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 seen in vivo^4,5 However, 3D culture alone does not fully recapitulate the many components of an in vivo microenvironment because it lacks contributions from other cell types⁶.

Direct (contact between cells) and indirect cell-cell interactions play a major role in the behaviour of cells in vivo^7,8. Recapitulating these interactions requires 3D co-culture models, where multiple cell types are cultured together in a 3D environment. Using these techniques, an in vivo-like growth environment can be established in vitro, improving the biological relevance of the results^9,10. However, the more specific the environment required for a particular experiment is, the more complex the methodology becomes. There is a lack of a standardized model system that can be used to study cell-cell interactions between cells separated by a basement membrane matrix. As such, this protocol was designed to simulate cell-cell interactions that occur across a basement membrane matrix in a robust, reproducible manner. Adapted from a previously established 3D culture model¹¹, the technique uses a layer of Matrigel, hereby referred to as ECM gel, to physically separate two different cell types in culture while allowing secreted factors to pass through. It was originally designed to study the indirect impact of adipocytes on mesenchymal breast cancer cells, but can easily be modified to accommodate a variety of cell types. Careful consideration should be given to whether this method is appropriate for a given experimental design. This method does not permit direct cell-cell interaction and should therefore be limited to modelling in vivo scenarios that involve indirect communication between cells across a basement membrane matrix. For example, this system would not be ideal to model the interactions between cytotoxic T cells and tumour cells, as they commonly interact directly. Instead, consider biological scenarios where a basement membrane naturally separates the two cell types of interest. For instance, this method could be applied to the study of interactions between tumour cells and vascular endothelial cells prior to intravasation.

A common alternative 3D co-culture method involves the use of specialized microfluidics chips, or "organ-on-a-chip (OoC)". OoC systems are powerful and can be tailored to individual experiments, but require significant time and resources committed to chip design, material selection, peripheral equipment, optimization, and data collection^12,13. In addition, there is currently no standard material or method that can provide the biological and physical cues of the basement membrane matrix while not interfering with microfluidic flow¹⁴. As such, applying OoC technology to studies of cell-cell interactions across the basement membrane presents further challenges. The method outlined in this protocol seeks to offer a standardized alternative that maintains biological relevance and accessibility.

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

NOTE: The outlined protocol is intended as a representative application of the 3D co-culture method using 3T3-L1 adipocytes and MDA-MB-231 breast cancer cells. The use of different cell types will require optimization of cell seeding density and pre-testing of ECM gel lots to ensure efficacy. This protocol addresses the use of a no-adipocyte control and conditioned media control. More controls may be required depending on the experimental goal.

