CA2201315A1 - Direct introduction of foreign materials into cells - Google Patents
Direct introduction of foreign materials into cellsInfo
- Publication number
- CA2201315A1 CA2201315A1 CA 2201315 CA2201315A CA2201315A1 CA 2201315 A1 CA2201315 A1 CA 2201315A1 CA 2201315 CA2201315 CA 2201315 CA 2201315 A CA2201315 A CA 2201315A CA 2201315 A1 CA2201315 A1 CA 2201315A1
- Authority
- CA
- Canada
- Prior art keywords
- microprobes
- cells
- biological material
- support
- microns
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0335—Genetically modified worms
- A01K67/0336—Genetically modified Nematodes, e.g. Caenorhabditis elegans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
- C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
Disclosed is a simple, economical, and precise method of introducing a biological material into a predetermined target cell population. The method comprises the steps of providing (a) a plurality of biologically inert microprobes positioned on a support, (b) a solid or quasi-solid mass of the target cells defining an interface with the microprobes, and (c) a biological material at the interface, and then physically contacting the cells with the microprobes to cause the microprobes to non-lethally pierce the cell walls and/or membranes of the cells. The microprobes are preferably integral with the support, and are prepared by etching a single crystalline wafer material such as silicon. The mircoprobes are preferably pyramidally shaped. The target cells can be contacted with the microprobes in vitro or in situ. The method is applicable to virtually all cell types, and any biological material capable of being introduced into cells described herein.
Description
W096/1~0 1~1~$9S1l~8l .
D~CRIPTION
DTR~CT INTRODUCTION OF FOR~IGN MAT~I~T~ INTO c~r H~I I C~!T- FTP~r.n The present invention relates to methods for S the il.L~Gd~ction of biological materials into cells.
Rp~cKGRouND .~RT
The rapid advancement of recomhin~nt DNA
tech~ology has created a wide-ranging need for biological ccientists to transfer biological subst~nc~s from one cell to another, and to transfer synthetic biological material into living cells to exert their activity therein. Such materials include proteins,-e.g., antiho~;es or enzymes, pharmaceutically active agents, and more co~only, nucleic acids such as RNA or DNA. Thus, a variety of chemical and mec~nical cell transformation methods have emerged. Some of the more widely practiced methods include direct micro-injection, eleuL~ u~G~ ation, liposome-mediated transformation, cell fusion, cell implantation, biolistics, and viral- and bacterial-mediated transformation. -In the case of biolistic t~hniques~ metal mi~lu~ojectiles are coated with the foreign substance, and disposed on a miulo~-ojectile which is accelerated toward the cell target. These techniques ~uffer from several deficiencies. For example, most of the existing biolistic apparatus is bulky, generally immobile and cannot be hand held such as is nPo~D~ for some veterinary or medical applications. In addition, they are not flexible as to mode of use; rather, they tend to be optimal for only a single mode of use or for a single application. ~urther, they do not provide the degree of repeatable results that is desired.
Biolistic devices have also been criticized in terms of 3S inadequate velocity ~u-lL.ol, and excessive gas blast, acoustic shock, velocity debris, and heat and radiant energy. Moreover, extensive cell damage can occur due to eYc~-csively high velocity of the mi~Lo~ojectiles WO g6/10630 P~ I12381 _ -2-when they strike the cells and/or by high-pressure gas impinging directly on the cells. Most biolistic apparatus is limited to one-shot at the target. That i8, it is not possible to fire multiple shots of the S foreign biological cubstance at the same target or at different targets in rapid sll~coscion.
Cell implantation t~hn i ques generally involve creating a small hole in a single living cell with the aid of a fine needle under an optical microscope, thereby allowing DNA fragments to ènter the cells through the hole. This method, however, requires skilled manipulation of the needle, and is quite tedious and laborious. Another implantation method involves the steps of precipitating DNA in a culture medium, and making use of the phagocytotic properties of living cells to inco~olate the DNA. While this method is capable of handling a great number of cells simultaneously, the sllcceCc rate is quite low.
Virus-induced and chemical-in~l~ce~ fusion methods also have many shortcomings, including fusion yield and severe side effects on the fused cells.
Further, not all cell types can be fused with the same ease.
The ability to transfer exogenous genetic material into higher plants promises to provide PnhAnre~ o~o~Lul.ities for agricultural scientists to increase food production. Microinjection of DNA has been practiced in both animal and plant cells. This tPchnique, however, can be applied to only one cell at a time. Other plant transformation t~c~niques have taken advantage of the plant pathogen Aqrobacterium tumefaciens, which has the ability to transfer a portion of the DNA from an endogenous Ti (tumor-inducing) plasmid into an infected plant cell.
3S Aqrobacterium-mediated plant cell transformation has worked reasonably well in many model crop species, such as tobacco, petunia and carrot. Nonetheless, it has significant limitations. The first is that the W096/1~0 ~ S P~l/U~9S/1~81 _ -3-mediation can be only done on an individual cellular level, typically with somatic tissues, which then must be regenerated artificially into a whole plant.
~econ~, the natural host range of Aqrobacterium includes only dicotyledonous plants, and a limited number of monocot species.
In sum, most available cell transformation te~niques share common disadvantages. Most of the ~hni ques are painstakingly ~low; they use methods o which transport materials into, at moct~ only a few cells at a time; and they lack precise ~u~,L~ollability.
Most t~hniques are not universally applicable to a large number of different organisms in different biological classes or kingdoms. For example, chemical lS methods are generally applicable to procaryotic systems, whereas in eucaryotic systems, both chemical and mec~nical methods are used. Existing methods are further dependent upon both the gene which is to be transferred as well as the type of recipient cell.
Hence, a strong need remains for a method of transferring foreign biological materials into cells which is fast, uncomplicated, efficient, and which can be routinely and universally applied to large number~
of cells simultaneously.
SU~ARY OF TH~ lNV ~llON
The present invention provides a method of introducing a biological material of interest into a predetermined target cell population. The method comprises the steps of providing (a) a plurality of mi~Lo~obes positioned on a support, (b) a solid or quasi-solid mass of the target cells defining an interface with the microprobes, and (c) a biological material at the interface, and then physically contacting the target cells with the microprobes to cause the mi~ O~L obes to non-lethally pierce the cell membranes of the cells. The biological material is introduced into the cytosol of the cells directly via the mi~Lo~obes, or by passage through the opD~ings or WO g6/10630 ~ JS9~/12381 perforations in the cell walls and/or m mbranes created by the mi~L G~ obes.
In a preferred emhoAiment, a plurality of biologically inert mi~Lo~obes are integral with the S ~ G~ ~, and are prepared by etching a wafer, preferably silicon. The wafer is etched in such a manner 80 as to produce truncated, pyramidal mi~ G~l obes having a height from abou* lo microns to about 300 microns, preferably a height of from about 20 to about 90 microns, a tip width of from about 0.05 to about 10 microns, a base width of from about 30 to about 80 microns, and a distance between any two adjacent mi~.u~lobes of from about 1.0 to about 20 times the height of the mi~u~obes. The mi~o~,obes lS can be applied to the target cells in vitro or in situ.
Another embodiment of the present invention provides a method of introducing a biological material of interest into a predetermined target cell population, comprising the steps of providing in a liquid medium a target cell population, the biological material, and a plurality of microprobes positioned on a ~u~GLL, the mi~Lop~obes and support being integral with one another and having been prepared by etching a single crystalline wafer material, and subjecting the liquid medium containing the target cells, the biological material, and the mi~u~obes to physical motion under conditi-ons sufficient to cause the microprobes to non-lethally pierce the cell walls and/or membranes of the cells, whereby the biological material enters the cytosol of the cells.
The present invention is applicable to virtually all cell types, and can be practiced with any biological material capable of being intro~ltre~ into cells in the manner described herein.
3S BRIEF D~.~CRIPTION OF THE DRAWINGS
In the accompanying drawings, the preferred embodiments of the invention are illustrated in which:
WOg6/~ 3 ~ gs1l2381 _ -5-FIG. lA is a perspective view of a plurality of pyramidal mi~-u~obes positioned on a ~u~G~ L;
FIG. lB i~ an ele~o.. photomi~u~Laph of a single pyramidally-~h~pe~ mi~lo~obe;
S FIGS. 2A and 2B are cross-sectional views of a preferred system used for carrying out of the present invention;
FIG. 3 is a ~e.~pective view of another preferred system for carrying out the present invention; and FIG. 4 is a ~e~e-tive ~iew of a plurality of barbed-shaped microprobes positioned on a ~ OL -BEST MODE OF CAKK~l~G OUT lNv~NllON
Referring now to the drawings, and more lS specifically to FIG. lA, there is shown a structure 10 cont~ining a plurality of pyramidal mi~lu~obes 12 positioned or di~v_cd on a silicon wafer ~ o~L or substrate 1~. It is preferred that the mi~loy~obes are biocompatible. The mi~L U~l obes and the substrate preferably constitute an integral, i.e., one-piece, structure.
In a preferred embodiment, the illustrated structure is prepared by one of many variations of bulk micro-maohi ni ng. A st~n~rd silicon wafer is cut so 2S the plane specified by the Miller indices ~100) is coincident with the top ~urface. The wafer is thoroughly cleaned according to st~Ard pro~edu~es, such as a RCA clean. Next, a protective layer is grown or deposited on the top surface of the wafer. For example, a one-micron thick silicon dioxide layer could be grown on the wafer by wet oxidation at approximately 1000 C for six to twelve hours. In the alternative, a ~hinn~r layer of oxide could be grown, and silicon nitride deposited on top of the oxide. Photoresist is 3S then placed on top of the wafer. For example, the wafer can be spun, and several drops of photoresist placed on the surface. By correctly controlling the spinning of the wafer, a thin (e.g., usually a micron W096l1~0 ~ 3 ~ 5 ~ S~/1~81 _ -6-or less) layer of photoresist is left on the top surface of the wafer. The photoresist is then soft baked, at approximately 100 C for ten to thirty minutes, to strengthen the photoresist for h~n~l ing and S ~h-?quent steps.
