For a three-dimensional collagen lattice with a final volume of 150 µl, the following protocol is recommended. The collagen lattice is prepared in two parts: 100 µl collagen preparation and 50 µl cell suspension

Part A. Collagen Preparation

5 µl bicarbonate (e.g. Sigma-Aldrich S8761-100ML)

10 µl 10x concentrated minimum essential Eagle’s medium (e.g. Sigma-Aldrich M0275-100ML)

85 µl PureCol (e.g. Inamed, Fremont, USA, catalog #5005-100ML)

Mix components at room temperature under sterile conditions.

Links to recommended reagents:

https://www.sigmaaldrich.com/catalog/product/sigma/s8761?lang=en&region=US

https://www.sigmaaldrich.com/life-science/cell-culture/classical-media-salts/mem-media.html

https://www.advancedbiomatrix.com/collagen-type-i/purecol-bovine-collagen-product-3-mgml/

Part B. Cell Suspension

Use regular cell culture medium without serum that is recommended for the cell line or use PBS.

Adjust amount of cells to 1.5x105 for tumor cells (final concentration 5x104 tumor cells) or 4.5x105 for leukocytes (final concentration 1.5x105 leukocytes) in 50 µl. When using mixed cell cultures, lower number of each cell type by half. Suspend each cell type in 25 µl and mix then. Further adjustments of cells numbers might be necessary due the size of the cells.

When a pharmacological substance is tested, suspend cells in 35 µl and add 15 µl of solubilized drug at 10fold of the desired final concentration (cells generally tolerate a final concentration of 0.1% ethanol or 0,05% DMSO, if the substance is not soluble in PBS-buffer).

Mix 50 µl cell suspension and 100 µl collagen preparation, pipette into chamber. Let it form a collagen lattice for 30 min in an incubator at 37° C in a humidified atmosphere with 5% CO2.

Fill remaining compartments of the chamber as per recommendation of the supplier. Seal chamber before recording.

Recommended Chambers: