Laser-based directed release of array elements for efficient collection into targeted microwells

Abstract

A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25-400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%.

Fig. 1

Examination of various pallet release angles and travel distances upon laser-based release. A) Schematic of pallet release by a single laser pulse. B) Schematic demonstrating the central location of the laser focal point used for (C, D). C) Scatter plot of release angle and distance travelled for 50 µm × 50 µm × 50 µm pallets (n = 35) released as in “B”. D) Vector diagram of release angle and distance travelled for pallets shown in “C”. E) Schematic showing the focal point on central left edge of pallets as used for (F, G). F) Scatter plot of release angle and distance travelled for 50 µm × 50 µm × 50 µm pallets (n = 35) released as in “E”. G) Vector diagram of release angle and distance travelled for pallets shown in “F”. H) Schematic of the focal point used for (I, J). I) Scatter plot of release angle and distance travelled for 50 µm × 50 µm × 50 µm pallets (n = 35) released as in “H”. J) Vector diagram of release angle and distance travelled for pallets shown in “I”.

Fig. 2

Examination of the impact of pallet height on release angle and travel distance. A) Scatter plot of release angle and distance travelled for 50 µm × 50 µm × 100 µm pallets (n = 35). B) Vector diagram of release angle and distance travelled for pallets shown in “A”. C) Scatter plot of release angle and distance travelled for 50 µm × 50 µm × 150 µm pallets (n = 35). D) Vector diagram of release angle and distance travelled for pallets shown in “C”.

Fig. 3

Description of the collection system. A) Schematic of a microwell array showing four of the 16 sub-arrays each containing 25 individual microwells. B) Electron micrograph of one sub-array containing 25 microwells (450 µm × 450 µm × 300 µm, scale bar is 450 µm). C) Schematic of rotary stage for the microwell array aligned with the pallet array holder.

Fig. 4

Determination of accuracy, efficiency, and cross-talk during pallet collection. A) Schematic demonstrating release and collection technique of a pallet aligned with the edge of a microwell and released with a laser pulse focused near the pallet edge. B) Image of 5×5 microwell sub-array containing 25 pallets demonstrating pallets collected in target wells, an uncollected pallet (above “1I”), and microwells with cross-talk (“1H&2H” and “4H&5H”). Empty wells resulting from cross-talk and an uncollected pallet are seen in “1H&I” and “5H”. (scale bar is 450 µm).

Fig. 5

Demonstration of large-scale pallet release and collection. Image of 400 microwells (addresses omitted) containing 401 pallets collected to demonstrate feasibility of large-scale pallet release and collection (scale bar is 900 µm) with inset of magnified view of microwells (scale bar is 450 µm).

Fig. 6

Cell viability after release and collection. Overlaid bright field and fluorescence images of a pallet containing a GFP-expressing HeLa cell at 0 h (A) and at 72 h (B) after collection (scale bar is 100 µm).