Patent Application
20110076734
Tissue Engineering (TE) is evolving as a potential solution for the repair and reconstruction of diseased or damaged tissues (Langer and Vacanti, 1993, Science, 260:920-926). In the US alone, about eight million surgical procedures are performed each year to treat tissue-related maladies. Furthermore, over 70,000 patients are waiting for the organs to be donated, and more than 100,000 people die with tissue related disorders.
A variety of methods have been developed for manufacturing 3D scaffolds with embedded cells and growth factors for soft tissue engineering. These methods can be generally classified as two categories: non-automation methods and Solid Freeform Fabrication (SFF). Non-automation methods have been used for simple research purposes, while computer-aided SFF has been used to better control scaffold architecture with cells and growth factors embedded.
Non-automation methods used to manufacture scaffolds fall into several general classes. First, cells can be cultured and attached to flat surfaces, where they will synthesize extracellular matrix (ECM). Upon reaching confluence, a sheet of cells and ECM can be peeled from the substrate and rolled to form a tubular blood vessel media (L'Heureux et al., 1998, FASEB J, 12:47-56) or stacked to form a thicker block of tissue (Baar et al., 2005, FASEB J, 19:275). Second, cells can be mixed with ECM proteins such as collagen or Matrigel, or with hydrogel precursors that are allowed to gel with cells entrapped. Gels can then be compacted and shaped by a mechanical means such as centrifugation. Third, porous polymer scaffolds can be formed in bulk and then seeded with cells. Typical bulk synthetic scaffolds can be fibrous or cellular. Fibrous scaffolds can be woven or nonwoven mats, and can be preferentially oriented by strain or centrifugal forces. Cellular scaffolds can be formed by porogen leaching, gas foaming, gel casting, solution casting and gel freeze drying, among other methods. In each case, cells are seeded onto the prepared scaffold. Fourth, a cellularized tissue such as small intestinal submucosa can be used as a non-synthetic scaffold material and then seeded with cells.
Non-automation methods have the advantages of simplicity and low cost, but the control over the microarchitecture is limited to approximate control of pore size for cast hydrogels, and fiber size and planar orientation (linear or random mats) for fibrous networks. Further, control of seeding is limited to cell density control and heterogeneous cell patterning cannot be achieved. In most non-automation methods, an internal porous structure is generated by randomly packed porogen and cannot be controlled precisely or flexibly. For example, the pore size and porosity at different sections of the scaffold should be different in many cases, and all the pores should be interconnected; however, these requirements cannot be obtained or guaranteed. The biggest limitation with these methods is their incapability for making complex 3D multicellular constructs, as well as their incapability for incorporating a vascular network.
SFF is a newer manufacturing technology, involving a group of technologies that are together capable of producing complex freeform parts directly from a computer aided design (CAD) model of an object, without part-specific tooling or fixture. The CAD model can be designed using 3D CAD software or obtained through reverse engineering, through the reconstruction of three dimension models from the data produced by Computed Tomography (CT), Magnetic Resonance Imaging (MRI) or a 3D coordinate measuring machine. The CAD model is then transferred into sliced layers. Based on these layers, numerical control codes are generated to control the machine in building the part.
SFF possesses several unique advantages that make it a powerful manufacturing tool for 3D scaffolds. First, a three-dimensional CAD model of the tissue can be reconstructed precisely by reverse engineering software based on the data of CT or MRI system. The SFF machine can subsequently make the scaffold with any complex geometry. Second, SFF technology makes parts in an additive fashion through a layer-by-layer process. In each layer, materials can be added line by line, even dot by dot, so the internal structure of the porous scaffold can be controlled directly and precisely to meet any special requirements, including relatively complex and curved shapes such as myocardial microvascular network. Third, a wide range of biomaterials of hydrogels are available to make scaffolds for soft tissue using SFF technology.
SFF methods look promising for manufacturing scaffolds for bone tissue engineering (Yeong et al. 2004, Trends in Biotechnology, 22), but the potential of SFF has not yet been fully exploited for soft tissue engineering yet. Of several SFF methods being studied, the most popular uses commercially available inkjet printers, with slight modifications, to print solutions of cells and of growth factors, with a resolution as small as 200 μm. Also, extrusion-based SFF techniques have been reported for making hydrogel scaffolds with embedded cells, which are dispensed in lines, deposited in parallel and layer by layer to create 3D tissue constructs with grid-like architecture. Also, a pulsed-laser-based printing technique has been reported for printing individual cells from a slide coated with a laser-absorptive layer. This method has some advantages, such as its ability to print single cells and to print very small size of droplets (100 nm-10 μm) (Barron et al., 2005, Ann Biomed Eng 33:121-130). Also, photopolymerisable hydrogels have been used in combination with the computer aided methods to create cell encapsulated hydrogel scaffolds.
