Granted Patent B2

US 12,454,674 · App. 18/039,848 · Granted Oct 28, 2025

Inventors: Hsueh-Chia Chang (South Bend, IN); Zehao Pan (South Bend, IN); Vivek Yadav (South Bend, IN); Loan Bui (South Bend, IN); Donny Hanjaya-Putra (South Bend, IN)

Assignee: UNIVERSITY OF NOTRE DAME DU LAC

C12N5/0012C12N5/0652C12N5/0693C12N13/00C12N2533/30C12N2533/54C12N2533/74C12N2537/10

Abstract

Claims (27)

1. A method for high-throughput single-cell encapsulation and tip streaming, the method comprising:

(a) combining a plurality of cells with a hydrogel and oil to generate a water-in-oil emulsion;

(b) agitating the emulsion to drive single cells into the center of individual hydrogel beads to generate single-cell encapsulated hydrogel beads;

(c) crosslinking the hydrogel;

(d) extracting the crosslinked single-cell encapsulated hydrogel beads from the emulsion into cell media;

(e) transferring the extracted single-cell encapsulated hydrogel beads into a micropipette;

(f) applying a back pressure and alternating current (AC) electric potential to the micropipette to generate an AC Taylor cone encompassing a droplet containing the single-cell encapsulated hydrogel bead at the micropipette tip; and

(g) ejecting the droplet containing the single-cell encapsulated hydrogel bead from the micropipette tip.

2. The method of claim 1 , wherein the plurality of cells comprises human cancer cells or human multipotent mesenchymal stromal cells (hMSCs).

3. The method of claim 1 , wherein agitating comprises the use of a shaker at about 500-5000 rpm for at least about 1 minute, and wherein agitating does not affect droplet monodispersity.

4. The method of claim 1 , wherein the hydrogel comprises a natural hydrogel comprising alginate or collagen, or wherein the hydrogel comprises a synthetic hydrogel comprising hyaluronic acid functionalized with norbornene groups (NorHA).

5. The method of claim 4 , wherein the alginate is present at about 2 wt %.

6. The method of claim 4 , wherein the alginate encapsulated hydrogel beads have an average thickness of about 4-5 μm, or wherein the collagen encapsulated hydrogel beads have an average thickness of about 4-6 μm.

7. The method of claim 4 , wherein the alginate encapsulated hydrogel beads have a cell viability of about 80-85% after 24 hours in vitro, or wherein the collagen encapsulated hydrogel beads have a cell viability of about 70-75% after 24 hours in vitro.

8. The method of claim 4 , wherein the crosslinking of alginate comprises mixing alginate with a complex of divalent cations, ethylenediaminetetraacetic acid (EDTA), and 0.01% acetic acid for about 2 minutes, or wherein the crosslinking of collagen comprises increasing the temperature of the collagen to 37° C. at a neutral pH for about 30 minutes.

9. The method of claim 4 , wherein extracting the single-cell encapsulated alginate beads into cell media comprises mixing the emulsion with 10% w/w 1H,1H,2H,2H-perfluorooctanol (PFO) followed by centrifugation at about 100-500×g for at least about 2 minutes, or wherein extracting the single-cell encapsulated collagen beads into cell media comprises mixing the emulsion with 10% w/w 1H,1H,2H,2H-perfluorooctanol (PFO) followed by centrifugation at about 100-500×g for at least about 2 minutes.

10. The method of claim 4 , wherein the NorHA hydrogel is prepared by mixing 1.2% w/v NorHA macromer with dithiothreitol (DTT) at a 0.8 ratio in PBS and adding 0.2% w/v of the water soluble photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

11. The method of claim 4 , wherein the NorHA encapsulated hydrogel beads have an average thickness of about 2-3 μm.

12. The method of claim 4 , wherein the NorHA encapsulated hydrogel beads have a cell viability of greater than 80% after 24 hours in vitro.

13. The method of claim 4 , wherein the crosslinking of NorHA comprises irradiating the NorHA with UV light (10 mW cm −2 , 365 nm) for at least about 5 seconds, and wherein extracting the single-cell encapsulated NorHA beads into cell media comprises mixing the emulsion with 25% w/w 1H,1H,2H,2H-perfluorodecanol (PFD) followed by centrifugation at about 100-500×g for at least about 2 minutes.

14. The method of claim 4 , wherein the NorHA hydrogel preserves the osteogenic and adipogenic differentiation potential of hMSCs.

15. The method of claim 1 , wherein the droplet encompassed by the AC Taylor cone and containing the single-cell encapsulated hydrogel bead is electroneutral, and wherein the AC Taylor cone has a half angle less than 49°.

16. The method of claim 1 , wherein the micropipette tip has a diameter of about 30 μm, and wherein the droplet has a diameter size range of about 1 μm to about 10 μm.

17. The method of claim 1 , wherein the AC electric potential comprises a frequency greater than the inverse charge relaxation time of the micropipette tip orifice.

18. The method of claim 1 , wherein the AC electric potential comprises a frequency of about 10 KHz to about 300 KHz and a voltage of about 300 V to about 500 V, and wherein the back pressure is set slightly below a critical pressure of 2.4 kPa.

19. The method of claim 1 , wherein the tip streaming has a flow rate of about 5 nL/min.

20. The method of claim 1 , wherein the method generates an encapsulation efficiency greater than 80%, and wherein greater than 85% of the single-cell encapsulated hydrogel beads have a concentric index (CI) below 0.8, indicating that most encapsulated single cells are centered within the hydrogel bead droplet.

Assignments (2)

Continuity (2)

References Cited (115)