WO2002058847A2 - Transformation cellulaire a l'aide d'un reseau microfabrique de micropuces en silicium comprenant des injecteurs de micro percage integres - Google Patents

Transformation cellulaire a l'aide d'un reseau microfabrique de micropuces en silicium comprenant des injecteurs de micro percage integres Download PDF

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WO2002058847A2
WO2002058847A2 PCT/US2001/044289 US0144289W WO02058847A2 WO 2002058847 A2 WO2002058847 A2 WO 2002058847A2 US 0144289 W US0144289 W US 0144289W WO 02058847 A2 WO02058847 A2 WO 02058847A2
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cells
microdevice
microchambers
substances
micro
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WO2002058847A9 (fr
WO2002058847A3 (fr
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Makarand Paranjape
Mark A. Esrick
John F. Currie
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Georgetown University
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    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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

Definitions

  • This invention relates to methods for transforming or transfecting a plurality of individual cells with nucleic acids, small drug doses or other molecules of interest, and particularly to microfabricated devices for performing such transformations and other minute operations on a high throughput level.
  • Cell transformation is a procedure often used by researchers in genetics, cell biology, and molecular biology that results from the introduction of specific molecules, such as DNA, RNA, and low-dose drugs into the nucleus or cytoplasm of a recipient cell. Transformation allows for the identification of novel genes, the isolation of genetically modified cells, and the screening of potential drugs, and much effort has been focused on ensuring that transformation efficiencies are optimized in order to increase the retrieval of transformed cells and/or decrease the number of cells which must be treated.
  • Retroviral retro viruses may be used to accurately integrate a single copy of a gene into the genome of a target cell.
  • such vectors cannot infect non-dividing or fully differentiated cells, e.g., neurons or hepatic cells, unless they are stimulated to divide, and can only accommodate genes and other nucleic acids that are less than 8 kilobases long.
  • purification and concentration of retroviruses without loss of infectivity is difficult, and stable transfectants which exhibit long-term expression of transfected genes are rare.
  • Recombinant adenoviruses are advantageous in that they infect non-dividing cells and may be concentrated without significant loss of infectivity.
  • these viruses can only incorporate genes which are less than 5 kilobases, and their genome does not integrate into the target cell's genome but rather remains episomal, resulting in transient gene expression.
  • Electroporation employs an electric pulse applied to a cell/DNA suspension, and is believed to induce local areas of reversible membrane breakdown thereby creating pores through which the DNA enters the cell.
  • electroporation is effective for a large number of different cell types and is relatively reproducible and easy to perform, it requires more cells and DNA than chemical methods and there is typically a large variation in optimizing parameters between different cell lines, e.g., field strength, pulse duration.
  • efficiency may vary depending on the cell type, and the non-physiological conditions required in the medium limit cell viability.
  • Hollow micro-capillaries have also been designed which permit controlled injection of DNA and other materials into cells, whereby the hollow micro- capillaries are inserted into individual cells trapped in microchambers within the surface of a silicon wafer and the material of interest is injected.
  • McAllister et al Three- Dimensional Hollow Micro-Needle and Micro-Tube Arrays, Transducers '99, 1098-1101, Sendai, Japan; see also Chun et al, An Array of Hollow Microcapillaries for the Controlled Injection of Genetic Materials into Animal/Plant Cells, 12 th IEEE Int'l Conf. on Microelectromechanical Systems, Orlando, FI, Jan. 1999, pp. 406-411.
  • Microprobes or short points covered with genetic material have also been used to pierce cells and introduce the genetic material into an array of individual cells.
  • microfabricated devices are advantageous in the control they provide over transfection conditions, the ease of operation, and the level of transfection efficiency achieved, the apparatus requires two separate devices (micro-capillary array for injection and micro-chamber array for holding the cells).
  • Such a system inherently possesses critical alignment problems resulting in variations in injection efficiency, and typically requires a further device for positioning the first two devices accurately with respect to one another. See Leighton and Brownstein, U.S. Patent 5,262,128.
  • the micro-capillary needles are fragile and therefore not very durable, and require a complex and specialized fabrication process.
  • the present invention overcomes the deficiencies of the prior art by providing a cost effective, durable apparatus that enables essentially simultaneous transformation (into the cytoplasm) or transfection (into the nucleus) of a plurality of cells with a wide variety of molecules and substances. More particularly, the present invention provides a silicon wafer containing an array of micro-cavities fitted with hollow needle like protrusions allowing individual cells to be simultaneously trapped and pierced for material injection or extraction.
  • the micro-device of the present invention is advantageous over devices of the prior art in that a large number of cells may be easily transformed or transfected in a single step with minimal loss of cell viability, requiring minimal expertise and handling at an optimum cost.
  • the microdevices of the present integrate micro- piercing injectors into the design of the micro-chambers, the present devices are simpler to make, e.g., are made using standard silicon processing and micromachining technologies, and are more durable than the silicon chip injection units of the prior art.
  • the micro- devices of the present invention are one-piece injection modules that require no critical alignment procedures.
  • Cell transformation, transfection and fusion may be performed in a parallel process with the potential for high throughput using multi-level stacked elements.
  • Transformed and transfected cells produced by the methods of the invention are also included, as are a variety of supplementary devices and connections which facilitate collection and analyses of the transformed cells.
