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Bioengineering

Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates

Published: June 22, 2022 doi: 10.3791/63420

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.

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Protocol

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Nebraska-Lincoln (Project ID: 1046). Primary astrocytes were prepared from 1-3 day-old Sprague-Dawley rat pups in compliance with UNL's IACUC protocol 1046 and according to protocol with slight modifications7,11.

1. Preparations

  1. Obtain a master pattern of desired geometry before beginning this protocol.
    NOTE: Commercially available silicon wafers prepared using standard photolithography techniques are used in this protocol. The recommended starting pattern is 100-200 µm lines.
  2. Prepare buffers and media.
    1. Prepare poly(diallylmethylammonium chloride) (PDAC) and sulfonated poly(sterene) (SPS) polymer solutions to coat plates.
      1. PDAC coating solution is 0.3 wt. % PDAC and 0.1 M NaCl in ddH2O.
      2. SPS coating solution is 30 µM SPS and 0.1 M NaCl in ddH2O.
        NOTE: 1 L of PDAC coating solution and 1 L of SPS coating solution is sufficient to coat six 6-well plates. Solutions can be reused 2-5 times. Replace the solutions when they become cloudy.
    2. Prepare standard cell culture media, if needed.
      1. Typical astrocyte and GBM media are, respectively, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in Dulbecco's Modified Eagle Medium (DMEM).
  3. Obtain, prepare, or thaw cells.
    NOTE: This protocol is described for primary rat astrocytes isolated from 1-3 day neonatal rats and U87MG or A172-MG glioma cells. This protocol can be expanded to similar cell lines after confirmation of differential attachment of cell lines of interest on polymer surfaces.
    1. Due to cell loss during staining, use a minimum of 200,000 GBM cells on Day 0 and 400,000 live primary rat astrocytes on Day 1 per well in a 6-well plate. Adjust cell numbers if using a different size well or plate.
      ​NOTE: Primary astrocytes from passages 2-4 prepared as described by Wilson and coworkers12 are recommended for this protocol. Glioma cells were commercially obtained (see Table of Materials).

2. Polydimethylsiloxane (PDMS) stamp molding

  1. In a disposable container, weigh 12 parts (by weight) of PDMS pre-polymer and 1 part (by weight) of curing agent. Mix vigorously for 2-4 min until the entire mixture is filled with bubbles.
    NOTE: 26 g of the mixture is sufficient for one 100 mm Petri dish.
  2. Place fluorosilane into the fluorosilane desiccator. Place the disposable container with the polymer mixture into the desiccator to degas for 20 min.
    NOTE: This allows bubbles to rise out of the mixture. More time may be needed if large bubbles are still present after 20 min.
  3. Place the pre-patterned structure master into a Petri dish. Pour the PDMS mixture slowly into the Petri dish over the pre-patterned structure master, ensuring to completely cover the master. The optimal depth of the PDMS layer is about 2-3 mm thick.
    NOTE: Avoid creating air bubbles by slowly pouring the mixture. Ensure that the silicone master is lying flat in the Petri dish.
  4. Keep the Petri dish in the fluorosilane desiccator until all the bubbles are removed. Cure the Petri dish in an oven at 60 °C for 1 h. If 60 °C is not available, cure the Petri dish at 37 °C overnight.
  5. Using a sharp scalpel, evenly and gently cut around the master.
    CAUTION: Use caution when using a scalpel. Never leave the exposed blade unattended. Scalpels are sharp. Use appropriate safety precautions to prevent injury.
  6. Remove the stamp using a tweezer and place it on a cutting surface such as a cutting board. Using a scalpel, cut the stamps into a size small enough to fit into 6-well plates (approximately 2.5 cm x 2.5 cm).

3. Building Polyelectrolyte Multilayers (PEMs)

NOTE: This protocol describes plasma coating with an attached pressurized oxygen tank and gas mixing accessory (see Table of Materials). Any method of evenly plasma-cleaning the tissue culture polystyrene (TCPS) surface is suitable. Time and intensity may need to be optimized by the user when using a different plasma cleaning method.

