Guidelines for recovering cells and spheroids from GrowDex® using GrowDase™ enzyme

​GROWDASE IS USED FOR THE ENZYMATIC DEGRADATION OF GROWDEX

GrowDase is a purified mixture of cellulase enzymes that have been specifically prepared to degrade the nano-cellulose fibrils present in GrowDex, reducing them to soluble glucose. This simple enzymatic removal procedure allows cells to be recovered from the matrix efficiently, without adverse effect and retain any 3D structure such as a spheroid or organoid fully intact. The degradation process is simple; enzyme is mixed with cell culture media and incubated with GrowDex at 37°C until the hydrogel has been dissolved away (Fig. 1).

The amount of GrowDase required for cell recovery is dependent on the amount of GrowDex (cellulose) present in the experimental well. It is recommended that GrowDase is used at a working concentration of 300 µg/mg (µg enzyme / mg cellulose). An equal volume of working concentration GrowDase to GrowDex/cell matrix volume present in the experimental well should be used, i.e. if 100 µL of GrowDex/cell matrix is present in the experimental well then 100 µl of GrowDase should be added to that well.

 Figure 1.png

PROCEDURE FOR REMOVING GROWDEX BY ENZYMATIC DEGRADATION

1.Calculate the amount of cellulose present in the sample well using the following equation. NOTE:  100 µL of 1 % GrowDex = 1 mg GrowDex

Sample well volume (µL)×GrowDex concentration/100=mg GrowDex/sample well

2.Prepare the working concentration of GrowDase by diluting the stock solution with culture media. 

3.Pipette the diluted GrowDase onto the top of the sample in the microplate.

4.Incubate the plate at 37°C until the hydrogel has fully degraded.

5.Recover the cells from the well using standard techniques.

EXAMPLE EXPERIMENTAL PROCEDURE

 

SAMPLE:  80 µL of 0.9 % GrowDex/cell mix per well in a 96-well microplate

 

1.Amount of GrowDex present in the sample:

       80 µL  x  0.9 / 100   =  0.72 mg GrowDex/ sample well

2.Amount of GrowDase needed to degrade the GrowDex in the sample :

       0.72 mg  x  300 µg/mg  =  216 µg GrowDase enzyme

3.Volume of GrowDase stock solution (10mg/mL) needed:

       216 µg   / 10 µg/µL  =  21.6 µL GrowDase enzyme stock solution

4.Prepare the working concentration of GrowDase by diluting the stock solution with culture media:

       80 µL  -  21.6 µL  =  58.4 µL culture media for dilution  

5.Pipette the diluted GrowDase onto the top of the sample, incubate at 37°C until the hydrogel has fully degraded and recover cells from the well using standard techniques.

DILUTION TABLE

 Table 1.png

Volumes of GrowDase enzyme stock solution (10 mg/mL) and cell culture medium required for the preparation of 100 µL of 300 µg/mg enzyme working solution for the degradation of 100 µL GrowDex in different concentrations from 0.3% to 1%.

Table 2.png