  • Figure 1 Diagram of a preferred cell transformation microdevice of the present invention.
  • Figure 2 Enlarged view of a single micro-piercing injector.
  • Figure 3 Cross-sectional view of cavity formation using a masking layer with an isotrophic etchant.
  • Figure 4 Top and cross-sectional view of the annulus masking pattern.
  • Figure 5 Cross-sectional views of the etch profile of isotropic etching for varying times.
  • Figure 6 Cross-sectional view of the micro-injector with inlet port.
  • FIG. 7 Fabrication process flow for (a) ultra-thin wafer, and (b) SOI wafer. The only difference in the fabrication sequence shown above is in the first step where front-side lithography is done on the SOI wafer, while back-side lithography on the ultra-thin wafer.
  • Figure 8 An SEM of the test structure for determining RIE isotropy.
  • Figure 9 An SEM of the annuli test structure.
  • Figure 10 SEM of one annulus test structure.
  • Figure 13 Results of trial at 95/0 W with varied percentage O 2 (a) RIE etch rate; (b) Si/SiO 2 selectivity; and (c) Silicon etch anisotropy.
  • Figure 14 SEM's illustrating the anisotropy and etch profiles for the trial of Figure 13 using (a) 10% O 2 and (b) 20% O 2 .
  • Figure 15 Results of trial at 205/5 W with varied percentage O 2 (a) RIE etch rate; (b) Si/SiO 2 selectivity; and (c) Silicon etch anisotropy.
  • FIG. 17 Plexiglass, water tight CTM macro-model for pressure analysis used in conjunction with one embodiment of the invention.
  • Figure 18 Cross-section of micro-injector with inlet and venting ports in accordance with a preferred embodiment of the invention.
  • Figure 19 Device according to a preferred embodiment of the invention using polydimethylsiloxane silicone rubber (PDMS).
  • PDMS polydimethylsiloxane silicone rubber
  • Figure 20 SEM's of (a) SU-8 mold and (b) PDMS layer made therefrom.
  • the present invention encompasses microfabricated array/injection devices for transforming simultaneously a plurality of cells.
  • a preferred microdevice structure is based on a single chip module containing a large array of microchambers or micro-wells in a silica-based or silicon substrate that house recipient cells to be transformed (Fig. 1).
  • Inlet ports located at the bottom of each microchamber are integrated into the device in such a way as to simultaneously render micro-piercing injectors during micro-chamber fabrication (Fig. 2).
  • the silicon structure is bonded with a capping substrate on the top in order to introduce recipient cells into the micro-chambers and hold them in place, preferably by the application of hydrostatic pressure, and also with a bottom substrate that contains micro-fluidic channels for biological and molecular component transport (Fig. 1).
  • the cells are allowed to settle into the bottom of each micro- chamber, and when all cavities are filled, hydrostatic pressure is applied from above to trap and hold them in place.
  • the cell membrane is perforated mechanically by the micro-piercing structures.
  • the inlet ports act as micro-injectors through which biological material or molecules are introduced into the cell using positive pressure applied beneath the micro-injector array. This pressure is applied shortly after the trapping hydrostatic pressure in order to prevent the escape of cell cytoplasm through the perforation.
  • the present invention encompasses a microdevice for introducing molecules or substances into a plurality of cells, comprising (a) a single microfabricated array substrate having a plurality of individual microchambers, wherein each microchamber holds at least one cell and incorporates an integrated micro-piercing injector; (b) a top planar substrate for entrapping individual cells in microchambers; and (c) a bottom planar substrate enclosing flow channels which run beneath said microchambers; wherein said microchambers and said flow channels are connected by individual inlet ports through said micro-piercing injectors.
  • Manufacturing the microdevice elements into the substrate may be carried out using microfabrication techniques known in the art including photolithography etching, plasma etching or wet chemical etching. Alternatively, micromachining methods such as laser drilling, micromilling and the like may be employed.
  • the molecules or substances to be introduced into the plurality of cells may be introduced into the cytoplasm (transformation) or the nuclei (transfection).
  • the molecules or substances to be introduced may be any molecules or substances of interest, but are typically selected from the group consisting of DNA, RNA, ribozymes, molecular probes, hormones, growth factors, enzymes, proteins, drugs, organic chemicals, inorganic chemicals, viruses and expression vectors.
  • Organelles such as nuclei, mitochondria, chloroplasts and the like may also be introduced into the cytoplasm of the target cells using the disclosed devices. Introduction of nuclei, for example, is particularly useful for technologies like cloning which employs nuclear transfer into a recipient oocyte.
  • the cells to be transformed by the present invention may be any cells of interest.
  • the cells may be selected from the group consisting of somatic cells, oocytes, stem cells, mammalian cells, spleen cells, myeloma cells, and plant cells. Transfection of oocytes and stem cells may have particular use in the transfection of transgenes in the design of transgenic animals.
  • the recipient cell is an oocyte
  • the material to be inserted may also be another cell, such as a sperm cell or a stem cell in the case of nuclear transfer into an enulceated oocyte. Methods of nuclear transplantation are well known in the art as evidenced by U.S. Patent No.
