Methods, tip assemblies and kits for introducing material into cells

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.

1 . A tip assembly for introducing a composition in a fluid into cells comprising:
 an attachment portion open to the atmosphere and proximally fitted to a flow device that generates at least one of a positive pressure and a negative pressure for directing the fluid;   a channel portion contiguous to and distal to the attachment portion and the flow device;   a constriction portion contiguous with the channel portion, wherein a constriction portion inner diameter and a constriction portion cross sectional area are smaller than a channel portion inner diameter and a channel portion cross sectional area, wherein a distal end of the constriction portion comprises an opening for ejecting or drawing the fluid, wherein increased fluid velocity and a decreased pressure in the fluid in the constriction portion compared to velocity and pressure in the channel portion enhances formation of membrane pores in the cells, whereby the tip assembly introduces the composition into the cells through the membrane pores.   
     
     
         2 . The tip assembly according to  claim 1 , wherein the tip assembly is characterized in that it is at least selected from: disposable, modular, reusable, transparent, and translucent. 
     
     
         3 . The tip assembly according to  claim 1 , wherein at least one of the attachment portion, the channel portion, and the constriction portion comprises a substance selected from the group of: a glass, a metal, a plastic, a polymer, a nano-based composition, a composite material comprising at least two different types of substances, and the like. 
     
     
         4 . The tip assembly according to  claim 1 , wherein an inner diameter or outer diameter of the attachment portion of the tip assembly fits the flow device in a male to female arrangement respectively. 
     
     
         5 . The tip assembly according to  claim 1 , wherein the channel portion cross sectional area or the constriction portion cross sectional area is bounded by a circle, an ellipse, a rectangle or a square, and wherein the distal end of the constriction portion comprises an inner diameter that is less than or substantially equal to an inner diameter of the opening. 
     
     
         6 . The tip assembly according to  claim 1 , wherein the tip assembly contains a volume which is: about 2 microliters (μl), about 20 about 50 μl, about 200 μl, about 400 μl, about 500 μl, about 1 milliliter (ml), about 5 ml, and about 10 ml, and wherein the channel portion inner diameter is about 1.0 millimeter (mm) to about 10.0 mm, and the diameter of the constriction inner diameter is about 0.05 mm to about 2.0 mm, wherein the constriction inner diameter is smaller than the channel portion inner diameter. 
     
     
         7 . (canceled) 
     
     
         8 . The tip assembly according to  claim 1 , further comprising a shoulder extending laterally from of an outward surface of the attachment portion, for example a lower ejector section of the flow device removes the tip assembly from the flow device. 
     
     
         9 . A method for introducing a composition in a fluid into cells, the method comprising:
 contacting the cells in a reservoir with a fluid comprising the composition;   inserting a tip assembly into the reservoir, wherein the tip assembly comprises: an attachment portion open to the atmosphere and proximally fitted to a flow device that generates at least one of a positive pressure and a negative pressure for directing the fluid; a channel portion contiguous to and distal to the attachment portion and the flow device; a constriction portion contiguous with the channel portion, wherein a constriction portion inner diameter and a constriction portion cross sectional area are smaller than a channel portion inner diameter and a channel portion cross sectional area, wherein a distal end of the constriction portion comprises an opening for ejecting or drawing the fluid, wherein increased fluid velocity and a decreased pressure in the fluid in the constriction portion compared to velocity and pressure in the channel portion enhances formation of membrane pores in the cells; and,   passaging a mixture of the cells, the fluid and the composition at least once through the tip assembly using a flow device, whereby passaging the mixture forms the membrane pores in the cells and introduces the composition into the cells.   
     
     
         10 . The method according to  claim 9 , wherein the cells are eukaryotic cells. 
     
     
         11 . The method according to any of  claim 9 , further comprising after passaging, observing localization of the composition to at least one subcellular compartment selected from: 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. 
     
     
         12 . The method according to  claim 11 , wherein observing the localization further comprises at least one of: visualizing the composition with a detectable marker selected from the group consisting of: detectable, fluorescent, colorimetric, enzymatic, radioactive; and quantifying directly the product of the composition that entered the cell from the group consisting of: mRNA, DNA, RNA, and protein. 
     
     
         13 . The method according to  claim 9 , wherein passaging the mixture further comprises redirecting the mixture to the reservoir, or wherein passaging the mixture further comprises dispensing the mixture into a receptacle. 
     
     
         14 . The method according to  claim 9 , wherein the cells comprise a population of a plurality of living cells, and the method further comprises observing that cell viability is not substantially reduced, wherein observing that the cell viability is not substantially reduced comprises assaying that the cell viability of at least about: 1%, 10%, 30%, 50%, 75%, 90%, or 95% of control cells not so passaged through the tip assembly. 
     
     
         15 . The method according to  claim 9 , wherein the fluid comprises a Ca +2  concentration less than about 200 nanomolar (nM), about 150 nM, about 100 nM, or about 75 nM, and wherein the fluid comprises a Mg +2  concentration less than about 200 nanomolar (nM), about 150 nM, about 100 nM, or about 75 nM. 
     