Many types of micro-droplet generators that do not control surface tension (ST) have been used for bio-engineering applications. Each type has its own advantages and limitations (see
One tool for manipulating tiny amounts of liquids on surfaces based on ST manipulation is Electrowetting On Dielectric (EWOD) (see U.S. Pat. Nos. 6,989,234 and 6,911,132). EWOD has not been used in TE. Current EWOD applications include biomedical diagnostics, adjustable lenses, ‘lab-on-a-chip’ systems for applications such as DNA and protein analysis, and new kinds of electronic displays (Cho et al., 2003, J Microelectromechanical Systems 12:70-80; Fair et al., 2003, IEEE International Electronic Devices Meeting, pp. 32.5.1-32.5.4).
EWOD uses the electrocapillary principle: ST is a function of electric potential across an interface, and the change in liquid-solid ST, γSL, changes the contact angle at the liquid-solid-gas interfaces (
All of the SFF methods for soft TE are at an early stage of development and continue to face many challenges. Inkjet printing methods depend on commercial inkjets, which were designed to dispense ink. These systems can function only in a narrow, low viscosity range, which limits the type and strength of solutions that can be printed. In addition, inkjet printers have problems with cells clogging the jets, have a resolution limited to about 200 μm and are not well-suited for dispensing living cells, based on the 25% cell death that has been reported (Wilson & Boland, 2003, Anat Rec A Discov Mol Cell Evol Biol 272:491-496). This process also includes an extra step of using an intermediate substrate “bio paper” to support cells, because the process cannot jet out hydrogel along with cells, which limits the prospects of this method for 3D printing.
Extrusion-based SFF methods produce a limited range of scaffold architectures, with parallel linear elements stacked in layers, at a resolution of around 100 μm, and do not enable heterogeneous cell patterning (precise arrangement of multiple cell types). Laser-based SFF methods expose cells to high stress, UV light, and heat, which must be carefully controlled to avoid damaging cells. SFF methods also may not scale up easily to 3D manufacturing because cells are usually delivered from 2D arrays. Although TE has recently been used successfully to replace a blood vessel in pediatric surgery (Yuji, 2001, J Thorac Cardiovasc Surg Volume 125:419-420), TE is still relatively new and has a long way to go to effectively and efficiently manufacture biocompatible and bioactive tissue substitute. To become effective and efficient, TE must overcome several challenges. First, manufacturing techniques that mimic tissue and extra cellular matrix (ECM) architecture, with high resolution (less than 10 μm) for tissues such as, for example, myocardium (heart muscle), blood vessels, bone or nerves are needed. Second, innovative multiple-jet printing methods for the delivery of cells and growth factors into scaffolds are needed. Third, manufacturing techniques for building complex structures (e.g., vascular structure) enabling nutrient transport are needed. The current invention fulfills these needs.
The invention disclosed herein relates to apparatuses and methods for engineering tissue using electrowetting techniques. The apparatuses and methods of the invention allow for the manufacturing of tissue by the deposition of a variety of biomaterials with a single innovative tissue manufacturing system. The invention includes a system and methods for manufacturing 3D, bioactive, tissue scaffold fabrications with embedded cells and bioactive materials, such as growth factors, using biomimetic structure modeling, SFF, biocompatible hydrogel material, and EWOD-based multi-microarray printing.
In one embodiment, the invention is an apparatus for engineering tissue by actuating droplets using electrowetting techniques. In various embodiments, the apparatus of the invention can have at least four microarray print heads, but can also have at least two, or three, or more than four print heads. Each of the microarray print heads has a first conductive layer having an array of control electrodes covered by a first hydrophobic insulator surface; a second conductive layer having a second conductive layer surface facing the first hydrophobic surface, the second conductive layer spaced from the first conductive layer to define a gap there between, and having an actuation voltage thereon of 20 to 100 volts; and a wire traction system having at least one conductive elongate wire element disposed in the gap between the first and second conductive layers and having a second hydrophobic surface, and having a voltage thereon less than the second conductive layer actuation voltage; and a voltage source communicating with the second conductive layer and the elongate wire element that provides an actuation voltage to the second conductive layer of 20 to 100 volts, wherein the droplet is caused to move along a pathway extending around the conductive elongate wire element and from the first conductive layer towards the second conductive layer. In embodiments of the invention having at least four microarray print heads, one of the print heads actuates droplets containing a hydrogel, one of the print heads actuates droplets containing a crosslinker, one of the print heads actuates droplets containing a cell suspension, and one of the print heads actuates droplets containing a growth factor.
