Method, system and apparatus for the introduction of materials across lipid membranes using modifications in hydrostatic pressure

Abstract

The present invention is directed to methods, systems and apparatuses for the introduction of materials across lipid membranes, particularly the cellular transfection of nucleic acids and compounds aimed at modifying cell function and the treatment of disease. Pressure-jump-poration (PJP) is a method using rapid depressurization to transfect even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner. Finally, PJP-induced transfection in primary versus transformed cells may reveal a surprising dichotomy between these classes which may provide further insight into the process of cellular transformation.

Description

[0001] METHOD, SYSTEM AND APPARATUS FOR THE INTRODUCTION OF MATERIALS ACROSS LIPID MEMBRANES USING MODIFICATIONS IN HYDROSTATIC PRESSURE

[0002] FIELD OF THE INVENTION

[0003] 0001 The present invention relates to methods, systems, and apparatuses for the introduction of materials across lipid membranes, particularly the cellular transfection of nucleic acids and compounds aimed at modifying cell function or in the treatment of disease.

[0004] BACKGROUND OF THE INVENTION

[0005] 0002 Transient and stable modification of cell signaling may be one of the most fundamental tools of modem molecular biology. It may be used for a variety of purposes including, but not limited to direct modification of cellular DNA, examination and / or modification of cell signaling processes, tracking of endogenous or transplanted cells, modification of cellular differentiation or response to external injury and a host of others.

[0006] 0003 At present there are a number of approaches to the transduction, transfection or introduction of materials across lipid and phospholipid membranes. Essentially, these fall into two basic mechanisms for getting such materials across lipid or phospholipid membranes. The first involves the use viruses (such as but not limited to adeno- associated virus, lentiviruses or retroviruses), while the second involves mechanical / biophysical means including but not limited to: electroporation, laser-poration, microinjection, biolistics, cell squeezing, or the application of lipid or calcium containing nanoparticles

[0007] 0004 Viral approaches (even those using non-integrating AAV vectors) continue to pose significant clinical concern, and the development of such vectors can be both expensive and time consuming; particularly when large numbers of different DNA constructs need to be examined. This need is becoming pressing with current advances to genetic modification (for example through the use of CRISPR and similar nuclease assisted approaches). 0005 Biophysical methods on the other hand are free of many of the concerns plaguing viral approaches and can be performed rapidly in a very cost effective manner with a minimum of specialized equipment and training. Presently there exist several biophysical methods for performing manipulations to introduce exogenous materials. These include electroporation, microinjection, biolistic transduction, laser or sonoportaion, and through direct deformation of the cell. Direct transfection / transduction may also be accomplished by a variety of means known to the art by charge neutralization through the introduction of DNA-divalent cation / DNA- lipid complexes (calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene or polyethyleneimine-mediated transfection, lipofection, liposome vesicle fusion and protoplast fusion represent well-characterized examples of these).

[0008] 0006 Such current physical methods however exhibit a notoriously poor efficiency (e.g. degree of transfection) at modifying a variety of "difficult to transfect" cell populations, such as but not limited to, primary cells, stem cells and cell lines. Notable examples of these include embryonic stem (ES) cells, induced pluripotent stem (IPS) cells, primary cells from a variety of animal tissues, some cancers including cancer stem cells, primary neurons from a variety of species, etc. As a result, there is a significant academic, clinical, and industrial need to identify cheap, safe and effective systems to introduce agents particularly at large scale (large volumes and / or large numbers of cells), and for means to ensure sterility between individual (transfection) applications, using methods and apparatuses to perform such alterations in these populations. An additional limitation to current methods used to transfect ' ' difficult to transfect' ' cell populations may be that they are frequently difficult to scale up to large operational volumes.

[0009] 0007 The delivery of biologically active and / or tracer materials across lipid membranes may provide many diagnostic and therapeutic benefits in the understanding both normal and disease states. Though the initial motivation was often the analysis and treatment of cell signaling disorders and the like, it is becoming increasingly apparent that such gene therapy / modification may also be useful for academic and clinical analysis, diagnosis, and treatment of a broad range of normal and disease states including but not limited to cancer, infectious viral disorders, heart disease, arthritis and acute and chronic neurodegenerative disease (stroke, Parkinson’s, Huntington’s, Alzheimer's). In addition to providing therapeutic benefit, nucleic acid / small molecule / diagnostic tracer delivery may be also beneficial to the study of normal cellular and gene function. Not only can functional genes and modified polynucleotides be delivered to repair / alter specific genetic deficiencies, but such agents can also be delivered to activate, modify, or inhibit gene expression. The new method can thus be used to inhibit gene expression as dictated by the introduction of antisense polynucleotides, small interfering RNAs (siRNA), micro RNAs (miRNA), through introduction of small molecules inhibitors, or via similar such agents.

[0010] 0008 A basic challenge for therapeutic or research purposes however remains in the development of approaches to deliver materials in a manner that is rapid, efficient, specific, and safe.

[0011] SUMMARY OF THE INVENTION

[0012] 0009 An embodiment of the present invention is directed to systems, methods and apparatuses directed to the transient application of high hydrostatic pressure to deliver efficiently and effectively (e.g., transfect) DNA and RNA nucleic acid polymers, compounds aimed at modifying biological signaling, and / or large organic molecule complexes, vehicle tracers or tagging reagents to transfect structures to incorporate the material therein.

[0013] 0010 The present invention is directed to what can be referred to pressure-jump- poration (“PJP”), a process using pressurization and rapid depressurization to transfect cells, including even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers or other compounds in a safe, sterile manner.

[0014] 0011 Embodiments of the present invention are directed to processes for transfecting cells, including primary cells, cell lines, eukaryotic cells and prokaryotic cells, etc., the processes comprising: (a) mixing cells of the desired isotype with a material to be transfected in a solution compatible with cell viability; (b) applying hydrostatic pressure (preferably 60-80 MPa for mammalian cells, but may extend beyond this) to the mixture of step (a) and maintaining the mixture at this hydrostatic pressure for a predetermined period (typically 30 seconds to 10 minutes); (c) within a second predetermined time (typically less than 5 seconds, preferably less than 1 second, and more preferably, as rapidly as possible), rapidly / suddenly reducing the hydrostatic pressure to a lower hydrostatic pressure (in a preferred embodiment by a pressure drop of about 80 MPa, typically achieved by a drop to ambient pressure); and (d) extraction of the transfected cells for subsequent use.

[0015] 0012 In a further preferred embodiment, the predetermined period after step (a) may vary, but may typically include 5 to 30 sec. The longer a cell stays at elevated pressure the greater the chance of pressure-induced death, as cell death is a function of both pressure and time (e.g. cells can remain higher pressure for a short period, or a lower level of elevated pressure for a longer period with similar levels of death). The invention further provides that cells can be maintained at sufficient pressure for sufficient time to allow phase transitions to stabilize (typically gel transitions in the cell membrane), followed by the pressure drop of the present invention.

[0016] 0013 In a preferred embodiment, the pressure drop of the present invention (preferably 40-80 MPa) may be used for cell transfection events unaided by other transfection modifying agents. It will be understood by a person skilled in the relevant art that higher or lower pressures may be used, particularly in combination with pressure treatment in the presence of transfection modifying agents (e.g. pressure used in combination with other ‘transfection agents’ like nanoparticles, lipid agents etc.)

[0017] 0014 A preferred embodiment of the present invention is directed to the process noted above wherein the first hydrostatic pressure is greater than 40 MPa for approximately 30 seconds.

[0018] 0015 Another preferred embodiment of the present invention is directed to the process noted above wherein the first hydrostatic pressure is approximately 60 MPa to approximately 80 MPa.

[0019] 0016 Yet another preferred embodiment of the present invention is directed to the process noted above wherein the first predetermined time is preferably between approximately 10 seconds to approximately 10 mins, more preferably approximately 5 to 30 seconds. 0017 Yet another preferred embodiment of the present invention is directed to the process noted above wherein the second predetermined time was immediate or almost instantaneous so as to cause a rapid drop in pressure, preferably less than 30 seconds, more preferably less than 10 seconds, yet more preferably less than 5 seconds, even more preferably less than 1 sec.

[0020] 0018 Yet another preferred embodiment of the present invention is directed to the process noted above wherein there is a pressure drop (e.g., a differential pressure drop) of at least about 40 MPa, more preferably of about 40 MPa to about 80 MPa, even more preferably about 60 MPa to about 80MPa.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 0019 The foregoing and other objects, features and advantages of the present invention should become apparent from the following description when taken in conjunction with the accompanying drawings (see Figure 1). The patent or patent application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) may be provided by the office upon request and payment of the necessary fee.