  • microdevices of the present invention are also useful for fusing spleen cells and myeloma cells, i.e., hybridoma technology, for the purpose of making monoclonal antibodies.
  • U.S. Patent No. 4,822,470 of the Baylor College of Medicine discloses a method for the poration and fusion of cells using radiofrequency electrical pulses in hybridoma technology and is herein incorporated by reference. Such applications could readily be accomplished using the micropores created by the present transformation devices.
  • spleen cells from an immunized mammal could be isolated and entrapped into individual microwells of the disclosed devices, thereby being perforated and fused with a myeloma cell essentially simultaneously.
  • the fused hybridoma cells could then be collected and screened for the production of antibodies having specific binding characteristics.
  • microdevices of the present invention may be designed having microchambers of varying size to accommodate specific cell types.
  • Preferred individual microchambers range in size from approximately 5 microns and above.
  • individual inlet ports may be designed in order to accommodate a specific molecule, substance or cell to be introduced into the target recipient cell in order to better control the amount of material entering cells during transformation. More specifically, inlet ports may range in size from about 1 micron and above.
  • the microfabricated array module of the present invention made be made of any material commonly used in the micromachining art.
  • such materials include silica, silicon, silicon carbide and gallium arsenide to name a few.
  • the array substrate should be made of a microfabrication facilitating substance which may also be heated or cooled depending on the use of the device.
  • the top and bottom planar substrates are most preferably glass, but may also be made of any suitable micromachining material such as silica, silicon, silicon carbide, gallium arsenide, glass, silicon elastomer (silicone), fused quartz, plastics and photo-etchable glass (Foturan ).
  • the microdevice is designed with micro- fluidic flow channels beneath the microchambers that deliver biological and other molecules to the entrapped cells on the array.
  • the device may be specially designed wherein independent, unconnected flow channels feed different groupings of microchambers for simultaneous targeting of different molecules or substances to different cells on the array.
  • the microdevice may also include multiple layers of arrays, bottom and top substrates for high throughput, and also for delivering different molecules simultaneously through independent, unconnected flow channels.
  • the cross-sectional dimension of an individual flow channel will depend on the particulr application of the microdevice. For instance, if cells are to be transformed simultaneously with the same molecule or substance, one flow channel may feed more than one well in the array. Alternatively, individual unconnected flow channels may be designed for the purpose of feeding individual inlet ports. Flow channels may also be fluidly connected to a fluid feeding and/or direction system for introducing and/or directing said molecules or substances into said microdevice.
  • the microdevices of the present invention entrap cells into microchambers using applied hydrostatic pressure.
  • the devices may be further equipped with a pressure means for pressurizing or applying pressure to the top substrate, whereby the amount of pressure may be easily controlled by the operator. Suction from below the trapped cells could also be used to assist in entrapment of cells, either independently or in conjunction with hydrostatic pressure.
  • the microdevices of the present invention are preferably designed with an array substrate that conducts heat, so that that cells may be heated or cooled depending on the micro-operation to be transformed.
  • the array substrate may be further connected to a heater element, and said heater further connected to an adjustable power source.
  • a temperature sensor and monitoring means would also be incorporated so that the operator could readily adjust and monitor temperature levels.
  • it may be advanageous for the array substrate to be connected to a voltage supply which provides an adjustable electrical pulse, for instance, for nuclear transfer applications.
  • Miniaturized devices such as heaters and voltage devices for carrying out a variety of synthetic and diagnostic operations are described in U.S. Patent No.
  • the top planar substrate of the microdevices described herein may incorporate openings for washing away untrapped cells, and/or supplying or washing away medium or specific molecules or chemicals or radioactive labels to or from entrapped cells (see Fig. 1).
  • the array substrate and/or top substrate may be optionally connected to a sample handling system that permits the transfer of cells from microchambers to outside analytical or collection devices.
  • a particularly useful sample handling system comprises individual exit ports for each microchamber, or groups of microchambers, wherein said exit ports are connected to individual flow channels.
  • Cells may be collected or routed into sample handling devices using any convenient means known in the art.
  • the sample handling system may be further connected to a vacuum or pressure means for effectuating movement of said cells from said microchambers into said exit ports and/or said exit channels. Electrical currents and thermal expansion may also be used to effectuate sample movement.
  • U.S. Patent No. 5,872,010 Northeastern University
  • U.S. Patent No. 5,872,010 Northeastern University
  • Similar techniques may be applied to the chips of the present invention following cell transformation using exit port adaptors and flow channels. Cell-sorting using magnetically tagged cells and an external magnetic field could also be used.
  • outside analytical or collection devices which may be used in conjunction with the microdevices described herein include secondary microfabricated arrays of microchambers or multiwell plates, e.g., for culturing cell populations from individual transformed cells; filters or films for conducting hybridization, e.g., Southern, Northern and Western analyses; apparatus for receptor/ligand analyses, e.g. screening transformed cells for those which express a particular ligand or receptor and bind to another molecule or protein of interest; apparatus for immunological screening, e.g., of antibody producing hybridoma cells; devices for radioactivity measurements,e.g., of transformed cells labeled with a radioactive isotope; flow cytometry or FACS apparatus, e.g. to screen cells for the expression of surface proteins encoded by transfected genes or for other changes in gene expression; mass spectrometry or nuclear magnetic resonance analyses, chromatography, and fluorescence imaging.
