Here you can find frequently asked troubleshooting questions and answers related to the practical use of GrowDex hydrogels in 3D cell culture.
Handling and dilution issues
Q: It is difficult to push GrowDex out of the syringe, it bursts out and how can I measure it more accurately?
A: Before opening the syringe screwcap, please pump the syringe plunger back and worth few times. The seal on the syringe plunger may have gotten stuck to the syringe barrel, which may cause the sudden outburst of GrowDex during dispensing. Alternatively, GrowDex can be measured by weighing. The density of GrowDex equals the density of water.
Q: Pipetting of GrowDex seems difficult. Gel is stuck to the pipette tip and I cannot transfer it to tube. Do you have any suggestions?
A: Pipetting of viscous materials like GrowDex in stock concentration, needs to be done slowly. Currently, we provide GrowDex hydrogels in syringe to avoid the pipetting of hydrogel in stock concentration. GrowDex can be dispensed directly from the syringe to the tube containing cell culture medium. Low-retention pipette tips should be used to avoid GrowDex sticking to the tip, e.g. TipOne RPT 1250ul CAT NO S1161-1820. Also, the use of wide-bore pipette tip, or cutting the pipette tip to make the hole larger, makes the pipetting easier. Alternatively, positive-displacement pipettes work very well in pipetting GrowDex.
Q: I get air bubbles in GrowDex when diluting it with cell culture medium. Any advice how to prevent air bubble formation or how to remove the bubbles from GrowDex?
A: Aspirating and dispensing GrowDex should be performed slowly to avoid air bubbles. Care needs to be taken that air is not introduced into the hydrogel. Pipette should not be pushed completely empty when the pipette tip is inside the hydrogel. Serum in the culture medium increases the risk of getting air bubbles in the hydrogel. If the air bubbles are observed, they can be removed: Leave the diluted hydrogel in fridge overnight and bubbles will disappear. If bubbles do not disappear, then centrifuge the diluted hydrogel shortly. If phase separation occurs during centrifugation, please mix the hydrogel before use.
Q: I tried to incubate (stock) GrowDex for a longer time at 37C but did not observe gel formation - it rather remained in the initial viscous and fluid state. This contrasts with the BME matrices that we use routinely. Is this expected?
A: Yes, it is expected that GrowDex does not 'gel' because of temperature. It is stable in temperatures between 0-121°C, and it is delivered as a ready-to-use hydrogel. It consists of water and cellulose nanofibers that are not covalently linked together, but physically tangled together. Due to the shear-thinning property of the material, the hydrogel starts to flow when force is applied (e.g. pipetting or injecting), and the viscosity is retained when force is stopped.
Gel Clumps
Q: After diluting the GrowDex hydrogel, I see gel clumps in the tube. Is this normal and is there any advice how to avoid the clumps?
A: Clumps in the hydrogel are likely due to insufficient mixing. It is recommended to mix by pipetting up and down for a minimum of 90 seconds. If there are any visible clumps remaining after 90 sec mixing, the mixing should be continued until the hydrogel is homogenous.
Q: I have plated the cells in GrowDex to a 6-well plate. After overnight incubation at +37C, there are gel clumps in the well.
A: We recommend using smaller well plates, such as 96-well plates for GrowDex cultures. The smaller well provides more wall support for GrowDex and it remains more stable. The lack of adequate wall support in larger wells may cause the clumping of the hydrogel leading to non-homogenous matrix. Please make sure also that the mixing was done properly before plating the hydrogel to the well plate. GrowDex should be mixed by pipetting up and down for a minimum of 90 seconds.
Cells on the bottom of the well
Q: The cells were embedded in GrowDex but after 24h incubation they were on the bottom of the well. How to avoid the 2D culture of the cells on the well bottom?
A: GrowDex provides the cells physical support to remain in 3D. The cells do not sink to the well bottom by gravity, but they are able to migrate in GrowDex. If adherent cells are embedded in GrowDex in a normal cell culture plate, they may migrate to the bottom and adhere there. Low-attachment microplates are recommended for adherent cells to prevent the 2D cell growth on the bottom of the well, but not all cells need the low-attachment plates. For example, pluripotent stem cells and A549 lung cancer cells have been cultured on standard cell culture treated plates (Nunc Microwell #167008) for PSC, and Greiner #655180 for A549). It is dependent on the cell type, if low-attachment plates are needed. For adherent cells such as HepG2, e.g. Corning Costar flat bottom ultra-low attachment (ULA) plates and BRANDplates inertGrade plates work well. It is also possible to pre-coat the standard cell culture treated plates with a layer of 0.4% poly-HEMA to prevent the 2D culture.
It may be also possible that if the GrowDex hydrogel was not mixed thoroughly before embedding the cells, the cells may have sunk to the bottom of the well via the liquid channels in GrowDex. To avoid this, careful and thorough mixing of GrowDex should be done to have a homogenous culture matrix.
Changing culture media
Q: Do you have any hints and tips for handling GrowDex during medium change? I would want to avoid disturbing the cell-hydrogel when replacing the medium.
A: Please see our ‘GrowDex 3D cell culture’ -video showing the whole assay protocol including also the culture medium change. The plate can be tilted to an angle and medium removed from the top layer, above the layer of GrowDex. Applying the fresh culture medium on top of GrowDex should be done carefully against the well wall. If you are working with GrowDex concentration lower than 0.5%, you may centrifuge the plate 200g for 5 min before medium exchange to pack the hydrogel slightly to the bottom of the well. To reduce the risk of accidentally removing the hydrogel during medium removal, it is recommended to replace only 50-75% of the medium.
The water separates from the gel
Q: GrowDex hydrogel in the syringe does not appear to be homogenous. It seems that water has separated from the hydrogel, see image below. Is this normal and can I mix the water back to the hydrogel?
A: No, it is not normal, and the product should not be used. Water should not be separated from GrowDex. It is likely that the product has been frozen at some point. During the freezing, the water separates from the nanofibrillar cellulose and it cannot be mixed back to the hydrogel anymore. Please do not use the product. GrowDex hydrogels should be stored always above 4°C degrees.