[0023] 0020 Figure 1. Hydrostatic pressure application to mammalian cells. (A) High hydrostatic pressure generating device used for experimental pressures of 1 - 200 MPa. Pressure line with valve seat (black arrow) and pressure chamber (red arrow) are indicated. Total chamber capacity 10 mL. (B) Example of cell suspension isolated in borosilicate capillary. (C) Closeup of capillary petrolatum seal. Scale bars in figures B and C equal to 2 mm. (D) Schematic of the Pressure Jump-Poration. Phase 1 : Sample is pressurized from ambient to the desired pressure at a rate of ~65 MPa / min; Phase 2: pressure is maintained at the desired static pressure for the indicated period (0.5 - 10 min); Phase 3: sample pressure is immediately (preferably less than 1 second, more preferably 0.05 seconds, or as quickly as possible) and / or rapidly dropped by at least 40 MPa or more preferably returned to ambient pressure, such as, for example, in which the sample is generally returned to ambient pressure by opening valve. (E) Schematic of pressure-induced transfection of genetic material. (F) Detection of propidium iodine uptake in select cells (borosilicate capillary) immediately following acute depressurization. Scale bar denotes 100 pm. 0021 Figure 2. Efficacy of transfection of ES cells using Pressure-jump-poration.

[0024] (A) Transfection rate for the pressures indicated compared to standard electroporation conditions following transfection. NDN: (-) is the DNA control; DNE: (+) DNA, no electroporation; E.STD: (+) DNA, (+) electroporation; ND80: (-) DNA at pressure (P) = 80 MPa; 0 - 100: (+) DNA, (+) pressure treatment; 70SL: (+) DNA, (+) pressure treatment, but with slow (3 min) pressure release. All other samples were held at indicated pressures for 90 seconds followed by acute depressurization. Samples were performed in triplicate for n = 9 / pressure condition, and n = 5 / electroporation condition.

[0025] (B) ES cells on gelatin at 24 hrs following pressure treatment at 60 MPa (no selection). Scale bar denotes 150 pm. (C) Clonal ES cell colony on fibroblast bed layer following puromycin selection and growth. Scale bar denotes 200 pm. Results shown ±S.D. *- Denotes significant enhancement at < 0.01 over electroporation. Transfection plasmid shown in (B) and (C) expresses dTomato / puromycin.

[0026] 0022 Figure 3. Expression of DNA constructs in mammalian cells. ES cells were transfected with plasmids containing several selection markers to assess expression competency. (A-C) Fluorescence photomicrographs of pressure treated EYFP- expressing ES cells 24 hours following treatment at the pressures indicated in the absence of selection. Relative numbers of transfected, dTomato-expressing cells are indicated (red arrow). Transfected cells exhibit reduced levels of cytoplasmic citrine compared to non-transfected cells (green arrow). Scale bar denotes 100 urn. (D-F) Betagalactosidase expression in R1 transfected ES cells 48 hours following pressure treatment. Scale bar denotes 300 mm. (D) (+) DNA, (-) pressure treatment (1, (E) (-) DNA (+) 80 MPa pressure, (F) (+) DNA (+) pressure treatment. Beta-galactosidase positive cells are indicated (blue arrow), as are examples of dead cells following pressure treatment (box).

[0027] 0023 Figure 4. Characteristics of pressure-induced DNA transduction. (A) Transfection response profiles of six ES cell lines to pressure treatment. *-Denotes significant enhancement at p < 0.01 over values seen at 100 MPa. (B) Initial plating density dependence on numbers of resulting transformants in pressure-treated ES cells. Lines: 1 -R1 , 2-Citrine, 3-Casp3KO, 4-RipKlKO, 5-RIPK3KO, 6-Casp8KO. *-Denotes significant enhancement at p < 0.01 over values seen at 2000 cells / well. (C) Cotransfection incidence in transfected ES cells treated at 70 MPa with different reporter plasmid ratios: 0 ug dTomato:5 pg EGFP; 2.5 pg dTomato:2.5 ug EGFP; 3 .75 pg EGFP: 1.25 pg dTomato; 4 pg EGFP: 1 pg dTomato. For each of the indicated series, samples were performed in triplicate with n = 3 independent experiments / condition. Results shown ±S.D.

[0028] 0024 Figure 5. Properties of pressure-treated cells. (A) Differences in pressure- mediated transduction efficiency in immortalized versus primary cells. Shown are relative transduction efficiencies of primary fibroblasts (blue) vs. mouse L-cells (red) by electroporation and pressure-mediated transduction. Samples were performed in triplicate with n = 3 independent experiments / condition. Results shown ±S.D. (B-H) Cellular features and morphology of pressure-treated R1 ES cells. Citrine-expressing ES cells were treated with 1.5 pM Hoechst 33342, 100 nM TMRM and 0.75 pM propidium iodide prior to pressure treatment. (B) Untreated cells; (C) Typical appearances of ES cells following 1 minute of pressure treatment at 100 MPa. (D) A small subgroup of these cells become PI+ but retain cellular features. (E) A major portion of these PI+ cells go on to exhibit features of cellular destruction in the immediate (30 min) post-treatment period. (F-H) ES cells following 5 minutes at 100 MPa. The majority of these cells demonstrate features shown in F-G. (G) A portion of recovered cells exhibit features of reduced cellular volume. (H) The great majority of cells that become PI+ following treatment at 100 MPa for 5 minutes exhibit features of cellular degeneration. For A-I scale bar indicate in (A) represents 20 pm. (I-K) Electron photomicrographs of ES cells. (I) Following rapid depressurization, a population of ES demonstrated the presence of intracellular voids proximal to the cell membrane (red arrows), frequently associated with protuberances of the cell membrane (blue arrows). ES cell held at ambient pressure is shown for comparison (J). By contrast ES cells subjected to slow pressure release, (K) often demonstrated extensive extrusions (blue arrowheads). For I-K scale bar represents distance of 1 pm.

[0029] 0025 Figure 6. Table 1. Differential survival of primary and transformed cells after pressure treatment. For each of the pressure treatments listed, the average percentages of surviving cells (D = dead) were determined from the initial number plated at 24- and 72-hours post-treatment, 3 wells / condition with n = 3 independent experiments / treatment. Results are indicated < ±3.5%. Determinations were performed in triplicate for 3 independent replicates with general survival characteristics indicated for each of the cell line. Indicated below are brightfield photomicrographs of (A) ES R1 cells 24 hrs post-treatment, (B) HEK293T cells 24 hrs post-treatment, (C) T24 cells 24 hrs post-treatment, and (D) L-cells 72 hrs post-treatment for each of the conditions indicated. For A-D the scale is the same, the scale bar in A represents 100 pm.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] 0026 The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present invention. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the invention.

[0032] 0027 It should also be appreciated that the present invention can be implemented in numerous ways, including as a process, method, an apparatus, a system, a device, or a method. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention. The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present invention. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the invention.

[0033] 0028 In this disclosure, a number of terms and abbreviations are used. The following definitions of such terms and abbreviations are provided. It will be understood by a person skilled in the relevant art that in different geographical regions and jurisdictions these terms and definitions used herein may be given different names but relate to the same respective systems.

[0034] 0029 Preferred embodiments of the present invention can be implemented in numerous configurations depending on implementation choices based upon the principles described herein. Various specific aspects are disclosed, which are illustrative embodiments not to be construed as limiting the scope of the disclosure. Although the present specification describes components and functions implemented in the embodiments with reference to standards and protocols known to a person skilled in the art, the present disclosures as well as the embodiments of the present invention are not limited to any specific standard or protocol.

[0035] 0030 As used herein, a person skilled in the relevant art may generally understand the term “comprising” to generally mean the presence of the stated features, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, steps, components, or groups thereof.

[0036] 0031 As used herein, a person skilled in the relevant art may generally understand the terms “nucleic acid molecule encoding”, “DNA sequence encoding,” “RNA sequence encoding,” “mRNA sequence encoding,” “an oligonucleotide having a nucleotide sequence encoding a gene” “polynucleotide having a nucleotide sequence encoding a gene,” “DNA encoding”, “RNA encoding” and similar terminology to generally refer to the order or sequence of nucleotides along a single or double strand of nucleic acid comprising the coding region of a gene or, in other words, the nucleic acid sequence that encodes a gene product. The order of these nucleotides determines the order of amino acids along the polypeptide chain. The coding region may be present in a cDNA, genomic DNA, or RNA form. The oligonucleotide or polynucleotide may be singlestranded (e.g., the sense strand) or double-stranded (e.g. antisense and sense strands). Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close molecular and / or sequence proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in expression vectors may contain endogenous enhancers / promoters, splice junctions, intervening sequences, poly adenylation signals, etc. or a combination of both endogenous and exogenous control elements.