  • secondary microfabricated arrays of microchambers or multiwell plates e.g., for culturing cell populations from individual transformed
  • the present invention also includes methods of using the disclosed microdevices for introducing molecules or substances simultaneously into a plurality of cells, and also the transformed, transfected or fused cells produced thereby. Methods of using the transformed cells for diagnostic applications and further analyses as proposed above are also included. For instance, the transformed cells of the present invention could be used to identify genes of interest, for high throughput hybridoma screening and efficient identification and isolation of monoclonal antibodies, for the production of useful proteins, for the screening of drugs and pharmaceuticals, and for the production of transgenic animals.
  • the present invention may be distinguished from silicon chip-based micro-injection techniques of the prior art by the single module nature of the microchamber/injection apparatus. Accordingly, the present invention includes a method for simultaneously positioning and perforating a plurality of cells, comprising (a) positioning cells on a single microfabricated array substrate that incorporates integrated micro-piercing structures within microchambers; and (b) entrapping cells in said microchambers using hydrostatic pressure applied from above such that said cells are perforated by said micro-piercing structures. Molecules, substances, organelles or other cells of interest may be introduced into or extracted from said plurality of cells during perforation.
  • the microdevice may also be used to remove cellular contents for the purpose of isolating cell membranes, e.g., erythrocyte ghosts.
  • the plurality of cells is exposed to said molecules, substances, organelles or other cells of interest by way of a flow channel encased by a bottom substrate underneath said array substrate.
  • kits comprising the disclosed microdevices, which may optionally comprise accessory devices such as a heater and power source for altering the temperature of the array substrate.
  • accessory devices such as a heater and power source for altering the temperature of the array substrate.
  • Kits of the present invention may further comprise a sample handling system comprising individual exit ports and flow channels for each microchamber or group of microchambers, which may be optionally connected to a vacuum or pressure means for effectuating movement of said cells from said microchambers into said exit ports and/or said exit channels for subsequent analysis or collection.
  • a sample handling system comprising individual exit ports and flow channels for each microchamber or group of microchambers, which may be optionally connected to a vacuum or pressure means for effectuating movement of said cells from said microchambers into said exit ports and/or said exit channels for subsequent analysis or collection.
  • the microdevice structure (Fig. 1) is based on a single chip module containing a large array of microchambers in a silicon substrate that houses recipient cells to be transformed (Fig. 1). Inlet ports located at the bottom of each microchamber are integrated into the device in such a way as to simultaneously render micro-piercing injectors during microchamber fabrication (Fig. 2).
  • the silicon structure is bonded with a glass substrate on the top in order to introduce recipient cells into the microsystem, and to allow the application of hydrostatic pressure.
  • a glass substrate is also bonded to the bottom of the device, which contains the micro-fluidic channels for biological and molecular component transport (Fig. 1).
  • the cells are allowed to settle onto the bottom of each microchamber, and when all microchambers are filled by the cells, hydrostatic pressure is applied from above to trap and hold them in place.
  • the cell membranes are perforated mechanically by the micro-piercing structures.
  • the inlet ports then act as micro-injectors through which biological material and/or molecules can be introduced into the cell using positive pressure. This pressure is applied shortly after the application of the trapping hydrostatic pressure from above in order to prevent the escape of cell cytoplasm through the perforation.
  • the fundamental fabrication steps involve silicon micromachining, from simple bulk micromachining to deep reactive ion etching (DRIE) procedures. Similarly, for the top and bottom glass substrates, wet chemical etching will be necessary to create fluidic channels for cell and material transport.
  • DRIE deep reactive ion etching
  • the core aspect of this microdevice is the creation of the injector structure during the same time that the micro-chambers are being formed.
  • the use of isotropic etchants is ideal for the formation of the desired profile in silicon.
  • the ability to etch silicon crystal planes at the same rate in all directions is the defining characteristic of isotropic etchants.
  • the masking pattern should therefore be designed in order to forni a circular cavity within the silicon substrate to hold the recipient cell, with a sharp protrusion at the bottom of each cavity to form the injector.
  • This etch profile can be accomplished with an annulus or donut-shaped masking pattern where the area between the two concentric circles is bare silicon, and therefore, the region to be etched.
  • the inner circle acts as a mask over which the isotropic etchant would remove the silicon, by means of undercutting, to create the protrusion while the outer circle will provide the radial dimension of the resulting cavity.
  • the top and cross-sectional views of the masking pattern have been shown in Fig. 4.
  • Either wet or dry isotropic etching can be performed, where the former consists of wet chemistries, typically a mixture of hydrofluoric acid (HF), nitric acid (HNO 3 ), and acetic acid (CH 3 COOH).
  • This etchant referred to as "HNA” has some limitations in its use because the resulting etch profile is highly agitation-dependant and sensitive to temperature (Madou 1997).
  • HNA can etch the masking layer very quickly. This makes it quite difficult to control lateral undercutting as well as vertical etch depth.
  • Dry isotropic etching is further divided into plasma-assisted etching and gas-phase etching.
  • Plasma-assisted etching involves creating an area of high energy electric and magnetic fields in a vacuum chamber that cause a gas to dissociate to form highly energetic ions, photons, electrons, and reactive radicals and molecules, which establish the etching process.