The photoresist is then patterned. An optical mask having the correct features is used to ~Yro~e the photoresist. For example, the mask can be brought in contact with the top surface of the photoresist, and ultraviolet light shown through the mask onto the photoresist. The photoresist is then developed, using procedures specified by the particular manufacture of the photoresist. Then the photoresist is har~h~ke~ for approximately one-half hour at approximately 120 C. At this point, photoresist only covers those portions of the wafer that were not exposed to ultraviolet light (or in some cases, only those regions that were exposed to ultraviolet light).
The wafer is then placed in buffered hydrofluoric acid for several ~inutes to remove the oxide layer present on the wafer, except where the wafer is protected by the remaining photoresist. If the wafer is also protected by silicon nitride, a method of removing the silicon nitride, such as 2S reactive ion etching, needs to be used.
The remaining photoresist is removed off the top surface of the wafer, leaving the silicon oxide and/or silicon nitride (sio2 / SiN) protecting selected regions of the wafer. For the micro-point fabrication, these protected regions of SiO2 / SiN are squares with their sides orientated along the ~llO] directions.
Alternatively, the protected regions could be squares orientated along the [lO0] directions with corner compensation arms of sio2 / SiN ext~n~ing away from the 3S corners of the squares. This corner compensation allows taller micro-probes to be fabricated.
The wafer is placed in an anisotropic etching solution. Isotropic etching could also be used. A
wog6/~ 3 ~ 9~/l238l -solution of approximately one part KOH and three parts of distilled water is preferred. The wafer is etched until the SiO2 / SiN protective regions ~tart to float off the wafer into the water. This indicates the S protective coating has been completely under etched.
The wafer is now cleaned, and cut into a~rup,iately shaped chips.
In the a more preferred emho~iment, the illustrated structure is prepared by the anisotropic etr~ing of {100} silicon wafers substantially as disclosed in Offereins et al., S~nrors and Actuators A, 25-27:g-13 (1991), with modifications.
The result of cutting (etc~ing) the top surface of the silicon wafer in the {100} direction is lS the formation of discrete, four-sided truncated pyramids formed by {411} planes, i.e., the mi~-u~Lobes.
Upon completion of the etchj~g process, the substrate 14 preferably has a final thir~cs of from about 200 to about 550 microns.
Microprobes 12 have a -half angle h of about 13 (not illustrated). That is, each face is disposed at an angle of about 13 to the central axis of the pyramid. Stated differently, the included angle between any two oppositely directed faces of the 2S pyramidal structure is about 25. In general, the dimensions of the mi~. u~L DbeS and the distance between any two adjacent mi~.u~lobes on support 1~ are selected so as to maximize the number of cells in the target cell population that are pierced by the mi~Lo~lobes in a single application. As illustrated in FIG lB, the height h of the mi~.oylobes will usually range from about 10 microns to about 300 microns. The preferred height is from about 20 to about 90 microns, more preferably about 80 microns. The tip width t of the mi~.o~obes will usually vary from about 0.05 to about 10 microns, and is preferably no more than about 1 micron. The base width, b, i.e., the linear dimension along an edge of the microprobes at the substrate or sgsll238 V~O g6/10630 su~u L 1~, is from about 30 to about 80 microns, and the distance between microprobes is from about 1.0 to about 20.0 times the height of the microprobes, e.g., from about 1o to about 3000 microns, and preferably S from about 80 to about 800 microns. Thus, a 5 millimeter square ~ilicon wafer typically will contain about Soo mi~o~obes. These dimensions can be varied by adjusting the process parameters,-e.g., dimensions of mask, etching time, etchant type concentration of etchant and etr~inq time, accordingly. See Petersen, "Silicon as a Mec~nical Material", Proc. IEEE
70(5):420-457 (1982).
Turning now to FIG. 2A, system 20 features opposing plates 22 and 22', connected by flexible lS linkages 25 and 25'. Plate 22' contains a recess 23 bounded by a wall having an edge 29. This recess is adapted to receive structure ~0 and a solution of biological material 24. Thus, when structure lo is di~y& ^~ in recess 23, substrate 14 is recessed from edge 29. However, the tips of mi~lo~-obes ~2 ~luL~de beyond edge 29. The surface of plate 22 facing the microprobes is attached to flexible material 26. The flexible material is easily deformed (deformable), highly elastic, and resilient so as to conform to the surface of the solid or quasi-solid mass of the target cells. Examples include silicon rubbers and gels, and foam rubbers. The biological material, in this case a tobacco plant leaf 28, is disposed atop the microprobes, thus defining an interface between the mi~lo~obes and the tobacco cells 28'. A solution of biological material 24 is present at this interface.
Alternatively, the leaf may be attached to flexible material 26.
As illustrated in FIG. 2B, upon the 3S application of sufficient force urging plates 22 and 22' towards one another, the mi~o~obes are caused to pierce individual cells 28' of leaf 28, rendering the cells temporarily permeable such that biological W0 ~/1~0 ~ 9S11~81 ._ _g_ material solution 2~ is caused to enter (e.g., diffuse into) the cytosol of the cells. Of course, to the extent that biological material is retained on the mi~,o~obes (as described in greater detail below), the S material is delivered into the cytosol by the mi~L~lobes per se. By the term "sufficient" force, it is meant that the flexible material 26 presses the biological material until the tips of the mi~L~Lobes penetrate the cells and force leaf 28 approaching the ~urface of substrate l~. This process can be repeated several times, each time changing the position the leaf 28 relative to the probes 12 so that different injection locations are proA~lreA, thus maximizing the number of cells pierced.
In FIG. 3, structure 30 contains a handle 31 attached at an appropriate angle to ~UP~L L 33. The handle and SU~OLL can also be integral with one another. That is, an a~p~p.iately shaped structure can be made from one piece. Substrate 3~ having pyramidal microprobes 32 di~ e~ thereon is attached to the bottom surface of ~U~pGl L 33. This emhoA;ment of the present invention is advantageously used to apply the microprobes directly to the target cells in situ, e.g., in vivo . Examples of target cells particularly well ~uited to the application of the mi~L O~L obes in this fashion include animal skin (epidermal cells), animal and human internal tissues, and plant tissue. In the case of internal tissues, structure 30 can be used during the course of a surgical or endoscopic proceduLe as an adjunct in gene therapy. In this embodiment, the biological material may be applied directly to the target cell population prior to the application of the microprobes, or onto the microprobes themselves as described herein. Those 3S skilled in the art will appreciate that size and shape of the handle and support can be varied to accom_odate the particular use.
W096/1~0 ~ 9SI1~81 FIG. 4 illustrates another preferred embodiment of the present invention, wherein a plurality of barbed microprobes ~2 are positioned on silicon substrate ~. Each barb ~2 is comprised of a S ~u~G~L ~2', and a head ~2'' which is disposed upon the ~y~uLL ~2'. The barbed mi~,o~obes are prepared by the isotropic etching of [100~ oriented silicon wafers as described in ~an et al., "Mating and-Piercing Micro-mech~nical Structures for Surface Ron~;ng lo Application~," in The Procee~in~s of the IEEE Micro Electro ~c~nical Systems Workshop, January, 1991, Nara Japan, pp. 253-258. Briefly, the silicon wafer is cleaned and oxidized, and then patterned into an array of small squares. The wafer is then etched in XOH and lS isopropyl alcohol. The oxide mask is then stripped and a ~econ~ silicon dioxide film is grown. This film is patterned into an array of Greek crosses, and a second KOH etch is applied to remove a portion of the underlying silicon. An isotropic etch is then used to remove additional material from under the thus-formed microbarbs. The barbed microprobes ~2 have a height equal to about twice that of the microprobes 12 illustrated in FIG. 1. The dimensions i.e., tip width, base width and height, of each of the heads ~2'' and 2S support ~2', are approximately equal to those of a single pyramidal miu~o~obe described above. When the method of the present invention is practiced using barbed miulu~obes, cell death is minimized by providing a lateral force to substrate ~ while the heads of the miu~o~lobes are in contact with the cytosol of the target cells, so as to break off heads ~2 " from supports ~2'. In this manner, the step of removing the barbed mic~ u~L obes from the cells and the concomitant risk of causing cell death is minimized or - 35 obviated. The lateral force is typically in the range of from about 0.001 to about l.o lb/mic~o~obe.
There are numerous permutations of the preferred embodiments. The miulo~obes can be prepared Wo~ o ~ 3 ~ PCT~S9~1~81 using any substance which can be formed into a desired shape and which is capable of piercing cell walls and cell membranes, and preferably which will maintain its ctructural integrity over the course of several S applications to the target cell~. Biocompatible materials are preferred for these ~h~ ~G CS, in addition to their non-reactivity with a~d non-toxicity to cells.
In regard to etchable materials, the mi~ r 0~ obes and substrates are prepared from any single crystalline material that can be isotropically etched or anisotropically etched relative to crystallographic planes. Examples include quartz and gallium. The etching process can also be varied to produce needle-like or Wh; ck~r-like mi~Loplobes. The Petersen et al ., lS supra. Those skilled in the art will appreciate, however, that the su~o.~-based mi~u~obes can be prepared or replicated in accordance with art-recognized techniques other than etching such as molding or metal plating, using non-etchable materials such as ceramics, plated metals and plastics. See, e.g., Erfeld et al., Fabrication of Microstru~Lule_ Usinq the TTGA Process, Kernforschungszentrum Xarlsruhe GmbH, Karlsruhe, Federal Republic of Germany.
The biological materials can be applied to the microprobes and/or support or substrate in several ways. For example, the substrate or support can be adapted, e.g., recessed, to hold a liquid solution of the biological material. A film or coating of the biological material may also be deposited or otherwise applied directly onto the microprobes in accordance with st~nA~rd t~rhn; ques. Such a composition may be prepackaged. Moreover, surface tension alone typically will hold the biological material in place between the probes. Although the invention is not limited to any 3S particular theory of operation, it is believed that because the microprobes are closely spaced, any liquid containing the material which wets the substrate surface will be trapped as a men;~r~lC between the WO g6/10630 ~ 9~/l2381 _ -12-mi~lu~obes. This effect can be enhanced by adding a wetting agent to the solution of biological material, or to the medium in which the method is carried out.
