Cell Assembly to Generate Multiple Cell Type Containing Spheroids or Tissues
Vial A – Grey cap
Vial B- Red cap
Before cell assembly is attempted the following concerns must be addressed beforehand to ensure a successfully assembled tissue construct is synthesized. First there must an adequate amount of cells to create a 3D tissue in the prescribed area of the culture or well the tissue will be seeded into. For wells with an approximately 1cm2 surface area a combined cell density of approximately 9 million cells will be required to obtain a tissue of approximately 25-35µm or 3-4 cell layers. Cells which are not seeded with high enough cell density will not result in a continuous tissue by rather separate deposited spheroids on the seeded surface.
Vial A – Cell population 1
Vial B – Cell population 2
- Remove the pair of vials from 4oC storage and allow to warm to room temperature before use.
- Vortex both vials for 20 seconds.
- Aspirate the growth media from the cell plates and wash once with PBS or other suitable cleaning solution.
- Aspirate the PBS or cleaning solution and replace with a minimal amount of typical growth Ex. 10mL in a 10 cm culture plate.
- To the 10mL of growth media add 500uL (5% v/v)of Vial A solution to Cell population 1 culture plates and add Vial B solution to Cell population 2 culture plates.
- Incubate the cells under optimal growth conditions for 4 hours. 37oC and 5% CO2
- Aspirate the media from the treated culture plates and wash twice with PBS or other cleaning solution.
- Remove the cells from the culture plate surface. Treat with 0.25% trypsin (Sigma Aldrich) for 5 minutes, add 6mL of growth media, centrifuge, decant and resuspend cells.
- The cells should be resuspended with high cell density in minimal growth media.
- With the two resuspended cell populations (1 and 2) in separate tubes the cell density should be measured accurately to the Cell 1 and Cell 2 populations when combined produce a desired tissue composition ratio. Ex. 4.5M/mL Cell population 1 and 4.5M/mL Cell population 2 (1mL each) will produce a tissue with 50:50 ratio of 9 million total cells.
- The desired amount of cell suspensions are combined in the growth well of interest and gentled agitated to thoroughly mix the cell populations
- Allow the cells to incubate in optimal conditions (Ex. 37oC and 5% CO2) undisturbed for minimum 4 hours to allow proper adhesion of cells.
- Check cells under brightfield to confirm the cells have adhered to one-another and have begun spreading out on each other.
- Incubate the cells for a further 12 hours or overnight for complete adherence.
- Cells can be used for assays with no further special manipulation.