Biointerfaces Between Cells and Substrates in Three Dimensions - Biointerfaces: Where Material Meets Biology - page 146

6.3.4 Metabolic Assays and Other Functional Experiments
Probing metabolic and functional activity in three dimensions is generally
less challenging than imaging cells in three dimensions. Many commercial
assays rely on analysing the culture media, where the detection of secreted
metabolites, proteins, hormones and other biomolecules can simply be
performed by taking samples of the culture media without disrupting the
culture. However, for substrates such as hydrogels this may be more chal-
lenging due to slower media diffusion through the gel (see Section 6.2.5).
Similarly, simulating cell function in two and three dimensions is much the
same, as most stimulants can be added to the culture media (e.g., dissolving
a drug in the culture media would be the same for both 2D and 3D cultures).
Other metabolic assays, such as the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay, can also be easily applied to a 3D cul-
ture to gain preliminary insights into the culture's viability over the growth
period.
Often with metabolic and functional data it is important to employ some
form of normalisation to cell number. For example, it might be important to
know how many cells have contributed to a total measured protein level
obtained from a media sample. This then requires measuring cell number,
which is usually a challenge in three dimensions. For conventional 2D cul-
tures, normalisation is fairly straightforward as cells can either be counted
directly on the plastic, or subsequently extracted and counted with tools
such as a haemocytometer or a flow cytometric assay. However, retrieving
and counting cells from 3D cultures can be more dicult (see Section 6.3.6).
Cells in three dimensions will form tight junctions with their neighbours, as
well as secrete their own ECM which anchors them to one another and the
3D interface. To bypass this, it is possible to normalise functional data to the
level of double-stranded deoxyribonucleic acid (dsDNA) using a commercial
assay kit (PicoGreen s ; Invitrogen). Double-stranded DNA is much easier to
extract than entire cells as it can be obtained by simply lysing cells, rather
than having to disrupt the strong protein complexes anchoring the cell to the
substrate. Once the DNA is collected, a PicoGreen s calibration curve with
known cell numbers can be used to extrapolate the number of cells in the 3D
population. A brief protocol/example of PicoGreen s analysis of cells cul-
tured on Alvetex s is given below:

d n 3 r 4 n g | 3

.

1. Hepatocyte cells were first cultured on Alvetex s for 5 days.
2. Culture medium was analysed for total albumin secretion, represen-
tative of the entire 3D population.
3. Culture medium was then removed and the scaffolds were then treated
with a standard lysis solution. The mixture was then vortexed and
homogenised to extract all DNA from the scaffold culture into the lysis
solution.
4. The lysis solution was then incubated with the PicoGreen s reagent and
the absorbance measured. In parallel, known cell numbers from 2D