In the case of silicon, again while not being bound to S any particular theory of operation, it is believed that when the silicon curfaces of the microprobes and ~ub~trates are ~Yro~-~ to air, an oxide is formed which is l.y~lu~hilic in nature and thus causes any aqueous biological material solution to more strongly adhere lo thereto. Further, the mi~-opLobes and/or substrate can be physically pretreated, e.g., roughened or chemically pretreated, e.g., with a porous material to enh~nce the "adhesion" between the biological material solution and the probes and substrate.
lS The conditions in which the physical contact between the mi~ obes and target cells o~u~ can be varied widely For example, the solid or quasi-solid target cell population may be provided in a liquid, i.e., hydlo~onic, or a misting, i.e., aeroponic environment. In addition, the injection location and the penetration depth of the mi~ G~L obes can be accurately controlled on both two dimensional and three dimensional planes. Two dimensional controllability is achieved simply by conducting multiple applications of 2S the probes to the cells, each time positioning the target cell population relative to the mi~Lop~obes such that different injection locations are produced. Three dimensional controllability can be used to deliver biological material to exact locations in the cells.
The ~ol.L~ol of the biological material delivery can be achieved based on force or displacement feedback. In the case of force feedback, the force required to penetrate a target cell membrane or cell wall to a specific depth is determined and this force is then applied to the mi~u~obe-mediated injection system such as the transfusion clamp illustrated in Figs. 2A
and 2B. On the other hand, if the thickness or cell membrane of cell wall of a target cell is known, then WO g6110630 ~ r~ ss/l23s -the displacement of the microprobes can be manipulated to stop immediately or at any desirable depth after the mi~Lo~obe tips are exposed to cytosol of the target cells. To improve the biological material delivery S efficiency of the microprobes, the penetration depth of the mi~.u~obes can be controlled based on the knowledge of cell size and/or ctructure or ~trength of target cell membrane or cell wall. - Although the invention is not limited to any particular theory of o operation, it is believed that in the case where contoured, e.g., pyramidal or barbed, mio~u~obes are used, displacement control is facilitated because force increases with the displacement of the probes in the cells.
l~ To further increase the uptaXe of the biological material by the target cells, electric pulses of a voltage sufficient to ~nh~nce the reversible permeabilization of the cells can be applied to the mi~o~ûbes (which- may also include the substrate) and/or the medium. Typical voltages are in the range of about 3 kilovolts to about 20 kilovolts.
See, e.g., U.S. Patent No. 4,849,3S5. The method can also be conducted in an osmotic gradient. Thus, the present invention may be practiced in conjunction with 2S other techniques for introducing biological materials into cells, or with te~h~;ques which facilitate the i"L~o~ction of the material once the cells are permeabilized by the mi~op~obes. Thus, the present invention may be used in conjunction with other techniques for introducing biological materials into cells, or t~chniques which facilitate the i"L~od~ction of the material once the cells were permeabilized by the mi-~o~obes.
Any biological material, which when delivered to the cytosol of a cell is capable of exerting its intended effect, can be used in the present method.
Suitable biological materials include non-proteinaceous subst~nces such as organic and inorganic WO9611~ r~ 9S11U81 chemicals, e.g., insecticides, diagnostic chemicals, e.g., detectable lAhels such as radio- or fluorescent-labeled molecules and pharmaceutically active subst~nc~c~ e.g., drug such as anti-inflammatories and S antibiotics; proteins such as anti ho~ ies~ hormones, growth factors and enzymes; and nucleic acids such as DNAs, particularly DNAs which encode a protein of interest. Nucleic acids are preferred.
The present method may also be practiced with virtually any cell type, including both procaryotic, e.g. bacterial, and eucaryotic ce~ls, e.g., animal, human, plants and yeast. Examples of eucaryotic cells include animal and plant cells, the latter including both monocotyledonous and dicotyledonous cells, and 1~ protoplasts thereof. Preferred plant ~pecies include cereal crop species such as rice, corn, wheat, sorghum, barley, soybean, potato, tomato and legumes. In general, the method is applicable to any cell type whose cell membranes (and in the case of plants, cell walls) can be pierced by the instant mic~o~obes. The method of the present invention has been illustrated using target cells in vitro. However, the microprobes can also be applied to the cells in situ ~ as described above. In addition, the definition of "solid or quasi-2~ solid target cells" for purposes of the presentinvention is meant to include multi-cellular living organisms and tissues of living organisms.
In yet a further emhsAiment of the present invention ~ a method of introducing a biological material of interest into the cytosol of a target cell is provided wherein a solid or quasi-solid mass of cells is not required. In this embodiment, the target cellc, biological material and the support-based miu~u~Lobes are provided in a liquid medium, with the support being anchored or secured, e.g. to an interior surface of a contAin~r holding the liquid mixture. The 8U~G~ -based mi~ O~L obes are prepared by etching a wafer, preferably silicon, as described above. The wo gc/10630 rcrlusssll23sl liquid medium containing the target cells, the biological material, and the microprobes is then subjected to physical motion for a period of time and under conditions ~ufficient to cause the microprobes to S non-lethally pierce the cell walls and/or cell membranes of the cells, whereby the biological material is caused to enter the cytosol of the cells. The liquid medium may be agitated by stirring, vorteYi~g, or by an appropriate mech~nical, magnetic or electrical 10 force as described, for example, in U.S. Patent 5,302,523 to Coffee et al.
The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration 15 only, and are not intPn~P~ to be limiting unless otherwise specified. Unless otherwise indicated, all percentages are by weight.
E~AMPLE 1 F~brication ~ G ~S for Mi~l6y~bes Polished {lOO} oriented silicon wafers were cleaned in a mixture of hydrochloric acid (HCl) and hydrogen peroxide (H202) to remove ionic contaminants.
The wafers were dipped in a 10:1 hydrofluoric acid:water solution to remove the native oxide layer, 25 then treated with sulfuric acid (H2S04) and H22 to grow a thin chemical oxide film. The samples were oxidized for 8 hours in an electrically-heated quartz furnace tube maintained at 1000C in pure oxygen bubbled through deionized water resulting in an sio2 film 30 approximately 1.0 ~m thick. A single-mask lithographic sequence was employed to pattern the wafers.
Hexamethyl disilazane was applied to the obverse face of the samples followed by AZ4210 photoresist spun at 6000rpm for 60 ~eCon~-C. The photoresist solvent was 35 expelled with a 20 minute bake at 90C. An emulsion mask cont~;ning 10 ~m2 patterns on a clear field was used in a contact aligner to expose the photoresist.
The edges of the patterns aligned to the {011}
WOg6/~ 9 ~ ~ ~ }~ SI12381 directions. After e~G~e, the samples were developed in a 3:1 solution of deionized water and AZ400K for 60 ~?.CQn~, then har~h~ke~ for 30 minutes at 120C. The developed photoresist pattern was then transferred to S the SiO2 film by etching in buffered hydrofluoric acid.
Etch completion was determined by observation of the hydrophobic surface transformation on the wafer backsides. The wafers were subjected to ~ A~P
rinsing in deionized water followed by photoresist lo removal in ~llrce~cive baths of acetone, methanol, and deionized water. -The microbarbs are formed by anisotropicsilicon etc~ing in an aqueous solution of XOH at 85C.
The etchant attacks the ex~G~cd {100} silicon surface, but etches the protective Sio2 pattern very slowly.
These etching conditions result in undercutting of the convex corners of the mask, exposing {411} oriented planes. These facets etch rapidly until they meet the center resulting in a sharp point and mask liftoff.
The etch timing is monitored to ensure that the microbarbs are not further eroded.
E~AMPLE 2 Transient Espre~Yion of DNA in Nicotiana Tabacum After Microprobe-Ne~iate~ Transformation 2~ To demonstrate that a biological material can be deposited into cells by this method, an experiment was carried out using Tobacco (Nicotiana Tabacum W3) leaf tissues. The DNA of interest was the plasmid pB
121 which contains a kanaymycin resistance gene and a GUS assay sensitive protein (glucuronidase). The mi~. G~ obes were prepared according to Example 1. The silicon substrate had a dimension of 5 mm by 8 mm and cont~i~P~ approximately 200 25-~m height mi~o~obes.
The probes were soaked in 95% ethanol for 20-30 minutes and washed 3 times with autoclaved distilled deionized H20, then air dried thoroughly. A DNA mix was made using 5 ~g DNA in TE buffer, 12.5 ~1 of 2.5 M CaC12, and 5 ~1 of 0.1 M spermidine free base. The epidermal wogc/10630 ~ 3 ~ ~ P~ 2381 layer of the severed tobacco leaf was aseptically removed using f Gl ~ . To prepare the mi~uy~bes for the plant material, 2.5 ~g of DNA preparation were placed on the mi~Lo~lobes and the plant materiaI was S placed on the mi~ L obes. A cotton swab was used to rub the back-side of the leaf sample to encourage the penetration by the mi~u~obes. The plant material was then removed from the mi~,o~bes and then cultured for 72 hours on a regenerative tobacco medium.
To evaluate the effectivene~s of the technique, one experimental group and three control yLo~ were conducted. The experimental group was applied to the DNA coated mi~Lop~obes. The first control group was applied to microprobes that were without DNA solution. The ~?con~ control group was made to contact the DNA solution, but without the mi~lo~obe treatment. The third control group was neither exposed to DNA solution nor was treated with the mi~u~obes. Tobacco tissues were examined by applying a GUS assay to evaluate the amount of DNA
taken up by the tobacco cells, following the procedures described in Jefferson, GUS Gene Fusion SYstem User's Manual, Cambridge (July 1987) Successful plasmid uptake by the cells results in~the presence of typical blue st~; ni ng. The experimental group showed positive transient symptoms (i.e., dark blue-green dots), while an AhcPnc~ of such stAining was observed in all three control ~ ou~=.