  • RIE reactive ion etching
  • various etch profiles can be achieved by adjusting the chemistry and flow rates of the gases involved.
  • Etch cavities can range from isotropic to profiles with near- vertical sidewalls.
  • the RIE species to be used for the proposed microdevice will be sulfur hexafluoride (SF 6 ), which will etch silicon but that does not adversely affect an aluminum masking layer.
  • XeF 2 xenon difiuoride
  • Xenon difiuoride is a white crystalline solid at room temperature and atmospheric pressure, having a vapor pressure of about 4 Torr at these conditions [Ann].
  • XeF 2 exhibits a high selectivity to silicon over such common masking materials as silicon-oxide (SiO 2 ), silicon-nitride (Si N 4 ), aluminum, and photo-resist.
  • the etch cavities are touching, which would indeed create a protruding micro-injector.
  • the height of the micro-injector should ideally be contained entirely within the confines of the micro-chamber in order to avoid the possibility of damage during either device fabrication or operation. Therefore, by allowing the two etch cavities to merge, as shown in Fig. 5c, a recessed micro-injector is formed at the same time the micro- chamber is defined.
  • the inlet port must be incorporated into the micro-piercing injectors in order to allow the transfer of biological materials or molecules.
  • the inlet port would simply need to be a small access tube starting at the back-side of the silicon wafer, terminating at the tip of the micro-injector.
  • the technique used to fabricate such a hole relies on dry anisotropic etching using highly reactive ion species to chemically attack the silicon substrate.
  • wet isotropic etching will also be used to create the micro- fluidic channels in the top and bottom glass substrates.
  • the substrates will then be aligned and bonded to the silicon wafer using high temperature and high voltage anodic bonding techniques.
  • the glass will be either a standard pyrex-7740 wafer or the newer Foturan photo-etchable glass substrate. Both have thermal expansion coefficients similar to silicon and therefore introduce no stress when anodically bonded at high temperatures. Both 7740 and Foturan are etched in hydrofluoric (HF) acid.
  • Anisotropic etching of silicon is a fundamental technology required in the fabrication of both the inlet ports, and of the back-side reservoirs where the biological material or molecules will be stored prior to insertion into the recipient cells.
  • the quaternary alkaline silicon etchant known as tetra-methyl ammonium hydroxide (TMAH) will be used to create the back-side reservoirs.
  • TMAH tetra-methyl ammonium hydroxide
  • wet anisotropic solutions have crystallographic-dependant etch rates, and for TMAH, the ⁇ 111> planes of the silicon crystal lattice etch the slowest with respect to ⁇ 100> and ⁇ 110> planes.
  • etch profiles are usually in the form of inverted pyramidal cavities aligned with the wafers' ⁇ 110> primary flat.
  • the slopes of the pyramidal pit correspond to the ⁇ 111> planes, which intersect the ⁇ 100> plane at 54.7°. This is the reason why the back-side of the microdevice in Figs. 1 and 2 have sloping sidewalls, which correspond to the ⁇ 111> planes.
  • RIE reactive ion etching
  • DRIE deep reactive ion etching
  • Fabrication of the microsystem will follow two parallel processes, with one involving an ultra-thin 4' silicon wafer wlrier the other employing a 4" SOI, or silicon- on-insulator wafer. Testing of specific process flow steps will be performed on standard 4" silicon wafers.
  • the reason for using either ultra-thin or SOI wafers is because the overall diameter of the cavity is being designed for 10 ⁇ m, to accommodate cells of comparable sizes. Therefore, the isotropic etch that creates such a cavity will etch downwards by only 5 ⁇ m, since the diameter is formed by undercutting the masking layer on all sides of the circular annulus. Deeper cavities to accommodate the cells should be made by performing a relatively anisotropic RIE process.
  • the thickness of the ultra-thin wafer will be approximately 20 ⁇ m whereas the SOI wafer will be that of standard thickness, typically 500 ⁇ m. It is clear that by a fabrication standpoint, the SOI wafer will be easier to handle and process as compared with the ultra-thin wafer, which can prove to be difficult to handle due to their fragility.
  • the fabrication process for both types of wafer has been given in Fig. 7. Both processes are somewhat equivalent, beginning with a silicon surface oxidation followed by a DRTE step to create the inlet port. However, for the ultra-thin wafer, the lithography defining the pattern for the inlet port using DRIE is done on the back-side of the wafer, while for the SOI, it is done on the front.
  • the original silicon-dioxide layer is removed and a new layer of SiO 2 is grown on all exposed silicon surfaces, including within the inlet port hole.
  • Both wafers undergo the second lithographic step on the front-side that is used to define the annuli, each being centered and aligned with the inlet port hole.
  • an anisotropic RIE step can be applied in order to increase the depth of the resulting micro-cavity, followed by an isotropic etch.
  • This dry etch procedure can be made in two ways, and investigations will be made with either xenon- difluoride gas-phase etching or with sulfur hexafluoride plasma etching.
  • KIC software developed at the University of California at Berkeley
  • the masks were printed on a linotronic output transparency at a local desktop publishing company resulting in annuli with a resolution of about 20 microns, sufficient for initial testing of etching procedures.
