A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.
Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4.
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.
1. Establishment of fibroblast cultures from skin explants
2. Stage I – Preparation of collagen I from rat tails
Note: Protocol for approximately 12-14 adolescent (fresh or frozen) rat tails
3. Stage II – Setting up the 3D matrix with embedded fibroblasts (allow to contract for 8 days).
Note: The collagen concentration should be adjusted to suit the application. The more dilute the collagen solution, the faster it will be contracted by the fibroblasts. Slight differences in contraction rate will be experienced with different batches of collagen at the same concentration. Similarly, different fibroblast cultures will contract the collagen gels at different rates, and the more fibroblasts present, the faster the rate of contraction. Collagen concentration and fibroblast density can therefore by adjusted to modify the rate of contraction, and the final density of the collagen gel.
4. Stage II – Plating cells of interest on top of the matrix
Note: Sterilize all forceps and equipment with ethanol before use.
5. Stage III – Transferring the matrix to a grid for invasion (approximately 0-21 days)
Note: With respect to quantification of invasion, the day on which matricies are placed on the grids defines Day 0. Placement on grids generates a gradient of cell culture media that promotes invasion into the matrix. Samples can be imaged over the next 1 – 21 days (or longer) to assess biological processes such as invasion, proliferation, survival or differentiation (See reference list).
6. Stage IV – Fixation
7. Representative results:
Figure 1 Example of fibroblast-contracted collagen I. Mixture of fibroblast and rat tail collagen detached from the petri dish and allowed to contract over 6-8 days.
Figure 2 Organotypic assay progression. A, schematic of organotypic set up and progression. B, Example of collagen/fibroblast matrix on top of stainless steel, sterile grid to create air/liquid interface.
Figure 3 Applications of organotypic assay. A, B non-invasive and invasive pancreatic ductal adenocarcinoma (PDAC) cells invading over time with organotypic matrix. C, living skin equivalent showing a stratified epidermis on a fibroblast-contracted collagenous dermal component. D, C8161 melanoma cells invading in to a fibroblast-contracted collagenous dermal equivalent. E, invasive PDAC cells (green) interacting and invading with fibroblasts (red) within the organotypic matrix. F, invasive PDAC cells (green) interacting with a degrading surrounding extracellular matrix (purple) visualised using multiphoton -based second harmonic generation.
Here we present a method for production of a 3-dimensional matrix suitable for studies of invasive cell migration 6-8. The matrix consists of collagen type 1 fibrils, which are contracted over a period of several days by primary human epidermal fibroblasts. The collagen is prepared by acid extraction, not enzymatic digestion, which preserves reactivity at the poly-peptide ends and promotes cross-linking of collagen fibrils into larger aggregates upon neutralization of buffer conditions 9. This results in a re-constituted gel which more closely mimics the features of collagen in vivo and has important consequences for the invasive properties of cells cultured in the gels. It does not matter whether fresh or frozen starting material is used, but use of adolescent rat tails is important because collagen cross-linking is more labile in younger animals. Collagen I from younger animals is therefore easier to extract, and re-constitutes with higher fidelity.
The collagen matrix can be fixed and stained with antibodies directed against either invasive tumor cells or stromal fibroblasts 2,10 and are highly useful for testing the efficacy of drugs which might inhibit invasion 5,6.
Although this protocol suggests the use of primary human skin fibroblasts, it is feasible to use primary fibroblasts from other tissue types, according to the type of tissue under investigation. It is also possible to establish cultures using immortalized fibroblasts, including cells which have been stably transfected with fluorescent protein conjugates. In this way both stromal and tumor cells can be labelled to visualise cell-cell interactions during invasion 11.
The authors have nothing to disclose.
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
10x MEM | Gibco | 21430 | – |
NaOH | Sigma | 367176-500G | Prepare 0.22 M stock in water |
FCS | PAA Laboratories | A15-101 | – |
35 mm dishes | Falcon | 353001 | For step 3.4 |
60 mm dishes | Falcon | 353004 | For step 5.2 |
Spring forceps blunt | Samco | E003/02 | Toothed, not smooth |
16% paraformaldehyde | Electron Microscopy Services | 15710 | Dilute to 4% in PBS prior to use |
Screens for cd-1, size 40 mesh | Sigma | S07707-5EA | Stainless steel grids, 5 per pack |
Dialysis tubing | Medicell International | 7607 2295 | 12 – 14 kD |
PBS | Oxoid | BR0014G | – |
Acetic Acid | Sigma | 242853 | – |
24 well dish | Falcon | 353047 | For step 4.1 |
Fungizone | In vitrogen | 15290018 | – |