E~ANP~E 3 Nemato~e Transformation The mi~lu~obe-mediated transformation was used to generate transgenic nematodes. pPCZl and pRF4 were the DNAs used. pPCZ1 contained the C. eleqans 16kDa heat shock promoter fused to the E. coli B-- 35 galactosidase gene. pRF4 contained a 4Kb EcoRl genomic fragment of C. elegans encoAing the rol-6 (Su1006) collagen gene as described in Rramer et al., Mol. Cell.
Biol. 10:2081-89 (1990). The rol-6 gene was provided Wos6/10630 ~ PCIIUS9SI12381 by Peter Candide, University of British Columbia, Vancouver, B.C.
HeL~u~habd~tis bacterihora HP88 were collected at 4-6 eggs stage from lipid agar seeded with S r~hotorhabdus ltlminescence in double distilled sterile water and washed three times with sterile water. The miu.u~lobes of Example 1 were placed with the microprobes pointing up on the ~urface of a lipid agar plate seeded with ~hotorhabdus luminescence. A DNA mix lo containing 5 ~g DNA in TE buffer, 12.5 ~1 of 2.5 M
CaC12, and 5 ~ 1; of 0.1 M spermidine-free base. Ten ~ 1 of DNA mix were pipetted onto the mi~;Lop~obes to coat the tips with DNA. After 3 minutes, 10 ~1 of a highly concentrated nematode suspension (approximately 200-250 lS nematodes) were pipetted on top of the microprobes and left at room temperature for 8 to 10 minutes. Most nematodes crawled off the mi~;~u~be array onto the media. The array was then removed from the plate, and the agar plate contAini~g nematodes was inc~-hAted at 20 25C, until the injected nematodes proAIlr~i progeny.
Control experiments, wherein a mi~;.c,p~be array without DNA was used, were also conducted.
After the incubation period, hermaphrodite progeny were examined for roller phenotypic expression 25 of the transformants. Progeny were assayed for expression of the hs~-16-lacZ fusion gene by heat shocking for 2 hours at 33C as described in Stringham et al., Mol. Biol. Cell. 3:221-33 (1992). The nematodes were then permeabilized by lyophilization and 30 acetone treatment and incubated at 25C in a stArl~lArd histochemical contA i n i ng 3% X-gal as described in Fire EMBO J. 5:2673-80 (1986).
Nematodes were scored for the presence of typical blue S1-A i ni rlg of b-galactosidase; 8% of the 35 total progeny showed b-galactosidase expression in the first (Fl) generation. No stAining was observed in the control groups. Although the nematodes were heritable transformed individuals exhibited a non-Men~lPliAn Wos6/~ PCT~S9511~81 pattern of inheritance similar to those of C. eleqans (extrachromosomal transformants), there was no genome integration.
INDUSl~TAT. APpT-TcABI~-TTy S The present invention provides a simple, economical and precise method for i.-Llod~cing a biological material of interest into a predetermined target cell population, in ~tro or in s~tu. Thus, it is u~eful in both animal and plant applications, lo including therapeutics and plant biotechnology. The present invention can accommodate virtually all cell types and biological materials, and offers ceveral additional advantages over known biological material, e.g., gene, transfer methodologies. First, the 15 location of the introduction of the foreign biological materials and the penetration depth can be precisely controlled by varying the dimensions of the mi~opLobes, the injection pattern or both, thus "customizing" the method to any given target cell 20 population. Second, cell damage is minimized; the degree of penetration of each probe into the cell, and the size of the holes or perforations in the cell membrane, are controlled by the geometry of the microprobes. Third, the method is simple; it does not 2S require complicated preparatory steps associated with biolistic t~rhniques. Fourth, the method is economical--the mi~. U~L obe-substrate structure is inexpensive to manufacture, and can be easily sterilized and/or disposed of. Fifth, a larger number 30 of cells are treated simultaneously, compared with prior art microinjection tech~iques which are limited to the treatment of single cells at a time.
All publications cited in the specification are indicative of the level of ~kill of those skilled 3S in the art to which this invention pertains. All these publications are herein in~o ~o.ated by reference to the same extent as if each individual publication were W0961106~ ~ rCT~S9S/1 ~pecifically and individually indicated to be in~oL~o~ated by reference.
Further modifications of the invention described herein become apparent to those skilled in S the art. Such modifications are intended to fall within the scope of the appended claims.
D~CRIPTION
DTR~CT INTRODUCTION OF FOR~IGN MAT~I~T~ INTO c~r H~I I C~!T- FTP~r.n The present invention relates to methods for S the il.L~Gd~ction of biological materials into cells.
Rp~cKGRouND .~RT
The rapid advancement of recomhin~nt DNA
tech~ology has created a wide-ranging need for biological ccientists to transfer biological subst~nc~s from one cell to another, and to transfer synthetic biological material into living cells to exert their activity therein. Such materials include proteins,-e.g., antiho~;es or enzymes, pharmaceutically active agents, and more co~only, nucleic acids such as RNA or DNA. Thus, a variety of chemical and mec~nical cell transformation methods have emerged. Some of the more widely practiced methods include direct micro-injection, eleuL~ u~G~ ation, liposome-mediated transformation, cell fusion, cell implantation, biolistics, and viral- and bacterial-mediated transformation. -In the case of biolistic t~hniques~ metal mi~lu~ojectiles are coated with the foreign substance, and disposed on a miulo~-ojectile which is accelerated toward the cell target. These techniques ~uffer from several deficiencies. For example, most of the existing biolistic apparatus is bulky, generally immobile and cannot be hand held such as is nPo~D~ for some veterinary or medical applications. In addition, they are not flexible as to mode of use; rather, they tend to be optimal for only a single mode of use or for a single application. ~urther, they do not provide the degree of repeatable results that is desired.
Biolistic devices have also been criticized in terms of 3S inadequate velocity ~u-lL.ol, and excessive gas blast, acoustic shock, velocity debris, and heat and radiant energy. Moreover, extensive cell damage can occur due to eYc~-csively high velocity of the mi~Lo~ojectiles WO g6/10630 P~ I12381 _ -2-when they strike the cells and/or by high-pressure gas impinging directly on the cells. Most biolistic apparatus is limited to one-shot at the target. That i8, it is not possible to fire multiple shots of the S foreign biological cubstance at the same target or at different targets in rapid sll~coscion.
Cell implantation t~hn i ques generally involve creating a small hole in a single living cell with the aid of a fine needle under an optical microscope, thereby allowing DNA fragments to ènter the cells through the hole. This method, however, requires skilled manipulation of the needle, and is quite tedious and laborious. Another implantation method involves the steps of precipitating DNA in a culture medium, and making use of the phagocytotic properties of living cells to inco~olate the DNA. While this method is capable of handling a great number of cells simultaneously, the sllcceCc rate is quite low.
Virus-induced and chemical-in~l~ce~ fusion methods also have many shortcomings, including fusion yield and severe side effects on the fused cells.
Further, not all cell types can be fused with the same ease.
The ability to transfer exogenous genetic material into higher plants promises to provide PnhAnre~ o~o~Lul.ities for agricultural scientists to increase food production. Microinjection of DNA has been practiced in both animal and plant cells. This tPchnique, however, can be applied to only one cell at a time. Other plant transformation t~c~niques have taken advantage of the plant pathogen Aqrobacterium tumefaciens, which has the ability to transfer a portion of the DNA from an endogenous Ti (tumor-inducing) plasmid into an infected plant cell.
3S Aqrobacterium-mediated plant cell transformation has worked reasonably well in many model crop species, such as tobacco, petunia and carrot. Nonetheless, it has significant limitations. The first is that the W096/1~0 ~ S P~l/U~9S/1~81 _ -3-mediation can be only done on an individual cellular level, typically with somatic tissues, which then must be regenerated artificially into a whole plant.
~econ~, the natural host range of Aqrobacterium includes only dicotyledonous plants, and a limited number of monocot species.
In sum, most available cell transformation te~niques share common disadvantages. Most of the ~hni ques are painstakingly ~low; they use methods o which transport materials into, at moct~ only a few cells at a time; and they lack precise ~u~,L~ollability.
Most t~hniques are not universally applicable to a large number of different organisms in different biological classes or kingdoms. For example, chemical lS methods are generally applicable to procaryotic systems, whereas in eucaryotic systems, both chemical and mec~nical methods are used. Existing methods are further dependent upon both the gene which is to be transferred as well as the type of recipient cell.
Hence, a strong need remains for a method of transferring foreign biological materials into cells which is fast, uncomplicated, efficient, and which can be routinely and universally applied to large number~
of cells simultaneously.
SU~ARY OF TH~ lNV ~llON
The present invention provides a method of introducing a biological material of interest into a predetermined target cell population. The method comprises the steps of providing (a) a plurality of mi~Lo~obes positioned on a support, (b) a solid or quasi-solid mass of the target cells defining an interface with the microprobes, and (c) a biological material at the interface, and then physically contacting the target cells with the microprobes to cause the mi~ O~L obes to non-lethally pierce the cell membranes of the cells. The biological material is introduced into the cytosol of the cells directly via the mi~Lo~obes, or by passage through the opD~ings or WO g6/10630 ~ JS9~/12381 perforations in the cell walls and/or m mbranes created by the mi~L G~ obes.
In a preferred emhoAiment, a plurality of biologically inert mi~Lo~obes are integral with the S ~ G~ ~, and are prepared by etching a wafer, preferably silicon. The wafer is etched in such a manner 80 as to produce truncated, pyramidal mi~ G~l obes having a height from abou* lo microns to about 300 microns, preferably a height of from about 20 to about 90 microns, a tip width of from about 0.05 to about 10 microns, a base width of from about 30 to about 80 microns, and a distance between any two adjacent mi~.u~lobes of from about 1.0 to about 20 times the height of the mi~u~obes. The mi~o~,obes lS can be applied to the target cells in vitro or in situ.
Another embodiment of the present invention provides a method of introducing a biological material of interest into a predetermined target cell population, comprising the steps of providing in a liquid medium a target cell population, the biological material, and a plurality of microprobes positioned on a ~u~GLL, the mi~Lop~obes and support being integral with one another and having been prepared by etching a single crystalline wafer material, and subjecting the liquid medium containing the target cells, the biological material, and the mi~u~obes to physical motion under conditi-ons sufficient to cause the microprobes to non-lethally pierce the cell walls and/or membranes of the cells, whereby the biological material enters the cytosol of the cells.