In another embodiment of the apparatus for engineering tissue by manipulating droplets, each of the microarray print heads has a substrate having a substrate surface; an array of drive electrodes disposed on the substrate surface; a dedicated array of reference elements settable to a common reference potential and disposed in at least substantially co-planar relation to the electrode array, wherein the array of reference elements is electrically and physically distinct from the drive electrode array and further wherein each drive electrode is adjacent to at least one of the reference elements; a dielectric layer disposed on the substrate surface to cover the drive electrodes; and an electrode selector for sequentially activating and de-activating one or more selected drive electrodes of the array to sequentially bias the selected drive electrodes to an actuation voltage, whereby a droplet disposed on the substrate surface moves along a desired path defined by the selected drive electrodes. In embodiments of the invention having at least four microarray print heads, one of the print heads actuates droplets containing a hydrogel, one of the print heads actuates droplets containing a crosslinker, one of the print heads actuates droplets containing a cell suspension, and one of the print heads actuates droplets containing a growth factor.
In still another embodiment of the apparatus for engineering tissue by manipulating droplets, each of the microarray print heads has a substrate having a substrate surface; an array of electrodes disposed in at least substantially co-planar relation on the substrate surface, wherein the array of electrodes comprises drive electrodes and dedicated reference electrodes; a dielectric layer disposed on the substrate surface and covering the array of electrodes; an electrode selector for dynamically creating a sequence of electrode pairs, each electrode pair having a selected one of the drive electrodes biased to a first voltage and a selected one of the reference electrodes disposed adjacent to the selected drive electrode and biased to a second voltage less than the first voltage, whereby a droplet disposed on the substrate surface moves along a desired path running between the electrode pairs created by the electrode selector; and whereby manipulation of the droplet is accomplished by electrowetting actuation wherein the droplet overlaps a selected one of the drive electrodes and a selected one of the reference electrodes continuously. In embodiments of the invention having at least four microarray print heads, one of the print heads actuates droplets containing a hydrogel, one of the print heads actuates droplets containing a crosslinker, one of the print heads actuates droplets containing a cell suspension, and one of the print heads actuates droplets containing a growth factor.
In yet another embodiment, the invention is a method of tissue engineering by actuating droplets by electrowetting, comprising the steps of horizontally actuating a first group of droplets containing a hydrogel to position each droplet of the first group of droplets at a discrete target location on a microfluidic chip, and vertically actuating the first group of droplets to deposit the first group of droplets onto a tissue growth surface; horizontally actuating a second group of droplets containing a crosslinker to position each droplet of the second group of droplets at a discrete target location on a microfluidic chip and vertically actuating the second group of droplets to deposit the second group of droplets onto the tissue growth surface; horizontally actuating a third group of droplets containing a cell suspension to position each droplet of the third group of droplets at a discrete target location on a microfluidic chip vertically actuating the third group of droplets to deposit the third group of droplets onto the tissue growth surface; horizontally actuating a fourth group of droplets containing a growth factor to position each droplet of the fourth group of droplets at a discrete target location on a microfluidic chip and vertically actuating the fourth group of droplets to deposit the fourth group of droplets onto the tissue growth surface. In various embodiments of the methods of the invention, the methods steps described herein are repeated to deposit a subsequent layer of droplets onto the tissue growth surface to engineer the desired tissue.
The foregoing summary, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. In the drawings:
The invention disclosed herein overcomes the existing limitations for fabricating bioactive tissue scaffolds, by combining the advantages of the SFF method with a new application of electrowetting-based microstructure printing, permitting the deposition of various biomaterials with a single innovative tissue manufacturing system. The invention includes a system and methods for manufacturing 3D, bioactive, soft-tissue scaffold fabrications with embedded cells and bioactive materials, such as growth factors, using biomimetic structure modeling, SFF, biocompatible hydrogel material, and EWOD-based multi-microarray printing. In some embodiments, the system and methods of the invention integrate an EWOD array design with a computer-controlled motion system for positioning the EWOD chip for the delivery of hydrogel, crosslinker, cells, and bioactive materials, such as growth factors, on CAD models. The inventive system employs the principle of EWOD multiple microfluidics array printing, which is capable of creating and depositing droplets with sizes of less than about 10 μm, can work with a variety of hydrogels, and can deposit cells and bioactive materials, such as growth factors, during scaffold fabrication.
