Intracellular delivery using microfluidics-assisted cell screening (macs)

Abstract

Described herein are methods inducing the uptake of an agent by a cell. Aspects of the invention relate to physically compressing the cell to induce perturbations (e.g., holes) in the cell membrane or wall. An agent is taken up by the cell through induced perturbations.

1 .- 77 . (canceled) 
     
     
         78 . A microfluidic device for uptake of an agent into a cell or population comprising:
 a first flow channel, the flow channel being at least partially bounded on a first side by a flexible layer of the microfluidic device and being at least partially bounded on an opposing second side by a rigid layer of the microfluidic device, the first flow channel comprising a first flow fluid including a plurality of a first type of cell and a first agent;   a control channel formed in the flexible layer of the microfluidic device such that the flexible layer physically compresses at least one of the first type of cell in the first flow fluid against the rigid layer to induce at least one temporary perturbation in a membrane of the at least one of the first type of cell,   wherein the at least one temporary perturbation causes a portion of the first agent to be taken up by the at least one of the first type of cell through the at least one temporary perturbation in the membrane of the at least one of the first type of cell.   
     
     
         79 . The device of  claim 78 , wherein the flow rate of the first flow channel is adjustable. 
     
     
         80 . The device of  claim 78 , wherein when no pressure is applied to the control channel, the flow channel is open such that the flow fluid can flow freely in the flow channel. 
     
     
         81 . The device of  claim 78 , wherein the control channel comprises a valve. 
     
     
         82 . The device of  claim 78 , wherein the control channel comprises a dead end channel running in a perpendicular direction to the flow channel. 
     
     
         83 . The device of  claim 78 , wherein the control channel can be pressurized. 
     
     
         84 . The device of  claim 79 , wherein the flow rate of the first flow channel is adjusted when the control channel pressurized. 
     
     
         85 . The device of  claim 79 , wherein the adjusting includes adjusting the flow rate of the flow fluid in the flow channel to zero flow. 
     
     
         86 . The device of  claim 78 , wherein a pressure differential between a pressure inside the at least one of the plurality of cells and a pressure of the flowing fluid causes the at least one of the plurality of cells to take up the portion of the agent through the at least one temporary perturbation of the membrane. 
     
     
         87 . The device of  claim 78 , wherein prior to the at least one of the plurality of cells being physically compressed, a pressure differential between a pressure inside the at least one of the plurality of cells and a pressure of the flowing fluid is insufficient to cause the at least one of the plurality of cells to take up the portion of the agent. 
     
     
         88 . The device of  claim 78 , wherein the agent is selected from the group consisting of: a DNA staining dye, a fluorescent molecule, a plasmid, a vector, a protein, a nucleic acid, a polypeptide, a recombinant RNA, an RNA, a siRNA, an shRNA, a miRNA, a compound, a small molecule, an antibody, a virus, a quantum dot (Qdot), a chromosome, a sequence encoding a chimeric antigen receptors (CAR) for CAR-modified T-cells (CAR-T), a drug, a therapeutic, an anti-sense oligonucleotide (ASO), an mRNA, an RNA aptamers, a protein aggregate, a protein fibril, a nanoparticle, a polysaccharide, a lipid, an organelle, a mitochondrion, a prokaryote, a microbial cell, and a bacterial cell. 
     
     
         89 . The device of  claim 78 , wherein the fluid includes an extracellular growth factor. 
     
     
         90 . The device of  claim 78 , wherein the cell is selected from the group consisting of: a prokaryote cell, a eukaryotic cell, a mammalian cell, a human cell, a microbial cell, a bacterial cell, a yeast cell, an artificial cells, a liposome, an oocyte, a red blood cell, a white blood cell, a cell having a generally rod shape with a diameter that is less than two micrometer and a length that is less than ten micrometers, a cell having a generally rod shape with a diameter that is between about 0.2 micrometers and about two micrometer, a cell having a generally biconcave shape with a diameter that is between about two micrometers and about twenty micrometers, and a cell having a diameter that is between about one micrometers and about twenty micrometers. 
     
     
         91 . The device of  claim 78 , wherein the first type of cell is a prokaryotic cell and the second type of cell is a eukaryotic cell; or
 wherein the first type of cell and the second type of cell are a different prokaryotic cell; or   wherein the first type of cell and the second type of cell are a different eukaryotic cell; or   wherein the first type of cell and the second type of cell are a different diameter; or   wherein the first type of cell and the second type of cell are a different shape.   
     
     
         92 . The device of  claim 78 , wherein a second flow fluid is passed through the flow channel at a time point after the first fluid. 
     
     
         93 . The device of  claim 78 , wherein the first flow fluid is passed through the flow channel at a first time point. 
     
     
         94 . The device of  claim 92 , wherein the second flow fluid is passed through the flow channel at a second time point. 
     
     
         95 . The device of  claim 94 , wherein the second time point is a least 0.1 seconds following the first time point.