Patent Publication Number: US-2019177677-A1

Title: High-throughput system and method for the temporary permeablization of cells

Description:
RELATED APPLICATION 
     This Application claims priority to U.S. Provisional Patent Application No. 62/377,572 filed on Aug. 20, 2016, which is hereby incorporated by reference in its entirety. Priority is claimed pursuant to 35U.S.C. § 119 and any other applicable statute. 
    
    
     TECHNICAL FIELD 
     The technical field generally relates to devices and methods that are used to deliver molecules or other cargo into cells at clinically relevant scales. The technical field has particular suitability for the delivery of gene-editing constructs or biomolecules into large numbers of cells. In particular, the invention relates to microfluidic devices that use omniphobic and/or fouling-resistant microchannels that have constrictions therein to temporarily permeabilize cells that aid in the introduction and transfer of molecules or other cargo from the surrounding fluid into the cells. 
     BACKGROUND 
     Gene therapy and gene modification technologies are increasingly being studied, investigated, and used for clinical applications. In order to modify or alter genes, the gene-editing biomolecules or other constructs need to be delivered into cells. Currently, a standard technique for gene modification uses virus-based delivery systems that utilize, for example, lentiviruses, adenoviruses, adeno-associated viruses, or herpes virus. Lentiviruses, for instance, can deliver a significant amount of genetic information into DNA of the host cell so they are one of the most effective and commonly used methods of a gene delivery vector. The use of viral transfection, while effective as a vector system, is expensive and has potential serious adverse side effects. Principal among the possible dangers with virus-based delivery systems is the fact that integration of genetic modifications occurs semi-randomly, leading to concern for potential genotoxicity and carcinogenesis through off-target effects. 
     Electroporation, in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, is another technique that has been used to transfect cells for gene therapy based on targeted endonucleases. Conventional electroporation, however, suffers from toxicity problems as well as technical limitations in using this method in scaled-up clinical applications. Chemical transfection methods may also be used for gene-editing applications based on targeted endonucleases. 
     Still other approaches for the intracellular delivery of biomolecules involving nanoparticles or nanostructures (e.g., nanostraws, carbon nanotubes, or needles) have been demonstrated but have not been commercialized or scaled up for clinical use. Intracellular delivery of biomolecules by cell membrane deformation within microfluidic channels has been demonstrated. For example, U.S. Patent Application Publication No. 2014/0287509discloses a microfluidic system for causing temporary pertubations in the cell membrane using a cell-deforming constriction in the microfluidic channel. In another approach, a series of microconstrictions are generated by a pattern of protuberances that extend from a polydimethylsiloxane (PDMS) to apply shear and compressive forces on cells passing therethrough. See Han et al., CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation., Sci. Adv., pp. 1-8 (2015). 
     While the intracellular delivery through cell membrane deformation is beginning to emerge, current embodiments of this technology suffer from issues with fouling or clogging, which affects the long-term reliability of the device and efforts for translation towards clinicaly relevant applications. For example, in clinical gene therapy, large numbers of cells need to be transfected (e.g., billions of cells) rapidly. Current technologies are not adapted for such large scale processing because they tend to become quickly fouled or clogged. For example, it is not uncommon for a microfluidic device to become clogged with cells after just seconds or minutes of operation. 
     SUMMARY 
     In one embodiment, a microfluidic-based system for the intracellular transport of molecules or other cargo is disclosed. The system includes a microfluidic substrate or chip that includes therein a plurality of microchannels that contain one or more constrictions that are dimensioned to induce a transient increase in the permeability of cells that pass through the constrictions. The microchannels may be arranged in parallel in the substrate or chip (or multiple substrates or chips) (e.g., an array) so that cells may be processed in parallel fashion in a plurality of microchannels. In this regard, large numbers of cells may be processed so that useful quantities of transfected cells may be used for clinical applications. 