The present invention is applicable to virtually all cell types, and can be practiced with any biological material capable of being intro~ltre~ into cells in the manner described herein.
3S BRIEF D~.~CRIPTION OF THE DRAWINGS
In the accompanying drawings, the preferred embodiments of the invention are illustrated in which:
WOg6/~ 3 ~ gs1l2381 _ -5-FIG. lA is a perspective view of a plurality of pyramidal mi~-u~obes positioned on a ~u~G~ L;
FIG. lB i~ an ele~o.. photomi~u~Laph of a single pyramidally-~h~pe~ mi~lo~obe;
S FIGS. 2A and 2B are cross-sectional views of a preferred system used for carrying out of the present invention;
FIG. 3 is a ~e.~pective view of another preferred system for carrying out the present invention; and FIG. 4 is a ~e~e-tive ~iew of a plurality of barbed-shaped microprobes positioned on a ~ OL -BEST MODE OF CAKK~l~G OUT lNv~NllON
Referring now to the drawings, and more lS specifically to FIG. lA, there is shown a structure 10 cont~ining a plurality of pyramidal mi~lu~obes 12 positioned or di~v_cd on a silicon wafer ~ o~L or substrate 1~. It is preferred that the mi~loy~obes are biocompatible. The mi~L U~l obes and the substrate preferably constitute an integral, i.e., one-piece, structure.
In a preferred embodiment, the illustrated structure is prepared by one of many variations of bulk micro-maohi ni ng. A st~n~rd silicon wafer is cut so 2S the plane specified by the Miller indices ~100) is coincident with the top ~urface. The wafer is thoroughly cleaned according to st~Ard pro~edu~es, such as a RCA clean. Next, a protective layer is grown or deposited on the top surface of the wafer. For example, a one-micron thick silicon dioxide layer could be grown on the wafer by wet oxidation at approximately 1000 C for six to twelve hours. In the alternative, a ~hinn~r layer of oxide could be grown, and silicon nitride deposited on top of the oxide. Photoresist is 3S then placed on top of the wafer. For example, the wafer can be spun, and several drops of photoresist placed on the surface. By correctly controlling the spinning of the wafer, a thin (e.g., usually a micron W096l1~0 ~ 3 ~ 5 ~ S~/1~81 _ -6-or less) layer of photoresist is left on the top surface of the wafer. The photoresist is then soft baked, at approximately 100 C for ten to thirty minutes, to strengthen the photoresist for h~n~l ing and S ~h-?quent steps.
The photoresist is then patterned. An optical mask having the correct features is used to ~Yro~e the photoresist. For example, the mask can be brought in contact with the top surface of the photoresist, and ultraviolet light shown through the mask onto the photoresist. The photoresist is then developed, using procedures specified by the particular manufacture of the photoresist. Then the photoresist is har~h~ke~ for approximately one-half hour at approximately 120 C. At this point, photoresist only covers those portions of the wafer that were not exposed to ultraviolet light (or in some cases, only those regions that were exposed to ultraviolet light).
The wafer is then placed in buffered hydrofluoric acid for several ~inutes to remove the oxide layer present on the wafer, except where the wafer is protected by the remaining photoresist. If the wafer is also protected by silicon nitride, a method of removing the silicon nitride, such as 2S reactive ion etching, needs to be used.
The remaining photoresist is removed off the top surface of the wafer, leaving the silicon oxide and/or silicon nitride (sio2 / SiN) protecting selected regions of the wafer. For the micro-point fabrication, these protected regions of SiO2 / SiN are squares with their sides orientated along the ~llO] directions.
Alternatively, the protected regions could be squares orientated along the [lO0] directions with corner compensation arms of sio2 / SiN ext~n~ing away from the 3S corners of the squares. This corner compensation allows taller micro-probes to be fabricated.
The wafer is placed in an anisotropic etching solution. Isotropic etching could also be used. A
wog6/~ 3 ~ 9~/l238l -solution of approximately one part KOH and three parts of distilled water is preferred. The wafer is etched until the SiO2 / SiN protective regions ~tart to float off the wafer into the water. This indicates the S protective coating has been completely under etched.
The wafer is now cleaned, and cut into a~rup,iately shaped chips.
In the a more preferred emho~iment, the illustrated structure is prepared by the anisotropic etr~ing of {100} silicon wafers substantially as disclosed in Offereins et al., S~nrors and Actuators A, 25-27:g-13 (1991), with modifications.
The result of cutting (etc~ing) the top surface of the silicon wafer in the {100} direction is lS the formation of discrete, four-sided truncated pyramids formed by {411} planes, i.e., the mi~-u~Lobes.
Upon completion of the etchj~g process, the substrate 14 preferably has a final thir~cs of from about 200 to about 550 microns.
Microprobes 12 have a -half angle h of about 13 (not illustrated). That is, each face is disposed at an angle of about 13 to the central axis of the pyramid. Stated differently, the included angle between any two oppositely directed faces of the 2S pyramidal structure is about 25. In general, the dimensions of the mi~. u~L DbeS and the distance between any two adjacent mi~.u~lobes on support 1~ are selected so as to maximize the number of cells in the target cell population that are pierced by the mi~Lo~lobes in a single application. As illustrated in FIG lB, the height h of the mi~.oylobes will usually range from about 10 microns to about 300 microns. The preferred height is from about 20 to about 90 microns, more preferably about 80 microns. The tip width t of the mi~.o~obes will usually vary from about 0.05 to about 10 microns, and is preferably no more than about 1 micron. The base width, b, i.e., the linear dimension along an edge of the microprobes at the substrate or sgsll238 V~O g6/10630 su~u L 1~, is from about 30 to about 80 microns, and the distance between microprobes is from about 1.0 to about 20.0 times the height of the microprobes, e.g., from about 1o to about 3000 microns, and preferably S from about 80 to about 800 microns. Thus, a 5 millimeter square ~ilicon wafer typically will contain about Soo mi~o~obes. These dimensions can be varied by adjusting the process parameters,-e.g., dimensions of mask, etching time, etchant type concentration of etchant and etr~inq time, accordingly. See Petersen, "Silicon as a Mec~nical Material", Proc. IEEE
70(5):420-457 (1982).
Turning now to FIG. 2A, system 20 features opposing plates 22 and 22', connected by flexible lS linkages 25 and 25'. Plate 22' contains a recess 23 bounded by a wall having an edge 29. This recess is adapted to receive structure ~0 and a solution of biological material 24. Thus, when structure lo is di~y& ^~ in recess 23, substrate 14 is recessed from edge 29. However, the tips of mi~lo~-obes ~2 ~luL~de beyond edge 29. The surface of plate 22 facing the microprobes is attached to flexible material 26. The flexible material is easily deformed (deformable), highly elastic, and resilient so as to conform to the surface of the solid or quasi-solid mass of the target cells. Examples include silicon rubbers and gels, and foam rubbers. The biological material, in this case a tobacco plant leaf 28, is disposed atop the microprobes, thus defining an interface between the mi~lo~obes and the tobacco cells 28'. A solution of biological material 24 is present at this interface.
Alternatively, the leaf may be attached to flexible material 26.
As illustrated in FIG. 2B, upon the 3S application of sufficient force urging plates 22 and 22' towards one another, the mi~o~obes are caused to pierce individual cells 28' of leaf 28, rendering the cells temporarily permeable such that biological W0 ~/1~0 ~ 9S11~81 ._ _g_ material solution 2~ is caused to enter (e.g., diffuse into) the cytosol of the cells. Of course, to the extent that biological material is retained on the mi~,o~obes (as described in greater detail below), the S material is delivered into the cytosol by the mi~L~lobes per se. By the term "sufficient" force, it is meant that the flexible material 26 presses the biological material until the tips of the mi~L~Lobes penetrate the cells and force leaf 28 approaching the ~urface of substrate l~. This process can be repeated several times, each time changing the position the leaf 28 relative to the probes 12 so that different injection locations are proA~lreA, thus maximizing the number of cells pierced.
In FIG. 3, structure 30 contains a handle 31 attached at an appropriate angle to ~UP~L L 33. The handle and SU~OLL can also be integral with one another. That is, an a~p~p.iately shaped structure can be made from one piece. Substrate 3~ having pyramidal microprobes 32 di~ e~ thereon is attached to the bottom surface of ~U~pGl L 33. This emhoA;ment of the present invention is advantageously used to apply the microprobes directly to the target cells in situ, e.g., in vivo . Examples of target cells particularly well ~uited to the application of the mi~L O~L obes in this fashion include animal skin (epidermal cells), animal and human internal tissues, and plant tissue. In the case of internal tissues, structure 30 can be used during the course of a surgical or endoscopic proceduLe as an adjunct in gene therapy. In this embodiment, the biological material may be applied directly to the target cell population prior to the application of the microprobes, or onto the microprobes themselves as described herein. Those 3S skilled in the art will appreciate that size and shape of the handle and support can be varied to accom_odate the particular use.
W096/1~0 ~ 9SI1~81 FIG. 4 illustrates another preferred embodiment of the present invention, wherein a plurality of barbed microprobes ~2 are positioned on silicon substrate ~. Each barb ~2 is comprised of a S ~u~G~L ~2', and a head ~2'' which is disposed upon the ~y~uLL ~2'. The barbed mi~,o~obes are prepared by the isotropic etching of [100~ oriented silicon wafers as described in ~an et al., "Mating and-Piercing Micro-mech~nical Structures for Surface Ron~;ng lo Application~," in The Procee~in~s of the IEEE Micro Electro ~c~nical Systems Workshop, January, 1991, Nara Japan, pp. 253-258. Briefly, the silicon wafer is cleaned and oxidized, and then patterned into an array of small squares. The wafer is then etched in XOH and lS isopropyl alcohol. The oxide mask is then stripped and a ~econ~ silicon dioxide film is grown. This film is patterned into an array of Greek crosses, and a second KOH etch is applied to remove a portion of the underlying silicon. An isotropic etch is then used to remove additional material from under the thus-formed microbarbs. The barbed microprobes ~2 have a height equal to about twice that of the microprobes 12 illustrated in FIG. 1. The dimensions i.e., tip width, base width and height, of each of the heads ~2'' and 2S support ~2', are approximately equal to those of a single pyramidal miu~o~obe described above. When the method of the present invention is practiced using barbed miulu~obes, cell death is minimized by providing a lateral force to substrate ~ while the heads of the miu~o~lobes are in contact with the cytosol of the target cells, so as to break off heads ~2 " from supports ~2'. In this manner, the step of removing the barbed mic~ u~L obes from the cells and the concomitant risk of causing cell death is minimized or - 35 obviated. The lateral force is typically in the range of from about 0.001 to about l.o lb/mic~o~obe.