  1. Plasma-clean a 6-well plate for 7 min.
    1. Bleed the chamber by turning the three-way valve on the front of the door to the right until the hiss from the air release can be heard. Turn the three-way valve back to the vertical position once the pressure reading is above 1800 mTorr.
    2. Open the door and put the 6-well plate in the chamber. Close the door and turn on the vacuum pump. Evacuate the chamber until the pressure is stable around 100 mTorr.
      NOTE: While the chamber is evacuating, confirm that the oxygen tank is open, and the output pressure of the oxygen is 10 psi.
    3. Open the three-way valve to the left to align the line with the oxygen input hose. Allow the oxygen gas to bleed into the chamber until the pressure stabilizes between 400 and 450 mTorr.
      NOTE: Confirm that the input pressure on the flow meter is 10 mm and adjust if needed.
    4. Turn on the RF power. Wait for the plasma to form (~15 s), and then start the timer for 7 min.
    5. After 7 min, turn off the RF power and turn the three-way valve back to its vertical position. Allow the chamber to evacuate to 150 mTorr.
    6. Turn off the vacuum pump and wait for the pressure to rise to 1500 mTorr. Vent the chamber by turning the three-way valve to the right until the hiss from the air release can be heard. When the pressure reading is above 1800 mTorr, turn the three-way valve back to its vertical position, open the door and retrieve the sample.
  2. Place plasma coated plates in room temperature baths as follows: PDAC for 20 min, DI water for 5 min, DI water for 5 min, SPS for 20 min, DI water for 5 min, and DI water for 5 min. Ensure that the entire plate surface is submerged.
    NOTE: Optionally, an automated slide stainer may be used to reduce active time. Use gentle agitation, if available.
  3. Repeat the previous step a total of 10 times. Allow the plates to air dry.
    ​NOTE: The dried plates can be stored at room temperature for up to several weeks before use.

4. PEM patterning via micromolding in capillaries

  1. Wash the PDMS stamps with gentle lab soap followed by DI water. Dry the stamps using an air compressor and place them on a flat, movable surface such as an unused 6-well plate lid.
    OPTIONAL: The stamps can also be air-dried. The stamps must be covered and protected from dust while drying.
  2. Plasma coat the stamps, as described in step 3, for 1 min. Promptly remove the stamps from the plasma cleaner and place them face down on the prepared 6-well plates.
  3. Pipette 10 µL of PDAC solution along the bottom edge of the stamp, steadily dispensing the polymer along the length of the stamp. Ensure to add the polymer to the side of the stamp with openings from the line patterns to allow capillary action to take place.
  4. Place a 350 g weight on top of each stamp for 20 s to help enforce the pattern. Allow the stamps to rest on the plates for 20 min.
  5. Dip the plate into DI water and peel off the stamps in the direction of the line patterns. Wash the plate twice for 5 min each in DI water.
  6. Wash the stamps with soap and DI water and dry with an air compressor. Allow the plate to air dry.
  7. If ready for cell culture, sterilize the plates under UV light in a Class II biosafety hood for a minimum of 8 h immediately prior to use. The minimum recommended total UV-C dose is 400 mJ/cm2 at 265 nm. If visualizing patterns, skip the UV sterilization step, and perform step 5. If ready for cell culture, perform step 6 after UV sterilization.

5. Visualizing stamped patterns with carboxyfluorescein (CFSE)

NOTE: Perform this procedure to visualize stamped patterns or skip this step and instead prepare patterns for cell culture. Once the patterns are stained, they cannot be used for cell culture. Once sufficient confidence in stamping is gained, the patterns can be used for cell culture. Carboxyfluorescein is light-sensitive. Stain and transport in the dark.

  1. Place dried, stamped patterns in CFSE pattern staining solution (0.1 µM CFSE in 0.1 M NaOH) for 60-90 min. Wash stamped patterns for 5 min in DI H2O two times.
    NOTE: If using glass slides, place the entire slide into a 50 mL tube containing the staining solution. If using well plates, add enough staining solution to cover the entire pattern.
  2. Visualize patterns using an appropriate fluorescence microscope.
    1. Turn on the fluorescent bulb to allow it to warm up for 10 min. Since patterns are stained with CFSE, use the interference blue filter (IB) to visualize the green patterns. View the patterns and take photos.

6. Staining and seeding glioblastoma cells with carboxyfluorescein

NOTE: All cell culture work should be performed in a suitable Class II biosafety cabinet.