     The dimensions of the constrictions may vary but is typically between around 30% to around 90% smaller than the diameter or largest dimension of the cell of interest that is flowed through the microchannel. In one particular embodiment of the invention, the constriction has a width within the range between about 4 μm to 10 μm. In order to prevent fouling and/or clogging of the microchannels at the constriction, the constriction contains a surface with omniphobic, superhydrophilic, superhydrophobic, or anti-fouling characteristics or properties. For example, one particular embodiment may utilize microchannels having slippery liquid-infused porous surfaces (SLIPS). In SLIPS, a porous or textured solid contains an immobilized lubricant film that exhibits omniphobic properties. For example, a porous substrate formed from a polymeric or elastomeric material may be infused or loaded with a chemically-matched fluid such as an oil to create a SLIPS interface. An aqueous-based fluid that contains the cells is then run through the microchannels. The aqueous fluid that carriers the cells may contain the molecules or other cargo that is to be intracellularly delivered to the cells during the transitory state in which the cell membrane becomes permeable. As one particular example, biomolecules or gene-editing cargo materials are delivered into the permeable cells from the surrounding solution, which may be mixed with the cells or delivered separately. 
     In another embodiment, the constriction regions in the microfluidic device may contain on their inner or contact surfaces a plurality of nanofeatures that are sculpted or otherwise formed within the microchannels. The nanofeatures, which in some embodiments, may include sharp nanometer-sized structures, can be used in conjunction with a SLIPS layer to impart better anti-fouling properties. In particular, in some embodiments, the thickness of this lubricant layer can be adjusted to selectively expose or mask entirely the nanofeatures disposed on the surface to alter the surface characteristics of the microchannel. 
     In another embodiment, a microfluidic device for processing cells includes a substrate or chip having a plurality of microchannels disposed therein, the microchannels being fluidically coupled to an inlet configured to receive a solution containing the cells as well as molecules or other cargo to be delivered intracellularly to the cells, each of the plurality of microchannels containing a constriction region therein, wherein the microchannels including the constriction region comprise an omniphobic, superhydrophilic, or superhydrophobic surface. In some embodiments, the omniphobicity, hydrophilicity, or hydrophobicity may be created by a film or layer of lubricant that is disposed on the inner surface of the microchannel forming the constriction region. In some embodiments, the constriction may also include a plurality of nanofeatures that extend or project into the flow path created in the constriction region. The nanofeatures may, in some embodiments, comprise sharpened or pointed tips to aid in permeabilizing the cells. 
     In another embodiment, a method of delivering gene-editing molecules to cells includes flowing a solution containing the cells and the gene-editing molecules or other cargo through a plurality of microchannels formed in a microfluidic device or chip, wherein each of the microchannels comprises one or more constriction regions, wherein the one or more constriction regions comprise a surface rendered superhydrophobic, superhydrophilic, or omniphobic. The surface may be rendered resistant to fouling. This may be accomplished by rendering the surface superhydrophobic, superhydrophilic, or omniphobic using any of the methods or techniques described herein. Further, in some embodiments, cells and gene-editing molecules or other cargo are flowed through a plurality of microfluidic devices or chips to increase the number of cells that can be processed. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  schematically illustrates a microfluidic-based system for the intracellular transport of molecules or other cargo into cells. Also illustrated is a constriction or constriction region that is located in one of the microchannels. 
         FIG. 2  is a side view of one illustrative construction of the microfluidic substrate or chip that is used as part of the microfluidic-based system.  FIG. 2  illustrates a two-layer device although it should be appreciated that additional layers may be employed in other embodiments. 
         FIG. 3  schematically illustrates a microfluidic-based system for the intracellular transport of molecules or other cargo into cells. In this embodiment, separate pumps are used to pump the cells and the molecules or other cargo that is to be transported into the cells. 
         FIG. 4  illustrates one embodiment of a constriction or constriction region located in a microchannel of the microfluidic substrate or chip. In this embodiment, an omniphobic surface is created by the presence of a lubricant on the inner surface of the microchannel. 