[0037] 0032 As used herein, the term “vector” is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.

[0038] 0033 The term “expression vector” as used herein refers to a recombinant nucleic acid molecule that contains a desired nucleic acid target sequence and appropriate nucleic acid sequences necessary for the expression of nucleic acid or amino acid sequence in a host. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0039] 0034 As used herein, the term “host cell” refers to any nucleated cell which has no cell wall in the cell's native state, including all vertebrate cells, such as mammalian cells, avian cells, reptilian cells, amphibian cells, and fish cells, cells of multicellular invertebrate animals, such as insect cells, crustacean cells, and mollusk cells, cells of protozoans, etc., and to any nucleated cell which has had its native cell wall removed or is in a natural or artificially induced state wherein no cell wall is present, including all plant cells that are capable of forming protoplasts or are capable of being treated to form protoplasts, whether located in vitro or in vivo. For example, host cells may be located in a transgenic animal.

[0040] 0035 The terms “transfection” , “introduction” or “transduction” as used herein refers to the introduction of foreign material, including an extracellular biologically active component such as, but not limited to, nucleic acid polymers (natural and man-made RNA, DNA, hybrids thereof, etc., other macromolecules (e.g. proteins and peptides), drugs / small molecules, lipids, liposomes, cellular constituents of eukaryotic or prokaryotic cells and / or other entities employed for purposes of modifying cellular response, and agents utilized for purposes of cellular tagging / tracing, into a membranous structure having lipid / phospholipid membrane constituents or components. Currently known methods include, but are not limited to, calcium phosphate co-precipitation, viral transduction, liposome and modified lipid fusion, microinjection, microparticle bombardment, and electroporation. The terms "uptake of extracellular components by a host cell", "taking up of extracellular components by a host cell", "uptake of particles comprising extracellular components by a host cell", and "taking up of particles comprising extracellular components by a host cell" denote processes, including embodiments of the present invention, wherein an extracellular component, with or without accompanying material, enters a host cell. Such foreign material may consist of the variety of compounds noted above, preferably having a size of preferably less < ~2 micrometres, more preferably < 100 nm.

[0041] 0036 As used herein, the term “transfection” or derivatives thereof shall be understood to refer to means of contacting a cell or lipid membrane containing structure with a molecule, wherein the molecule is internalized by the cell or passed across the lipid membrane. As used herein, “transformation” or derivatives thereof shall be understood to refer to the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings across or through the cell membrane, typically through viral means. As noted above, the terms "transformation" and “transfection” both denote introducing extracellular components into a host cell so that the extracellular component is replicable (such as, for example, in the case of nucleic acids, either as a chromosomal integrant or as an extrachromosomal element), showing reproducible modification of a given cellular response (e.g. modification of cell signaling in the case of entities such as drugs), observable modification of the cell’s appearance (as observed by tight, fluorescence or electron microscopy, or other method), cell tracing / tagging, or the introduction of macromolecular cellular constituents. A person skilled in the relevant art will understand that the process of the present invention typically can be referred to as transfection.

[0042] 0037 The term “sample” as used herein is used in its broadest sense to denote components of the methods disclosed herein that are subjected to the process of the present invention.

[0043] 0038 As used herein, the term "nucleic acid" refers to any DNA, RNA or DNA / RNA hybrid, either naturally occurring or synthetic nucleotide variants.

[0044] 0039 As used herein, the term "DNA" is defined as any polydeoxyribonucleotide, including, e.g., double stranded DNA, single stranded DNA, double stranded DNA wherein one or both strands is (are) composed of two or more fragments, double stranded DNA wherein one or both strands has (have) an uninterrupted phosphodiester backbone, DNA containing one or more single stranded portion(s) and one or more double stranded portion(s), double stranded DNA wherein the DNA strands are fully complementary, double stranded DNA wherein the DNA strands are only partially complementary, circular, covalently closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically synthesized DNA, semisynthetic DNA, biosynthetic DNA, naturally isolated DNA, enzyme digested DNA, sheared DNA, plasmid DNA, chromosomal DNA, labelled DNA, such as radiolabelled DNA and fluorochrome- labelled DNA, DNA containing one or more nonnaturally occurring species of nucleic acid, etc., that is selected for transfecting a host cell. 0040 As used herein, the term "RNA" is defined as any polyribonucleotide, including, e.g., single stranded RNA, double stranded RNA, double stranded RNA wherein one or both strands is (are) composed of two or more fragments, double stranded RNA wherein one or both strands has (have) an uninterrupted phosphodiester backbone, RNA containing one or more single stranded portion(s) and one or more double stranded portion(s), double stranded RNA wherein the RNA strands are fully complementary, double stranded RNA wherein the RNA strands are only partially complementary, covalently cross-linked RNA, enzyme digested RNA, sheared RNA, mRNA, hnRNA, tRNA, including both charged and uncharged tRNA, rRNA, all forms of viral genomic RNA, chemically synthesized RNA, semisynthetic RNA, biosynthetic RNA, naturally isolated RNA, labelled RNA, such as radiolabelled RNA and fluorochrome-labelled RNA, RNA containing one or more nonnaturally occurring species of nucleic acid, etc., that is selected for transfecting a host cell.

[0045] 0041 Any desired nucleic acid for use in the methods of the invention can be prepared by a variety of methods known in the art. Suitable quantities of DNA template for use herein can be produced by amplifying the DNA in manners and means well known in the art, including, for example, amplified by polymerase chain reaction (PCR) as described by Saiki et al., Science, 230:1350 (1985), Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263 (1986), Mullis and Faloona, Methods Enzymol., 155:335 (1987), and Saiki et al., Science, 239:487 (1988). Any desired RNA for use in the methods of the invention can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis of RNA, and in vitro translation of a DNA template as described generally in Current Protocols in Molecular Biology, Wiley Interscience, New York (1990). Alternatively, the desired RNA can be isolated from total cellular RNA extracted from a host cell culture. Total cellular RNA can be isolated from the host cell culture by any method known in the art such as, in the case of RNA produced in mammalian host cells, the methods described by Favaloro et al., Methods Enzymol., 65:718 (1980), Stallcup and Washington, J. Biol. Chem, 258:2802 (1983), Bimboim, Nucleic Acids Res., 16:1487 (1988), Gilsin et al., Biochemistry, 13:2633 (1974), Ullrich et al., Science, 196:1313 (1977), Strohman et al., Cell, 10:265 (1977), and MacDonald et al., Methods Enzymol., 152:219 (1987). 0042 As used herein, the term “genetic modifier” can be used to refer to any nucleic acid sequence that can alter the function or appearance of a cell into which such genetic modifier has been transfected.

[0046] 0043 As used herein, the term “cell” will be understood to encompass any cell. The term “primary cells” typically refers to cells directly isolated or otherwise extracted from tissues / organs of interests by physical, mechanical, or enzymatic dissociation. Primary cell models are well established, relatively easy, and inexpensive to operate and offer several key advantages over the use of natural tissue slice models. The main advantage of primary cells is that primary cells retain functions and phenotypes close to those of in vivo tissues / organs, and the effect of drugs / toxins can be investigated on isolated primary cells at the cellular level. Examples of primary cells include, but are not limited to, epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic and stem cells.

[0047] 0044 Existing prior art methods for transfecting cells have a number of limitations. Electroporation, for example, has experienced problems with respect to scale-up, as well as transfection techniques such as biolistics, laser poration, microinjection, etc. Because of the nature of the transfection method, many of the potential target cells are currently destroyed in this process, a problem which becomes greater for many current transfection methods at scale. Additionally, a number of current biophysical transfection methods induce breakage in genomic DNA, an effect known to have significant consequences including potential mutagenesis; issues of particular concern in many clinical and research settings. By contrast genomic DNA as well as all other natural and synthetic nucleic acids are insensitive to pressure effects within the described range, thus based on existing literature there is strong reasons believe that the method described herein will be significantly less prone to such DNA mutagenic effects.