  1. Detach the cells from the culture surface with 0.25% trypsin-EDTA, centrifuge at 200 x g for 4 min at 4 °C, and remove the supernatant.
  2. Resuspend the cells in 1 mL of cell culture media without serum (DMEM and 1% penicillin-streptomycin), centrifuge at 200 x g for 4 min at 4 °C, and remove the supernatant.
  3. Resuspend the cells in 1 mL of PBS and transfer to sterile 1.5 mL tube. Add 10 µL of 10 µg/mL CFSE and immediately mix thoroughly by pipetting up and down. Incubate at room temperature for 10 min and transfer into a 15 mL sterile tube.
  4. Add a minimum of 1 mL of cell culture medium (see step 1.2.2.1) to quench the CFSE dye reaction. Centrifuge at 400 x g for 10 min at 4 °C and remove the supernatant. Resuspend the cells in 5 mL of cell culture medium and transfer to a fresh 15 mL sterile tube.
  5. Centrifuge at 400 x g for 5 min at 4 °C, remove the supernatant, and resuspend the cells in 5 mL of cell culture medium.
  6. Repeat the previous wash step twice.
  7. Seed the stained glioma cells (100 cells/mm2 or 100,000 cells/well) in the patterned 6-well tissue culture plate from step 4 with astrocyte culture media. Culture the cells in an incubator (37 °C, 5% CO2) for 24 h before adding stained astrocytes in step 7.

7. Staining and seeding primary astrocytes with PKH26

NOTE: All of the cell culture work should be performed in a suitable Class II biosafety cabinet.

  1. Detach the cells from the culture surface with 0.25% trypsin-EDTA, centrifuge at 200 x g for 4 min at 4 °C, and remove the supernatant.
  2. Resuspend the cells in 1 mL of cell culture media without serum (DMEM and 1% penicillin-streptomycin), centrifuge at 200 x g for 4 min at 4 °C, and remove the supernatant.
  3. Prepare a 2x cell suspension by resuspending the cell pellet in 0.5 mL of room-temperature Diluent C from the PKH26 kit.
  4. Prepare a 2x dye solution (4 x 10-6 M) by adding 2 µL of PKH26 to 0.5 mL of Diluent C in a sterile 1.5 mL tube. Prepare the dye solution right before use.
  5. Add 2x cell suspension to 2x dye solution and immediately mix by pipetting up and down.
  6. Incubate the cell suspension for 1-5 min.
    NOTE: The staining happens quickly, and Diluent C is harmful to cells. Longer incubation does not mean better dyeing.
  7. Quench the staining by adding a minimum 5x volume of the cell culture medium and incubating for 1 min to bind the excess dye.
  8. Centrifuge at 200 x g for 10 min at 4 °C and carefully remove the supernatant. Resuspend the cells in 5 mL of cell culture media and transfer to a new sterile 15 mL tube.
  9. Centrifuge at 200 x g for 5 min at 4 °C, remove the supernatant and resuspend in 5 mL of PBS.
  10. Centrifuge at 200 x g for 5 min at 4 °C, remove the supernatant and resuspend in 5 mL of cell culture media.
  11. Repeat steps 7.9 and 7.10 for a total of three washes.
  12. Seed the stained astrocytes (200 cells/mm2 or 200,000 cells/well) with astrocyte media (see step 1.2.2.1) in the patterned 6-well tissue culture plate pre-seeded with glioma cells from step 6. Culture the cells in 37 °C, 5% CO2 incubator for the time experiments are carried out.

8. Fluorescence imaging of patterned mono-culture and co-culture

  1. Turn on the fluorescent bulb to allow it to warm up for 10 min.
  2. Remove the cell culture from the incubator and set it on the stage of an inverted microscope capable of fluorescence imaging.
  3. Insert or attach the appropriate filter. If the cells being imaged are stained with CFSE, use the interference blue filter (IB) to visualize the green cells. If the cells being imaged are stained with PKH26, use a Rhodamine filter to visualize the red cells.
  4. View the specimen and take photos.
    NOTE: The cells can now be separated using flow cytometry to analyze the biological effects of contact-mediated communication.

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Representative Results

The protocol here describes the engineering of direct contact patterned co-culture of glioma cells and astrocytes. This platform provides a biomimetic multicellular model to study the role of direct contact in the communication between astrocytes and glioma cells in the progression of glioblastoma multiforme (GBM). Figure 1 provides a scheme of the step-by-step surface modification and cellular introduction outlined above. Step one is to obtain a culture platform (glass coverslip, tissue culture plate, etc.) coated with a self-assembled PEM coating of PDAC/SPS in 10 bilayers and a PDMS stamp of the desired spatial pattern for the co-culture. Ten bilayers of PDAC/SPS were established previously by Kidambi et al. to provide a uniform PEM coating useful for the spatial control of fibroblasts, neurons, and breast cancer cells6,7,8. The PDMS stamp formed from a silicon master provides a versatile, reusable tool for applying a variety of patterns to study various cellular interactions, including those requiring physical contact to explore the importance of specific communication methods in tumor progression.