  • the annuli had inner radii ranging from 10 to 30 microns in increments of 10 microns, and outer radii ranging from 20 to 60 microns in increments of 10 microns.
  • XeF 2 etching was attempted at first but proved unsuccessful, possibly due to the formation of a polymer-like film on the silicon surface during the etch procedure. Baking the silicon at 140°C for a short time (-10 minutes) may remove the unwanted surface adherents prior to etching which should produce more favorable results. This experiment will be re-attempted in the future.
  • Sulfur hexafluoride etching proved more successful. Initially, tests were conducted using an oxide masking layer to determine the relative isotropy of the RIE recipe. Simple test structures were etched to determine etch profile, as shown in the scanning electron micrograph (SEM) micrograph of Fig. 8. Aluminum was deposited on a silicon wafer, followed by photoresist, placement of the mask, UN illumination, aluminum etch, and finally SF 6 etch. An SEM of a portion of the resultant array of annuli is shown in Fig. 9. SEMs of two micro-cavities in the array are shown in Figs. 10 and 11. Their surface topography scans are also given in Figs. 12a and b, respectively.
  • the depth of the two micro-cavities are equal because both were parts of the same array that were etched for equal times. But since their dimensions (inner and outer diameters), are different due to the different annuli masking dimensions, the inner projection that forms the micro-injector has a different height. None of the smallest diameter annuli (10 micron inner diameter) patterned successfully, due to the limited resolution of the mask. It should be evident that the annuli profiles are not clean surfaces and exhibit tremendous roughness. This is due to the aluminum masking layer that was used since the RIE procedure affects the aluminum causing it to precipitate onto the etching silicon surface. As a result, the aluminum on the etch front produces a micro-masking effect leading to excessive surface roughness.
  • This problem can be alleviated by the use of an oxide masking layer. Indeed, as seen in Fig. 8, the etch profile is clean and there is no surface roughness on the silicon that is etched.
  • the aluminum masking layer was used since it is a quick procedure to deposit, and could also be used for masking in a XeF 2 environment.
  • the test structure shown in Fig. 2, consisted of a series of annuli patterned in silicon, masked by a thermal oxide layer.
  • the annuli had inner radii ranging from 30 to 60 microns in increments of 10 microns, and outer radii ranging from 50 to 100 microns in increments of 10 microns, with a resolution of about 20 microns, sufficient for initial testing of etching procedures. All etches were performed for 5 minutes at 85 mTorr, and etch depths were measured using a Dektak profilometer.
  • the mask underetch data was visually determined using an optical microscope while calculations were performed to arrive at the selectivity, etch rate, and anisotropy. Graphical results for only three trials are discussed below.
  • the inlet port in the micro-injector structure can be fabricated through a commercial MEMS service provider possessing Deep-RIE (DRIE) technology ideal for etching a 1-2 ⁇ m diameter holes in the silicon substrate to a depth of about 30 ⁇ m.
  • DRIE Deep-RIE
  • the added pressure would displace the air in the micro-needle downward due to the piston-like action of the free moving syringe (D).
  • D free moving syringe
  • the material to be injected could be pre-loaded in the micro-needles (fluid-filled) prior to injection, however, due to the lack of venting below the cells to be trapped, the design does not allow for the downward displacement of cells by the action of increased hydrostatic pressure. The result is that the cells are not seated in the micro-chambers due to the incompressibility of fluid below the cell.
  • the shortcoming of the design is that the micro- injector is used during both cell trapping and material injection phases.
  • the initial design was modified to allow for venting of the fluid trapped below the cells as they are pushed down onto the micro-needles. Ports were considered for the design (Fig. 18) to provide a means for fluid escape during the cell trapping operation, allowing the micro-injectors to be pre-loaded with the material to be introduced.
  • the 30-40 ⁇ m deep vent and inlet ports would be made using DRIE, an optimistic mimmum diameter for each hole, taking into account some unavoidable lateral etch, would be about 1.5 ⁇ m.
  • micro-chamber is a lO ⁇ m diameter cup, designed to be slightly less than the size of a host cell, performing lithography and processing in such a confined space would be difficult.
  • a new design incorporating fluidic channels for venting and suction was developed, and is described in the next section.
  • Second Embodiment device based on microchambers obtained by molding PDMS substrate.
  • a design was conceived that eliminated the etched micro-chambers so the micro-needle array could be formed on the silicon surface.
  • An intermediate structural layer made of polydimethylsiloxane (PDMS) silicone rubber, which uses standard silicon micro-technologies and a molding method for processing, was added between the silicon and top glass substrate.
  • PDMS polydimethylsiloxane
  • This new layer contains an array of micro-tubes lO ⁇ m in diameter that are aligned above the micro-needle array on the silicon.
  • the micro-tubes are the seating locations for the host cells, which are drawn into these holes using a combination of applied hydrostatic pressure applied and suction through the fluidic channels formed in the PDMS layer, as depicted in Fig. 19.
  • This design alleviates the need of isotropically etched micro-chamber fabrication, thereby eliminating the need for costly ultra-thin or silicon-on-insulator (SOI) wafers, which were in the original proposal. Since the microsystem is fabricated with standard silicon wafers, backside anisotropic etching is used to access the micro-needle injector ports. PDMS will also be used to backfill this large etch cavity to reduce its volume thereby reducing the amount (cost) of material to be injected. Fabrication of the top silicone layer relied on casting uncured PDMS onto a double-spun epoxy-based thick SU-8 photoresist mold that was patterned with the fluidic channels and micro-chamber.
