Microtable arrays for culture and isolation of cell colonies

Abstract

Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new three-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10-μJ pulses to each of the four support structures. In contrast, microstructures with a 150 × 150-μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie-Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array.

Figure 1

Release of large micropallets. A, B) A micropallet (150 × 150 μm) released using a single 212 μJ laser pulse: (A) side view (B) bottom view. Note the deep fissure created by the focused laser pulse (arrow). C) A micropallet (200 × 200 μm) released using multiple 10 μJ laser pulses (30 pulses): the patterning on the undersurface of the micropallet is due to local damage to the 1002F surface by each laser pulse. (D) Brightfield image of a colony of HeLa cells on a 200 × 200 μm micropallet 24 h after release by a single laser pulse. All cells adherent to the micropallet have taken up trypan blue indicating that they are nonviable.

Figure 2

(A) Microtable fabrication process. (B) Brightfield image of a microtable. (C) SEM of microtables released from the array showing top and bottom views of the structure. (D) SEM of a 2 × 3 portion of an array. One microtable has been released and is shown in side view.

Figure 3

(A) Minimum release energy for microtables of various dimensions and for two-layer micropallets with 50 μm sides. (B) Brightfield image of a released microtable. The focal plane is at the base of the legs. Small defects in each leg base show the site of the laser pulsed used to dislodge the leg from the glass substrate. (C) Brightfield image of the substrate surface after microtable release. Remnants of 1002F polymer are present where each leg was attached to the substrate.

Figure 4

The stability of the virtual air wall in solutions of varying concentrations of ethanol on (A) micropallet arrays and (B) microtable arrays. Shown on the x-axes are the distances between individual microstructures on the arrays. The y-axes display the fraction of the surface area between the microstructures covered with air. The error bars represent the standard deviation of the data points.

Figure 5

Brightfield images of HeLa cells cultured for 144 h on (A) arrays of micropallets (200 × 200 μm) and (B) arrays of microtables (200 × 200 μm). The virtual walls were absent on both arrays in the area imaged. Cells migrated across the inter-pallet region on the micropallet array, but remain localized to the culture sites on the microtable array.

Figure 6

Confocal images of HeLa cells with fluorescent nuclei cultured on a microtable array for 72 h. Shown is a single microtable at different image planes: (A) top surface of tabletop, (B) undersurface of tabletop, (C) glass substrate, (D) 3-D reconstruction of the series. (E) Brightfield image of a microtable array after 168 h in culture. The cells on the underlying glass surface (arrow) are adjacent to the table legs.

Figure 7

(A) Brightfield image and (B) fluorescence image 3 days after a colony of cells on a microtable was released and collected from an array. The colony expanded as the cells divided and grew off the structure.