Figure 1
Figure 1: Schematic overview for employing PEMs and micro-molding in capillaries to create a patterned co-culture platform of glioma cells and cerebellum astrocytes. (A) PDMS pre-polymer and curing agent are mixed 12:1 and poured over silicon wafers with desired nano-features, commercially prepared using standard photolithography techniques (1). (B) Cell culture substrate is treated with oxygen plasma to become negatively charged (2). The substrate is sequentially dipped in poly(diallydimethylammoniumchloride) (PDAC) for 20 min, deionized (DI) H2O for 5 min, DI H2O for 5 min, poly(styrene sulfonate) (SPS) for 20 min, DI H2O for 5 min, and DI H2O for 5 min. This process of six baths is repeated 10 times. This can be done manually or with the assistance of a programmable dip coater (3). (C) The substrate now has 10 stable bilayers of PDAC and SPS. The PDMS stamps (from A) are cut apart to usable sizes, treated with oxygen plasma, and placed (with the pattern-side facing down) on 6-well plates (4). (D) A pipette is used to dispense ~10 µL of PDAC along the edge of the PDMS stamp, shown in red (5). Slow and steady movement along the entire edge of the stamp will ensure even coverage. Patterns can be verified using charged dyes (6) or can be prepared for cell culture. (E) Patterned substrate surfaces are UV sterilized overnight. (F) Glioma cells are stained with CFSE and are seeded onto the patterned substrate. The glioma cells are given 1-2 days to attach to SPS surfaces within the patterns on the substrate. (G) Primary cerebellum astrocytes are stained with PKH26 and are backfilled onto the patterned substrate. Images and analysis can be taken once the co-culture matures on day 4. Created with BioRender.com Please click here to view a larger version of this figure.

Various patterns can be utilized in this platform. Figure 2 shows micro-patterns visualized with carboxyfluorescein demonstrating the versatility of shape and size achieved by using micromolding in the capillaries. For example, heterogeneous physical interaction of cells will only occur at the interface between PDAC and SPS of the patterns. Therefore, the square pattern in the lower-left corner of Figure 2 demonstrates the ability to control the spatial organization of the patterns, which can be further extended to controlling the specific cell-cell interaction based on the cell placements within these patterns. Comparison of co-cultures in these patterns would allow for investigation into the overall influence of direct communication as opposed to soluble factors and non-direct communication, through paracrine signaling, with greater precision13. Furthermore, this platform allows the investigator to design patterns for controlling the number of heterogeneous cellular interactions mimicking the various stages of GBM tumor growth and positions within the tumor to optimally mimic in vivo observations14.

Figure 2
Figure 2: PEMs with different patterns stained with carboxyfluorescein. PEMs allow the investigator to adjust surface conditions (chemistry, shape, and size) to optimize the culture. Scale bar 500 µm. Please click here to view a larger version of this figure.

Figure 3 shows monocultures of glioma cells selectively attached to the SPS regions in the patterned PDAC/SPS surface after 24 h. The representative images demonstrate the ability of A172 and U87 to selectively adhere to specific regions leaving space for the backfill of astrocytes. The U87MG cell line is commonly used for glioblastoma studies as it is an invasive, highly tumorigenic glioma cell line. A172 was also chosen for this study as an invasive, non-tumorigenic glioblastoma cell line. Despite the differences in these cell lines, they appear to behave similarly when introduced onto the patterned PDAC/SPS surface, being spatially distributed according to the available pattern. This highlights the versatility of this platform in studying varied stages of glioblastoma15. As glioma cells have a relatively quick attachment and growth rate compared to the astrocytes, the introduction of astrocytes into the culture is performed one day after glioma seeding. This is to prevent the overgrowth of glioma into regions specifically meant for astrocyte attachment.

Figure 3
Figure 3: Patterned glioma mono-cultures stained with carboxyfluorescein (green) and PKH26 (red) on PEM patterned surfaces. Scale bar: 500 µm. Please click here to view a larger version of this figure.

