ViaGlue™ Mixed 2 cell-type Tissue Assembly Protocol
ViaGlueTM Mixed 2 cell-type Tissue Assembly Protocol
Reagent A – Grey cap Reagent B – Red cap
- Have two populations of cells grown for cell assembly into a tissue in high densities.
- Remove the pair of vials from 4°C storage and warm to room temperature.
- Aspirate the cell growth media and wash once with PBS or fresh media or other suitable solution.
- Aspirate the cleaning solution and replace with a minimal amount of typical growth media. Eg. 5 mL in a 10 cm culture plate, 2 mL in 6 well plate.
- To the 5 mL of growth media (10 cm culture plate), add 250 µL of Reagent A (5% v/v) to cell population. To cell population 2 (10 cm cell plate) add 5 mL of growth media, then add 250 µL of Reagent B (5% v/v). Swirl the plates gently to mix.
- Incubate the cells under optimal growth conditions for 1 hour. Eg. 37°C and 5% CO2.
- Aspirate the growth media containing the Reagents from the treated cell populations and wash once with PBS, fresh media or other cleaning media.
- Immediately remove the cells from the culture plate surface. Eg. Treat with 3 mL 0.25% trypsin for 3-5 minutes, quench with 6 mL of serum containing growth media, centrifuge and decant media.
- With the two cell pellet populations (A and B) in separate tubes, the cell density should be measured accurately (to obtain a desired cell A: cell B ratio) to produce a tissue with specific cell composition ratios.
- The calculated volume of cell suspensions are combined and gently agitated.
- Deposit the cells onto the desired surface, allowing the cells to incubate in optimal conditions (Eg. 37°C and 5% CO2) undisturbed for 4-6 hours to allow cells to create natural adhesion connections.
- Check cells under Brightfield microscopy to confirm the cells have adhered and have begun spreading out on each other.
- Add additional amounts of media to keep tissues healthy and covered.
- Cells can be used for assays or experiments with no further special manipulation.
Tissue Assembly Considerations:
- High amounts of cells are necessary to produce 3D tissue in the prescribed area of the culture well.
- For wells with approx. 1 cm2 surface area, a combined 2 cell populations of approximately 9 million cells are required for 4-5 cell layers (25-35 µm thick tissue).
- Cells not seeded with high enough density will not result in continuous layers but rather separate spheroids on the culture surface.