         FIG. 5  illustrates another embodiment of a constriction or constriction region located in a microchannel of the microfluidic substrate or chip. In this embodiment, a plurality of nanofeatures are formed on constriction or constriction region of the microchannel. 
         FIG. 6  illustrates another embodiment of a constriction or constriction region located in a microchannel of the microfluidic substrate or chip. In this embodiment, a lubricant is located on the inner surface of the microchannel that also contains a plurality of nanofeatures formed in the constriction or constriction region of the microchannel. 
         FIG. 7  illustrates another embodiment of a constriction or constriction region located in a microchannel of the microfluidic substrate or chip in which the lubricant thickness is adjustable to selectively expose or fully mask the plurality of nanofeatures that are present in the constriction in the microchannel. 
         FIG. 8  schematically illustrates a microfluidic-based system for the intracellular transport of molecules or other cargo into cells that uses a plurality of microfluidic substrates or chips in parallel to process large numbers of cells. 
     
    
    
     DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS 
       FIG. 1  illustrates a microfluidic-based system  10  for the intracellular transport of molecules or other cargo  100  into cells  110 . The system  10  includes a microfluidic substrate or chip  12  that includes therein a plurality of microchannels  14  that contain one or more constrictions  16  or (constriction regions) that are dimensioned to induce a transient increase in the permeability of cells  110  that pass through the constrictions  16 . The microfluidic substrate or chip  12  includes at least one inlet  18  and at least one outlet  20  that are fluidically coupled to a plurality of microchannels  14  that are formed within the microfluidic substrate or chip  12 . Tubing  19  may be connected to the at least one inlet  18  and the at least one outlet  20  as illustrated. The microchannels  14  form a fluidic path through the microfluidic substrate or chip  12 . Generally, the microchannels  14  are rectangular or square in cross-sectional shape and have cross-sectional dimensions that are less than about 1 mm, although it should be understood that other geometric shapes may be used in the microfluidic system  10  described herein. Typically, the cross-sectional dimension of the microchannels  14  at their largest dimension is less than about 250 μm. More typically, the microchannels  14  have a diameter or width that is less than about 50 μm in some embodiments (e.g., around 25 μm×25 μm). The microchannels  14  are dimensioned so as to accommodate the passage of cells  110  contained within a carrying fluid  102 . The cells  110  are typically eukaryotic cells and more specifically eukaryotic cells obtained from a mammal. Cells  110  may have a range of sizes but typically have a diameter or largest dimension within the range of around 5 μm to around 20 μm. The length of the microchannels  14  may also vary. The length of the microchannels  14  may be tens or hundreds of microns in length or up to several or tens of centimeters in length. 
     The microchannels  14  may be linear in shape as illustrated in  FIG. 1  or they have other configurations such as being curved, spiraled, serpentine, or the like. As seen in  FIG. 1 , a plurality of microchannels  14  are provided in a single microfluidic substrate or chip  12  to enable parallel processing of cells  110 . As seen in  FIG. 1 , each microchannel  14  contains one or more constrictions  16  located along a length of the microchannel  14 . The width (W) of the constriction  16  ( FIG. 4 ) is formed so as to subject the cells  110  to a transient compression or stretching of the cell  110  that temporarily increases the permeability of the cellular membrane of the cells  110  such that the cells  110  uptake the extracellular molecules or cargo  100  that are contained in the surrounding fluid  102 . The uptake of the extracellular molecules or cargo  100  is vector-free and is diffusion based. The width (W) of the constriction  16  may vary but is generally less than about 10 μm. For example, the width of the constriction  16  may include 4 μm, 5 μm, 6 μm, 7 μm, or 9 μm. Of course, for larger cells  110 , the width (W) of the constriction  16  may be larger and above 10 μm. The key aspect is that the constriction impart upon the passing cells  110  a rapid and temporary stretching or compression that increases the permeability of the cellular membrane. Typically, the constriction  16  may have a width (W) that is about 30% to about 90% smaller than the diameter of the cell  110  of interest. The length (L) ( FIG. 4 ) of the constriction  16  may vary but is typically within the range of about 10 μm to about 100 μm. 