1. Cell culture and solution preparation
   1. Acquire and establish cultures of 3T3-L1 pre-adipocyte and MDA-MB-231 breast cancer cell lines. Grow and independently maintain all cells using aseptic technique in a class II biological safety cabinet and standard adherent cell culture methods¹⁵. Maintain 3T3-L1 cells in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% (v/v) Newborn Calf Serum (NBCS) and 1% Penicillin-Streptomycin (10,000 U/mL). Maintain MDA-MB-231 cells in RPMI 1640 supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (10,000 U/mL).
      ​NOTE: Never allow 3T3-L1 cells to exceed 80% confluence during maintenance, and do not use cultures past the 20^th passage number.
   2. Acquire or prepare a stock solution of 1x phosphate-buffered saline (PBS) with 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4.
   3. Prepare 50 mL of PBS-glycine wash solution by adding 0.375 g of glycine to 50 mL of PBS for a final glycine concentration of 100 mM. Store solution at 4 ˚C for up to 12 months.
   4. Prepare 10 mL of permeabilization buffer by adding 50 µL of Triton X-100 to 9.5 mL of PBS. Fill to 10 mL for a final concentration of 0.5% (v/v) Triton X-100. Store at 4 ˚C and use within 1 month.
   5. Prepare 50 mL of immunofluorescence (IF) buffer by adding 50 µg of bovine serum albumin (BSA), 100 µL of Triton X-100, and 25 µL of Tween-20 to 45 mL of PBS. Fill to 50 mL for a final concentration of 0.1% (w/v) BSA, 0.2% (v/v) Triton X-100, and 0.05% (v/v) Tween-20. Store at 4 ˚C and use within 1 month.
   6. Prepare 10 mL of blocking buffer by adding 1 mL of animal serum to 9 mL of IF buffer for a final concentration of 10% serum. Store at 4 ˚C and use within 1 month.
      ​NOTE: Species identity of serum depends on the host species of the secondary fluorescent antibodies used (see 2.2.4).
   7. Thaw ECM gel (total protein concentration ~9-9.5 mg/mL) at 4 °C overnight. Prepare 500 µL aliquots in 1.5 mL micro-centrifuge tubes and store at -20 °C.
      NOTE: ECM gel is highly variable and must be verified to induce the expected behaviour in each cell type. Different cell types may require different protein concentrations or matrix stiffness.
      NOTE: Always use pre-chilled pipette tips and containers when handling ECM gel to avoid polymerization at room temperature. Always keep the ECM gel on ice during handling.
2. Adipocyte differentiation
   1. Seed 50,000 to 80,000 3T3-L1 pre-adipocytes in 0.5 mL of standard growth medium into 2 wells of a 4-well chamber slide. The empty wells will be used for no-adipocyte and conditioned media controls.
      ​NOTE: Chamber slide cell cultures are prone to mechanical disruption. Avoid vacuum aspiration of media and use a gentle pipetting technique with manual pipettors.
   2. Seed 50,000 to 80,000 3T3-L1 pre-adipocytes in 0.5 mL of standard growth medium into a single well of a 24-well tissue culture plate. This will be used to collect conditioned media for the conditioned media control.
   3. Allow both cultures to reach 100% confluence over 24 h.
   4. Initiate adipogenesis by replacing growth medium with adipogenesis initiation medium, consisting of DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 1 µM dexamethasone. Incubate the cells for 48 h at 37 °C in a 5% CO₂ incubator.
   5. After 48 h, replace the spent media with fresh adipogenesis progression medium, consisting of DMEM with 10% FBS and 10 µg/mL insulin. Incubate cells for another 48 h at 37 °C in a 5% CO₂ incubator.
   6. Before continuing with 3D co-culture, visually confirm the presence of lipid droplets in >60% of cells. If lipid droplet accumulation is slow, replace progression media with DMEM supplemented with 10% FBS and monitor lipid content every 24 h.
3. Overlay and maintenance of 3D co-culture
   1. Thaw ECM gel at 4 °C overnight before continuing with overlay.
      ​NOTE: To avoid effects of lot-to-lot variability, it is recommended to use the same lot of ECM gel for all experiments within a single study.
   2. After successful differentiation of 3T3-L1s, remove the spent culture media, leaving a small buffer of ~50 µL of media.
   3. Carefully add 110 µL of ECM gel to each adipocyte-containing well of the chamber slide, being careful not to disturb the adipocyte monolayer.
      NOTE: ECM gel solidifies rapidly at room temperature. To avoid premature matrix polymerization, always keep the ECM gel on ice and use tips pre-cooled at -20˚C.
   4. Add 110 µL of ECM gel to the empty no-adipocyte control well. To ensure an even spread of Matrigel, add dropwise into the corners of the well and the middle of the well. Use the pipette tip to gently spread the Matrigel across the culture surface without scraping.
   5. Incubate the slide for 1h at 37 °C to allow the ECM gel to polymerize.
   6. During this incubation step, collect and count MDA-MB-231 cells
      OPTIONAL: See step 1.4 for details on fluorescently labelling these cells.
   7. Prepare a diluted suspension of MDA-MB-231 cells at 30,000 cells/mL in mammary growth medium containing 2% ECM gel (v/v).
      NOTE: Optimal cell seeding density varies across cell types and must be optimized prior to experimentation.NOTE: Mammary growth medium is a commercially available cell culture medium optimized for mammary epithelial cells. It contains 0.4 % Bovine pituitary extract, 10 ng/mL Epidermal growth factor (recombinant human), 5 µg/mL Insulin (recombinant human), and 0.5 µg/mL Hydrocortisone. The co-culture medium must be chosen carefully to suit the needs of both cell types.
   8. Once the ECM gel has polymerized, gently add 450 µL of MDA-MB-231 suspension to each well. This results in a total cell count of 13,500 cells/well.
      NOTE: An uneven overlay of the cell suspension over the gel is a possibility at this step. Visually assess chamber sides and tap gently on all sides of the chamber to distribute evenly.
   9. To the adipocyte culture on the 24-well plate, add 450 µL of mammary growth medium containing 2% ECM gel (v/v). Harvest and change media every 48 h.
   10. Maintain the co-culture for a maximum of 5 days, with media changes every 48 h.
       ​NOTE: Refrain from vacuum aspiration and use gentle pipetting. Detachment of the ECM gel is a possible issue that may arise during co-culture maintenance.
   11. For the conditioned media control well only, dilute harvested adipocyte (from step 1.3.9) conditioned media 1:1 with fresh media before replacing spent co-culture media on days 2 and 4.
       NOTE: At this point, the co-culture period is finished and must be analyzed either by fixing and staining the cultures to ascertain morphology and marker expression (see section 2) or by extracting cells from the co-culture environment and preparing cells for downstream analysis (see section 3).
4. Fluorescent cell labelling (Optional)
   ​NOTE: Labelling of overlaid cells can be performed prior to step 1.3.7 to help identify overlaid cells during immunofluorescence image capture.
   1. Prepare a 5 mM stock solution of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) by dissolving 10 mg of CFSE in 4.22 mL of DMSO. Store in 500 µL aliquots at -20 °C, protected from light.
   2. After counting cells from step 1.3.6, wash cells once with 5 mL of PBS. Resuspend the resulting pellet in 1x PBS to a final concentration of 1,000,000 cells/mL.
   3. Add 1 µL of 5 mM CFSE per mL of cell suspension to achieve a final working concentration of 5 µM. Incubate the cell suspension for 20 min at room temperature, protected from light.
   4. Quench the CFSE staining by adding 5 volumes of culture medium. Incubate an additional 5 min at room temperature.
   5. Pellet cells and wash twice with PBS to remove residual CFSE. Resuspend cells in pre-warmed mammary growth medium. Incubate for a further 10 min at room temperature to ensure no dye remains.
   6. Seed fluorescently labelled cells as described in step 1.3.7. The remainder of the protocol can be carried out as described.

