ViaGlue™ 3+ Cell type Spheroid Cell Assembly Protocol
ViaGlueTM 3+ Cell-type Spheroid Cell Assembly Protocol
Reagent A – Grey cap Reagent B – Red cap
- Have three or more populations of cells grown for spheroid assembly with high density.
- Remove the pair of reagent vials from 4°C storage and warm to room temperature.
- Aspirate the cell growth media from the cells 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 L of Reagent A (5% v/v) to cell population 1. To cell population 2 (10 cm cell plate) add 5 mL of growth media, then add 250 L of Reagent B (5% v/v). To cell population 3 add 5 mL of growth media (10 cm culture plate), then add 250uL of Reagent A (5% v/v). Swirl the plates gently to mix. (To add another cell type for a total of 4 cell lines, use an aliquot of Reagent A with cell type 4).
- 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 or fresh media or other cleaning media.
- Remove the cell populations from the culture 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 three cell pellet populations (1 and 2 and 3) in separate tubes, re-suspend and measure cell density accurately (to obtain desired cell type A: cell type B ratios) to produce a desired cell composition ratio. Eg. 1:1:1.
- In a 1.5 mL micro-tube add the calculated volumes of suspended cells together to reach a cell ratio of 1:1:1 combined.
- Under optimal culture conditions incubate the combined suspension of 1 and 2 and 3 cells for 45 minutes to adhere through applied reagents.
- Very gently re-suspend the pellet and dilute the formed spheroid suspension in media (recommended 100-1000X dilution).
- Deposit the spheroids onto a 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.
- Cells can be used for assays or experiments with no further manipulation.
Spheroid Assembly Considerations:
- High density depositing of cells will result in continuous tissues (further aggregation) rather than discrete spheroids.