     Generally, the increased permeability of cellular membrane of the cell  110  lasts hundreds of seconds to several minutes (e.g., about 4-10 minutes is common). As the molecules or other cargo  100  travel with the cells  110  through the microchannels  14 , they are incorporated intracellularly via diffusion across pores formed in the cell membrane established as the cells  110  pass through the constrictions  16 . 
     As seen in  FIG. 1 , the molecules or other cargo  100  are initially present within a carrier fluid  102  and are located outside or extracellular with respect to the cells  110 . The molecules or other cargo  100  may be added to a culture medium or buffer solution that surrounds the cells  110  and this mixture may be delivered via a common inlet  18  such as that illustrated in  FIG. 1 . Alternatively, as seen in the embodiment of  FIG. 3 , the microfluidic substrate or chip  12  may have a first inlet  18   a  that is that is used to deliver cells  110  and a second inlet  18   b  that is used to deliver the molecules or other cargo  100 . As seen in  FIGS. 1 and 3 , the microfluidic substrate or chip  12  is coupled to one or more pumps  30  that are used to pump the cells  110  and the molecules or other cargo  100  through the microchannels  14 . Any number of types of pumps  30  known to those skilled in the art may be used including, for example, syringe pumps, peristaltic pumps, and the like. The pumps  30  may be controlled or adjustable to modify the flow rate of fluid through the microchannels  14 . Generally, the flow rate of fluid  102  through the microchannels  14  is less than 1 mL/minute per microchannel  14 . Higher flow rates will produce higher throughputs through the system  10 . According to one preferred embodiment of the invention, flow rates that achieve cell processing rates between about 50 and about 100,000 cells/sec/microchannel are used. 
     The molecules or other cargo  100  may include any number of biomolecules. These include, by way of example, proteins, enzymes, nucleic acids (e.g., DNA, RNA), plasmids, and viruses. Molecules or other cargo  100  may also include one or more labels or dyes that may be used to target individual cell types or intracellular organelles or cell products. In one particular embodiment, the molecules or other cargo  100  include gene-editing molecules that alter the genetic makeup of the cells  110 . One particular example of gene-editing molecules includes the CRISPR-Cas9 nuclease system that includes single-guide RNA (sgRNA) and the enzyme Cas9. The sgRNA directs the Cas9 nuclease to introduce sequence-specific targeted insertions, deletions, and genetic edits at specific genetic targets. 
       FIG. 2  illustrates the construction of the microfluidic substrate or chip  12  according to one embodiment. In this embodiment, the microfluidic substrate or chip  12  is formed from a laminate structure having multiple layers that adhered or otherwise bonded to one another. As seen in  FIG. 2 , a first layer  32  of the device has the microchannels  14  with constrictions  16  formed therein that is bonded or adhered to a second layer  34  that serves as the bottom (or top) of the device. The at least one inlet  18  and at least one outlet  20  are also formed in the first layer  32 . Tubing  19  may be connected to the inlet  18  and outlet  20  as illustrated. In one embodiment of the microfluidic substrate or chip  12 , both the first layer  32  and the second layer  34  are formed from the same material. In another embodiment, the first layer  32  may be formed from a first material while the second layer  34  is formed from a second, different material. 
     In some embodiments of the microfluidic substrate or chip  12 , one or more the surfaces  22  of the microchannel  14  that are exposed to the fluid  102  environment containing the cells  110  are characterized as superhydrophobic. Superhydrophobic is meant to indicate that the surface has a contact angle with water that is 150° or greater and exhibits low contact angle hysteresis. In some other embodiments, the microfluidic substrate or chip  12  includes one or more surfaces  22  of the microchannel  24  that are characterized as superhydrophilic. Superhydrophilic is meant to indicate that that the surface has a contact angle with water that is equal to about 5-10° or less. Superhydrophilic surfaces may be created by deposition, modification of surface chemistry, surface roughening or the like. 
