Frequently Asked Questions

We are here to help you enjoy your scientific exploration. Please read these FAQs before contacting us. If your question still has not been answered, send us an email at

For first time user of VitroGel 3D system, please click to read the “first-time user note” to ensure optimal results.

If you use adherent cell lines, we recommend you use VitroGel 3D-RGD. If you use non-adherent cell lines or you want more flexibility in controlling the growth factors/proteins, we recommend you use VitroGel 3D. Please contact at if you need a customized product.
VitroGel 3D is stable at 4-40°C.  For storage, we recommend storing 4-8°C.  Do not freeze the product or heat the hydrogel solution over 80°C. The product is stable for 12 months unopened at 4°-8°C or 6 months unopened at room temperature. Once opened, please seal the cap, keep at 4 °C to prevent contamination and use within 1 month.
The hydrogel formation time highly depends on the mixing ratio of the hydrogel solution and the trigger solution (cell culture media/PBS). We recommend mixing the hydrogel solution and media/PBS at 4: 1 (v/v) ratio for an injectable hydrogel. The hydrogel should be stable within 30 minutes after mixing at this ratio. A higher mixing ratio (hydrogel solution: media > 4: 1) would cause a longer hydrogel formation time. A low mixing ratio (hydrogel solution: media < 1: 1) might lead to a non-injectable hydrogel.
The hydrogel formation time is dependent on a few factors such as gel concentration, the ionic strength of culture media and mixing ratio. If the hydrogel formation is too fast, we recommend to dilute the hydrogel solution with DI water before mixing with the cell media for gelation. After dilution, try to keep the 4:1 ratio (diluted hydrogel solution : cell medium = 4:1) for mixing as this is our optimal ratio for most media to use with our hydrogel system.
The bubble issue is related to the increased solution viscosity after mixing the gel solution with cell medium. Here are some suggestions that can help to reduce the formation of bubbles:
1) Warm up the hydrogel solution to 37° C to reduce the viscosity of the gel solution.
2) Gently mix the gel solution and cell medium and pipette slowly along the wall of the well plate without introducing bubbles. (some users prefer to use Vortex instead of pipette for mixing to avoid bubble)
3) Dilute the gel solution with DI water before mixing with cell medium.
4) Cut the pipette tips for a better flow.
5) If only small bubbles form after mixing, add a layer of medium on the top of hydrogel and incubate overnight.
The stiffness of the final hydrogel can be adjusted by diluting the hydrogel solution before mixing with cell culture media/PBS. Please use sterilized DI water to dilute the hydrogel solution. Do not use PBS or other ionic solutions. If you need a higher hydrogel stiffness than the original product, please contact us.
We recommend to use 200,000-1,000,000 cells/mL for this hydrogel system. You might need to replace the covering culture media every 1-2 days accordingly.
We have tested the growth of cells in the hydrogel system for up to two weeks.  Depended on the cell line, the 3D cell culture can last even longer. You might need to change the cover media more frequently once the number and size of cells increases.
Normally, spheroid formation requires about 5 – 10 days after culture in the hydrogel system. The formation time may vary depending on the cell line.
Yes, the cells can be harvested after 3D culture by using a simple pipetting and centrifuge protocol. Please download the full using handbook and read the section on harvesting cells from hydrogel for details.
The issue might because of the following few reasons:
1) using the non-treated tissue culture plate, which has a more hydrophobic surface which reduces the attachment of hydrogel/cells on the surface of well-plate. For better performance, we suggest using treated tissue culture plate with the VitroGel 3D system.
2) adding the hydrogel as a dome instead of covering the whole bottom of the well plate might cause this issue. We would suggest tilting the well plate after adding the hydrogel to make sure the whole bottom of the well plate is covered by the gel.
3) not enough waiting time before adding the additional medium on the top of the hydrogel. After transferring the hydrogel to the well plate, please wait 15-30 min for hydrogel stabilization before adding the top medium. Adding the medium before the hydrogel is stable would disrupt the structure of hydrogel. The low concentration of the hydrogel, the longer waiting time needed.
Yes. If the hydrogel is prepared in a proper mixing ratio, eg. 4:1 (hydrogel solution : cell medium = 4:1), the hydrogel would perform a special reversible mechanical property-shear-thinning and self-healing, which means the hydrogel can be transferred into liquid under the shearing force such as pipetting or injection and recovery as a hydrogel again once the shearing force ceased. Beside this mechanism, we can also manipulate the hydrogel to be reversible towards changes in temperature or other methods. Please contact us at for the customized solution.
Yes. Cells can be harvested from the VitroGel 3D hydrogel system and sub-culture for an additional period by using fresh VitroGel 3D or other 2D or 3D culture methods.
Co-culture of two or more different cell types can be done with VitroGel 3D hydrogel system. Besides adding different cell types together with hydrogel solution for co-culture, each cell type can be also mixed with hydrogel solution and added layer by layer. After the first layer of hydrogel become stable, carefully overlay the second layer of cells/hydrogel mixture on top of the first layer of cells/hydrogel .
Yes. Extracellular matrix proteins or other molecular compounds can be added into the VitroGel 3D hydrogel system before or after hydrogel formation. Before hydrogel formation, add the proteins or the molecular compounds into the cell culture media and then mix with VitroGel 3D hydrogel solution. After hydrogel formation, the proteins or molecular compounds can be added on the top of the hydrogel which penetrates into the nanofiber matrix. Please note that the hydrogel formation time and the final gel stiffness might change due to the salts contained in the proteins or chemical compounds. If the hydrogel properties are changed, we recommend washing the protein or chemical compounds with a sucrose solution to remove the salt before mixing with VitroGel 3D hydrogel solution.
Yes. Cells can be stained within the hydrogel or harvested from the hydrogel and then stain. Most fluorescent dyes and immunological reagents can be used at standard protocols. Please read the full using handbook of VitroGel 3D for more details. In addition, VitroGel 3D hydrogel is transparent and compatible on different imaging systems for cell observation.
Yes. After cultured in VitroGel 3D hydrogel, cells can be harvested from the hydrogel and used to perform molecular analyses according to standard procedures.
Yes, once the hydrogel becomes stable, you can take it out from culture well for sectioning. The hydrogel should be stable in 30-60 min after the top medium is added.
Yes. The VitroGel 3D can be injected before or after hydrogel formation for in vivo study. Before hydrogel formation, the VitroGel 3D solution can be injected directly into the animal which later becomes a hydrogel when it contacts with the ionic compounds of the physiological environment. Also, VitroGel 3D hydrogel has an advanced injectable property after hydrogel formation. Mixing the hydrogel solution and cell culture media/PBS at a proper ratio (we recommend hydrogel solution: media/PBS = 4: 1 v/v), the final hydrogel becomes injectable for in vivo studies. Using this method, cells or other chemical compounds can mix well in the hydrogel before injection.
We test the elastic modulus by a dynamic rheometer. The G’ is depended on the dilution and the cell culture medium used. For example, diluting the hydrogel with 0.5X PBS and then mixing with DMEM to further dilute the hydrogel solution at 4:1 ratio, the G’ is around 1500Pa for 1-0 dilution, 600-700 Pa for 1-1 dilution, 300-400 Pa for 1-2 dilution, 200-300 Pa for 1-3 dilution and 100 Pa for 1-4 dilution.