2. Immunofluorescence

1. Fixation
   NOTE: To avoid depolymerization of ECM gel and loss of samples during washing, allow all reagents to equilibrate to room temperature for ~30 min before use.
   1. After 5 days of co-culture, replace the media with 500 µL of PBS.
      ​NOTE: Steps 2.1.2 to 2.3.7 can be carried out in non-sterile conditions.
   2. Remove PBS and replace with 2% paraformaldehyde (PFA) in PBS (prepared fresh) at room temperature for 10 min.
      ​NOTE: ECM gel is sensitive to PFA and may lead to detachment of the gel layer.
      ​CAUTION: PFA decomposes into formaldehyde over time. Avoid inhalation of fumes, ensure sufficient ventilation, and always wear proper personal protective equipment (i.e., gloves, lab coat, eye protection) when handling PFA.
   3. Remove PFA and wash wells with 500 µL of PBS-Glycine wash solution for 15 min at room temperature with gentle agitation.
      NOTE: The use of PBS-Glycine wash is critical to prevent free aldehydes from producing background fluorescence. Do not wash in PBS without glycine.
   4. Repeat step 2.1.3 twice for a total of three washes. See 2.1.5 for storage conditions, skip to step 2.2.2 if not needed.
   5. At this point, the protocol can be paused for up to 1 week, and the slide can be stored at 4 °C. To store the slide, fill all wells with 1 mL of PBS, and seal the cover of the chamber slide with a thin ribbon of parafilm. Place the slide flat in a light-proof container, on top of a wetted delicate tissue to ensure cultures do not dry out.
2. Antibody staining
   1. Bring previously stored slides to room temperature, remove PBS, and wash wells with 500 µL of fresh PBS for 15 min with gentle agitation. Skip this step if continuing straight from step 2.1.4.
   2. Permeabilize cells with 500 µL of permeabilization buffer for 15 min at room temperature without agitation.
   3. Wash wells with 500 µL of PBS, with 15 min of gentle agitation. Repeat twice for a total of 3 washes.
   4. Add 500 µL of blocking buffer to each well and incubate for 1 h at room temperature with gentle agitation.
      ​NOTE: Ensure the source species of serum in the blocking buffer matches the host species of secondary antibody. In this protocol, we use donkey serum and donkey secondary antibodies.
   5. Dilute primary antibodies of interest in IF buffer, according to the manufacturer's recommendations. Remove blocking buffer and add 300 µL of diluted antibody solution to each well.
   6. Seal the cover of the chamber slide with parafilm and place the slide in a light-proof container. Incubate overnight at 4 °C with gentle agitation.
   7. Wash wells with 500 µL of IF buffer, with 15 min of gentle agitation. Repeat twice for a total of 3 washes.
   8. Dilute appropriate fluorescently labeled secondary antibody in IF buffer, according to manufacturer recommendations. Add 300 µL of secondary antibody dilution to each well and incubate for 1h at room temperature with gentle agitation.
      ​NOTE: Avoid prolonged exposure to light after this step to limit photobleaching.
   9. Repeat step 2.2.7.
   10. Dilute a 300 µM stock solution of 4',6-diamidino-2-phenylindole (DAPI) 1:800 in PBS. Add 300 µL to each well and incubate for 20 min, at room temperature, with gentle agitation.
   11. Wash cells with 500 µL of PBS for 5 min with gentle agitation. Repeat once.
3. Slide mounting
   1. Remove as much residual PBS as possible without disturbing the cultures. Replace the lid and separate the chamber from the glass slide using the tool provided by the chamber slide manufacturer.
   2. Using a delicate task tissue, wipe the edges of the slide to remove residual PBS without disturbing the ECM gel.
   3. Slowly pipe silicone sealant around the edges of the slide using a syringe and 18G needle. Allow 3 min for the silicone to set.
   4. While the silicone sets, add 1-2 drops of antifade mounting media to each well. Avoid bubbles. If bubbles are formed, use a pipette to carefully remove them without harming the culture.
   5. After the silicone has set, gently lower a glass coverslip at a shallow angle. Use forceps or a pipette tip to expel air bubbles as the coverslip is being lowered. Use gentle pressure to avoid shattering the coverslip.
   6. Place the slide in a light-proof container at room temperature and let it set overnight. The slide can then be stored at 4 °C, and the protocol paused for up to 1 week.
      ​NOTE (Optional): To better seal slides, apply clear nail polish to the edges of the coverslip 1 h after coverslipping.
   7. Image slides using a confocal fluorescence microscope at 10-20x magnification. Capture five random fields of view to minimize operator bias.