     In another embodiment of the microfluidic substrate or chip  12 , one or more surfaces  22  of the microchannels  14  that are exposed to the fluid  102  environment containing the cells  110  are rendered omniphobic. Omniphobic refers to a microchannel  14  surface that repels both aqueous and oil-based fluids. For example, an omniphobic surface may display contact angles of 150° and low contact angle hysteresis with both polar and non-polar liquids. In one embodiment, the first layer  32  and/or second layer  34  are formed from a porous or textured polymer or amorphous material that is capable of being infused or loaded with a lubricant  36  (seen in  FIGS. 4, 6, and 7 ). The lubricant  36 , in some embodiments, is immiscible with the carrier fluid  102 . For example, the first layer  32  and/or the second layer  34  may be formed using polytetrafluoroethylene, polydimethylsiloxane (PDMS), copolymers of urea and PDMS (uPDMS), glass, silicon, polyester, carbon, polyethersulfone, polyvinylidenedifluoride (PVDF), aliphatic or semi-aromatic polyamides. In one particular embodiment, the lubricant  36  contained in the first layer  32  and/or the second layer  34  creates an omniphobic film or layer on the surface  22  of the constriction  16  (and also optionally in the microchannel  14 ) that reduces fouling and/or clogging of the constriction  16  and/or the microchannels  14 . The surface  22  is thus rendered omniphobic and may exist on all exposed surfaces of the microchannel  14  and constriction  16  (e.g., all four sides) or one fewer than all sides (e.g., three sides, two sides, or one side). The omniphobic rendered surface  22  may be present only in the region of the constrictions  16  or, alternatively, the omniphobic rendered surface  22  may be located on substantially the entire length of the microchannels  14 . In some embodiments, the porous or textured material used for the microfluidic substrate or chip  102  is functionalized with one or more chemical groups to improve the adherence or affinity to the lubricant  36  and aid in creating a stable, immobilized layer on the surface  22  as described herein. 
     The lubricant  36  that is infused or loaded in the first layer  32  and/or second layer  34  may include, in one embodiment, an oil-based material. These include by way of example, oils such as mineral oils, olive oil, canola oil, coconut oil, corn oil, rice-based oils, cottonseed oil, grape seed oil, hemp oil, mustard oil, palm oil, peanut oil, pumpkin seed oil, safflower oil, sesame oil, soybean oil, sunflower oil, tea seed oil, walnut oil, and mixtures of the same. Perfluorinated fluids may also be used as the lubricant. Examples include, tertiary perfluoroalkylamines such as perfluorotri-n-pentylamine, FC-70 Fluorinert™ by 3M, perfluorotri-n-butylamine FC-40 Fluorinert™, etc.), perfluoroalkylsulfides and perfluoroalkylsulfoxides, perfluoroalkylethers, perfluorocycloethers (like FC-77 Fluorinert™) and perfluoropolyethers (such as KRYTOX™ family of lubricants by DuPont; e.g., KRYTOX™ 103), perfluoroalkylphosphines and perfluoroalkylphosphine oxides as well as their mixtures. The lubricant  36  may also include ionic liquids, hydrocarbons, and silicone oil. 
     The lubricant  36  may be loaded into the first layer  32  and/or the second layer  34  using any number of methods. For example, the first layer  32  and/or the second layer  34  may be soaked or otherwise exposed to the lubricant  36  by submerging the same in a bath of lubricant  36 . This can be done prior to assembly or post-assembly. In addition, the first layer  32  and/or second layer  34  may be infused with the lubricant  36  by flowing the lubricant  36  through the microchannels  14  by pumping or introducing the lubricant  36  through the microfluidic device  12  using the one or more inlets  18 . For example, for a PDMS-based microfluidic device  12 , silicone oil (10 cSt) may be pumped through the microchannel  14  at a flow rate of 0.0001-0.0005 mL/min for about 2-20 hours. 
