Representative Results
Discussion
Critical steps to assure the successful assembly of a reproducible patterned co-culture include: 1) the successful patterning of the surface by micromolding in capillaries, 2) the successful washing of stained cells, and 3) the analysis of the co-culture in the "mature culture" window. First, the successful reproduction of patterns with micromolding in capillaries is critical to the reproducibility of interaction as this is what sets patterned co-culture apart from random co-culture. To assure this reproducibilit…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported, in whole or in part, by NIH grants 1R01AA027189-01A1 (to S.K.), P20 GM104320 (to the Nebraska Center for the Prevention of Obesity Diseases Pilot Grant to S.K.), P20 GM113126 (to the Nebraska Center for Integrated Biomolecular Communication-Project Leader S.K.); UNL Office of Research and Development Biomedical Seed Grant and Nebraska Research Initiative-Systems Grant (to S.K.). K.M.S. was funded through T32GM107001, a training grant.
Materials
| 0.25% Trypsin-EDTA | Fisher Scientific | 25200056 | |
| 15 ml Nunc Conical Sterile Polypropylene Centrifuge Tubes | Fisher Scientific | 12-565-268 | |
| 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester | Millipore Sigma | Cat#21888 | |
| 50 ml Nunc Conical Sterile Polypropylene Centrifuge Tubes | Fisher Scientific | 12-565-270 | |
| A172-MG GBM cell line | ATCC | CRL-1620 | |
| Bright-Line Hemacytometer | Sigma | Z359629 | Or other suitable cell counting device |
| Cell Incubator | N/A | N/A | |
| Cooled tabletop centrifuge for 15 mL tubes | N/A | N/A | |
| Dulbecco's Modified Eagle Medium (DMEM) | MP Biomedicals | ICN 1033120 | |
| Expanded Plasma Cleaner | Plasma Harrick | PDC-001-HP | With attached pressurized oxygen tank and PlasmaFlo Gas Mixer (PDC-FMG) accessory |
| Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11550H | |
| Fluorosilane | Sigma Aldrich | 667420 | Full chemical name: 1H,1H,2H,2H-Perfluorooctyltriethoxysilane |
| Inverted Tabletop Microscope | N/A | N/A | Microscope capable of fluorescent imaging with λex = 551 nm; λem 567 nm [e.g. Rhodamine filter] (PKH26 dye) and λex 492 nm; λem 517 nm [e.g. interference blue filter (IB)] (CFSE dye) |
| NaCl | Sigma Aldrich | S7653 | |
| NaOH | Sigma Aldrich | 567530 | |
| Penicillin-Streptomicen | Fisher Scientific | 15140122 | |
| PKH26 Red Fluorescent Cell Linker Mini Kit | Millipore Sigma | Cat#MINI26-1KT | |
| Poly(diallyldimethylammonium chloride) solution (PDAC) | Sigma Aldrich | 409014 | 20 wt. % in H2O |
| Poly(sodium 4-styrenesulfonate) (SPS) | Sigma Aldrich | 243051 | average MW ~70,000 |
| Primary Astrocytes, isolated from Srague Dawley rats | Charles River | Crl:SD | Rats from Charles River; Lab isolated Cells |
| Scalpel | N/A | N/A | |
| Sodium bicarbonate | Sigma Aldrich | S5761 | |
| Sylgard 184 Silicone Elastomer Kit | Dow Chemical | Cat#2646340 | |
| Trypan Blue Stain | Fisher Scientific | 15-250-061 | |
| TryplE | Fisher Scientific | Gibco TrypLE | |
| U87-MG GBM cell line | ATCC | HTB-14 | |
| Vacuum Desiccator | N/A | N/A |
References
- Ostrom, Q. T., et al. CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2012-2016. Neuro Oncology. 21, 1 (2019).
- Ostrom, Q. T., et al. CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro Oncology. 19, 1-88 (2017).
- Roos, A., Ding, Z., Loftus, J. C., Tran, N. L. Molecular and microenvironmental determinants of glioma stem-like cell survival and invasion. Frontiers in Oncology. 7, 120 (2017).
- Rath, B. H., Fair, J. M., Jamal, M., Camphausen, K., Tofilon, P. J. Astrocytes enhance the invasion potential of glioblastoma stem-like cells. PLoS One. 8 (1), 54752 (2013).
- Le, D. M., et al. Exploitation of astrocytes by glioma cells to facilitate invasiveness: a mechanism involving matrix metalloproteinase-2 and the urokinase-type plasminogen activator-plasmin cascade. The Journal of Neuroscience. 23 (10), 4034-4043 (2003).
- Daverey, A., Brown, K. M., Kidambi, S. Breast cancer/stromal cells coculture on polyelectrolyte films emulates tumor stages and miRNA profiles of clinical samples. Langmuir. 31 (36), 9991-10001 (2015).
- Kidambi, S., Lee, I., Chan, C. Primary neuron/astrocyte co-culture on polyelectrolyte multilayer films: A template for studying astrocyte-mediated oxidative stress in neurons. Advanced Functional Materials. 18 (2), 294-301 (2008).
- Kidambi, S., et al. Patterned co-culture of primary hepatocytes and fibroblasts using polyelectrolyte multilayer templates. Macromolecular Bioscience. 7 (3), 344-353 (2007).
- Kidambi, S., Lee, I., Chan, C. Controlling primary hepatocyte adhesion and spreading on protein-free polyelectrolyte multilayer films. Journal of the American Chemical Society. 126 (50), 16286-16287 (2004).
- Mohammed, J. S., DeCoster, M. A., McShane, M. J. Micropatterning of nanoengineered surfaces to study neuronal cell attachment in vitro. Biomacromolecules. 5 (5), 1745-1755 (2004).
- Beaudoin, G. M., et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nature Protocols. 7 (9), 1741-1754 (2012).
- Wilson, C. L., Hayward, S. L., Kidambi, S. Astrogliosis in a dish: substrate stiffness induces astrogliosis in primary rat astrocytes. Rsc Advances. 6 (41), 34447-34457 (2016).
- Bogdanowicz, D. R., Lu, H. H. Studying cell-cell communication in co-culture. Biotechnololgy Journal. 8 (4), 395-396 (2013).
- Chen, W., et al. Glioma cells escaped from cytotoxicity of temozolomide and vincristine by communicating with human astrocytes. Medical Oncology. 32 (3), 43 (2015).
- Belot, N., et al. Molecular characterization of cell substratum attachments in human glial tumors relates to prognostic features. Glia. 36 (3), 375-390 (2001).
- Sin, W. C., et al. Astrocytes promote glioma invasion via the gap junction protein connexin43. Oncogene. 35 (12), 1504-1516 (2016).
- Sin, W. C., Crespin, S., Mesnil, M. Opposing roles of connexin43 in glioma progression. Biochimica et Biophysica Acta. 1818 (8), 2058-2067 (2012).
- Oliveira, R., et al. Contribution of gap junctional communication between tumor cells and astroglia to the invasion of the brain parenchyma by human glioblastomas. BMC Cell Biology. 6 (1), 7 (2005).
- Duval, K., et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda). 32 (4), 266-277 (2017).
- Rape, A., Ananthanarayanan, B., Kumar, S. Engineering strategies to mimic the glioblastoma microenvironment). Advanced Drug Delivery Reviews. 79-80, 172-183 (2014).
.