There are numerous permutations of the preferred embodiments. The miulo~obes can be prepared Wo~ o ~ 3 ~ PCT~S9~1~81 using any substance which can be formed into a desired shape and which is capable of piercing cell walls and cell membranes, and preferably which will maintain its ctructural integrity over the course of several S applications to the target cell~. Biocompatible materials are preferred for these ~h~ ~G CS, in addition to their non-reactivity with a~d non-toxicity to cells.
In regard to etchable materials, the mi~ r 0~ obes and substrates are prepared from any single crystalline material that can be isotropically etched or anisotropically etched relative to crystallographic planes. Examples include quartz and gallium. The etching process can also be varied to produce needle-like or Wh; ck~r-like mi~Loplobes. The Petersen et al ., lS supra. Those skilled in the art will appreciate, however, that the su~o.~-based mi~u~obes can be prepared or replicated in accordance with art-recognized techniques other than etching such as molding or metal plating, using non-etchable materials such as ceramics, plated metals and plastics. See, e.g., Erfeld et al., Fabrication of Microstru~Lule_ Usinq the TTGA Process, Kernforschungszentrum Xarlsruhe GmbH, Karlsruhe, Federal Republic of Germany.
The biological materials can be applied to the microprobes and/or support or substrate in several ways. For example, the substrate or support can be adapted, e.g., recessed, to hold a liquid solution of the biological material. A film or coating of the biological material may also be deposited or otherwise applied directly onto the microprobes in accordance with st~nA~rd t~rhn; ques. Such a composition may be prepackaged. Moreover, surface tension alone typically will hold the biological material in place between the probes. Although the invention is not limited to any 3S particular theory of operation, it is believed that because the microprobes are closely spaced, any liquid containing the material which wets the substrate surface will be trapped as a men;~r~lC between the WO g6/10630 ~ 9~/l2381 _ -12-mi~lu~obes. This effect can be enhanced by adding a wetting agent to the solution of biological material, or to the medium in which the method is carried out.
In the case of silicon, again while not being bound to S any particular theory of operation, it is believed that when the silicon curfaces of the microprobes and ~ub~trates are ~Yro~-~ to air, an oxide is formed which is l.y~lu~hilic in nature and thus causes any aqueous biological material solution to more strongly adhere lo thereto. Further, the mi~-opLobes and/or substrate can be physically pretreated, e.g., roughened or chemically pretreated, e.g., with a porous material to enh~nce the "adhesion" between the biological material solution and the probes and substrate.
lS The conditions in which the physical contact between the mi~ obes and target cells o~u~ can be varied widely For example, the solid or quasi-solid target cell population may be provided in a liquid, i.e., hydlo~onic, or a misting, i.e., aeroponic environment. In addition, the injection location and the penetration depth of the mi~ G~L obes can be accurately controlled on both two dimensional and three dimensional planes. Two dimensional controllability is achieved simply by conducting multiple applications of 2S the probes to the cells, each time positioning the target cell population relative to the mi~Lop~obes such that different injection locations are produced. Three dimensional controllability can be used to deliver biological material to exact locations in the cells.
The ~ol.L~ol of the biological material delivery can be achieved based on force or displacement feedback. In the case of force feedback, the force required to penetrate a target cell membrane or cell wall to a specific depth is determined and this force is then applied to the mi~u~obe-mediated injection system such as the transfusion clamp illustrated in Figs. 2A
and 2B. On the other hand, if the thickness or cell membrane of cell wall of a target cell is known, then WO g6110630 ~ r~ ss/l23s -the displacement of the microprobes can be manipulated to stop immediately or at any desirable depth after the mi~Lo~obe tips are exposed to cytosol of the target cells. To improve the biological material delivery S efficiency of the microprobes, the penetration depth of the mi~.u~obes can be controlled based on the knowledge of cell size and/or ctructure or ~trength of target cell membrane or cell wall. - Although the invention is not limited to any particular theory of o operation, it is believed that in the case where contoured, e.g., pyramidal or barbed, mio~u~obes are used, displacement control is facilitated because force increases with the displacement of the probes in the cells.
l~ To further increase the uptaXe of the biological material by the target cells, electric pulses of a voltage sufficient to ~nh~nce the reversible permeabilization of the cells can be applied to the mi~o~ûbes (which- may also include the substrate) and/or the medium. Typical voltages are in the range of about 3 kilovolts to about 20 kilovolts.
See, e.g., U.S. Patent No. 4,849,3S5. The method can also be conducted in an osmotic gradient. Thus, the present invention may be practiced in conjunction with 2S other techniques for introducing biological materials into cells, or with te~h~;ques which facilitate the i"L~o~ction of the material once the cells are permeabilized by the mi~op~obes. Thus, the present invention may be used in conjunction with other techniques for introducing biological materials into cells, or t~chniques which facilitate the i"L~od~ction of the material once the cells were permeabilized by the mi-~o~obes.
Any biological material, which when delivered to the cytosol of a cell is capable of exerting its intended effect, can be used in the present method.
Suitable biological materials include non-proteinaceous subst~nces such as organic and inorganic WO9611~ r~ 9S11U81 chemicals, e.g., insecticides, diagnostic chemicals, e.g., detectable lAhels such as radio- or fluorescent-labeled molecules and pharmaceutically active subst~nc~c~ e.g., drug such as anti-inflammatories and S antibiotics; proteins such as anti ho~ ies~ hormones, growth factors and enzymes; and nucleic acids such as DNAs, particularly DNAs which encode a protein of interest. Nucleic acids are preferred.
The present method may also be practiced with virtually any cell type, including both procaryotic, e.g. bacterial, and eucaryotic ce~ls, e.g., animal, human, plants and yeast. Examples of eucaryotic cells include animal and plant cells, the latter including both monocotyledonous and dicotyledonous cells, and 1~ protoplasts thereof. Preferred plant ~pecies include cereal crop species such as rice, corn, wheat, sorghum, barley, soybean, potato, tomato and legumes. In general, the method is applicable to any cell type whose cell membranes (and in the case of plants, cell walls) can be pierced by the instant mic~o~obes. The method of the present invention has been illustrated using target cells in vitro. However, the microprobes can also be applied to the cells in situ ~ as described above. In addition, the definition of "solid or quasi-2~ solid target cells" for purposes of the presentinvention is meant to include multi-cellular living organisms and tissues of living organisms.
In yet a further emhsAiment of the present invention ~ a method of introducing a biological material of interest into the cytosol of a target cell is provided wherein a solid or quasi-solid mass of cells is not required. In this embodiment, the target cellc, biological material and the support-based miu~u~Lobes are provided in a liquid medium, with the support being anchored or secured, e.g. to an interior surface of a contAin~r holding the liquid mixture. The 8U~G~ -based mi~ O~L obes are prepared by etching a wafer, preferably silicon, as described above. The wo gc/10630 rcrlusssll23sl liquid medium containing the target cells, the biological material, and the microprobes is then subjected to physical motion for a period of time and under conditions ~ufficient to cause the microprobes to S non-lethally pierce the cell walls and/or cell membranes of the cells, whereby the biological material is caused to enter the cytosol of the cells. The liquid medium may be agitated by stirring, vorteYi~g, or by an appropriate mech~nical, magnetic or electrical 10 force as described, for example, in U.S. Patent 5,302,523 to Coffee et al.
The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration 15 only, and are not intPn~P~ to be limiting unless otherwise specified. Unless otherwise indicated, all percentages are by weight.
E~AMPLE 1 F~brication ~ G ~S for Mi~l6y~bes Polished {lOO} oriented silicon wafers were cleaned in a mixture of hydrochloric acid (HCl) and hydrogen peroxide (H202) to remove ionic contaminants.
The wafers were dipped in a 10:1 hydrofluoric acid:water solution to remove the native oxide layer, 25 then treated with sulfuric acid (H2S04) and H22 to grow a thin chemical oxide film. The samples were oxidized for 8 hours in an electrically-heated quartz furnace tube maintained at 1000C in pure oxygen bubbled through deionized water resulting in an sio2 film 30 approximately 1.0 ~m thick. A single-mask lithographic sequence was employed to pattern the wafers.
Hexamethyl disilazane was applied to the obverse face of the samples followed by AZ4210 photoresist spun at 6000rpm for 60 ~eCon~-C. The photoresist solvent was 35 expelled with a 20 minute bake at 90C. An emulsion mask cont~;ning 10 ~m2 patterns on a clear field was used in a contact aligner to expose the photoresist.
The edges of the patterns aligned to the {011}
WOg6/~ 9 ~ ~ ~ }~ SI12381 directions. After e~G~e, the samples were developed in a 3:1 solution of deionized water and AZ400K for 60 ~?.CQn~, then har~h~ke~ for 30 minutes at 120C. The developed photoresist pattern was then transferred to S the SiO2 film by etching in buffered hydrofluoric acid.
Etch completion was determined by observation of the hydrophobic surface transformation on the wafer backsides. The wafers were subjected to ~ A~P
rinsing in deionized water followed by photoresist lo removal in ~llrce~cive baths of acetone, methanol, and deionized water. -The microbarbs are formed by anisotropicsilicon etc~ing in an aqueous solution of XOH at 85C.
The etchant attacks the ex~G~cd {100} silicon surface, but etches the protective Sio2 pattern very slowly.
These etching conditions result in undercutting of the convex corners of the mask, exposing {411} oriented planes. These facets etch rapidly until they meet the center resulting in a sharp point and mask liftoff.