For engineering soft tissues, scaffolds can comprise any suitable synthetic or natural biopolymer that can provide a porous (from 50% up to greater than 90%) support structure, thus mimicking the natural ECM environment in which cells attach, multiply, migrate and function (see Zeltinger et al., 2001, Tissue Engineering 7:557-572). In some embodiments, scaffolds can be resorbable by the body at a rate that is similar to the rate that cells can produce their own natural ECM. In preferred embodiments, scaffolds suitable for use in the invention can provide mechanical support during the body's reconstruction process, can maintain the initially fabricated 3D shape, and can withstand handling during implantation and in vivo loading. In some embodiments, the pores of the scaffold can be interconnected to allow both the ingrowth of cells and the transport of nutrients. The transport of nutrients through the interconnected pores of the scaffold can occur passively or actively. In some embodiments, the scaffold can be biocompatible. In other embodiments, the scaffold can contain bioactive materials, such as growth factors that can, for example, support living cells or enhance new tissue ingrowth. In some embodiments, the scaffold can contain cells that will secrete new ECM that is bio-mechanically similar to the tissue-specific ECM that will be replaced by the body.
The scaffold can be manufactured to any suitable, germane, complex three-dimensional shape, that is similar in size and shape of the tissue or organ to be replaced, at both the microscopic and macroscopic levels. At the cell scale (e.g., on the order of about 10 μm), the microarchitecture of the scaffold pores or fibers can be designed to control cell orientation and migration, which can ultimately affect cell function. At a slightly larger scale (e.g., on the order of about 100 μm), many tissues would require a microvascular-type network to function optimally after implantation, can be designed to be present at the time of manufacture and implantation (see Yin et al., 2004, Am J Physiol Heart Circ Physiol 287:H1276-1285). Currently, most TE constructs do not include vascular networks and thus vascular ingrowth can only occur after implantation, a feature which limits the size and cellular content of implants, and can delay integration with the body. At the tissue scale, the overall size and shape can be designed to be similar to the tissue or organ to be replaced.
In preferred embodiments, scaffolds include hydrogels. Hydrogels are a class of highly hydrated polymer materials with water content over 30% by weight that can be made from either natural or synthetic components. Hydrogels have been widely used in various biomedical applications including TE, due to their biocompatibility, low toxicity and low cost. Hydrogels have excellent mechanical properties that are similar those of soft tissues. Hydrogel molecules can be modified or functionalized, to, by way of nonlimiting examples, promote cell proliferation, cell migration and cell adhesion. The pore size of the gel network can be designed to allow for optimal diffusion and transport of biological molecules (see Watanabe et al., 2004, Hydrogels, in Encyclopedia of Biomaterials and Biomedical Engineering). Further, hydrogels are highly permeable, which can facilitate the exchange of oxygen and other dissolved gases, nutrients, water-soluble metabolites, and the like. The hydrophilicity of some hydrogels prevents protein adsorption, which serves to minimize foreign body responses when implanted in vivo.
One non-limiting example of a hydrogel useful as a scaffold material is chitosan hydrogel. Chitosan is a natural polymer. Chitosan is a deacetylated derivative of chitin commonly found in the shells of crustaceans. One advantage of using a natural polymer, such as chitosan, is that they are generally more likely to mimic the ECM and provide bio-inductive properties desirable for tissue engineering. Chitosan has been extensively used in drug delivery and tissue engineering because of its biocompatibility, biodegradability, antibacterial properties, bioadhesion and low cost (see Adekogbe & Ghanem, 2005, Biomaterials, 2005. 26: p. 7241-7250). Further, chitosan is degraded by lysozyme, an enzyme present naturally in the human body, allowing resorption of the material during subsequent tissue replacement.
Chitosan can be combined with additional biomaterials such as, by way of non-limiting examples, alginate, collagen, gelatin, chitin, hydroxyapatite, PMMA, calcium phosphate cement, b-tricalcium phosphate, poly-lactic-co-glycolic acid (PLGA). The a variety of bioactive materials, such as, for example, growth factors, can be included in the scaffolds useful in the invention. Of the many possible additional bioactive materials, collagen-I is known to be secreted by cardiac fibroblasts in the ventral myocardium and appears to be a good choice for use in cardiac tissue engineering. Although the use of chitosan in the context of cardiac tissue engineering is novel, similar studies have demonstrated the feasibility of culturing fetal cardiac cells in vivo on a 3D alginate scaffold (See Leor et al., 2000, Circulation 102:56-61). Further, it has been demonstrated that a PGL scaffold seeded with fibroblasts can induce angiogenesis around damaged cardiac tissue in mice (see Kellar et al., 2001, Circulation 104:2063).
In the multi-jet printing process described herein, a hydrogel can be solidified (i.e., gelated) in a variety of ways known to those skilled in the art. By way of non-limiting examples, a hydrogel can be solidified by adding a crosslinker (i.e., crosslinking agent) after the hydrogel is deposited, or by photocrosslinking using, for example, UV light, or by using a thermoresponsive polymer.