[0048] 0045 Without being bound by any theory of operation or mechanism of action, the embodiments disclosed herein are based in part, on an unpredicted / unexpected discovery that using alterations in pressure to transfect cells, even difficult to modify cells, such as primary cell types (e.g., embryonic stem cells and others) demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner to effect transformation. 0046 Subsequent to Hite's description in 1899 of the pressure induced inactivation of microorganisms in milk1, the effects of hydrostatic pressure have been extensively studied in both normal and pathologic cells. Industrially, high hydrostatic pressure (HHP) up to 900 MPa (0.1 MPa = 1 bar = 0.987 atm) has been used to sterilize and preserve thermally sensitive foods without loss of nutritional content or organoleptic properties; a process begun commercially in the 1990s in Japan2,3. Clinically, high hydrostatic pressure has also been used to sterilize bone transplants and generate antitumor peptides4 6. Generally, this process involves subjecting samples to a defined very high hydrostatic pressure (higher than that for the method described) for 10 - 30 minutes followed by a return to atmospheric or ambient pressure.

[0049] 0047 Without being bound by any theory of operation or mechanism of action, the effect of hydrostatic pressure on biological processes may arise from changes induced in the conformational states of proteins, nucleic acids, lipid membranes and their interactions. At pressures below 100 MPa, the conformational structure of many proteins may be stabilized. The pressure ranges of the present invention provide that, despite brief pressure-induced interruptions in cellular function as a result of applied pressure, the likelihood of permanent injury to the cell may be reduced or may not be observed. Above -200 MPa tertiary and quaternary structures may be frequently altered, and above -400 MPa protein denaturation can occur7 l0. The conformational changes induced by hydrostatic pressure may arise from, among others, changes in the state of ionization of charged amino acids, changes in hydrophobic interactions, changes in hydration of the component amino acids and a loss of the void volumes present in the protein structure due to the imperfect packing of polypeptide chains within the protein interior. Secondary protein structures such as a-helices and -pleated sheets may be quite resistant to pressure, remaining stable at pressures up to 1000 MPa11. For proteins possessing quaternary structure, subunit interactions may be altered at significantly lower pressures. For example, pressures of 50 to 300 MPa have been shown to alter the equilibrium of microtubules toward their constituent subunits12 l4; an effect which is rapidly reversible upon return to atmospheric pressure9,15. Similarly while the conformational stability of some non-canonical nucleic acid structures such as the G- quadruplex can be destabilized by pressure, double helical DNA may be resistant to pressure-induced conformational changes up to several hundred MPa16. 0048 Of the cellular constituents, membranes may the most labile to pressure-induced changes11. Within the range of 0.1 - 100 MPa9,17, phospholipid bilayers may undergo a phase transition from a fluid-crystalline state to a more ordered gel-like conformation, with the lipid headgroups being relatively incompressible compared to the bilayer interior17,18. Elevated pressure increases the packing density of lipid chains, resulting in an increase in membrane thickness and a concomitant decrease in the cross-sectional area of the lipid chain.19’20. Thus, with increasing pressure phospholipid acyl chains may become more ordered, diminishing void volumes between lipids, giving rise to the negative volume changes observed for these transitions.21Such pressure-induced changes could affect cellular functions related to membrane fluidity, such as membrane permeability, ion transport, and signal transduction. An example of this are NMR studies demonstrating that membrane-associated Ras undergoes significant adaptations between 60 - 100 MPa with respect to membrane mobility and partitioning22. Indeed, the high relative barosensitivity of lipid membranes may be thought to be a prime factor in determining tolerable pressure limits for specific cell types, with lethality for eukaryotic cells typically observed at pressures between 100 - 200 MPa as a function of exposure time18. Interestingly cancer cells are typically capable of surviving exposure to higher hydrostatic pressures than those seen in primary cells17

[0050] 0049 Lower hydrostatic pressures in the range of 30 - 100 MPa have also been shown to induce alterations in morphology, function and even viability in some cell types1315. Significantly, changes induced in this pressure range appear reversible upon return to atmospheric pressure. For example upon return to ambient pressure following exposure to < 90 MPa for 10 - 30 minutes, pressure-induced disruptions of mitosis, protein synthesis and the actin cytoskeleton all demonstrate reversibilityl3-18-23-24. Thus exposure to pressures of this magnitude and time period do not typically affect cell viability25, but can induce reversible stress responses26. Consistent with this, examination of a broad number of eukaryotic cell types including embryonic stem cells demonstrate that exposure to pressures of < 100 MPa for periods of 10 minutes or less does not significantly alter cell viability9’27. Melanocytes, adipose stem cells, dermal fibroblasts, keratinocytes, and malignant melanoma cells similarly demonstrate no significant growth alterations upon return to atmospheric pressure after 2 mins at 150 MPa28. 0050 Even among sensitive populations such as mammalian gametes and stem cells, exposure to 20 - 80 MPa for 30 - 120 minutes results in no significant loss of cell viability17,29. Indeed, exposure of spermatozoa, oocytes, murine embryos, and embryonic stem cells to 60 MPa for 30 min resulted in enhanced viability with respect to subsequent cryopreservation, fertilizing ability, developmental competence, and differentiation29. In the case of mouse embryos and blastocysts, there is evidence this pressure-induced cryopreservation protection may in part be due to the induction of stress response proteins30. Similarly re-expansion rates following vitrification of bovine blastocysts and porcine mesenchymal stem cells have also been shown to be significantly higher following 1 h pressure treatment in the range of 40 - 60 MPa compared to controls31,32Such results demonstrate that even very sensitive eukaryotic cells (mammalian embryos and blastocysts, mesenchymal and embryonic stem cells) can withstand (and thrive at) pressures of 60 - 80 MPa for periods of 10 minutes or more without irreversible inhibition of cell function.

[0051] 0051 In a preferred embodiment of the present invention, the transient modification, altercation and / or application of hydrostatic pressure can be used to achieve the introduction (e.g., transfection) of the specific genetic modifiers, molecular isoforms, etc. (DNA, RNA, chemical entity, etc.) into lipid vesicles or living cells (e.g., such as, for example, embryonic stem cells), at efficiencies which rival the best current biophysical methods without many of the noted drawbacks.

[0052] 0052 In a further preferred embodiment, it was found that this rapid (< 3 minute) method of cell transfection could be achieved via a sudden decrease in hydrostatic pressure, with preferable pressure drops of at least 40 MPa, more preferably between about 40 to about 80 MPa, even more preferably about 60 to 80 MPa. The more preferrable pressure drops provide good levels of transfection efficiency for mammalian cell types such as embryonic stem cells. This effect has broadly been termed pressure-jump-poration (“PJP”). Sudden pressure decreases as set out herein were found to induce reproducible and reversible interruptions of cell membranes capable of ectopically modifying cellular properties in both primary and transformed cells. The use of such hydrostatic pressure alterations may provide many ideal features for such cell modifications including: low cost per unit, high levels of biological safety (due to surety of sample-to-sample cell sterilization via high pressure cycling), efficiency relative to existing methods, lack of required additional consumables, scalability to any cell number or volume even for difficult to transfect cell populations, etc. This approach may also allow for direct utilization of the widest possible array of existing genetic resources without need for additional modification.

[0053] 0053 In another embodiment, the method of the present invention is directed to the transient application of high (preferably in excess of 40 MPa or 395 atmospheres or 5800 pounds per square inch (PSI); more preferably 60 - 80 MPa equivalent to 592 - 987 atmospheres or 8,700 - 14,500 PSI) hydrostatic pressure in order to introduce / transfect the above materials into eukaryotic cells or lipid vesicles, followed by rapid decreases of pressure to ambient levels. While the method described can be performed at any scale (nanoliters to hundreds of liters) it may be particularly noteworthy compared to existing methods in that its efficiency may be in no way reduced with scale and in the case of living cells can be applied to cells in both free suspension and adherent cell cultures states.

[0054] 0054 In a preferred embodiment of the present invention, analysis of cells transfected with materials such as nucleic acids using the embodiment of the present invention demonstrates that this process does not introduce undesirable or unexpected functional abnormalities as a result thereof. Analysis for common treatment-induced genomic rearrangements or genetic recombination further demonstrates the presence of no cellular abnormities as a function of brief hydrostatic treatment.

[0055] 0055 One advantage of the embodiments of the present invention is that such embodiments are insensitive to operational scale or geometry. Embodiments of the present invention can work with plated cells and cells in suspension. This makes it of substantial interest to large clinical / industrial operations.

[0056] 0056 In addition, such operations (for example cellular modification of human cells for transplantation etc.) often possess a high requirement for field sterility. In the current embodiment, this may be easily achieved as an inherent part of the same overall process (fluctuation in high hydrostatic pressure) without the use of additional / outside agents which might potentially compromise field sterility or cause other delirious effects. Thus, it may be anticipated that manifestations of this method may be of significant clinical and industrial interest with respect to progressing developments in gene modification and gene therapy.

