In situ-generated microfluidic isolation structures, kits and methods of use thereof
In situ-generated microfluidic isolation structures incorporating a solidified polymer network, methods of preparation and use, compositions and kits therefor are described. The ability to introduce in real time, a variety of isolating structures including pens and barriers offers improved methods of micro-object manipulation in microfluidic devices. The in situ-generated isolation structures may be permanently or temporarily installed.
1. A method of isolating a micro-object in a microfluidic device, comprising:
introducing the micro-object into an enclosure of the microfluidic device, the enclosure comprising a flow region and a sequestration pen comprising an isolation region and a connection region, the connection region having a proximal opening to the flow region and a distal opening to the isolation region;
disposing the micro-object into the sequestration pen;
introducing a solution comprising a flowable polymer into the enclosure;
solidifying the flowable polymer thereby forming an in situ-generated isolation structure, in-situ at least in part in the sequestration pen, wherein the in situ-generated isolation structure is disposed within the connection region and configured to block passage of the micro-object between the sequestration pen and the flow region; and
isolating the micro-object using the in situ-generated isolation structure.
2. The method of claim 1 , wherein the enclosure is configured to receive a fluidic medium, and the in situ-generated isolation structure is porous to the fluidic medium.
3. The method of claim 1 , wherein the in situ-generated isolation structure substantially restricts passage of the micro-object into or out of the sequestration pen in a size dependent manner.
4. The method of claim 1 , wherein the in situ-generated isolation structure partially blocks the proximal opening of the connection region.
5. The method of claim 1 , wherein the in situ-generated isolation structure is formed within the sequestration pen.
6. The method of claim 1 , wherein the in situ-generated isolation structure is formed within the isolation region or the connection region.
7. The method of claim 1 , wherein the in situ-generated isolation structure is formed at the proximal opening of the connection region.
8. The method of claim 7 , wherein the connection region comprises a width of about 50 microns to about 100 microns at the proximal opening, and the in situ-generated isolation structure has a dimension of at least 50 microns.
9. The method of claim 8 , wherein the width of the connection region is about 50 microns to about 60 microns.
10. The method of claim 1 , wherein solidifying the flowable polymer comprises optically illuminating a selected area, and further wherein solidifying the flowable polymer comprises polymerizing polymers of the flowable polymer to form a polymer network.
11. The method of claim 1 , further comprising:
reducing or removing the in situ-generated isolation structure by: increasing flow of fluidic medium through the flow region;
introducing a hydrolytic agent into the flow region; introducing a proteolytic agent into the flow region;
increasing/decreasing osmolality of fluidic medium within the enclosure; changing a temperature of the in situ-generated isolation structure; or
optically illuminating the isolation structure, and releasing the micro-object from the in situ-generated isolation structure.
12. The method of claim 11 , further comprising: exporting the micro-object from the microfluidic device.
13. The method of claim 1 , further comprising: introducing a plurality of micro-objects into the enclosure of the microfluidic device.
14. The method of claim 13 , further comprising: exporting from the microfluidic device a remainder of the plurality of micro-objects not isolated by the in situ-generated isolation structure.
15. The method of claim 1 , wherein the enclosure comprises a plurality of sequestration pens.
16. The method of claim 15 , wherein the flow region comprises a microfluidic channel and a proximal opening of each of the plurality of sequestration pens opens off of one side of the microfluidic channel.
17. The method of claim 1 , wherein the flowable polymer comprises a synthetic polymer, a modified synthetic polymer, or a biological polymer.
18. The method of claim 17 , wherein the modified synthetic polymer comprises size modification motifs, cleavage motifs, reactive terminal moieties, and/or cell recognition motifs.
19. The method of claim 1 , wherein the solidified polymer network comprises at least one of a polyethylene glycol, modified polyethylene glycol, polylactic acid (PLA), modified polylactic acid, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone (PCL), modified polycaprolactone, fibronectin, modified fibronectin, collagen, modified collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or a co-polymer in any combination.
20. The method of claim 1 , wherein disposing the micro-object into the sequestration pen comprises using dielectrophoresis (DEP) forces.