3. Harvesting co-cultures for downstream analysis

NOTE: Harvesting cells or biomolecules is incompatible with immunofluorescence. It is recommended to grow co-cultures intended for other downstream analyses in standard 24-well culture dishes using the same cell concentrations and volumes as outlined in sections 1 and 2.

1. Cell extraction
   1. Remove spent media and gently wash wells with ice-cold PBS. Repeat once for a total of two washes.
   2. Using a wide-bore pipette tip, add 1 mL of ice-cold cell recovery solution, herein referred to as recovery solution. Gently pipette up and down to disrupt the ECM gel layer. Transfer liquid to a 1.5 mL microcentrifuge tube.
      ​NOTE: Cell recovery solution is a commercially available non-enzymatic solution designed to gently liberate cells embedded in ECM gel. It is a proprietary composition and may be incompatible with certain cell types. As an alternative, enzymatic depolymerization of the ECM gel may be considered using gentle proteinases like Dispase or Accutase. This will require 37 °C incubations along with optimization of incubation time.
   3. Add 250 µL of ice-cold recovery solution to each well to collect any remaining cells. Transfer into the corresponding microcentrifuge tube.
   4. Invert tubes 4-5 times to mix.
   5. Transfer tubes to ice and incubate for 60 min with gentle agitation. Every 10 min, invert tubes 2-3 times to prevent settling of tube contents.
   6. Centrifuge tubes at 300 x g for 5 min at 4 °C
   7. Verify the presence of a visible cell pellet. Carefully remove supernatant and add 1 mL of ice-cold 1x PBS. Centrifuge at 300 x g for 5 min at 4 °C. Repeat once for a total of two washes. Expect the pellet to contain 80-90% of initially seeded cells, with the proportions of each cell type remaining constant.
      ​NOTE: At this point, the cell pellet can be lysed to collect protein or nucleic acids. Lysed pellets can be stored at -80 °C, and the protocol can be paused for 3 months. Lysis is incompatible with 3.2.
2. Single-cell suspension preparation
   1. Resuspend pellet in 200 µL of 0.25% trypsin-EDTA. Then, incubate at 37 °C for 3-4 min.
   2. Add 600 µL of complete growth medium to quench trypsin digestion.
   3. Centrifuge at 300 x g for 5 min at room temperature. Remove supernatant and add 500 µL of room temperature PBS.
   4. Centrifuge at 300 x g for 5 min at room temperature. Repeat once for a total of two washes.
   5. Resuspend pellet in 80 µL of PBS with 0.04% BSA, filter suspension using a 40 µm cell strainer.
   6. Dilute 5 µL of suspension in 5 µL of trypan blue. Count cells and assess viability on a hemacytometer. Cells are now ready for downstream analysis.

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