Three-Dimensional Co-Culture Method for the Study of Indirect Interactions Between Different Cell Types Across a Reconstituted Basement Membrane

Summary

This article details a straightforward method for the three-dimensional co-culture of two cell types separated by a reconstituted basement membrane that requires little specialized equipment and can be readily analyzed by a variety of experimental techniques.

Abstract

Three-dimensional (3D) co-culture is a rapidly evolving technique for researchers looking to accurately study cell-cell interactions using in vitro experiments. The limitations of monolayer cell culture, including limited interactions between the cellular and extracellular environment and disturbed cell morphology, are addressed by simulating the in vivo cellular environment. Using a scaffold to provide structural support and including biologically active extracellular matrix components, 3D cultures display behaviours and morphologies more congruent with tissue. Incorporating multiple cell types into this kind of 3D environment allows for the study of cell-cell interactions inside a biomimetic model system. A wide range of 3D co-culture technologies has emerged, each with its own advantages and challenges. Often these technologies require specialized equipment, a complex setup, or specific technical knowledge. As well, there exist a few standardized methods for studying indirect cell-cell interactions between two cell types separated by a reconstituted basement membrane. Here, we describe a 3D co-culture method that requires only fundamental technical skills and uses more widely applicable materials to successfully recapitulate indirect cell-cell interactions across a basement membrane. A monolayer of cells is covered in a layer of extracellular matrix, in the form of Matrigel, and a second cell type is seeded on top. The resultant co-culture is maintained for five days, at which point cells are analyzed for morphological changes by immunofluorescence or extracted from the co-culture for more detailed genomic, transcriptomic, or proteomic analyses. This protocol is ideal for studying the impact of cell-cell communication on cell behaviour when physical contact is prohibited by a basement membrane. As researchers continue to opt for more in vivo-relevant cell culture methods, a streamlined approach is necessary to avoid high barriers to entry.

Introduction

In vitro cell culture is a robust, rapid, and widely used research technique. Though traditional cell culture methods are powerful, they differ greatly from an in vivo environment. The spatially restricted nature of monolayer cell culture leads to changes in gene expression, morphology, and biochemistry^1,2,3. Three-dimensional (3D) culture techniques address this disparity, as cells are grown in a scaffold that simulates the extracellular matrix (ECM) present in organs and tissues. In 3D culture, cells display morphology and behaviour more like that which is....

Protocol

This protocol (Figure 1A) outlines a 3D co-culture system designed to recapitulate cell-cell interactions across a basement membrane matrix. Downstream analysis is possible using confocal microscopy (Figure 1B) or by harvesting cells from within the co-culture (Figure 1C). By using a 'sandwich' construction, where a layer of ECM gel is sandwiched between two cell populations, the protocol does not require specialized apparatus and can be performed with foundational knowledge of modern cell culture techniques.

1. 3D Co-culture preparation

Results

To demonstrate this protocol's effectiveness for studying the effects of cell-cell interactions across a basement membrane, Figure 2 and Figure 3 have been repurposed from a previous publication (Pallegar et al.¹⁶) as representative outcomes. Figure 4 and Figure 5 are novel data and have been generated to show this protocol's adaptability to non-breast cancer cells and the abilit.......

Discussion

The lack of complex cell-cell interactions in traditional cell culture highlights the need for improved organ and tissue models to more accurately study drug responses and disease progression in vivo. The development of 3D co-culture systems has contributed to filling this gap by allowing study of the consequences of cell-cell interactions without the need for in vivo studies^9,12. However, many of these methods can be mechanically complex, requi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 Well Tissue Culture Plate	Corning	3524
24 x 60 mm Cover Glass	Ultident Scientific	170-C2460
4% Paraformaldehyde in PBS	Invitrogen	J61899.AK	Diluted to 2% in 1x PBS
4-Well Chambered Culture Slides	Falcon	354114
Alexa Fluor 647 AffiniPure Fab Fragment Donkey Anti-Goat IgG (H+L)	Jackson Immunoresearch Laboratories Inc.	705-607-003
Bright-Line Hemacytometer	Hausser Scientific	3120
CellTrace CFSE Proliferation Kit	Invitrogen	C34570
Cell Recovery Solution	Corning	354253
DAPI	Sigma-Aldrich Canada Co.	D9542
Donkey anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, DyLight 594	Invitrogen	SA5-10168
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Invitrogen	A31573
DMEM/F-12, no phenol red	Gibco	21041025
Dulbecco's Modified Eagle Media, High Glucose, Pyruvate	Gibco	11995-065
Fetal Bovine Serum, Canada Origin	Gibco	12483-020
Flowmi Cell Strainers, 40 µM	Bel-Art	136800040
GE Advanced Silicone Window and Door, Clear	Henkel International	2811093
Goat Polyclonal Anti ZO-1	SantaCruz Biotechnology	SC8146	Primary antibody, discontinued
Rat Monoclonal Anti ZO-1	SantaCruz Biotechnology	SC33725	Alternative primary antibody to replace discontinued SC8416 anti ZO-1
Mammary Epithelial Cell Basal Medium	Promocell	C-21210
Matrigel, Phenol Red-free, Growth Factor Reduced, LDEV-free	Corning	356231	Protein Concentration: 9-9.6 mg/mL
Mouse Monoclonal Anti-Vimentin	Sigma-Aldrich Canada Co.	V2258	Primary antibody
Newborn Calf Serum, New Zealand Origin	Gibco	16010159
Penicillin-streptomycin	Gibco	15140-122
Phosphate-Buffered Saline, pH 7.4	Gibco	10010023
ProLong Gold Antifade Mountant	Invitrogen	P36934
Purified Mouse Anti-E-Cadherin	BD Biosciences	610181	Primary antibody
Rabbit Polyclonal Anti-Claudin 7	Abcam	AB27487	Primary antibody
RPMI 1640, Glutamine	Gibco	11875-093
SupplementMix	Promocell	C-39115
Trypan Blue Stain (0.4%)	Gibco	15250-061
Trypsin 0.25%-EDTA	Gibco	25200056