     The microchannels  14  as well as the constriction  16  may be formed using any number of methods including three-dimensional printing, laser cutting, mechanical cutting, soft lithography, pipette pulling, or thermal molding. In one particular method of making the microchannels  14 , a direct casting method is employed. In the direct casting method of fabrication, a two-part liquid curable solution in a 1:1 vol/vol ratio is mixed and poured over a photolithographically prepared master mold (e.g., silicon mold, glass capillaries) that contains relief structures that that form the microchannels  14  and constriction regions  16  in the first layer  32 . As one illustrative example, the two-part mixture uses mixture SM47i-02 (Parts A and B) available from SLIPS Technologies, Cambridge, Mass. SM47i-02 Part A is a mixture that includes vinyl modified Q silica resin, vinyl terminated polydimethylsiloxane, trifluromethyl C1-4 alkyl dimethicone, platinum 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complexes, monovinyl terminated polydimethylsiloxane, and divinyltetramethyldisiloxane. Part B is a mixture that includes vinyl modified Q silica resin, vinyl terminated polydimethylsiloxane, methylhydrosiloxane dimethylsiloxane copolymer, trimethylsiloxane terminated, trifluromethyl C1-4 alkyl dimethicone. 
     The two-part mixture can be applied by conventional coating techniques such as drop-casting, draw-down, or cured in a mold. The liquid curable solution is cast directly onto the master mold and allowed to cure (e.g., cured for six (6) hours at room temperature or fifteen (15) minutes at 70° C.) to form a solid structure (i.e., first layer  32 ) that is then removed from the mold and then bonded to, adhered to, or encapsulated to a second layer  34  which may be made from the same or different material. For example, the first layer  32  may be made from PDMS while the second layer  34  may be made from glass. The first layer  32  may be secured to the second layer  34  using a clamp or by using one or more fasteners (e.g., bolts and nuts) that secure the two layers  32 ,  34  together. Alternatively, bonding through the use of an adhesive or through the use of other bonding techniques such as oxygen plasma. 
     In some embodiments, a sacrificial layer may be used whereby the two-part liquid curable solution is cast over the sacrificial layer wherein is then removed after curing using different stimuli or a removal agent (e.g., temperature or solvent). In this manner, the sacrificial layer is removed by melting or dissolving of the sacrificial material; leaving the microchannels  14  and constrictions  16 . Glass capillaries may also be used to create the microchannels. In this method, glass capillaries (VitroCom, World Precision Instruments, Inc.) are functionalized with fluorinated silanes (e.g., PFOTS) are used as an alternative strategy to pattern a microchannel  14  with a 5 μm constriction with the two-part liquid curable solution being cast over the capillary. Using a capillary puller (Sutter Instrument Co.), glass capillaries are pulled in such a way as to fabricate a constriction in the middle of the capillary. These glass capillaries are functionalized with fluorinated silanes by vapor disposition at elevated temperatures. After functionalization, the two-part liquid curable mixture was casted around the glass capillaries and molded, as described above. After curing, the glass capillaries can be removed from the mold by pulling both ends apart to retain the constriction  16 . Wires can be used in a similar process. 
     As an alternative to direct casting, the microfluidic substrate or chip  12  may be formed from a plurality of porous sheets or membranes are created and assembled to form the final structure. In this method, polytetrafluoroethylene (PTFE) porous membranes are used with polymethylmethacrylate (PMMA) sheet. The PTFE membranes have pore size &lt;5 μm and thickness &lt;1 mm. The patterns for the microchannel  14 , the inlet  18 , and the outlet  20  are micro-machined onto the PMMA sheet and the PTFE porous membranes via laser cutting. The inlet  18  and outlet  20  are assembled with the microchannel  14  and bound together using epoxy. Fasteners such as hex screws can be used to complete device assembly. Additional details regarding the use of multiple sheets or membranes to form the device may be found in International Patent Publication No. WO 2014/145528, which is incorporated herein by reference. 