The etch timing is monitored to ensure that the microbarbs are not further eroded.
E~AMPLE 2 Transient Espre~Yion of DNA in Nicotiana Tabacum After Microprobe-Ne~iate~ Transformation 2~ To demonstrate that a biological material can be deposited into cells by this method, an experiment was carried out using Tobacco (Nicotiana Tabacum W3) leaf tissues. The DNA of interest was the plasmid pB
121 which contains a kanaymycin resistance gene and a GUS assay sensitive protein (glucuronidase). The mi~. G~ obes were prepared according to Example 1. The silicon substrate had a dimension of 5 mm by 8 mm and cont~i~P~ approximately 200 25-~m height mi~o~obes.
The probes were soaked in 95% ethanol for 20-30 minutes and washed 3 times with autoclaved distilled deionized H20, then air dried thoroughly. A DNA mix was made using 5 ~g DNA in TE buffer, 12.5 ~1 of 2.5 M CaC12, and 5 ~1 of 0.1 M spermidine free base. The epidermal wogc/10630 ~ 3 ~ ~ P~ 2381 layer of the severed tobacco leaf was aseptically removed using f Gl ~ . To prepare the mi~uy~bes for the plant material, 2.5 ~g of DNA preparation were placed on the mi~Lo~lobes and the plant materiaI was S placed on the mi~ L obes. A cotton swab was used to rub the back-side of the leaf sample to encourage the penetration by the mi~u~obes. The plant material was then removed from the mi~,o~bes and then cultured for 72 hours on a regenerative tobacco medium.
To evaluate the effectivene~s of the technique, one experimental group and three control yLo~ were conducted. The experimental group was applied to the DNA coated mi~Lop~obes. The first control group was applied to microprobes that were without DNA solution. The ~?con~ control group was made to contact the DNA solution, but without the mi~lo~obe treatment. The third control group was neither exposed to DNA solution nor was treated with the mi~u~obes. Tobacco tissues were examined by applying a GUS assay to evaluate the amount of DNA
taken up by the tobacco cells, following the procedures described in Jefferson, GUS Gene Fusion SYstem User's Manual, Cambridge (July 1987) Successful plasmid uptake by the cells results in~the presence of typical blue st~; ni ng. The experimental group showed positive transient symptoms (i.e., dark blue-green dots), while an AhcPnc~ of such stAining was observed in all three control ~ ou~=.
E~ANP~E 3 Nemato~e Transformation The mi~lu~obe-mediated transformation was used to generate transgenic nematodes. pPCZl and pRF4 were the DNAs used. pPCZ1 contained the C. eleqans 16kDa heat shock promoter fused to the E. coli B-- 35 galactosidase gene. pRF4 contained a 4Kb EcoRl genomic fragment of C. elegans encoAing the rol-6 (Su1006) collagen gene as described in Rramer et al., Mol. Cell.
Biol. 10:2081-89 (1990). The rol-6 gene was provided Wos6/10630 ~ PCIIUS9SI12381 by Peter Candide, University of British Columbia, Vancouver, B.C.
HeL~u~habd~tis bacterihora HP88 were collected at 4-6 eggs stage from lipid agar seeded with S r~hotorhabdus ltlminescence in double distilled sterile water and washed three times with sterile water. The miu.u~lobes of Example 1 were placed with the microprobes pointing up on the ~urface of a lipid agar plate seeded with ~hotorhabdus luminescence. A DNA mix lo containing 5 ~g DNA in TE buffer, 12.5 ~1 of 2.5 M
CaC12, and 5 ~ 1; of 0.1 M spermidine-free base. Ten ~ 1 of DNA mix were pipetted onto the mi~;Lop~obes to coat the tips with DNA. After 3 minutes, 10 ~1 of a highly concentrated nematode suspension (approximately 200-250 lS nematodes) were pipetted on top of the microprobes and left at room temperature for 8 to 10 minutes. Most nematodes crawled off the mi~;~u~be array onto the media. The array was then removed from the plate, and the agar plate contAini~g nematodes was inc~-hAted at 20 25C, until the injected nematodes proAIlr~i progeny.
Control experiments, wherein a mi~;.c,p~be array without DNA was used, were also conducted.
After the incubation period, hermaphrodite progeny were examined for roller phenotypic expression 25 of the transformants. Progeny were assayed for expression of the hs~-16-lacZ fusion gene by heat shocking for 2 hours at 33C as described in Stringham et al., Mol. Biol. Cell. 3:221-33 (1992). The nematodes were then permeabilized by lyophilization and 30 acetone treatment and incubated at 25C in a stArl~lArd histochemical contA i n i ng 3% X-gal as described in Fire EMBO J. 5:2673-80 (1986).
Nematodes were scored for the presence of typical blue S1-A i ni rlg of b-galactosidase; 8% of the 35 total progeny showed b-galactosidase expression in the first (Fl) generation. No stAining was observed in the control groups. Although the nematodes were heritable transformed individuals exhibited a non-Men~lPliAn Wos6/~ PCT~S9511~81 pattern of inheritance similar to those of C. eleqans (extrachromosomal transformants), there was no genome integration.
INDUSl~TAT. APpT-TcABI~-TTy S The present invention provides a simple, economical and precise method for i.-Llod~cing a biological material of interest into a predetermined target cell population, in ~tro or in s~tu. Thus, it is u~eful in both animal and plant applications, lo including therapeutics and plant biotechnology. The present invention can accommodate virtually all cell types and biological materials, and offers ceveral additional advantages over known biological material, e.g., gene, transfer methodologies. First, the 15 location of the introduction of the foreign biological materials and the penetration depth can be precisely controlled by varying the dimensions of the mi~opLobes, the injection pattern or both, thus "customizing" the method to any given target cell 20 population. Second, cell damage is minimized; the degree of penetration of each probe into the cell, and the size of the holes or perforations in the cell membrane, are controlled by the geometry of the microprobes. Third, the method is simple; it does not 2S require complicated preparatory steps associated with biolistic t~rhniques. Fourth, the method is economical--the mi~. U~L obe-substrate structure is inexpensive to manufacture, and can be easily sterilized and/or disposed of. Fifth, a larger number 30 of cells are treated simultaneously, compared with prior art microinjection tech~iques which are limited to the treatment of single cells at a time.
All publications cited in the specification are indicative of the level of ~kill of those skilled 3S in the art to which this invention pertains. All these publications are herein in~o ~o.ated by reference to the same extent as if each individual publication were W0961106~ ~ rCT~S9S/1 ~pecifically and individually indicated to be in~oL~o~ated by reference.
Further modifications of the invention described herein become apparent to those skilled in S the art. Such modifications are intended to fall within the scope of the appended claims.
Claims (45)
1. A method of introducing a biological material of interest into a predetermined target cell population, characterized by the steps of:
providing (a) a plurality of microprobes positioned on a support (b) a solid or quasi-solid mass of the target cells defining an interface with the microprobes, and (c) the biological material at the interface; and physically contacting the cells with the microprobes to cause the microprobes to non-lethally pierce the cell membranes of the cells.
providing (a) a plurality of microprobes positioned on a support (b) a solid or quasi-solid mass of the target cells defining an interface with the microprobes, and (c) the biological material at the interface; and physically contacting the cells with the microprobes to cause the microprobes to non-lethally pierce the cell membranes of the cells.
2. A method according to claim 1, wherein the microprobes are pyramidally shaped.
3. A method according to claim 1, wherein the microprobes are needle-shaped.
4. A method according to claim 1, wherein the microprobes are barbed shaped.
5. A method according to any of claims 1-4, wherein the microprobes and the support are integral with one another, and are prepared by etching a wafer.
6. A method according to claim 5, wherein the wafer comprises a single crystalline material.
7. A method according to claim 5, wherein the etching is anisotropic.
8. A method according to claim 5, wherein the etching is isotropic.
9. A method according to claim 5, wherein the material is silicon.
10. A method according to claim 5, wherein the material has a top surface cut in the {1 0 0}
direction.
direction.
11. A method according to claim 5, wherein the microprobes are formed by etching to the {411}
planes of the wafer.
planes of the wafer.
12. A method according to any of claims 1-4, wherein the microprobes have a height of from about 10 microns to about 300 microns.
13. A method according to any of claims 1-4, wherein the microprobes have a height of from about 20 to 90 microns.
14. A method according to any of claims 1-4, wherein the microprobes have a tip size of from about 0.5 to about 10 microns.
15. A method according to any of claims 1-4, wherein the microprobes have a base size of from about 30 to about 80 microns.
16. A method according to any of claims 1-4, wherein the distance between the microprobes is from about 1.0 to about 20 times the height of the microprobes.
17. A method according to any of claims 1-4, wherein the support has a thickness of from about 200 to about 550 microns.
18. A method according to claim 1, wherein the biological material is an organic or inorganic chemical.
19. A method according to claim 18, wherein the biological material is a pharmaceutical agent.
20. A method according to claim 1, wherein the biological material is a protein.
21. A method according to claim 20, wherein the protein is an enzyme.
22. A method according to claim 1, wherein the biological material is a nucleic acid.
23. A method according to claim 22, wherein the nucleic acid contains a DNA molecule encoding a protein of interest.
24. A method according to claim 1, wherein the predetermined target cell population contains eucaryotic cells.
25. A method according to claim 24, wherein the eucaryotic cells are animal cells.
26. A method according to claim 25, wherein the animal cells are human cells.
27. A method according to claim 25, wherein the animal cells are epidermal cells.
28. A method according to claim 24, wherein the eucaryotic cells are plant cells.
29. A method according to claim 28, wherein the plant cells are monocotyledonous cells.
30. A method according to claim 28, wherein the plant cells are dicotyledonous cells.
31. A method according to claim 1, wherein the predetermined target cell population contains procaryotic cells.
32. A method according to claim 1, wherein said step of physically contacting characterized by the step of contacting the microprobes with the target cells in situ.
33. A method according to claim 1, wherein said step of contacting is repeated at least twice, wherein prior to each repetition the disposition of the target cells relative to the microprobes is changed such that upon said contacting step, a different injection pattern over the previous repetition is achieved.
