Biointerfaces Between Cells and Substrates in Three Dimensions - Biointerfaces: Where Material Meets Biology - page 147

cultures (e.g., cells pre-counted on a haemocytometer) were also lysed
and incubated with PicoGreen s . Absorbances of the known 2D cul-
tures were used to correlate 3D absorbance with cell number.
5. The total albumin level obtained from the initial media sample was
then divided by the 3D cell number to provide a reading of albumin
secretion per cell. This then allowed for a direct comparison with al-
bumin secretion of 2D cell cultures.

d n 3 r 4 n g | 3

6.3.5 Genomics and Proteomics
The study of genes and proteins is an important aspect of cell biology, es-
pecially in cancer biology. Hence being able to successfully extract mRNA
and total protein from 3D cultures is crucial. Encouragingly, the process is
straightforward for most 3D substrates. Usually all that is needed is the
application of a lysis buffer to burst open the cell membranes and release
the contents, including the cellular proteins and genetic material. Once
the lysate is collected, typical protein or RNA isolation and purification
procedures can be applied as normal. For Alvetex s , mRNA and total protein
are easily extracted using IGEPAL s CA-630 (Sigma), although a slightly more
aggressive homogenisation process compared to 2D cultures is normally
needed to ensure all genetic material is obtained from a 3D sample.

6.3.6 Cell Retrieval
One of the main issues with growing cells at the 3D interface is retrieving
cells at the end of the culture period. Cells that are densely packed into a 3D
structure will secrete their own ECM to tightly anchor themselves to one
another and to the 3D substrate. In addition, most 3D substrates cannot be
easily opened or disrupted, especially chemically cross-linked hydrogels,
electrospun fibres and polymeric scaffolds. As a result, the short incubation
times with proteases such as trysin-EDTA that were suitable for retrieving
cells from 2D cultures are much less effective in three dimensions.
Whilst full cell retrieval may not be an issue for tissue engineering or
regenerative medicine applications, where the biodegradable material is
intended to be implanted into the body along with the cells, it does pose a
potential issue for in vitro cell culture applications. Not being able to con-
tinually passage proliferative cells in three dimensions is a major challenge,
in that cells stocks need to be pre-cultured in two dimensions before being
applied to 3D experiments. Similarly, certain in vitro biological techniques
require cells to be extracted from the substrate, including flow cytometry or
using transfection kits.
Some 3D materials do allow for partial cell retrieval. For example, some
physically cross-linked hydrogels or polymeric scaffolds can be de-gelled or
broken down by certain external stimuli but, again, care needs to be
taken that this process does not harm the cells. For Alvetex s , which is a
chemically cross-linked material, partial cell retrieval

.

(ca. 30-50%) is