[0057] 0057 The embodiments of the present invention exploit the heretofore unidentified and / or unknown properties of hydrostatic pressure to facilitate the introduction of exogenous materials into cells and lipid vesicles. By its nature with the appropriate gene sequences this may allow for the integration of genetic sequences into host cells. As a result of the method’s ability to achieve complete non-thermal sterilization / destruction of living cells within the primary compartment, multiple cell types and transfection modes can be employed utilizing a single pressure transduction unit, with efficient transfection / introduction events achieved at microliter to multi-liter scale as a one-pass single source-sealed operation. In one embodiment of the present invention, hydrostatic pressure mediated transfection or PJP proceeds as follows: Cells of the desired isotype are mixed with material to be transfected (e.g., prepared nucleic acids sequences or other genetic modifiers) of sufficient purity to perform common cell transfection experiments, in a buffered salt solution (frequently, but not exclusively, containing calcium ions or equivalent to aid in phosphodiester - phospholipid charge neutralization) which may be sufficient to support cell viability during the time of the pressure transfection process (typically 2-10 minutes). Following introduction of the desired agent to be introduced into cells, the cell / agent mixture may be sealed in a pressure transducing container. This container may be then subjected to hydrostatic pressures specific to the pressure optimum identified for a given cell type and experimental procedure (for example, DNA / ES cell optimums typically fall in the range of about 60 - 80 MPa). Once the desired pressure is attained the pressure may be rapidly reduced or, in one embodiment, returned to normal atmospheric pressure (e.g., pressures typically returned to that found at sea level, also known as ambient pressure) in order to create a transient disturbance in the lipid / cell vesicle (cell vesicles may include, but are not limited to, exosomes, micro vesicles, etc.) or cell membrane. This process (rapid pressure drop) may be repeated more than once and may not be identical in each instance. In another embodiment, the material to be transfected may be used alone or could be mixed with other agents such as lipids to further enhance its efficacy.

[0058] 0058 Although the invention is not limited to any particular mechanism of action, it has been previously shown (e.g. see listed references) that mammalian cells can adapt to short periods (<10-30 minutes) of modified (increased and / or decreased) local hydrostatic pressure within certain limits (e.g. elevated pressure <100 MPa). However, a precipitous drop in elevated pressure over a short time period (1 second or less) may result in a non-uniform volume expansion, where different components of the cell are unequal in their response thereto. Among these responses may be a transient disruption of some lipid membranes, the degree to which differs as a function of the volume they contain. Thus the (outer) cell membrane of larger cells may experience greater relative pressure affect than smaller nuclear or mitochondrial membranes within that cell. Similarly larger cells may manifest different pressure optima from that for smaller cells assuming similar total lipid compositions and distribution between the two. Due to the nature of this effect, experimentally induced or natural differences in cell membrane factors including lipid composition, protein composition, or pretreatment with chemical or biological agents designed to alter intrinsic membrane stability may be expected to alter the desired conditions for a given procedure. Such differences in membrane lipid and protein composition and differences in intrinsic cellular volume are thought to underlie defenses in pressure optima for different cell types and growth conditions. That faults induced in membrane continuity as a result of changes in local pressure are transient may be evidenced by the continued viability of the majority (but not all) cells to optimal pressure jump conditions as determined by transfection efficiency.

[0059] 0059 The embodiments of the present invention provide similar or superior rates of transfection and transformation efficiency to other established biophysical methods such as, for example, electroporation. However, the preferred embodiment of the present invention may provide insensitivity to operational scale, unlike previous physical approaches, which may be of interest to large clinical / industrial operations. In addition, prior art operations (including, but not limited to modification of human cells for experimental and clinical purposes etc.) often possess a high requirement for field sterility. In a preferred embodiment of the present invention, this may be achieved without the use of additional / outside agents. Thus, it may be anticipated that manifestations of this method may be of significant clinical and industrial interest with respect to progressing developments in gene therapy and other applications, of which a person skilled in the art would appreciate. The embodiments set out herein, by their nature, may allow the integration of sequences of complete non-thermal sterilization / cell destruction within the cell-containing compartment, together with efficient cell transfection events at microliter to multi-liter scale as a single one-pass operation. As such multiple cell types and transfection modes can be employed utilizing a single pressure transduction unit.

[0060] 0060 Embodiments of the present invention are directed to the use of changes to hydrostatic pressure, including, but not limited to, sudden depressurization from pressures in the range of approximately 60 to 100 MPa to induce transfection in difficult to transfect cells (e.g., Pressure-jump-poration (PJP)). This method may provide a quick, economical means of producing a wide range of cell transfections at any scale using DNA-based plasmids, pharmaceuticals, or other transfection agents without need for further modification. In addition, the transfection takes place in an environment which ensures sample-to-sample sterility via the insertion of high pressure (sterilizing) interphases. Transfection of large plasmids is possible using the embodiments of the present invention in sensitive cell types such as embryonic stem cells. Similar to other biophysical methods such as electroporation, this method does not irreversibly alter cell function of successfully transfected cells as shown by their continued growth and function43 45. Indeed, prior work within this pressure range exposing cells, blastocytes and gametes for longer (> 5 min) periods with successful freeze / thaw recovery, growth, development and metabolic function suggests such treatments are well tolerated even in sensitive cell types223031-46-47|nfact. treatment of mouse blastocysts at 60 MPa for 30 min significantly improved their survival rates following traditional freezing and / or suboptimal culture conditions30,31. Thus, for the current study, an important factor in regulating successful transfection likely lies in the fraction of cells capable of recovering from the acute injury induced by a sudden drop in pressure. In this way, the embodiment of the present invention as outlined herein shares properties with other biophysical transduction approaches such as electroporation41.

[0061] 0061 In accordance with Le Chatelier’s principle, increasing pressure appears to induce cells to adapt to a smaller hydrodynamic volume11>48. With respect to the cellular transfection observed, such pressure-induced effects may act through the alteration of existing structural voids, particularly at sites where different biomolecular isotypes interdigitate; such as the integral membrane proteins and membrane phospholipids20,49Indeed the plasma membrane appears to be a key target of pressure-induced modification over the range of pressures examined, with increasing hydrostatic pressure promoting reduction in membrane fluidity via liquid crystal to gel transition of the lipid membrane10,20’21’49.

[0062] 0062 One advantage of PJP compared to other biophysical transfection methods may be the differential transfection potential seen between primary versus immortalized cells. In most such methods, transfection of primary cells is significantly more difficult compared to immortalized cell tines such as Hela and L-cells50’51. However, using PJP transfection efficiency was significantly enhanced in primary cells such as ES cells compared to other methods such as electroporation, yet was not observed in the immortalized cell lines examined. These results stand in striking contrast to prior observations obtained using a variety of biophysical transduction methods, suggesting differences in aspects of their underlying mechanism. Given that the differential transfection effects observed in primary versus immortalized cell lines examined may be related to their differential survival properties, the response of these cell lines to applied pressure was examined. In most cases, only modestly enhanced pressure resistance for immortalized versus primary cells was observed. These effects may reflect differences between primary and immortalized cells in the production or composition of membrane phospholipids, and / or differential configuration of their microtubules cytoskeleton or other factors. Interestingly, cytoskeletal microtubules have been shown to be affected at the pressures examined and may play a role in disrupting the cell membrane12-15,24’25’53.

[0063] 0063 As with other biophysical methods of cell transfection, a delicate balance exists between disruptive forces promoting viable transfection and irreversible damage leading to cell death. While both electroporation and PJP are based upon permeabilizing the plasma membrane only the latter is readily scalable. This is particularly true in the case of sensitive cell types given that applied voltage increases with increasing cathodeanode gap distances. Additionally, given its known response to applied hydrostatic pressure, the risk of pressure-induced modification of genomic DNA is significantly reduced compared to potential (polyanion) damage during with electroporation. While other methods such as microinjection and laser-poration may also avoid such damage, these methods cannot compare in terms of speed, cost, scalability, and simplicity. Similarly, although transformation methods that use calcium phosphate, polyethyleneimine and lipid-DNA complexes have proven effective transfection strategies for a variety of cell lines (particularly transformed ones), these methods are far less effective in their application toward primary cells. In addition, these methods require the ongoing use of consumables, several of which (e.g., modified lipids, nucleofection) impose significant costs particularly at scale. By contrast, transformation achieved by PJP typically requires no consumables. As practical note, although a stepper motor-controlled pressure generator was used in the current study, the pressures employed could readily be obtained manual devices that are commercially available.

