May 2011


Easy Interrogation of Three-Dimensional Cellular Arrays

SUMMARY: An easy approach is reported for experimentally interrogating living cells in a primitively realistic mimic of their native growth environment.
Many experimental studies of how cells work involve tearing apart a cell and extracting a particular biomolecule (e.g. an enzyme) for further study. Such an extraction removes the influence of other miscellaneous cellular material on biomolecular function, and may greatly simplify the study.

However, removing this miscellaneous cellular material may critically impact the biomolecule's function, including but not limited to eliminating macromolecular crowding effects. Thus, a misleading picture of the biomolecule's behavior in its native environment (within the cell) may result.

When feasible, the next best thing may be to study the biomolecule of interest within the cell itself. Again, depending on the experimental question, this may provide a misleading picture, because cells often do not exist in isolation.

An example that's probably the most familiar to clinical scientists is biofilms, adhered cellular aggregates common to microbial physiology and the bane of implanted medical devices. Assembly into biofilms can facilitate microbial survival, perhaps most impressively by imparting brief resistance to drain opener.

Furthermore, in a broad sense, tissues and organs can also be viewed as three-dimensional cellular assemblies. Imagine what would happen to your kidneys if they were sliced into thin two-dimensional sections (it goes without saying that they wouldn't work anymore).

Ordered, experimentally-tractable three-dimenional cellular assemblies are needed.

Many studies of cellular physiology could clearly benefit from interrogating cells in a three-dimensional state. However, this is far easier said than done.

Cells are much more easily grown and studied either in isolation or as an ordered two-dimensional sheet. Cells can be readily grown as an irregular three-dimensional clump.

However, it's not obvious how the behavior of individual cells can be distinguished from collective cellular behavior from such an experimental setup. Furthermore, this arrangement does not mimic typical in vivo three-dimensional cellular assembly.

Ratmir Derda, George Whitesides (Harvard University, United States), and coworkers have addressed this experimental cellular assembly challenge with their development of "cells in gels in paper." Their three-dimensional matrix provides structured support to the cells without killing them, but is nevertheless readily and gently disassembled for subsequent biochemical analysis.

Experimental design.

The scientists matrix is constructed by first printing wax (hydrophobic, cells don't grow here) patterns onto a piece of porous filter paper (hydrophilic, cells do grow here), generating 96 circular surfaces for subsequent cellular deposition. They found that they could sterilize the matrix prior to cellular deposition, as well as retain the wax pattern and hydrophobicity, by immersing in water while autoclaving at 120°C, followed by briefly (one minute) heating the dried paper at the same temperature.

Cells suspended in a mixture of gel and culture medium were then deposited on the paper. These two-dimensional patterns were quickly immersed in growth medium to prevent drying.

The gel penetrates the 20 micrometer diameter pores of the filter paper, enabling molecular transport between layers of paper, but restricting cellular transport between layers. Figure 1 in the original manuscript provides a clear illustration of the two-dimensional experimental setup, if this explanation isn't clear.

Stacking these two-dimensional sheets on top of one another yields a three-dimensional assembly, in which the cells are largely confined to one sheet, but molecular transport between sheets is unrestricted. Figure 2 of the original manuscript provides an illustration of some three-dimensional patterns possible with this setup, e.g. different cells or different concentrations of cells in different layers.

The paper sheets do not stick together, and must be physically clamped together. However, this means that individual layers can be easily removed from one another with tweezers, enabling the scientists to study specific cells in specific layers after a period of growth (nine days in these experiments) within the three-dimensional assembly.

Experimental highlights.

The scientists' cells (a breast cancer cell line) were not entirely prevented from moving between the paper sheets. However, the extent of cellular exchange between layers (a factor of less than 1.2) was less than the extent of cellular reproduction and death (a factor of 2-3) over the course of 9 days of cellular growth.

Interlayer cellular transport depends on many experimental conditions, such as the density of cells in one layer relative to an adjacent layer. Cells tend to migrate from regions of higher to lower cellular density.

The proximity of each layer to oxygen is also important; cells tend to migrate upwards to an adjacent layer if the oxygen levels are greater there. Such observations should be taken into consideration when the scientists' assemblies are used in subsequent experiments.

Another research highlight is that cell proliferation within a given layer depends on the total number of cells in the layer above it, as well as the position of the layer within the three-dimensional assembly. The specific results, again, depend on the number of cells in adjacent layers, such that the use of oxygen and nutrients in the top layers doesn't necessarily lead to uniform mass death in the lower layers.

Overall evaluation.

The scientists' setup is perfectly suited to the standard rapid-readout 96 well plate used in biochemistry and cell biology. Custom three-dimensional cellular assemblies are now accessible to scientists of relatively limited financial resources, which will greatly speed up detailed studies of cellular behavior in biofilms, tissues, and other three-dimensional environments.

NOTE: The scientists' research was funded by Vertex, the Wyss Institute of Biologically Inspired Engineering, Fulbright-Generalitat de Catalunya, the American Heart Association, the National Institutes of Health, and the Department of Defense. George Whitesides (one of the corresponding authors) may have financial interest in this research, based on the conpeting interest statement he provided for the original manuscript.
Derda, R., Tang, S. K. Y, Laromaine, A., Mosadegh, B., Hong, E., Mwangi, M., Mammoto, A., Ingber, D. E., & Whitesides, G. M. (2011). Multizone Paper Platform for 3D Cell Cultures PLoS ONE, 6 (5) DOI: 10.1371/journal.pone.0018940