     In the embodiments described above, the lubricant  36  forms a film or layer in the microchannels  14  to create the omniphobic film or layer on the surface  22  that acts as an anti-fouling and/or anti-clogging layer and prevents cells  110  from clogging in the constriction regions  16 . The film of lubricant  36  also prevents fouling of the surface through, for example, the adhesion of biological molecules (e.g., proteins) to the inner surface  22  of the microfluidic device  12 .  FIG. 4  illustrates an embodiment, where the omniphobic layer is formed or generated on the exposed inner surfaces  22  of the microchannels  14  and constriction  16 . In one embodiment, the lubricant  36  may completely fill the microchannels  14  (and constriction  16 ) where no or low flows of fluid are present. Upon flow of fluid  102  that contains the cells  110  and the molecules or other cargo  100 , the lubricant  36  is present in a thin film or layer as illustrated to create the omniphobic contact layer on the surface  22  that prevents fouling and/or clogging of the microfluidic device  12 . 
     In one alternative embodiment, as illustrated in  FIG. 5 , the constriction  16  in the microchannel  14  contains a plurality of nanofeatures  120  that extend or otherwise project inwardly from the surface of the microchannel  14 . Nanofeatures  120  are nanometer sized protrusions or protuberances that extend into the flow channel. Nanofeatures may extend into the constriction  16  for a distance of tens or hundreds of nanometers. Nanofeatures  120  may include any number of shapes of protuberances that extend into the flow path created in the constriction  16 . These may include pillars, posts, wires, tubes, cones, pyramids, needles, and the like. The nanofeatures  120  may be formed using lithographic techniques including electron-beam and nanosphere lithography. In nanosphere lithography, periodic arrays of self-assembled close-packed nanospheres are used as masks to pattern underlying substrate materials. Reactive ion etching or the like may also be used to form the nanofeatures  120  with appropriate masking. The nanofeatures  120  may be formed on all exposed surfaces of the microchannel  14  and/or in the constriction region  16 . Alternatively, less than all of the surfaces in the constriction  16  may contain nanofeatures  120 . For example, only a single surface or two of four surfaces may contain nanofeatures  120  (e.g., top and bottom). For example, the nanofeatures  120  may be formed on silicon or glass that are then used to form the top and/or bottom of the microfluidic device  12 . The nanofeatures  120  may be used without a lubricant as illustrated in  FIG. 5  or, alternatively, with a lubricant as illustrated in  FIG. 6 . The nanofeatures  120  may be used to aid in permeabilizing the cells  110  that pass through the constrictions  16 . For example, the tips or ends of the nanofeatures  120  may be sharpened to aid in physically disrupting the cell membranes of the cells  110 . The nanofeatures  120  may also be functionalized to attract or repel cells  110  of certain types. 
       FIG. 6  illustrates an embodiment in which the nanofeatures  120  are present in the constrictions  16  along with the lubricant-formed omniphobic or superhydrophobic surface  22 . In this embodiment, the lubricant on the surface  22  in conjunction with the nanofeatures  120  may impart better anti-fouling properties. Further, in one alternative embodiment, the thickness of the lubricant that is present on the surface  22  in the constriction region  16  may be adjusted to selectively expose or mask entirely the nanofeatures  120  to alter the surface characteristics or performance of the microfluidic substrate or chip  12 .  FIG. 6  illustrates, for example, a constriction region  16  in which the tips or ends of the nanofeatures  120  extend beyond the surface of the lubricant  36  located on the surface  22  of the constriction  16 . 
