(108c) High Aspect Ratio Hydrogel Microwell Arrays As a Novel Platform for the In Situ Cloning of Rare Cells

Introduction: The colony forming unit assay (CFU) for adherent cells is a well established
technique for assessing the prevalence and function of rare cells in complex
cell mixtures. However, cell migration and colony merger during expansion means
the clonality of individual colonies cannot be assured, and the effect of
overall plating density on the function, survival and proliferation of rare
cells cannot be assessed over a relatively low plating density. We address
these shortcomings in the CFU assay by designing high aspect ratio hydrogel
microwell arrays which provides an optically transparent and cytocompatible
cell growth surface for the attachment and proliferation of cells which are
separated by non-fouling PEG walls.  This
platform enables the in situ analysis
of single cell behavior during culture.

Materials and Methods: Non-fouling
hydrogel microwell arrays were fabricated through the UV-initiated free radical
polymerization of poly(ethylene glycol) dimethacrylate (M_n = 1000) using
soft lithography. The arrays have been fabricated on a variety of substrates: glass
via silane chemistry, plastics (Mylar and
polystyrene) via an argon plasma treatment, and layer-by-layer polyelectrolyte
films. Arrays were seeded with bone marrow-derived and expanded aMSCs. Time-lapse
video microscopy was used to study single cell migration and proliferation.

Results and
Discussion: Hydrogel microwell arrays are stable for greater than two
weeks in culture, and the dimensions of the microwells can be easily adjusted
by changing the geometry of the PDMS stamp resulting in a versatile cell
culture system. Interestingly, aMSCs were able to adhere to and migrate on otherwise
cell resistant hydrogels which contact PDMS during molding illustrating a
previously unreported phenomena  by which
micromolding with PDMS undermines the cell resistance
of non-fouling hydrogels.  We present
techniques for overcoming this unwanted cellular attachment.  Microwell arrays were fabricated on a range
of cell culture substrates (silanized glass,
plastics, and polyelectrolyte thin films) and we determined that the substrate
affected the fidelity of pattern transfer and the performance of the microwell
arrays in containing the seeded cells. Time lapse video microscopy was used to
verify that regions within the hydrogel array are able to confine cells and
their progeny, and these videos were used to quantify the individual cell
proliferation kinetics of aMSCs.

Figure 1. High aspect
ratio hydrogel microwell arrays: (A) Scanning electron micrograph showing high
fidelity patterning of microwell arrays to polystyrene and (B) confocal microscopy image of mesenchymal stem cells
cultured in the hydrogel microwell arrays showing varying proliferation
capacities. 

Conclusions: High aspect ratio hydrogel microwell arrays which are stable for up to 2
weeks in culture conditions can be fabricated via soft lithography. PDMS
stamping alters the non-fouling interfacial properties of PEG, and the cell
culture surface affects the quality of pattern transfer during micromolding. Regions of the microwell arrays are capable
of containing their cells and their progeny enabling the high resolution study
of individual cells behavior.