[0064] EXAMPLES

[0065] Methods

[0066] 0064 Reagents and cell assays. Reagents utilized include: Silicone oil (Fisher S159- 500), Borosilicate glass (Harvard Apparatus, type GC150T-10, 30-0062), Petrolatum (Sigma-Aldrich, 16415), Puromycin (BioShop, PUR333.100), Geneticin (Gibco, #11811023). Plasmids used for transfections: px459V2 (Addgene # 62988), pLVX- dTomato-Cl (Takara Bio), pEGFP-puro (Addgene #45561), or pBS / KSTau / LacZ (courtesy of Dr. Friedhelm Bladt, SLRI, Mt. Sinai Hospital, Toronto), modified from TauLacZ LTNL (Dr. Peter Mombaerts, Max Planck Institute, Frankfurt). Plasmids were prepared from bacterial overnight stock using EndoFree Plasmid Maxi Kit (Qiagen, #12362), with plasmid integrity verified using gel electrophoresis and DNA transfection quality independently determined using electroporation and polyethylenimine (Sigma Aldrich, 25 kDa MW, # 408727) mediated transduction of HEK 293T cells (ATCC CRL-3216) according to standard methods together with culture lines and cell media constituents as indicated below. Analyses for betagalactosidase activity in transfected cells was performed as previously described33. Cell viability was assessed using standard trypan blue exclusion assay (Gibco 15250061)34, propidium iodide (Sigma P4170)35or calcein AM / ethidium homodimer live / dead assay36(Molecular Probes L-3224) methods described previously.

[0067] 0065 Cell culture. Primary murine embryonic stem (ES) cell lines were maintained at 37 °C and 6% CO2 in growth media containing high glucose DMEM (Invitrogen 11960-044), 2 mM L-glutamine (Invitrogen 25030), 2 mM GlutaMAX (Invitrogen / #35050) with addition of 0.1 mM 2-mercaptoethanol (Sigma M7522), 0.1 mM MEM non-essential amino acid (Invitrogen 11140), 1 mM sodium pyruvate (Invitrogen 11360), 50 U / mL penicillin / streptomycin (Invitrogen 15140), 1000 U / mL LIF (Chemicon ESG1107) and 15% ES cell qualified fetal bovine serum (HyClone SH3007E03E). ES cells or sublines were maintained on mitomycin C inhibited multidrug resistant mouse embryonic fibroblasts (DR4 MEFs, ATCC SCRC-1045), or on 0.1% Gelatin (Millipore ES-006-B). Additional cell lines examined in this study include: T24 (human bladder epithelial carcinoma, ATCC HTB-4) cultured in McCoy’s 5 A and Lovo cells (human colorectal, ATCC CCL-229) cultured in F12 media (Gibco 11765054). HEK 293T (Human embryonic kidney, ATCC CRL-3216), Mouse L-cells (fibroblastic, ATCC CRL-2648), and primary mouse fibroblasts were all cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma, D5796). Each of the above media were supplemented with 10% fetal bovine serum (Invitrogen, 12483020), 2 mM glutamine (Invitrogen 25030081) and 50 U / mL penicillin / streptomycin (Invitrogen 15140). R1 ES cells (129Sl / SVImJ) were generated locally as previously described37, EYFP (citrine) expressing ES cells (129 / SVEvTac) were a kind gift from the laboratory of Dr. M. A. Magnuson, Vanderbilt University38.

[0068] 0066 Cell preparation, Pressure-jump-poration, and plating. Cultured cells for hydrostatic pressure experiments were first detached from their support matrices with 0.25% Trypsin-EDTA under sterile conditions in a biosafety cabinet, centrifuged at 300 xg for 4 minutes and gently re-suspend in Embry oMax electroporation buffer (Millipore ES-003-D, 4.5 g / L glucose, 2.3 g / L bicarbonate, pH 7.3, 327 mOsm) or Opti-MEM (ThermoFisher 31985062, 2.5 g / L glucose, 2.4 g / L bicarbonate, pH 7.4, 285 mOsm) as indicated, either with or without DNA to a final concentration of 20 mg / mL at a cell concentration of 4 x 106cells / mL.

[0069] 0067 Fifty -millimeter borosilicate capillary segments were pre-prepared following scoring with a diamond scribe and sterilized. Fifty microliters of cell resuspensions were then introduced to one end of a borosilicate capillary, drawing it to the far end of the tube where it was the sealed using sterilized petrolatum via syringe. Lateral displacement of cell media to the end of the tube was then performed via continued application of petrolatum, followed by petrolatum sealing on the opposing end to ensure total sample isolation from air (see Figure 1). Enclosed capillaries were then placed into a 2-mL high-pressure microreactor (Model MS-1, HiP, Inc., Erie, PA, USA) filled with silicone oil. After closing the microreactor, the process of PJP consists of three phases: 1. pressurization, 2. maintaining the sample at a static high pressure for a variable amount of time, and 3. sudden depressurization. In the pressurization phase, the pressure was increased to the desired level at a rate of ~65 MPa / min via a pressure generator driven by a stepping motor (Nova Swiss, Effretikon, Switzerland). Cells were held at the desired static pressure for periods of between 30 s to 10 mins, followed by sudden return to atmospheric (ambient) pressure. The sudden return to ambient pressure may be achieved by simply opening one of the valves on the apparatus to ambient pressure. Immediately upon reaching atmospheric pressure the pressure apparatus was disassembled, and sealed capillaries opened with a diamond scribe at which point cells were removed, counted, and plated. For the samples held for 30 seconds at static pressure, we estimate that the total time of the experiments was approximately 5 minutes. That is, after sealing the microreactor, the total time for completion of the three phases of the process and removal of the cells from borosilicate capillaries was typically 5 minutes.

[0070] 0068 Cell pressurization experiments were also performed using sterilized heat- sealed polyethylene tubing (McMaster-Carr #5233K111). However, borosilicate capillaries were preferred in order to eliminate the potential contribution of localized heat-sealing effects in altering cell membrane or solution properties. Pressure-treated cells were plated at a concentration of 2 - 10,000 cells / well in standard 24-well tissue culture plates (Sarstedt 83.3922), or 10 - 20,000 cells / well in 6-well tissue culture plates (Fisher CS003516). For numeric analyses / clonogenic assays of transfected cells, treated cells were allowed to recover for one day, followed by antibiotic selection for 3 - 5 days at concentrations predetermined to be lethal for each of the respective wildtype lines. Clonogenic assays were performed 7 days following removal of selecting agents.

[0071] 0069 Live cell imaging. Where indicated, pressure-treated cells were incubated with the following reagents after pressure treatment in order to monitor cellular compartments in real time: Hoechst-33342 (Life Technologies Hl 399, final concentration 1 microgram / mL), Tetramethyl rhodamine methyl ester (Setareh biotech. 6275, final cone. 5 nanograms / mL, excitation wavelength (ex.) 553 nm, emission wavelength (em.) 577 nm), Propidium iodide (Sigma P4170, final cone. 1 microgram / mL, ex. 535 nm, em. 615 nm), Calcein AM (Invitrogen C3100MP, final cone. 5 micromolar, ex. 494 nm, em. 516 nm). Citrin (ex. 513 nm, em. 528 nm) was imaged on the EGFP channel.

[0072] 0070 Microscopy. Cellular imaging was performed on a Zeiss Axio Imager.Ml equipped with standard DAPI, EGFP, FITC, Rhodamine, Texas Red and Cy5 excitation / emission filters using a Marzhauser Wetzlar motorized XY controller on 40x, 63x and lOOx (infinity / 0.17) Plan-Apochromat and 40x Neofluar Zeiss objectives. Images were captured prior to or ~30 mins following pressure treatments on a Hamamatsu ORCA-Flash 4.0 4MP sCMOS camera using ZEN blue 3.0 software. Images were acquired from at least five independent fields within n > 4 separate experiments. Relative fluorescence intensity was calculated by comparing mean fluorescence intensity of cells to the background fluorescence in each field. Significance was determined by ANOVA. Images were processed using Zen software and Adobe Photoshop CS.

[0073] 0071 Electron Microscopy. Cells prior to or following pressure treatment were infused with 100 mM cacodylate buffer at pH 7.4 containing 4% para-formaldehyde and 0.1% glutaraldehyde overnight at 4 °C. Samples were then washed in cacodylate buffer, treated with 1% osmium tetroxide and embedded in Spurr resin as previously described39. Blocks were sectioned at 70 nm onto Formvar-coated 200 mesh copper grids and stained with 2% uranyl acetate and 0.1% lead citrate. Imaging was performed using a Phillips CM 100 EM microscope.