In some embodiments, the hydrogel can be crosslinked using a variety of crosslinkers, including covalent or ionic crosslinkers. Covalently crosslinked hydrogels are bound by irreversible chemical links (see Berger et al., 2004, Eur J Pharmaceutics and Biopharmaceutics 57:19-34). By way of a non-limiting example, Genipin is a crosslinker useful in the invention. Genipin is a naturally occurring covalent crosslinking reagent which has been used in herbal medicine and food dyes, has been used as crosslinker of chitosan with little or no cytotoxic effect. In some embodiments, chitosan can be used as the hydrogel material and genipin can be used as the crosslinker. In other embodiments, ionic crosslinkers, such as, for example, tripolyphosphate (which has been used for simulation of permeability of drugs through skin), can be used. As one of skill in the art would understand, it is important to use care in selecting an appropriate crosslinker, as some have been reported to be relatively cytotoxic.
In other embodiments, the hydrogel can be crosslinked by using photocrosslinking by way of for example, the use of a photoinitiator. Photoinitiators have been used for the UV curing of systems comprising unsaturated monomers and prepolymers. Irgacure®2959, for example, has been used mixed with ethanol to create a photoinitiator stock. A photoinitiator stock can be mixed with a variety of different kinds of solutions to be gelled. Poly(ethylene glycol)-diacrylate (PEG-DA) dissolved in phosphate buffered saline (PBS), for example, has been mixed with a photoinitiator stock to create a hydrogel mixture. Also, PEG-Fibrinogen, for example, can be used to create a hydrogel that possess improved cell adhesion properties. (see, for example, Ciba® IRGACURE® 2959)
In still other embodiments, gelation can be achieve through using a thermoresponsive polymer, such as one that forms a free flowing solution in water at ambient temperatures (for example, at room temperature), and a gel at higher temperatures (for example, at body temperature). Such a thermoresponsive hydrogel solution undergoes a phase transformation at its lower critical solution temperature (LCST) to either form a gel or aqueous solution. An example of such a polymer is poly(N-isopropylacrylamide) (PNIPAAm). Another example of such a polymer, poly(ethylene glycol)dimethacrylate (PEGDM), was added to create a branched copolymer. Addition of a branching copolymer at varied ratios can be used to adjust the flexibility or rigidity of the hydrogel to achieve the desired properties (for example, a hydrogel that can better withstand manipulation). (See, for example, Vernengo, 2007, Journal of Biomedical Materials Research Part B: Applied Biomaterials, 23:653).
The present invention provides droplet-based handling and manipulation methods by implementing electrowetting-based techniques. The droplets can be moved by controlling voltages to electrodes. Generally, the actuation mechanism of the droplet is based upon surface tension gradients induced in the droplet by the voltage-induced electrowetting effect. The mechanisms of the invention allow the droplets to be transported. The chip can include an array of electrodes that are reconfigurable in real-time to perform desired tasks. The invention enables several different types of handling and manipulation tasks to be performed on independently controllable droplet samples.
Methods of the present invention form droplets for independently transporting, merging, mixing, and other processing of the droplets. The preferred embodiment uses electrical control of surface tension (i.e., electrowetting) to accomplish these manipulations. In one embodiment, the droplet is contained within a space between two parallel plates. One plate contains etched drive electrodes on its surface while the other plate contains either etched electrodes or a single, continuous plane electrode that is grounded or set to a reference potential. Hydrophobic insulation covers the electrodes and an electric field is generated between electrodes on opposing plates. This electric field creates a surface-tension gradient that causes a droplet overlapping the energized electrode to move towards that electrode. Through proper arrangement and control of the electrodes, a droplet can be transported by successively transferring it between adjacent electrodes. The patterned electrodes can be arranged in a two dimensional array so as to allow transport of a droplet to any location covered by that array. The space surrounding the droplets may be filled with a gas such as air or an immiscible fluid such as oil.
In another embodiment, the structure used for ground or reference potential is co-planar with the drive electrodes and the second plate, if used, merely defines the containment space. The co-planar grounding elements can be a conductive grid superimposed on the electrode array. Alternatively, the grounding elements can be electrodes of the array dynamically selected to serve as ground or reference electrodes while other electrodes of the array are selected to serve as drive electrodes.
Droplets can be combined together by transporting them simultaneously onto the same electrode. Droplets are subsequently mixed either passively or actively. Droplets are mixed passively by diffusion. Droplets are mixed actively by moving or “shaking” the combined droplet by taking advantage of the electrowetting phenomenon. In a preferred embodiment, droplets are mixed by rotating them around a two-by-two array of electrodes. The actuation of the droplet creates turbulent non-reversible flow, or creates dispersed multilaminates to enhance mixing via diffusion. Droplets can be split off from a larger droplet or continuous body of liquid in the following manner: at least two electrodes adjacent to the edge of the liquid body are energized along with an electrode directly beneath the liquid, and the liquid moves so as to spread across the extent of the energized electrodes. The intermediate electrode is then de-energized to create a hydrophobic region between two effectively hydrophilic regions. The liquid meniscus breaks above the hydrophobic regions, thus forming a new droplet.
