Air-liquid organotypic assays to investigate cellular crosstalk in the tumor microenvironment of cancer cells

Abstract

Air-liquid organotypic culture models enable the study of the cellular crosstalk in the tumor microenvironment. This 3D assay recapitulates the tumor niche more faithfully than 2D culture systems and represents a versatile platform that can be easily adapted to different types of cancer cells, stromal components, or ECM composition. Here, we detail the steps to build an organotypic culture including the preparation of the organotypic structure, organotypic gels, cell seeding, gel casting, membrane processing, and image and data analysis. For complete details on the use and execution of this protocol, please refer to Linares et al. (2022).

Keywords: Biotechnology and bioengineering; Cancer; Cell Biology; Cell culture; Tissue Engineering.

Graphical abstract

Figure 1

Organotypic structure Preparation of the structure for organotypic gel. (A) Stainless steel mesh labelled in red the areas to be cut and labelled in green the areas to be bent down. (B) Grid platform after cutting and bending, 0.5 cm height, 2.5 cm weight. (C) Nylon membrane on top of the grid as it will be placed for the organotypic culture.

Figure 2

Coating and organotypic gel mix material Materials for coating and organotypic gel mix. (A) Cold PBS (1), rat tail collagen type I (2), sterile 24 well plate (3), pre-cooled p1000 tips (4), micropipettes (5), residues beaker with 10% bleach (6). (B) DMEM 10× (1), DMEM 1× (2), rat tail collagen type I (3), FBS (4), Matrigel (5), 1 N NaOH (6), pre-cooled p1000 and p200 tips (7), residues beaker with 10% bleach (8), micropipettes (9). (C) Phenol red pH scale.

Figure 3

Coating material for the nylon membranes Materials for coating the nylon membranes. (A) 1% glutaraldehyde (1), FBS (2), DMEM 1× (3), DMEM 10× (4), rat tail collagen type I (5), Cold PBS (1), rat tail collagen type I (2), 1 N NaOH (6), sterile p100 plate with the desired amount of sterile 100 μm nylon mesh (7), pre-cooled p1000 tips (8), residues beaker with 10% bleach (9), micropipettes (10). (B) Collagen type I gel mix to form a drop on top of the mesh.

Figure 4

Gel casting Materials for gel casting and mounted organotypic gel culture. (A) Collagen type I pre-coated membranes (1), organotypic gels with cell coculture (2), sterile grid (3), sterile material, forceps and two spatulae (4), p1000 tips (5), residues beaker with 10% bleach (6), micropipettes (7). (B) Organotypic gel coculture on top of the structure, from the bottom, stainless steel grid, pre-coated 100 μm nylon mesh, organotypic gel. (C) Complete cell media incorporation starting from the vertex of the grid to avoid bubbles below the mesh.

Figure 5

Membrane fixation and embedding (A) Materials for membrane fixation and embedding (A) 10% neutral buffered formalin (1), organotypic gels with cell coculture (2), six-well plate (3), sterile forceps (4). (B) Schematic of membrane cutting and how to place the membrane perpendicularly to the cassette sponge before paraffin embedding.

Figure 6

H&E staining from organotypic sections (A) LNCap and LNCaP cocultures with WPMY-1. (B) PC3 and PC3 cocultured with WPMY-1. (C) MyC-Cap cocultured with mince WAT from a WT mouse. Scale bar 50 μm.

Figure 7

Quantification method A (A) Set scale. (B) 8-bit transformation of the image. (C) Set threshold for total area measurement. (D) Draw the area of invading cells using the free hand tool.

Figure 8

Quantification method B (A) Set scale. (B) 8-bit transformation of the image. (C) Straight freehand tool to draw the distance from the top of non-invading cells to the bottom of the invasive colony. (D) Set threshold for particles and total area measurement. (E) Analyze particles to count invasive colonies and obtain the total area of each one.