The scaffold does not wet properly…
20% ethanol should normally wet the scaffold within less than a minute. If you experience incomplete wetting, try a slightly higher concentration of ethanol, e.g. 25-30%. If that does not work either, please contact us.
There are air bubbles underneath the scaffold…
This happens especially for our 12-well format with removable scaffold discs. Try pipetting the cell medium up and down and see if this removes the bubble. If this does not help, a suction pump might do the trick.
The retaining rings have become loose…
Same as above, this happens especially for our 12-well and 6-well format with removable scaffold discs; scaffold discs can become loose during shipping and handling. To put them back in place, press the retaining ring down on two sides using tweezers or pipette tips. Please note: the retaining rings for the 6-well plates with hanging inserts are very slightly conical. If they have fallen out completely, make sure you put them back at the right orientation (admittedly, this can be a bit tricky to see initially!).
Cell Culture and Assays
My cells do not grow…
It is a well-known fact that cells generally proliferate slower in 3D than in 2D, so don’t start worrying straightaway – this could be a good thing! We have observed that it takes a bit longer for cells to reach a certain density than in 2D, but because of that (and the greater space available) they can be kept in culture for much longer. If you feel that something is completely wrong, your cells might require a coating to improve attachment. Please contact us if you feel that you need some help.
I cannot tell at what level of confluence my cells are…
Confluency and cell viability can be estimated with optical or fluorescence microscopy using appropriate dyes for cell staining (e.g. neutral red). Note that some hydrophobic dyes may adsorb onto the scaffold. Quantitative, colorimetric assays (see next section) are also compatible and can provide a more accurate measurement of cell numbers within a scaffold. We recommend recording a growth curve before using a new cell line in the Mimetix scaffold to get a feel for its growth behaviour in 3D and how long it takes for the cells to become half-confluent or confluent.
The 96-well plates have a high background signal in my platereader
The Mimetix scaffolds gives rise to a background signal in absorbance-based assays. We hence recommend the use of fluorescence-based assays to avoid this problem. Alternatively, well contents can be transferred to a new 96-well plate for an absorbance-based read-out.
I cannot get my cells out of the scaffold
In general, cells can be removed from the Mimetix scaffold by trypsinisation and agitation. As a rule of thumb, slighly longer incubation times are required than in 2D, but be careful that your cells are not compromised by doing that. However, some cell lines are particularly “sticky” and hard to remove. In most cases, we recommend finding analysis techniques which do not rely on retrieving the cells from the scaffolds, i.e. assays measuring analyte in the supernatant (MTS, LDH) or assays which lyse the cells in a first step (DNA and protein content).