According to one embodiment of the present invention, an apparatus for manipulating droplets comprises a substrate comprising a substrate surface, an array of electrodes disposed on the substrate surface, an array of reference elements, a dielectric layer disposed on the substrate surface, and an electrode selector. The reference elements are settable to a reference potential. The array of reference elements is disposed of in substantially co-planar relation to the electrode array, such that each reference element is adjacent to at least one of the electrodes. The dielectric layer is disposed on the substrate surface and is patterned to cover the electrodes. The electrode selector can be provided as a microprocessor or other suitable component for sequentially activating and de-activating one or more selected electrodes of the array to, sequentially bias the selected electrodes to an actuation voltage. The sequencing performed by the electrode selector enables a droplet disposed on the substrate surface to move along a desired path that is defined by the selected electrodes.
According to one method of the present invention, a droplet is actuated by providing the droplet on a surface that comprises an array of electrodes and a substantially co-planar array of reference elements. The droplet is disposed on a first one of the electrodes, and at least partially overlaps a second one of the electrodes and an intervening one of the reference elements disposed between the first and second electrodes. The first and second electrodes are activated to spread at least a portion of the droplet across the second electrode. The first electrode is de-activated to move the droplet from the first electrode to the second electrode.
According to one aspect of this method, the second electrode is adjacent to the first electrode along a first direction. In addition, the electrode array comprises one more additional electrodes adjacent to the first electrode along one or more additional directions. The droplet at least partially overlaps these additional electrodes as well as the second electrode. In accordance with this aspect of the method, the first direction that includes the first electrode and the second electrode is selected as a desired direction along which the droplet is to move. The second electrode is selected for activation based on the selection of the first direction.
In accordance with another method of the present invention, a droplet is split into two or more droplets. A starting droplet is provided on a surface comprising an array of electrodes and a substantially co-planar array of reference elements. The electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to the medial electrode. The starting droplet is initially disposed on at least one of these three electrodes, and at least partially overlaps at least one other of the three electrodes. Each of the three electrodes is activated to spread the starting droplet across the three electrodes. The medial electrode is de-activated to split the starting droplet into first and second split droplets. The first split droplet is disposed on the first outer electrode and the second split droplet is disposed on the second outer electrode.
In yet another method of the present invention, two or more droplets are merged into one droplet. First and second droplets are provided on a surface comprising an array of electrodes in a substantially co-planar array of reference elements. The electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to the medial electrode. The first droplet is disposed on the first outer electrode and at least partially overlaps the medial electrode. The second droplet is disposed on the second outer electrode and at least partially overlaps the medial electrode. One of the three electrodes is selected as a destination electrode. Two or more of the three electrodes are selected for sequential activation and de-activation, based on the selection of the destination electrode. The electrodes selected for sequencing are sequentially activated and de-activated to move one of the first and second droplets toward the other droplet, or both of the first and second droplets toward each other. The first and second droplets merge together to form a combined droplet on the destination electrode.
According to one aspect of this method, the first droplet comprises a first composition, the second droplet comprises a second composition, and the combined droplet comprises both the first and second compositions. The method further comprises the step of mixing the first and second compositions together. In accordance with the present invention, the mixing step can be passive or active. In one aspect of the invention, the mixing step comprises moving the combined droplet on a two-by-two sub-array of four electrodes by sequentially activating and de-activating the four electrodes to rotate the combined droplet. At least a portion of the combined droplet remains substantially stationary at or near an intersecting region of the four electrodes while the combined droplet rotates. In another aspect of the invention, the mixing step comprises sequentially activating and de-activating a linearly arranged set of electrodes of the electrode array to oscillate the combined droplet back and forth along the linearly arranged electrode set a desired number of times and at a desired frequency. Additional mixing strategies provided in accordance with the invention are described in detail herein below.
According to another embodiment of the present invention, an apparatus for manipulating droplets comprises a substrate comprising a substrate surface, an array of electrodes disposed on the substrate surface, a dielectric layer disposed on the substrate surface and covering the electrodes, and an electrode selector. The electrode selector dynamically creates a sequence of electrode pairs. Each electrode pair comprises a selected first one of the electrodes biased to a first voltage, and a selected second one of the electrodes disposed adjacent to the selected first electrode and biased to a second voltage that is less than the first voltage. Preferably, the second voltage is a ground voltage or some other reference voltage. A droplet disposed on the substrate surface moves along a desired path that runs between the electrode pairs created by the electrode selector.