[0074] 0072 Statistics. Statistical comparisons between individual groups were performed using Student’s t test (unpaired, two tailed with assumption of equal variance) for examination of significance, determined at a minimum level of p < 0.05. Statistical analyses of greater than two groups with one independent variable were performed using one-way ANOVA and Tukey’s post hoc with significance defined at a minimum level of p < 0.05. Statistical measures were performed using Microsoft Excel and GraphPad Prism Software, version 6. All values are presented as mean values ± standard deviation. Results

[0075] 0073 As stated above, certain cell types, particularly transformed lines, can currently be efficiently transfected using several physical methods including electroporation, lipofection, and calcium phosphate-mediated uptake. By contrast, many primary cell types including embryonic stem cells are more difficult to transfect. The use of dynamic hydrostatic pressure modification for transfection overcomes these difficulties. In the method describe here (see for example Figure 1 A-C), borosilicate capillary segments were prepared, sterilized, and filled with single cell suspensions in electroporation buffer whereupon they were sealed with sterilized petrolatum to allow rapid transmission of the local hydrostatic pressure (see Methods). Capillaries were then placed into a sealed silicone oil-filled pressure vessel. The hydrostatic pressure was increased to the desired static pressure at a rate of approximately 65 MPa / min and maintained for periods of 0.5 - 10 minutes after which the pressure was abruptly or slowly returned to ambient (Figure ID).

[0076] 0074 It was surprisingly observed that sudden depressurization from a particular range of static high pressures resulted in a small number of cells incurring permeabilization of their outer cell membrane without overt cellular destruction (see Figure IE). An example of this can be seen in Figure IF, in which the cell impermeant marker propidium iodide (MW 668.4) has been added to a concentration of one micromolar to the resuspension solution. Immediately following depressurization (< 5 min) a minority of cells could be observed allowing uptake of propidium iodide suggesting such cells might also be capable of absorbing higher molecular weight entities such as plasmid DNA. As shown in Figure 2A, following pressure-induced transfection with an ~9.2 kb DNA plasmid inducing puromycin resistance (Addgene # 62988), numbers of puromycin-resistant colonies were observed at an initial plating density of 1042 cells / cm2(lOk / well of standard 6-well TC plate) to quantitatively assess plasmid uptake over a range of pressures. It was surprisingly found that static pressures within the range of 60 - 80 MPa followed by sudden depressurization resulted in a marked increase in plasmid uptake. Given that such uptake was assessed via puromycin-resistant clonogenic assay, this observation demonstrates faithful plasmid uptake together with continued cell survival, propagation, and function following cell transfection. It also demonstrates that transfected plasmids were accurately and actively transcribed. Plasmid uptake was found to be reduced if the static holding pressure was outside the range of 60 - 80 MPa (see Figure 2A).

[0077] 0075 To determine the relative efficiency of PJP, results with ES cells were compared in identical solutions and DNA concentrations to an optimized electroporation standard (E STD: Biorad 240 volts, 500 pF in 4 mm cuvette). A shown in Figure 2, the values obtained compare favorably to the current standards. No transfection was observed in the absence of added puromycin-encoding DNA in either the absence (ND) or presence (ND80) of applied hydrostatic pressure; similar to that seen for electroporation (NDE). As shown in Figure 2, even in the presence of added plasmid DNA (20 pg / mL), very low levels of transfection are observed at static pressures below 30 MPa. In addition, even at optimal pressures for transfection in ES cells, low levels of transfection were observed under conditions where the hydrostatic pressure applied was reduced slowly (70SL). Though the data presented here are for ES cells resuspended in Embry oMax electroporation buffer, results obtained using OptiMEM or serum-free basal media such as DMEM were not significantly different demonstrating similar transfection results (data not shown).

[0078] 0076 To assess post-treatment function, unselected ES cells were plated on gelatin and visualized 24 hrs following treatment with static pressures equal to 60 MPa (see Figure 2B). As indicated in Figure 2B, dTomato transfected cells were capable of appropriately transcribing and translating transfected DNA markers during this period. Similarly, pressure transfected ES cells plated onto DR4 multi drug resistant mitomycin- treated fibroblasts (Figure 2C), demonstrated continued growth characteristics like that seen for other methods of gene transduction such as electroporation and lipofection.

[0079] 0077 To further address the capabilities and characteristics of pressure transduction, experiments were conducted in a separate ES cell line expressing EYFP (citrine) from the ROSA26 locus38in the cytoplasmic cellular compartment. An advantage of this cell line is that it allows real-time examination for diffusion of the fluorescent 27.1 kDa EYFP protein from the cytoplasmic compartment in the event of interruption of the plasma membrane. As shown in Figure 3A-C (red arrows), transfection of EYFP- expressing ES cells using PJP at different static pressures and plated onto supporting DR4 fibroblasts (no puro selection applied) demonstrated competent expression of dTomato from the 9.5 kb plasmid vector in a minority of cells by 24 hours post- transfection. Consistent with these findings, the data in Figure 2A, show that the frequency of these events followed a general trend with respect to applied static hydrostatic pressure, with a reduction in the average intensity of EYFP expression within dTomato+versus dTomato-negative cell populations (Figure 3B). As shown in Figures 3D-F, similar experiments performed ES R1 cells using a 12.3 kb Lac-Z expression plasmid demonstrated beta-galactosidase expression 48 hours following pressure treatment. Plating at lOx higher densities (10,400 cells / cm2- Figures 3D-E) failed to show beta-galactosidase expression in the absence of either pressure treatment (Figure 3D) or pressure treatment in the absence of DNA vector addition (Figure 3E). By contrast, beta-galactosidase+cells (blue arrow) were observed even at reduced cell densities (1040 cells / cm2) under appropriate conditions of pressure treatment (Figure 3F). Pressure treatment did result in the induction of cell death within a subpopulation of cells (boxed). In summary, using two distinct embryonic stem cell lines and several different expression vectors we observed that upon sudden depressurization after maintaining appropriate levels of static pressure resulted in the uptake and expression of ectopic DNA in a subpopulation of surviving cells.

[0080] 0078 In order to better understand the features of this pressure responsivity in mammalian cells, parental and sublines of R1 or citrine-expressing ES cells were examined for their DNA transfection potential as a function of pressure as shown in Figure 4A. While absolute differences in the pattern and level of DNA uptake were noted between different cell lines, a general pattern of responsivity for the conditions utilized was observed for static pressures between 60 - 80 MPa. Compared to other types of primary cells, stem cells exhibit strong density -dependent effects with respect to survival and growth40. As with other biophysical methods of cell transfection, successful uptake of ectopic molecules represents a balance between sufficient and excessive injury to the cell membrane. Integral to this may be the level of support enabling injured cells to recover. Transfection efficiency was examined as a function of cell density. As shown in Figure 4B, higher initial plating densities resulted in significantly higher numbers of transfected clones over a pressure range of 60 - 80 MPa. A common feature of transfection methods which act through interruption of the cell membrane may be the creation of transient holes in the phospholipid bilayer, followed by resealing on the time scale of seconds to minutes whereupon the cell membrane recovers its intrinsic impermeability41. To assess the ability of PJP to allow multiple plasmid uptake, the ability of pressure treated cells to uptake multiple plasmid reporters was examined. As shown in Figure 4C, once cells are in a sufficient state to take up one plasmid, their propensity to take up an additional reporter appears to be high, as variance of the relative reporter within the ratios indicated did little to alter the overall rate of double positive cells. This suggests that the primary limitation to transfection may he in achieving a receptive or competent uptake state, and that once achieved, the probability of continued survival with further uptake remains high, at least on the time scale of plasmid uptake.

[0081] 0079 Given that several straightforward methods currently exist to efficiently transfect immortalized cell lines, initial efforts were focused on cell types which are more difficult to transfect such as ES cells. Thus, after determining the basic features of transfection using the PJP technique, the ability of PJP to transfect other cell types was examined. As shown in Figure 5 A, though PJP-induced transfection of primary fibroblasts demonstrated a pattern similar to that seen for ES cells, attempts to induce transfection in mouse L-cells using PJP were unsuccessful. That this effect was unrelated to the reporter plasmid utilized may be demonstrated by the successful transfection of both L-cells and primary fibroblasts using standard electroporation. Similar attempts to pressure transfect immortalized well-characterized cell lines of HEK293T and Cos-7 (lines efficiently transfected by electroporation, calcium phosphate mediated transfection and lipofection) were similarly unsuccessful (data not shown).

