3D Culture Troubleshooting

3D cell cultures are too sparse?  Cells grow too slowly?

Potential reasons

1. Starting number is too low.

2. Media is not optimal for growth.


1.We recommend optimizing cell number, starting with 2500 cells/well in a 96 well plate.

2.Check to make sure your media contains the necessary nutrients/growth factors/supplements for growth.

3D cell cultures become too dense too fast?  Spheroids grow into each other?

Potential reasons

Starting number is too high.


We recommend optimizing cell number, starting with 2500 cells/well in a 96 well plate.

Cells do not form consistent spheroids ?

Potential reasons

1. Matrix is responsible.

2. Cell type/cell line is responsible.


1. Some matrices such as Matrigel and collagen may affect the cell structures formed in 3D. It may be necessary to test different matrices, or different combinations of matrices to determine which one(s) are optimal for your experiment.

2.Some cell lines may form symmetrical or assymetrical spheroids depending on their transformative state. For example, normal or benign epithelial cell lines form symmetric spheroids. Malignant cell lines tend form assymmetric cell clusters. These are not technical problems but may be inherent characteristics of your cell type/cell line.

3D cell cultures were not viable ?

Potential reasons

1. Not enough media.

2. No serum in media or incompatible media.

3. Matrix not compatiable with cells.

4. Cells are contaminated.

5. Cells are senescent or overpassaged.

6. When adherent cells were being lifted off plate for seeding of 3D culture, they became over trypsinized.


1. We recommend submerging 3D cell cultures in media. The volume will depend on the size well that is being used.

2. We recommend incubating 3D cell cultures for 24 hours in serum containing media to allow cells to recover, before starting experiment.

3.Make sure the matrix you are using is biocompatible.

4. Check for mycoplasm, bacteria, fungal or yeast contamination. Signs of contamination include: visible signs of particles under the microscopy, cloudy media and abrupt changes in pH in phenol red containing media. Repeat experiment. Add antibiotics to medium to reduce chances of contamination. Check reagents and equipment.

5. Check passage number. Use earlier passage cells.

6. Reduce concentration of trypsin. Reduce duration of tryspinization. Use a different method for cell dettachment. There are other enzymatic approaches that are commercially available that too many to list here, but search "alternatives or replacement of trypsin."