According to yet another method of the present invention, a droplet is actuated by providing the droplet on a surface comprising an array of electrodes. The droplet is initially disposed on a first one of the electrodes and at least partially overlaps a second one of the electrodes that is separated from the first electrode by a first gap. The first electrode is biased to a first voltage and the second electrode is biased to a second voltage lower than the first voltage. In this manner, the droplet becomes centered on the first gap. A third one of the electrodes that is proximate to the first and second electrodes is biased to a third voltage that is higher than the second voltage to spread the droplet onto the third electrode. The bias on the first electrode is then removed to move the droplet away from the first electrode. The droplet then becomes centered on a second gap between the second and third electrodes.
According to still another method of the present invention, a droplet is split into two or more droplets. A starting droplet is provided on a surface comprising an array of electrodes. The electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to the medial electrode. The starting droplet is initially disposed on at least one of the three electrodes and at least partially overlaps at least one other of the three electrodes. Each of the three electrodes is biased to a first voltage to spread the initial droplet across the three electrodes. The medial electrode is biased to a second voltage lower than the first voltage to split the initial droplet into first and second split droplets. The first split droplet is formed on the first outer electrode and the second split droplet is formed on the second outer electrode.
According to a further method of the present invention, two or more droplets are merged into one droplet. First and second droplets are provided on a surface comprising an array of electrodes. The electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to the medial electrode. The first droplet is disposed on the first outer electrode and at least partially overlaps the medial electrode. The second droplet is disposed on the second outer electrode and at least partially overlaps the medial electrode. One of the three electrodes is selected as a destination electrode. Two or more of the three electrodes are selected for sequential biasing based on the selection of the destination electrode. The electrodes selected for sequencing are sequentially biased between a first voltage and a second voltage to move one of the first and second droplets toward the other droplet or both of the first and second droplets toward each other. The first and second droplets merge together to form a combined droplet on the destination electrode.
Referring now to
Control electrodes E.sub.1, E.sub.2, and E.sub.3 are embedded in or formed on a suitable lower substrate or plate 21. A thin lower layer 23 of hydrophobic insulation is applied to lower plate 21 to cover and thereby electrically isolate control electrodes E.sub.1, E.sub.2, and E.sub.3. Lower hydrophobic layer 23 can be a single, continuous layer or alternatively can be patterned to cover only the areas on lower plate 21 where control electrodes E.sub.1, E.sub.2 and E.sub.3 reside. Upper plane 14 comprises a single continuous ground electrode G embedded in or formed on a suitable upper substrate or plate 25. Alternatively, a plurality of ground electrodes G could be provided in parallel with the arrangement of corresponding control electrodes E.sub.1, E.sub.2 and E.sub.3, in which case one ground electrode G could be associated with one corresponding control electrode E. Preferably, a thin upper layer 27 of hydrophobic insulation is also applied to upper plate 25 to isolate ground electrode G. One non-limiting example of a hydrophobic material suitable for lower layer 23 and upper layer 27 is TEFLON® AF 1600 material (available from E. I. duPont deNemours and Company, Wilmington, Del.). The geometry of microactuator mechanism 10 and the volume of droplet D are controlled such that the footprint of droplet D overlaps at least two control electrodes (e.g., E.sub.1 and E.sub.3) adjacent to the central control electrode (e.g., E.sub.2) while also making contact with upper layer 27. Preferably, this is accomplished by specifying a gap or spacing d, which is defined between lower plane 12 and upper plane 14 as being less than the diameter that droplet D would have in an unconstrained state. Typically, the cross-sectional dimension of spacing d is between approximately 0.01 mm to approximately 1 mm. Preferably, a medium fills gap d and thus surrounds droplet D. The medium can be either an inert gas such as air or an immiscible fluid such as silicone oil to prevent evaporation of droplet D.
Ground electrode G and control electrodes E.sub.1, E.sub.2 and E.sub.3 are placed in electrical communication with at least one suitable voltage source V, which preferably is a DC voltage source but alternatively could be an AC voltage source, through conventional conductive lead lines L.sub.1, L.sub.2 and L.sub.3. Each control electrode E.sub.1, E.sub.2 and E.sub.3 is energizable independently of the other control electrodes E.sub.1, E.sub.2 and E.sub.3. This can be accomplished by providing suitable switches S.sub.1, S.sub.2 and S.sub.3 communicating with respective control electrodes E.sub.1, E.sub.2 and E.sub.3, or other suitable means for independently rendering each control electrode E.sub.1, E.sub.2 and E.sub.3 either active (ON state, high voltage, or binary 1) or inactive (OFF state, low voltage, or binary 0). In other embodiments, or in other areas of the electrode array, two or more control electrodes E can be commonly connected so as to be activated together.