  • SOI silicon-on-insulator
  • microdevice For testing the microdevice to successfully transfect cells, we proceed as follows (Maniatis 1989): A commercial vector designed for transfection of mammalian cells and which contains a gene for expression of green fluorescent protein will be loaded into the microdevice and injected into cells as described above. The injected cells will be assayed for viability by growth as clones on agar. Within a few hours of injection, single cells will be assayed for successful transfection and expression by assay under fluorescence microscopy following inoculation onto a polylysine coated coverslip. For expression of larger soluble proteins, cells will be injected with an expression vector for beta-galactosidase and clones assayed by a commercial color development assay.
  • cDNA that specifies a protein of interest, such as carboxypeptidase II or the metabotropic glutamate receptor subtype 1. Clones will be assayed by standard methods used in Professor Neale's laboratory, substrate hydrolysis for the enzyme and receptor mediated increase in intracellular calcium for the receptor.
  • the device will: require minimally skilled expertise, handling and expenditure of time transform large numbers of cells in a single step maintain cell viability in a high percentage of cells be cost-effective overcome limitations inherent in other transfection microfabricated devices such as complex fabrication processing and device fragility, by using a simplified device fabrication strategy developing a single component device inherently more durable and robust then the current two component devices.
  • Transducers 1098-1101 (June 7-10) Sendai, Japan.

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Abstract

La présente invention concerne une méthodologie améliorée d'introduction de molécules spécifiques dans des cellules, ou d'élimination de matière des cellules, par rapport à l'art de la technique antérieure. L'invention concerne, en particulier, un moyen efficace de mise en oeuvre de ces procédures à haut rendement, faisant appel à une technicité et à une manipulation moins spécialisée, par utilisation d'un substrat à réseau de silicium microfabriqué possédant une stratégie de fabrication de dispositif simplifiée, un composant de dispositif unique réalisant le processus de transformation permettant d'obtenir un dispositif plus durable et plus résistant.
PCT/US2001/044289 2000-11-28 2001-11-28 Transformation cellulaire a l'aide d'un reseau microfabrique de micropuces en silicium comprenant des injecteurs de micro percage integres WO2002058847A2 (fr)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1762608A1 (fr) * 2005-09-09 2007-03-14 Fujitsu Limited Une boîte de petri pour le piégeage d'une cellule
GB2479521A (en) * 2010-03-19 2011-10-19 Univ Leiden Array microinjection apparatus and methods for single cells or embryos
EP2394725A3 (fr) * 2003-02-27 2012-04-11 Seng Enterprises Limited Procédé et dispositif de manipulation des cellules
US9739699B2 (en) 2007-11-15 2017-08-22 Seng Enterprises Ltd. Device for the study of living cells
US9975118B2 (en) 2007-11-15 2018-05-22 Seng Enterprises Ltd. Device for the study of living cells
US10081816B1 (en) 2014-07-03 2018-09-25 Nant Holdings Ip, Llc Mechanical transfection devices and methods
US10190082B2 (en) 2003-06-26 2019-01-29 Seng Enterprises Ltd. Multiwell plate
CN109795978A (zh) * 2018-12-26 2019-05-24 华中科技大学 一种微型空心硅针管阵列及其制作方法
JP2019517273A (ja) * 2016-06-09 2019-06-24 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー 細胞トランスフェクション及び生存性を改善する為のナノストローウェルインサートデバイス
WO2019182631A1 (fr) * 2018-03-20 2019-09-26 Owl biomedical, Inc. Dispositif de manipulation de particules microfabriquées
EP3512802A4 (fr) * 2016-09-13 2020-03-18 The Board of Trustees of the Leland Stanford Junior University Procédés d'échantillonnage intracellulaire à nanopaille non destructif pour surveillance de cellule longitudinale
US10760040B1 (en) 2014-07-03 2020-09-01 NanoCav, LLC Mechanical transfection devices and methods
US10760060B2 (en) 2012-12-02 2020-09-01 Biomedcore Inc. Injection and incubation of circulating tumor cells from a cancer biopsy in zebrafish for accelerated prediction of cancer progression and response to treatment
US10815499B2 (en) 2017-07-19 2020-10-27 The Board Of Trustees Of The Leland Stanford Junior University Apparatuses and methods using nanostraws to deliver biologically relevant cargo into non-adherent cells
US11685897B2 (en) 2011-04-27 2023-06-27 The Board Of Trustees Of The Leland Stanford Junior University Nanotube structures, methods of making nanotube structures, and methods of accessing intracellular space
WO2023215325A1 (fr) * 2022-05-03 2023-11-09 Basilard Biotech, Inc. Dispositifs, systèmes et procédés de mécanoporation déterministe

