How do I prepare the cell suspension to mix with the hydrogel? Can I use basal medium only or add supplement/serum?

Cells can be prepared in a regular complete cell culture medium and directly mixed with VitroGel. On the other hand, cells embedded in the hydrogel need supplements to provide the nutrients. When mixing the hydrogel with cell suspension at 2:1 v/v ratio (e.g., 200 µL hydrogel solution and 100 µL cell suspension), the supplement concentration in the cell suspension has been diluted to 1/3 after mixing. This change in the nutrient concentration will cause a slow growth rate or even cell death in the first 24 hours; therefore, we recommend preparing the cell suspension with 3X supplement so after 2:1 mixing, the final supplement concentration in the gel-cell mixture is still 1X.

Two concepts need to be clarified:
1) The concentration of the cell medium (basal medium) and
2) The concentration of the supplement in the cell medium (growth factors, etc.).
We recommend preparing cell suspension with a high concentration of supplement (3X to 5X) WITHOUT changing the concentration of the basal medium.

The polymerization of our hydrogel is based on the ionic molecules in the cell culture medium, which is mainly in the basal medium. Therefore, we don’t recommend making 2X basal medium because that would increase the concentration of the ionic molecules, which will affect the gelation process. For example, a 1X basal medium has 20 mM CaCl2; if you make a 2X basal medium, the CaCl2 concentration will increase to 40 mM, which would make the gelation faster than usual and lead to a non-homogenous gel formation. (You can read further on “How Does Gelation Works With VitroGel Hydrogels?

For example, if 1X DMEM with 10% FBS is used for regular 2D cell culture.

A) Using a ready-to-use VitroGel hydrogel with a recommended 2:1 (v/v) mixing ratio:

  1. Prepare DMEM with 30% FBS first:  Use 1X DMEM with 30% FBS.  Don’t increase 3X DMEM with 30% FBS, as it would increase the concentration of the ionic molecules.
  2. When mixing the VitroGel hydrogel solution with the 1X DMEM with 30% FBS at a 2:1 (v/v) mixing ratio,  the final VitroGel-DMEM mixture will be 10% FBS in the hydrogel matrix.

B) Using a High-concentration VitroGel hydrogel with a recommended 4:1 (v/v) mixing ratio between diluted hydrogel solution and cell suspension:

  1. Prepare hydrogel solution at the desired dilution ratio by mixing the high-concentration VitroGel solution with VitroGel Dilution Solution (for example, mix 100 µL high-concentration VitroGel solution with 300 µL VitroGel Dilution Solution for 1:3 dilution ratio)
  2. Prepare DMEM with 50% FBS: Use 1X DMEM with 50% FBS.  Don’t increase 5X DMEM with 50% FBS, as it would increase the concentration of the ionic molecules.
  3.  When mixing the diluted VitroGel hydrogel solution (from step 1) with the 1X DMEM with 50% FBS at a 4:1 (v/v) mixing ratio,  the final VitroGel-DMEM mixture will be 10% FBS in the hydrogel matrix.

The serum or critical supplement(s) in the hydrogel affects cell growth, especially during the first 48 hours. Please see the example figures below.

Bone marrow cells (OP9) 3D cultured in VitroGel with 2% and 10% FBS. The cells were encapsulated in the hydrogel matrix with 2% and 10% final FBS concentrations, respectively. The images were taken 18 hours after cell seeding.