     
         16 . The method according to  claim 9  further comprising after passaging, centrifuging the mixture to obtain a cell pellet and a supernatant, removing the supernatant, adding culture medium to the reservoir, re-suspending the cell pellet in the medium, and culturing the cells. 
     
     
         17 . The method according to  claim 9 , 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. 
     
     
         18 . The method according to  claim 9 , wherein the genetic material comprises a DNA or an RNA, wherein the RNA is selected from the group of: mRNA, tRNA, rRNA, siRNA, RNAi, miRNA, and dsRNA, or a portion thereof, and the method further involves assaying transfection of the cells. 
     
     
         19 . The method according to  claim 9 , further comprising applying to the mixture at least one selected from the group of: an electric field, a light comprising a wavelength, and a sound pulse. 
     
     
         20 . A kit for introducing a composition in a fluid into cells comprising:
 a tip assembly for passaging a mixture of the cells and the fluid comprising: an attachment portion open to the atmosphere and proximally fitted to a flow device that generates at least one of a positive pressure and a negative pressure for directing the fluid; a channel portion contiguous to and distal to the attachment portion and the flow device; a constriction portion contiguous with the channel portion, wherein a constriction portion inner diameter and a constriction portion cross sectional area are smaller than a channel portion inner diameter and a channel portion cross sectional area, wherein a distal end of the constriction portion comprises an opening for ejecting or drawing the fluid, wherein increased fluid velocity and a decreased pressure in the fluid in the constriction portion compared to velocity and pressure in the channel portion enhances formation of membrane pores in the cells, whereby the tip assembly introduces the composition into the cells through the membrane pores; and,   a container.   
     
     
         21 - 24 . (canceled) 
     
     
         25 . A system for introducing a composition in a fluid into cells, the system comprising;
 a flow device that generates at least one of a positive pressure and a negative pressure for impelling the fluid; and,   a tip assembly for passaging a mixture of the cells and the fluid comprising: an attachment portion that connects to the flow device, a channel portion contiguous to and distal to the attachment portion and the flow device, a constriction portion contiguous with the channel portion, wherein a constriction portion inner diameter and a constriction portion cross sectional area are smaller than a channel portion inner diameter and a channel portion cross sectional area, wherein a distal end of the constriction portion comprises an opening for ejecting or drawing the fluid, wherein increased fluid velocity and a decreased pressure in the fluid in the constriction portion compared to velocity and pressure in the channel portion enhances formation of membrane pores in the cells, whereby the system introduces the composition into the cells through the membrane pores.   
     
     
         26 . The system according to  claim 25 , further comprising a receptacle adjacent to the opening for receiving the fluid passaged through the tip assembly. 
     
     
         27 . The system according to  claim 25 , wherein the tip assembly comprises at least one selected from the group consisting of: a glass, a metal, a plastic, a polymer, a nano-based composition, a composite material comprising at least two different types of substances; and the like. 
     
     
         28 . The system according to  claim 25 , wherein the flow device comprises at least one selected from the group consisting of: a syringe, a plunger, a bulb, a diaphragm, and a compressor. 
     
     
         29 . The system according to  claim 25 , wherein the flow device is controlled or operated manually. 
     
     
         30 . The system according to  claim 25 , wherein control and operation of the flow device is at least one selected from the group of: automated, electromechanical, and programmable. 
     
     
         31 . The system according to  claim 25 , wherein operation of the flow device comprises control of at least one selected from the group of: flow velocity; flow acceleration; mass flow rate; initial velocity ramp; starting velocity; maximum velocity; starting position; cutoff velocity period of time the fluid is under pressure generated by the flow device; temperature, and period of time the fluid is held in the channel portion, constriction portion, or both. 
     
     
         32 . The system according to  claim 31 , wherein the flow device generates a flow velocity along the length of the tip assembly, the flow velocity selected from about: about 0.1 cm/s to about 1 centimeter per second (cm/s), about 1 cm/s to about 5 cm/s, about 5 cm/s to about 15 cm/s, and about 15 cm/s to about 20 cm/s; wherein the flow device generates a mass flow rate selected from a group consisting of about: 0.01 milliliter per minute (ml/min), 1 ml/min, 10 ml/min, 25 ml/min, 50 ml/min, 100 ml/min, and 150 ml/min; and wherein the flow device generates an acceleration in the tip assembly of about 0.6 μl/s/s, about 1 μl/s/s, about 6 μl/s/s, about 10 μl/s/s, about 12 μl/s/s, about 24 μl/s/s, about 36 μl/s/s, about 48 μl/s/s, and about 60 μl/s/s. 
     
     
         33 . The system according to  claim 25 , wherein the tip assembly further comprises at least one valve. 
     
     
         34 . The system according to  claim 25  further comprising at least one selected from the group of: a power source; a connector for interacting with a user interface, a computer, a hand-held device, a transmitter, or a display; a conduit for connecting the flow device to the tip assembly; and a sensor. 
     
     
         35 . The system according to  claim 25  further comprising a housing.