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 diagnosis^1,2. 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 invasion^4,5. 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 PDAC^6,7,8,9,10. 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

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 modifications^7,11.

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.

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 cul...

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...

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