Figure 4 is representative of the mature co-culture taken 4 days after astrocyte seeding. Astrocytes are seeded at a high density to assure full attachment in the space unoccupied by glioma. After the addition of astrocytes, the culture is allowed to mature 4 days before analysis. The maturation time (day 1-4 of co-culture) allows the glioma cells to fill in the regions to which they attach and choke out astrocytes that may have attempted to attach in the regions the glioma cells are established. Furthermore, the maturation period allows for the establishment of physical interaction, such as gap junctions, which are commonly seen to mediate cellular interaction of astrocytes and glioma cells in vivo and in vitro16,17,18.

Figure 4
Figure 4: Fluorescent images on day 4 of co-culture. (A) Astrocytes co-cultured with U87 glioma cells and (B) astrocytes co-cultured with A172 glioma cells on day 4 of co-culture demonstrating the selective ability of these PEM surfaces. Scale bar: 500 µm. Please click here to view a larger version of this figure.

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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 reproducibility, it is important that the user develops the skill of patterning through practice or obtains an automated system that can recreate patterns precisely. Furthermore, although the PDMS stamps are reusable, the repeated modification and use of the stamp may wear down the integrity of the pattern over time. Therefore, it is advised to periodically test the pattern integrity with a microscope or visualize patterns with carboxyfluorescein. Second, successful washing of cells stained with PKH26 and carboxyfluorescein is crucial for the differentiation of cell types when first establishing the method and following the different cell types. Furthermore, cross staining due to unsuccessful washing may inhibit future ability to separate cells from the co-culture via flow cytometry. This protocol includes wash steps optimized for the flow cytometry equipment and successful creation of the co-culture; however, it may be necessary to increase the number of wash steps if the user consistently finds stain cross-over during co-culture. Finally, the time window for analyzing a mature culture is limited as the glioma cells will eventually fill the pattern and begin to invade astrocyte-rich regions of the pattern. Therefore, it is advisable to analyze the co-culture at consistent time intervals to assure reproducibility of the results.

Potential problems users could encounter include problems with CFSE saturation. If the images are saturated with fluorescence, reduce pattern staining time from 60-90 min to 30-60 min. If the problem persists, decrease the stain concentration to 0.075 µM in 0.1 M NaOH. Alternatively, if the images show no fluorescence, first confirm that the microscope settings and prism are appropriate for excitation and emission wavelengths of 492 nm and 517 nm, respectively (i.e., interference blue filter (IB)) and check a range of exposure times from 100 ms to 10,000 ms. If there is still no fluorescence, increase pattern staining time from 60-90 min to 90-120 min. If the problem persists, increase stain concentration to 0.15 µM in 0.1 M NaOH. Additional problems that users may encounter with CFSE-visualized patterns is blurred or fuzzy edges instead of desired crisp geometries, which can be a sign of overused PDMS stamps. In this case, make new PDMS stamps using the silicon master wafers. Blurry or fuzzy images can also be a sign of contaminated CFSE staining solution and, in this case, prepare a new CFSE staining solution.

This system does have certain inherent limitations. First, it is a two-dimensional monoculture which although useful for high through-put preliminary studies6,7,8 and comparable to the currently available interaction models falls short of the ideal mimicry of the three-dimensional in vivo tissue19. Furthermore, physical interaction is optimized in this co-culture model but interaction through soluble factors is not completely eliminated due to the static nature of this system. The static media can be an advantage as analysis of released soluble factors may be insightful for understanding cellular response20. Understanding of purely physical interaction requires minimized interaction through soluble factors, which can be achieved through frequent media replacement and maintaining a large media volume for factor diffusion.

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Disclosures

The authors declare no conflicts of interest.

Acknowledgments

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

Name Company Catalog Number Comments
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

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References

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Direct-Contact Co-culture Astrocytes Glioblastoma Cells Polyelectrolyte Multilayer Templates Glioblastoma Multiforme GBM Malignant Brain Cancer Invasive Nature Tumor Progression Molecular Understanding Patterned Co-culture Method Layer-by-layer Assembly Micro-capillary-force Driven Patterning Protein-free Cell Culture Environment Cellular Interaction Pattern Dimensions Versatile Protocol Economical Protocol Reproducible Protocol Cellular Interaction Between Glioma And Astrocytes Glioma Tumors Physical Contact Soluble Cofactor Communication
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Stanke, K. M., Kidambi, S.More

Stanke, K. M., Kidambi, S. Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates. J. Vis. Exp. (184), e63420, doi:10.3791/63420 (2022).

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