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5183744A (en) * 1988-10-26 1993-02-02 Hitachi, Ltd. Cell handling method for cell fusion processor
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
DE19841337C1 (de) * 1998-05-27 1999-09-23 Micronas Intermetall Gmbh Verfahren und Vorrichtung zur intrazellulären Manipulation einer biologischen Zelle
WO2000020554A1 (fr) * 1998-10-08 2000-04-13 Astrazeneca Ab Injecteur de cellules microfabrique
WO2001019978A1 (fr) * 1999-09-14 2001-03-22 Cornell Research Foundation, Inc. Microfabrication d'un jeu ordonne d'echantillons de transfert nucleaire pour clonage animal a haut rendement

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5183744A (en) * 1988-10-26 1993-02-02 Hitachi, Ltd. Cell handling method for cell fusion processor
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
DE19841337C1 (de) * 1998-05-27 1999-09-23 Micronas Intermetall Gmbh Verfahren und Vorrichtung zur intrazellulären Manipulation einer biologischen Zelle
WO2000020554A1 (fr) * 1998-10-08 2000-04-13 Astrazeneca Ab Injecteur de cellules microfabrique
WO2001019978A1 (fr) * 1999-09-14 2001-03-22 Cornell Research Foundation, Inc. Microfabrication d'un jeu ordonne d'echantillons de transfert nucleaire pour clonage animal a haut rendement

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHUN K ET AL: "An array of hollow microcapillaries for the controlled injection of genetic materials into animal/plant cells" MICRO ELECTRO MECHANICAL SYSTEMS, 1999. MEMS '99. TWELFTH IEEE INTERNATIONAL CONFERENCE ON ORLANDO, FL, USA 17-21 JAN. 1999, PISCATAWAY, NJ, USA,IEEE, US, 17 January 1999 (1999-01-17), pages 406-411, XP010321695 ISBN: 0-7803-5194-0 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2394725A3 (fr) * 2003-02-27 2012-04-11 Seng Enterprises Limited Procédé et dispositif de manipulation des cellules
US10190082B2 (en) 2003-06-26 2019-01-29 Seng Enterprises Ltd. Multiwell plate
EP1762608A1 (fr) * 2005-09-09 2007-03-14 Fujitsu Limited Une boîte de petri pour le piégeage d'une cellule
US9739699B2 (en) 2007-11-15 2017-08-22 Seng Enterprises Ltd. Device for the study of living cells
US9975118B2 (en) 2007-11-15 2018-05-22 Seng Enterprises Ltd. Device for the study of living cells
GB2479521A (en) * 2010-03-19 2011-10-19 Univ Leiden Array microinjection apparatus and methods for single cells or embryos
US11685897B2 (en) 2011-04-27 2023-06-27 The Board Of Trustees Of The Leland Stanford Junior University Nanotube structures, methods of making nanotube structures, and methods of accessing intracellular space
US10760060B2 (en) 2012-12-02 2020-09-01 Biomedcore Inc. Injection and incubation of circulating tumor cells from a cancer biopsy in zebrafish for accelerated prediction of cancer progression and response to treatment
US11046976B2 (en) 2014-07-03 2021-06-29 NanoCav, LLC Mechanical transfection devices and methods
US11549089B2 (en) 2014-07-03 2023-01-10 NanoCav, LLC Mechanical transfection devices and methods
US10081816B1 (en) 2014-07-03 2018-09-25 Nant Holdings Ip, Llc Mechanical transfection devices and methods
US10760040B1 (en) 2014-07-03 2020-09-01 NanoCav, LLC Mechanical transfection devices and methods
JP2019517273A (ja) * 2016-06-09 2019-06-24 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー 細胞トランスフェクション及び生存性を改善する為のナノストローウェルインサートデバイス
US11530378B2 (en) 2016-06-09 2022-12-20 The Board Of Trustees Of The Leland Stanford Junior University Nanostraw well insert devices for improved cell transfection and viability
EP3468533A4 (fr) * 2016-06-09 2020-01-15 The Board of Trustees of the Leland Stanford Junior University Dispositifs d'insertion de puits à nanopaille pour une meilleure transfection et une meilleure viabilité cellulaires
EP3512802A4 (fr) * 2016-09-13 2020-03-18 The Board of Trustees of the Leland Stanford Junior University Procédés d'échantillonnage intracellulaire à nanopaille non destructif pour surveillance de cellule longitudinale
US11149266B2 (en) 2016-09-13 2021-10-19 The Board Of Trustees Of The Leland Stanford Junior University Methods of non-destructive nanostraw intracellular sampling for longitudinal cell monitoring
US10815499B2 (en) 2017-07-19 2020-10-27 The Board Of Trustees Of The Leland Stanford Junior University Apparatuses and methods using nanostraws to deliver biologically relevant cargo into non-adherent cells
US11999966B2 (en) 2017-07-19 2024-06-04 The Board Of Trustees Of The Leland Stanford Junior University Apparatuses and methods using nanostraws to deliver biologically relevant cargo into non-adherent cells
WO2019182631A1 (fr) * 2018-03-20 2019-09-26 Owl biomedical, Inc. Dispositif de manipulation de particules microfabriquées
CN109795978A (zh) * 2018-12-26 2019-05-24 华中科技大学 一种微型空心硅针管阵列及其制作方法
WO2023215325A1 (fr) * 2022-05-03 2023-11-09 Basilard Biotech, Inc. Dispositifs, systèmes et procédés de mécanoporation déterministe

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