Methods tip assemblies and kits for introducing material into cells

Patent metadata

Field	Value
Publication number	US-10364441-B2
Application number	US-201816159185-A
Country	US
Kind code	B2
Filing date	Oct 12, 2018
Priority date	Mar 13, 2009
Publication date	Jul 30, 2019
Grant date	Jul 30, 2019

Abstract

Methods, tip assemblies and kits are provided for introducing material into cells. The tip assemblies include an attachment portion, a channel portion, and a constriction that function to reduce fluid pressure as a fluid passes through the constriction portion from the channel portion, whereby the tip assemblies form pores in the membranes of cells and introduce material into the cells. The material includes for example one selected from the group of: an inorganic compound, a drug, a genetic material, a protein, a carbohydrate, a synthetic polymer, and a pharmaceutical composition.

First claim

What is claimed is:

1. A method of introducing a composition in a fluid into cells, the method comprising:
a) passaging a suspension of cells in a fluid with the composition through a decrease in pressure, wherein the passaging occurs by the application of a positive pressure; and
b) drawing the suspension of cell back through the at least one constriction by the application of a negative pressure,

wherein the decrease in pressure occurs in a device comprising a channel with at least one constriction, wherein the positive pressure and a negative pressure are generated by a flow device, and wherein the decrease in pressure enhances temporary permeability in the cells, thereby introducing the composition into the cells.

2. The method of claim 1 , wherein the passaging of the suspension of cells in the fluid is performed continuously.

3. The method of claim 1 , wherein the passaging of the suspension of cells in the fluid is performed with a plurality of pauses.

4. The method of claim 3 , wherein each of the plurality of, pauses, are from about 1 ms to about 100 s in length.

5. The method of claim 1 , wherein the decrease in pressure is from about 0.1% to about 95% of the pressure of the suspension of cells in a fluid with the composition prior to passaging.

6. The method of claim 1 , wherein the constriction has a cross sectional area bounded by a shape selected from the group consisting of circular, ellipsoidal, rectangular, or square.

7. The method of claim 1 , wherein step a) and/or b) is performed more than once.

8. The method of claim 7 , wherein the passaging is for a period of time of about 0.1 minutes to about 30 minutes.

9. The method of claim 1 , further comprising observing localization of the composition to at least one subcellular compartment selected from the group consisting of a nucleus, a mitochondrion, a Golgi body, a chloroplast, a chromoplast, an endosome, a vesicle, a lysosome, an axon, a cytoplasmic membrane, a nuclear membrane, and a cytoplasm.

10. The method of claim 9 , wherein observing the localization of the composition comprises visualizing the composition with a detectable marker selected from the group consisting of a fluorescent marker, a chemiluminescent marker, a colorimetric marker, an enzymatic marker, and a radioactive marker.

11. The method of claim 9 , wherein observing the localization of the composition further comprises quantifying directly the amount of the composition that entered the cell.

12. The method of claim 1 , further comprising dispensing the suspension of cells in the fluid and the composition into a receptacle.

13. The method of claim 1 , wherein the cells comprise a population of a plurality of living cells.

14. The method of claim 13 , wherein cell viability of the population of a plurality of living cells is from about 1% to about 95% of control cells not passaged through the channel with at least one constriction.

15. The method of claim 1 , wherein the fluid comprises a Ca 2+ concentration from about 50 nM to about 500 nM.

16. The method of claim 1 , wherein the fluid comprises a Mg 2+ concentration from about 100 nM to about 10 mM.

17. The method of claim 1 , further comprising the steps of:
a) centrifuging the suspension of cells in a fluid with the composition to obtain a cell pellet and a supernatant;
b) removing the supernatant;
c) adding a culture medium;
d) re-suspending the cell pellet in the culture medium; and
e) culturing the cells.

18. The method of claim 1 , wherein the cells comprise a cell type selected from the group consisting of epithelial cells, hematopoietic cells, stem cells, spleen cells, kidney cells, pancreas cells, liver cells, neuron cells, glial cells, smooth or striated muscle cells, sperm cells, heart cells, lung cells, ocular cells, bone marrow cells, fetal cord blood cells, progenitor cells, peripheral blood mononuclear cells, leukocyte cells, lymphocyte cells, living postmitotic cells, physiologically inactive cells, inhibited cells, UV-inactivated cells, enucleated cells, anucleate cells, heat-killed cells, non-reproducing cells, and synthetic cells having an artificial membrane.

19. The method of claim 1 , wherein the composition comprises an inorganic compound, a drug, a genetic material, a protein, a carbohydrate, a synthetic polymer, or a pharmaceutical composition.

20. The method of claim 19 , wherein the composition comprises a genetic material.

21. The method of claim 20 , wherein the genetic material comprises a DNA or an RNA.

22. The method of claim 19 , wherein the composition comprises a protein.

23. The method of claim 1 , further comprising assaying transfection of the cells.

24. The method of claim 1 , further comprising applying to the suspension of cells in the fluid and the composition at least one treatment selected from the group consisting of an electric field, light comprising at least one wavelength, and a sound pulse.

25. The method of claim 1 , wherein the fluid further comprises at least one transfection agent selected from the group consisting of a nanoparticle, a liposome, a viral vector, a bacteriophage, and a detergent.

26. The method of claim 21 , wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, siRNA, RNAi, miRNA, and dsRNA, or a portion thereof.

Assignees

• Univ Tufts

Inventors

• Diefenbach Thomas J

Classifications

• C12N15/89 Primary

  using microinjection · CPC title

• C12M35/04

  Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli · CPC title

• C12M35/00

  Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion (machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87) · CPC title

• B01L3/021

  Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids · CPC title

• C12M33/04

  by injection or suction, e.g. using pipettes, syringes, needles (pipettes in general B01L3/02) · CPC title