Disruption and field enabled delivery of compounds and compositions into cells
A microfluidic system for causing perturbations in a cell membrane includes (a) a microfluidic channel defining a lumen and configured such that a cell suspended in a buffer can pass there through, and (b) source or emitter of an energy field. The microfluidic channel may include a cell-deforming constriction. A diameter of the constriction may be a function of the diameter of the cell. Related apparatus, systems, techniques, and articles are also described.
1. A method for delivering a compound or composition into a cell, the method comprising:
providing a cell in a payload-containing cell suspension;
passing the cell suspension through a microfluidic channel that includes a cell-deforming constriction;
passing the cell through the constriction such that a pressure is applied to the cell causing perturbations of the cell membrane large enough for the payload to pass through the cell membrane and into the cytosol of the cell; and
contacting the cell with an electric field,
wherein the electric field has a strength or pulse strength that is about 0.1-10 kV/cm and a pulse duration of about 50-2000 microseconds.
2. The method of claim 1 , wherein the step of contacting the cell with an electric field translocates the payload from a first location in the cell to a second location inside the cell after the payload has entered the cell.
3. The method of claim 1 , wherein the cell is contacted with a magnetic field, and the magnetic field is generated by at least one electromagnet.
4. The method of claim 1 , wherein the electric field is generated by one or more electrodes.
5. The method of claim 4 , wherein the cell is one of a plurality of cells, and each cell is passed through one of a plurality of parallel microfluidic channels, wherein each microfluidic channel of the plurality of parallel microfluidic channels includes a cell-deforming constriction, and wherein the plurality of cells is passed through the electric field.
6. The method of claim 4 , wherein the electric field is generated by two electrodes to drive the payload into the cell.
7. The method of claim 4 , wherein the electric field is generated by a plurality of electrode pairs in which electrode size varies between electrode pairs.
8. The method of claim 4 , wherein at least one of the one or more electrodes is driven by a function generator coupled to the electrode, the function generator driving the electrode to generate the electric field.
9. The method of claim 1 , wherein the cell is passed through the microfluidic channel in a first device and then removed from the first device and contacted with the electric field in a second device.
10. The method of claim 1 , wherein the microfluidic channel and the electric field are within one device.
11. The method of claim 10 , wherein
(a) the cell passes through the constriction to the field in a continuous flow, wherein after passing through said constriction, the cell contacts or passes through a portion of the electric field; or
(b) after passing through the constriction the cell flows into and remains within a zone of the device where the cell is contacted with the field.
12. The method of claim 1 , wherein the payload comprises one or more of
(a) a protein;
(b) a small molecule;
(c)a sugar;
(d) polymers of biological, synthetic, organic, or inorganic molecules;
(e) a charged molecule or composition comprising a charged molecule; or
(f) an uncharged molecule.
13. The method of claim 1 , wherein the payload is driven into one or more of
(a) the nucleus of the cell;
(b) a mitochondrion of the cell; or
(c) an organelle of the cell other than the nucleus or a mitochondrion of the cell.
14. The method of claim 1 , wherein
the diameter of the constriction is about 20-99% of the diameter of the cell passing therethrough.
15. The method of claim 1 , wherein the cell is a prokaryotic cell or a eukaryotic cell.
16. The method of claim 1 , wherein the diameter of the constriction is about 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 4 μm-10 μm, or 10 μm-20 μm.
17. The method of claim 1 , wherein the length of the constriction is about 10 μm, 15 μm, 20 μm, 24 μm, 30 μm, 40 μm, 50 μm, 60 μm, 10 μm-40 μm, 10 μm-50 μm, or 10 μm-60 μm.
18. The method of claim 1 , wherein the cell is contacted with the electric field about 0.0001 s, 0.001 s, 0.002 s, 0.003 s, 0.004 s, 0.005 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 0.001 s-0.005 s, or 0.0001 s-10 s after exiting the cell-deforming constriction, or within about 0.0001 s, 0.001 s, 0.002 s, 0.003 s, 0.004 s, 0.005 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 0.001 s-0.005 s, or 0.0001 s-10 after exiting the cell-deforming constriction.
19. The method of claim 1 , wherein the exposure time of the cell to the electric field is about 10 ms-50 ms, 50 ms , 100 ms, or 10 ms-100 ms.
20. The method of claim 1 , wherein the electric field is a pulsed direct electric current.
21. The method of claim 1 , wherein the electric field is pulsed at about 50 μs-200 μs.
22. The method of claim 1 , wherein the pulse strength of the electric field is about 1 kV/cm-3 kV/cm, 0.1 kV/cm-0.5 kV/cm, 0.1 kV/cm-1 kV/cm, 0.1 kV/cm-1.5 kV/cm, 0.1 kV/cm-2 kV/cm, 0.1 kV/cm-2.5 kV/cm, or 0.1 kV/cm-3 kV/cm.
23. The method of claim 1 , wherein a pressure of about 10 psi-100 psi is used to pass the solution through the microfluidic channel.
24. The method of claim 1 , wherein the cell passes through the microfluidic channel at a speed of about 300 mm/s, 100 mm/s-300 mm/s, 200 mm/s-700 mm/s, 250 mm/s-400 mm/s, 100 mm/s-1000 mm/s, or 1 mm/s-1000 mm/s.
25. The method of claim 1 , wherein said microfluidic channel comprises multiple cell-deforming constrictions in series.
26. The method of claim 1 , wherein said microfluidic channel comprises a single cell-deforming constriction.
27. The method of claim 1 , wherein the cell is one of a plurality of cells, and about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 90-95, or 80-100% of the cells are viable after passing through the electric field.
28. The method of claim 25 , wherein the electric field is pulsed at a duration of about 0.1 ms, at a period of 1 ms-20 ms, 0.1 ms-2000 ms, or 1-200 ms.
29. The method of claim 1 , wherein the cell passes through the electric field at a speed of about 100 mm/s, 170 mm/s, 300 mm/s, 100 mm/s-300 mm/s, 200 mm/s-700 mm/s, 250 mm/s -400 mm/s, 100 mm/s-1000 mm/s, or 1 mm/s-1000 mm/s.
30. The method of claim 1 , wherein the perturbations of the cell membrane include a maximum diameter of about 1 nm-20 nm, 1 nm-600 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 25 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, or 600 nm.
31. The method of claim 1 , wherein perturbations of the cell membrane having a maximum diameter of about 1 nm-20 nm, 1 nm-600 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 25 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, or 600 nm persist on the cell membrane for at least 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or 1 min-10 min.