       FIG. 7  illustrates another embodiment in which the tips or ends of the nanofeatures  120  are fully covered by the lubricant  36  on the surface  22  so that the nanofeatures  120  are fully masked by the lubricant  36 . The control of the thickness of the lubricant  36  may be adjusted in any number of ways including the choice of lubricant  36  and porosity of the material for the microfluidic device  12  as well as the flow rate through the microchannel  14 . Higher flow rates may produce thinner layers of lubricant  36  while slower flow rates may be used to generate thicker layers of lubricant  36 . Other approaches include incorporating a capillary or other fluidic network into the first or second layers  32 ,  34  that is coupled to a reservoir containing the lubricant  36  or other source and controlling the volume or pressure of lubricant  36  that is delivered to the via capillaries or fluidic network. In another approach, the level of lubricant  36  may be controlled by control of the porosity of the porous material making up the first or second layers  32 ,  34 . This porosity may be controlled by the selection of materials used in the microfluidic substrate or chip  12  or by adjusting the effective pore sizes by adjusting the compressive force (e.g., by adjusting fasteners, clamps, external pressure, or the like) that pinch or sandwich the layers  32 ,  34  (e.g., PTFE layer). In yet another alternative for controlling the thickness of the lubricant  36 , a capillary-stabilized liquid may be used as a reversible, reconfigurable gate to modulate the level of lubricant  36  in on the surface  22  of the constriction. Details regarding the liquid-based gating mechanism may be found in Hou et al., Liquid-based gating mechanism with tunable multiphase selectivity and antifouling behavior, Nature, Vol. 159 (March 2015), which is incorporated herein by reference. 
       FIG. 8  illustrates a schematic representation of a microfluidic-based system  10  for the intracellular transport of molecules or other cargo  100  into cells  110 . As seen in  FIG. 8 , the cells  110  and the molecules or other cargo  100  are run through one or more microfluidic substrates or chips  12 . In this particular embodiment, a plurality of microfluidic substrates or chips  12  (N is the total number of microfluidic substrates or chips  12 ) are employed in parallel so that large numbers of cells  110  may be processed. As explained herein, according to one preferred embodiment of the invention, flow rates that achieve processing rates of cells  110  between about 50 and about 100,000 cells/sec/microchannel may be achieved. Preferably, the microchannel  14  and the constriction region  16  remain unclogged after the passage and sustainable processing (i.e., the cells  110  remain live) of 1×10 6  cells, and more preferably more than 1×10 7 , 1×10 8 , and 1×10 9  cells through the microchannel  14 . 
     The cells  110  may be obtained from a mammalian subject, for example, a human. The cells  110  may include, as one example, stem cells or cells with stem like properties that are obtained for example, from the bone marrow of a subject. In one preferred embodiment, the cells  110  are living cells and remain living after intracellular delivery of the molecules or other cargo  100 . The cells  110  may also include immune cells that are obtained from a subject. An example includes T-lymphocytes that are obtained from the subject for adoptive cellular therapies. The invention is not, however, limited to use with stem cells or immune cells. In other embodiments, healthy cells  110  may also be run through the system  10 . As noted herein, the cells  110  are run through the microfluidic substrates or chips  12  along with the molecules or other cargo  100  that are to be intracellularly transported into the cells. 
     The permeablized cells that uptake the molecules or other cargo  100  are then captured or collected after passing through the microfluidic substrates or chips  12 . This is illustrated in operation  140  in  FIG. 8 . For example, the outlets  20  may be coupled to a collection container (not shown) or other receptacle (e.g., bag, vial(s), bottle(s) which may be used to enrich the concentration of collected cells  110  that are processed using the system  10 . In one embodiment, for example, where the molecules or other cargo  100  include gene-modification components, the collected cells  110  that have been modified genetically may then be introduced into a subject as seen in operation  150 . The subject that receives the processed cells  110  may be the same individual that provided the cells  110  that were initially processed with the system  10 . Alternatively, the recipient of the cells  110  may be a different subject from the source of cells  110  that are run through the system  10 . 
     While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited except to the following claims and their equivalents.