34. A method according to claim 1, wherein said step of providing the biological material is characterized by applying the biological material to the microprobes, the support or both the microprobes and the support.
35. A method according to claim 1, wherein said step of providing the biological material is characterized by applying the biological material to the target cells.
36. A method according to claim 1, wherein said step of physically contacting is characterized by applying a predetermined force to the support.
37. A method according to claim 1, wherein said step of providing is characterized by providing the plurality of microprobes positioned on a first support and the target cells positioned on a second support.
38. A method according to claim 37, wherein said step of physically contacting is characterized by applying a predetermined force to the first support, the second support, or to both the first and second supports.
39. A method according to claim 1, further characterized by the step of applying electric pulses to the microprobes.
40. A method of introducing a biological material of interest into a predetermined target cell population, characterized by the steps of:
providing in a liquid medium the target cells, the biological material, and a plurality of microprobes positioned on a support, the microprobes and support being integral with one another and having been prepared by etching a single crystalline wafer material, and subjecting the liquid medium containing the target cells, the biological material, and the microprobes to physical motion under conditions sufficient to cause the microprobes to non-lethally, pierce the cell membranes of the cells.
providing in a liquid medium the target cells, the biological material, and a plurality of microprobes positioned on a support, the microprobes and support being integral with one another and having been prepared by etching a single crystalline wafer material, and subjecting the liquid medium containing the target cells, the biological material, and the microprobes to physical motion under conditions sufficient to cause the microprobes to non-lethally, pierce the cell membranes of the cells.
41. A method according to claim 40, further characterized by the step of applying electric pulses to the medium.
42. A composition of matter, characterized by:
a plurality of microprobes positioned on the surface of a support, and a biological material disposed on the microprobes, the surface of the support, or both the microprobes and the surface of the support.
a plurality of microprobes positioned on the surface of a support, and a biological material disposed on the microprobes, the surface of the support, or both the microprobes and the surface of the support.
43. A composition of matter, according to claim 42, wherein said support and said microprobes contain silicon.
44. A composition of matter, according to claim 42 or claim 43, wherein said biological material contains DNA.
45. A nematode transformed with a recombinant DNA molecule, said molecule characterized by a promoter which in its native state is associated with a C. elegans heat shock protein gene, operably linked to an E. coli .beta.-galactosidase structural gene.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US31533694A | 1994-09-30 | 1994-09-30 | |
| US08/315,336 | 1994-09-30 | ||
| US33460694A | 1994-11-04 | 1994-11-04 | |
| US08/334,606 | 1994-11-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2201315A1 true CA2201315A1 (en) | 1996-04-11 |
Family
ID=26979842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2201315 Abandoned CA2201315A1 (en) | 1994-09-30 | 1995-09-29 | Direct introduction of foreign materials into cells |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0782614A1 (en) |
| AU (1) | AU3758595A (en) |
| CA (1) | CA2201315A1 (en) |
| WO (1) | WO1996010630A1 (en) |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9815819D0 (en) * | 1998-07-22 | 1998-09-16 | Secr Defence | Transferring materials into cells and a microneedle array |
| AU6216199A (en) | 1998-10-08 | 2000-04-26 | Astrazeneca Ab | Microfabricated cell injector |
| EP1333880B1 (en) | 2000-10-26 | 2009-04-15 | Alza Corporation | Transdermal drug delivery devices having coated microprotrusions |
| DE60307640T2 (en) * | 2002-06-28 | 2007-09-13 | Alza Corp., Mountain View | Administration and optimization of the transdermal delivery of an agent through the skin |
| KR20120087197A (en) * | 2002-07-19 | 2012-08-06 | 쓰리엠 이노베이티브 프로퍼티즈 컴파니 | Microneedle device, method of using microneedle device and method of delivering microneedle device |
| MXPA06000281A (en) | 2003-06-30 | 2006-07-03 | Johnson & Johnson | Formulations for coated microprojections containing non-volatile counterions. |
| WO2005020912A2 (en) | 2003-08-25 | 2005-03-10 | 3M Innovative Properties Company | Delivery of immune response modifier compounds |
| US7132242B2 (en) * | 2003-11-10 | 2006-11-07 | Hewlett-Packard Development Company, L.P. | Method and device for targeted delivery of materials to selected single cells |
| WO2006055799A1 (en) | 2004-11-18 | 2006-05-26 | 3M Innovative Properties Company | Masking method for coating a microneedle array |
| US8057842B2 (en) | 2004-11-18 | 2011-11-15 | 3M Innovative Properties Company | Method of contact coating a microneedle array |
| ATE504328T1 (en) | 2004-11-18 | 2011-04-15 | 3M Innovative Properties Co | MICRONEEDLE ARRANGEMENT APPLICATOR AND HOLDER |
| JP5015787B2 (en) | 2004-11-18 | 2012-08-29 | スリーエム イノベイティブ プロパティズ カンパニー | Contact coating method of microneedle array |
| AU2005314151B2 (en) | 2004-12-07 | 2011-09-08 | 3M Innovative Properties Company | Method of molding a microneedle |
| JP5301985B2 (en) | 2005-04-07 | 2013-09-25 | スリーエム イノベイティブ プロパティズ カンパニー | System and method for tool feedback sensing |
| EP1904158B1 (en) | 2005-06-24 | 2013-07-24 | 3M Innovative Properties Company | Collapsible patch with microneedle array |
| CA2613111C (en) | 2005-06-27 | 2015-05-26 | 3M Innovative Properties Company | Microneedle array applicator device and method of array application |
| AU2006261899B2 (en) | 2005-06-27 | 2012-05-10 | Kindeva Drug Delivery L.P. | Microneedle cartridge assembly and method of applying |
| WO2007061964A1 (en) | 2005-11-18 | 2007-05-31 | 3M Innovative Properties Company | Methods for coating microneedles |
| US20070142781A1 (en) * | 2005-12-21 | 2007-06-21 | Sayre Chauncey B | Microinjector chip |
| US8632801B2 (en) | 2005-12-28 | 2014-01-21 | Alza Corporation | Stable therapeutic formulations |
| US9119945B2 (en) | 2006-04-20 | 2015-09-01 | 3M Innovative Properties Company | Device for applying a microneedle array |
| JP4984736B2 (en) * | 2006-08-18 | 2012-07-25 | 凸版印刷株式会社 | Exposure apparatus and method |
| JP5070764B2 (en) * | 2006-08-18 | 2012-11-14 | 凸版印刷株式会社 | Microneedle patch manufacturing method |
| WO2008076465A1 (en) * | 2006-12-21 | 2008-06-26 | Primegen Biotech, Llc | Microinjector chip |
| DE102009005003A1 (en) * | 2008-08-01 | 2010-03-25 | Frankenförder Forschungsgesellschaft mbH | Device for mechanical disintegration of cells, comprises elements movably arranged to each other, where one element has micro-mechanical structure for cell perforation and other element has hollow space to receive cells, and adjusting unit |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4849355A (en) * | 1985-01-08 | 1989-07-18 | Wong Tai Kin | Method of transferring genes into cells |
| US5302523A (en) * | 1989-06-21 | 1994-04-12 | Zeneca Limited | Transformation of plant cells |
| US5262128A (en) * | 1989-10-23 | 1993-11-16 | The United States Of America As Represented By The Department Of Health And Human Services | Array-type multiple cell injector |
| US5457041A (en) * | 1994-03-25 | 1995-10-10 | Science Applications International Corporation | Needle array and method of introducing biological substances into living cells using the needle array |
-
1995
- 1995-09-29 EP EP95935656A patent/EP0782614A1/en not_active Withdrawn
- 1995-09-29 WO PCT/US1995/012381 patent/WO1996010630A1/en not_active Application Discontinuation
- 1995-09-29 AU AU37585/95A patent/AU3758595A/en not_active Abandoned
- 1995-09-29 CA CA 2201315 patent/CA2201315A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU3758595A (en) | 1996-04-26 |
| WO1996010630A1 (en) | 1996-04-11 |
| EP0782614A1 (en) | 1997-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2201315A1 (en) | Direct introduction of foreign materials into cells | |
| US5137817A (en) | Apparatus and method for electroporation | |
| US5457041A (en) | Needle array and method of introducing biological substances into living cells using the needle array | |
| Van West et al. | Oomycete plant pathogens use electric fields to target roots | |
| EP1522578B1 (en) | Transferring materials into cells using porous silicon | |
| US6686299B2 (en) | Nanosyringe array and method | |
| JP3035608B2 (en) | Microcapillary array, method for manufacturing the same, and substance injection device | |
| EP0444882B1 (en) | Plant transformation process with early identificaton of germ line transformation events | |
| JP4842512B2 (en) | Combined parallel drug delivery and electroporation of cellular structures and uses thereof | |
| JP2002325572A (en) | Method for introducing exogenous substance | |
| US6563017B2 (en) | In vivo electroporation method for early stage embryo of chickens | |
| AU2008275064A1 (en) | Methods and devices for charged molecule manipulation | |
| Trimmer et al. | Injection of DNA into plant and animal tissues with micromechanical piercing structures | |
| EP3563673A1 (en) | Method for introducing substance into plant | |
| WO2007076458A1 (en) | Microinjector chip | |
| JPH06343478A (en) | Micro-injection method and apparatus | |
| JPH11511974A (en) | Electroporation-mediated molecule transfer into intact plants | |
| US6617153B2 (en) | Portable magneto-mechanical delivery device and method for delivery of reactive substances | |
| WO2001009297A1 (en) | A method for selective electrofusion of at least two fusion partners having cell-like membranes | |
| CA2020265C (en) | Method and apparatus for the acceleration of a propellable material | |
| O’ Brochta et al. | Transformation systems in insects | |
| Banerjee et al. | Transient expression of β-glucuronidase in embryo axes of cotton by Agrobacterium and particle bombardment methods | |
| US20040182706A1 (en) | Microinjection method and device based on electroosmosis | |
| Paik et al. | A highly dense nanoneedle array for intracellular gene delivery | |
| Kościańska et al. | Electroporated intact BY-2 tobacco culture cells as a model of transient expression study. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |