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 und....

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,

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