[0082] 0080 While previously there has been extensive characterization of several cell types in response applied hydrostatic pressure, the present invention differs in that the exposure to pressure terminates with a sudden drop to ambient pressure. In order to better understand how this process affects cellular dynamics, several intracellular features were examined. For these experiments, citrine-tagged ES cells expressing monomeric EYFP from the Rosa26 locu38were used, which were incubated with Hoechst 33342, TMRM and propidium iodide at final concentrations of 1.5 pM, 100 nM and 0.75 pM respectively. As shown in Figure 5B, cells untreated by pressure transduction exhibited intact nuclei with granules of TMRM fluorescence, indicative of dye accumulation in active mitochondria with an intact membrane potential. In Figure 5C, the samples were held at a static pressure of 100 MPa for 1 minute followed by sudden depressurization. The ES cells continued to demonstrate TMRM fluorescence, however with some reduction in the number of distinct granules suggestive of mitochondrial fusion. In addition, a small number of cells (-1%) began to demonstrate the features indicative of entry of the cell impermeant marker propidium iodide identifying localized membrane disruption, Panel 5D. Additionally, a significant number (-30%) of cells developed morphologies like those shown in Figure 5E over 5 - 30 minutes post-treatment. Such cells presented with overt morphologic cell disruption with membrane-bound cell fragmentation reminiscent of apoptotic cell death. Where the static pressure may be maintained longer (5 min at 100 MPa, Figure 5F-H), then the majority of cells continue to present intact plasma and nuclear membranes, with cytoplasmic compartments exhibiting granules of TMRM fluorescence albeit with a small decrease in the average signal intensity. A fraction of these cells (Panel 5G) exhibited features consistent with significant reduction in cellular volume. Extended pressure treatment resulted in an increased number of cells exhibiting overt features of cellular degeneration with very few structurally intact cells exhibiting propidium iodide entry (see Figure 5H).

[0083] 0081 Given the apparent reduction in cellular volume seen under conditions of PJP, cell size was examined, and volumes estimated based upon area measurements through the cell diameter from free-floating cells as determined by optical sectioning. For ES cells, an interpolated volume of 161 ± 15 pm3prior to the pressure cycling process and 131 ± 17 pm3after exposure to 100 MPa for 5 minutes followed by sudden depressurization (n > 60) were calculated. This result appears consistent with an average reduction of volume equal to 18.6% after pressure treatment. However, many immortalized lines demonstrate little change in volume, for example, HEK293T cells revealed interpolated volumes of 187 ± 14 pm3prior to pressure cycling, and 208 ± 20 pm3after exposure to 100 MPa for 5 minutes followed by sudden depressurization (n >40); an apparent increase of 10.1%. Thus, the volume change appears to depend on the cell type.

[0084] 0082 To examine in greater detail the structural features resulting from PJP, TEM imaging was performed on ES cells fixed 5 minutes following exposure to one of three treatments: (1) non-pressurized cells resuspended in Embry oMax electroporation buffer and loaded into borosilicate capillaries cells in the presence of reporter DNA for 10 minutes; (2) ES cells resuspended in EB loaded into capillaries in the presence of reporter DNA for 10 minutes followed by pressurization to 80 MPa, maintained at this pressure for 1 minute, followed by sudden depressurization to initial starting conditions (e.g. ambient pressure); (3) ES cells resuspended in EB and loaded into capillaries in the presence of reporter DNA for 10 minutes followed by pressurization to 80 MPa, maintained at this static pressure for 1 minute, followed by slow depressurization over the course of 3 minutes to ambient pressure. As shown in Figure 51, while several treated cells exhibited features of overt rupture, a sub-population of cells experiencing sudden depressurization presented with the appearance of distinct cellular voids proximal to the cell membrane (red arrows), often in conjunction with irregular protrusions of the cell membrane and associated cytoplasm (blue arrows) compared to untreated cells (Figure 5 J). Notably little disturbance of the nuclear membrane or material was observed under these conditions. ES cells experiencing a slow reduction in static pressure frequently exhibited significantly greater disruption of cell constituents, with gross disruption of cytoplasmic, membrane and nuclear constituents (see Figure 5K).

[0085] 0083 Based upon the functional criteria of continued survival and growth, the results demonstrated that even sensitive cell lines such as ES cells could survive for short periods at 100 MPa with minimal reductions in survival. Yet a number of transformed cell lines appear resistant to pressure-induced transfection (Figure 5A). Given that the cellular response to pressure is known to be a function of both absolute pressure and the length of time to which cells are exposed23,25’28, the potential nature of such differences among transfection-positive and transfection-negative lines were reviewed by examining the survival properties of cell lines at different pressures and exposure periods. For each cell line investigated, equivalent cell densities were maintained at a specified pressure and time, followed by sudden return to ambient pressure. To determine relative levels of cell survival, treated cells were then plated and cultured in the manner described above. As shown in Table 1, cell types such as ES cells and primary fibroblasts demonstrated lower upper pressure limits with respect to survival compared to cell lines that exhibit transfection resistance such as mouse L-cells, exhibiting greater survival at significantly higher static pressures. A similar pattern was observed for other transfection resistant cell lines such as HEK293T, T24 and Lovo cells. Brightfield photomicrographs of these cell lines at three different levels of pressure treatment are shown in Figures 6A-D.

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[0142] 0084 Even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and function of the invention, the disclosure is illustrative only. Changes may be made in detail, especially in matters of shape, size and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.

Claims

We claim:

1. A process for transfecting a membranous structure having lipid or phospholipid membrane constituent, the process comprising:(a) mixing the cells of the desired isotype with a material to be transfected in a solution to support cell viability;(b) applying a first hydrostatic pressure to the mixture of step (a) and maintaining the mixture at the first hydrostatic pressure statically for a first predetermined time;(c) within a second predetermined time, reducing the first hydrostatic pressure to a second hydrostatic pressure; and(d) extracting the transfected cells.

2. The process of claim 1 wherein the first hydrostatic pressure is greater than 40 MPa.

3. The process of claim 2 wherein the first hydrostatic pressure is approximately 60 MPa to approximately 100 MPa.

4. The process of claim 3 wherein the first hydrostatic pressure is approximately 60 MPa to approximately 80 MPa5. The process of claim 4 wherein the first predetermined time is between approximately 1 second to approximately 10 mins.

6. The process of claim 4 wherein the second predetermined time less than 5 seconds.

7. The process of any one of claims 1 to 6 wherein the membranous structure is a cell.

8. The process of claim 7 wherein the cell is a eukaryotic cell.

9. The process of any one of claims 1 to 8 wherein the material is an extracellular biologically active component.

10. The process of claim 9 wherein the extracellular biologically active component is selected from the group consisting of a component used for purposes of modifying cellular response and an agent utilized for purposes of cellular tagging / tracing.

11. The process of claim 9 wherein the extracellular biologically active component is selected from the group consisting of nucleic acid polymers, proteins, peptides, drugs, small molecules, lipids, liposomes, cellular constituents12. The process of any one of claims 9 to 11 wherein the material has a size of < approximately 2 micrometres.

13. The process of claim 12 wherein the material has a size of < approximately 100 nm.

14. An apparatus for transfecting a membranous structure having lipid or phospholipid membrane constituent, the apparatus comprising:(a) a container for mixing the cells of the desired isotype with a material to be transfected in a solution to support cell viability; and(b) a device for applying (i) a first hydrostatic pressure to the mixture in container (a) and maintaining the mixture at the first hydrostatic pressure statically for a first predetermined time; and (ii) within a second predetermined time, reducing the first hydrostatic pressure to a second hydrostatic pressure.

15. The apparatus of claim 14 wherein the first hydrostatic pressure is greater than 40 MPa.

16. The apparatus of claim 15 wherein the first hydrostatic pressure is approximately 60 MPa to approximately 100 MPa.

17. The apparatus of claim 16 wherein the first hydrostatic pressure is approximately 60 MPa to approximately 80 MPa.

18. The apparatus of claim 17 wherein the first predetermined time is between approximately 1 second to approximately 10 mins.

19. The apparatus of claim 18 wherein the second predetermined time less than 5 seconds.

20. The apparatus of any one of claims 14 to 19 wherein the membranous structureis a cell.

21. The apparatus of claim 20 wherein the cell is a eukaryotic cell.

22. The apparatus of any one of claims 14 to 21 wherein the material is an extracellular biologically active component.

23. The apparatus of claim 22 wherein the extracellular biologically active component is selected from the group consisting of a component used for purposes of modifying cellular response and an agent utilized for purposes of cellular tagging / tracing.

24. The apparatus of claim 22 wherein the extracellular biologically active component is selected from the group consisting of nucleic acid polymers, proteins, peptides, drugs, small molecules, lipids, liposomes, cellular constituents25. The apparatus of any one of claims 22 to 24 wherein the material has a size of < approximately 2 micrometres.

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