The structure of electrowetting microactuator mechanism 10 can represent a portion of a microfluidic chip, on which conventional microfluidic and/or microelectronic components can also be integrated. As examples, the chip could also include resistive heating areas, microchannels, micropumps, pressure sensors, optical waveguides, and/or biosensing or chemosensing elements interfaced with MOS (metal oxide semiconductor) circuitry.
Referring now to
Referring now to
The voltage dependence of the interfacial energy reduction is described by:
where .epsilon. is the permittivity of the insulator, d is the thickness of the insulator, and V is the applied potential. The change in γ.sub.SL acts through Young's equation to reduce the contact angle at the interface between droplet D and energized control electrode E.sub.2. If a portion of droplet D also overlaps a grounded electrode E.sub.1 or E.sub.3, the droplet meniscus is deformed asymmetrically and a pressure gradient is established between the ends of droplet D, thereby resulting in bulk flow towards the energized electrode E.sub.1 or E.sub.3. For example, droplet D can be moved to the left (i.e., to unit cell C.sub.1) by energizing control electrode E.sub.1 while maintaining control electrodes E.sub.2 and E.sub.3 at the ground state. As another example, droplet D can be moved to the right (i.e., to unit cell C.sub.3) by energizing control electrode E.sub.3 while maintaining control electrodes E.sub.1 and E.sub.2 at the ground state.
Referring now to
Turning to
In the present discussion, the terms MERGE and MIX have been used interchangeably to denote the combination of two or more droplets. This is because the merging of droplets does not in all cases directly or immediately result in the complete mixing of the components of the initially separate droplets. Whether merging results in mixing can depend on many factors. These factors can include the respective compositions or chemistries of the droplets to be mixed, physical properties of the droplets or their surroundings such as temperature and pressure, derived properties of the droplets such as viscosity and surface tension, and the amount of time during which the droplets are held in a combined state prior to being moved or split back apart. As a general matter, the mechanism by which droplets are mixed together can be categorized as either passive or active mixing. In passive mixing, the merged droplet remain on the final electrode throughout the mixing process. Passive mixing can be sufficient under conditions where an acceptable degree of diffusion within the combined droplet occurs. In active mixing, on the other hand, the merged droplet is then moved around in some manner, adding energy to the process to effect complete or more complete mixing. Active mixing strategies enabled by the present invention are described herein below.
It will be further noted that in the case where a distinct mixing operation is to occur after a merging operation, these two operations can occur at different sections or areas on the electrode array of the chip. For instance, two droplets can be merged at one section, and one or more of the basic MOVE operations can be implemented to convey the merged droplet to another section. An active mixing strategy can then be executed at this other section or while the merged droplet is in transit to the other section, as described herein below.
Referring now to
To create a droplet on the electrode array, the control electrode directly beneath the main body of liquid LQ (control electrode E.sub.1) and at least two control electrodes adjacent to the edge of the liquid body (e.g., control electrodes E.sub.1 and E.sub.3) are energized. This causes the dispensed body of liquid LQ to spread across control electrodes E.sub.1 and E.sub.2 as shown in
The aspects of the invention thus far have been described in connection with the use of a droplet actuating apparatus that has a two-sided electrode configuration such as microactuator mechanism 10 illustrated in
Referring now to
Importantly, the conducting line grid is coplanar or substantially coplanar with the electrode array. The conducting line grid can be embedded on lower plane 512 by means of microfabrication processes commonly used to create conductive interconnect structures on microchips. It thus can be seen that microactuator mechanism 500 can be constructed as a single-substrate device. It is preferable, however, to include an upper plane 514 comprising a plate 525 having a hydrophobic surface 527, such as a suitable plastic sheet or a hydrophobized glass plate. Unlike microactuator mechanism 10 of
In the use of microactuator mechanism 500 for electrowetting-based droplet manipulations, it is still a requirement that a ground or reference connection to droplet D be maintained essentially constantly throughout the droplet transport event. Hence, the size or volume of droplet D is selected to ensure that droplet D overlaps all adjacent drive electrodes E as well as all conducting lines G surrounding the drive electrode on which droplet D resides (e.g., electrode E.sub.2 in
To operate microactuator mechanism 500, a suitable voltage source V and electrical lead components are connected with conducting lines G and drive electrodes E. Because conducting lines G are disposed in the same plane as drive electrodes E, application of an electrical potential between conducting lines G and a selected one of drive electrodes E.sub.1, E.sub.2, or E.sub.3 (with the selection being represented by switches S.sub.1-S.sub.3 in
It will thus be noted that microactuator mechanism 500 with its single-sided electrode configuration can be used to implement all functions and methods described hereinabove in connection with the two-sided electrode configuration of
Referring now to
In operation, selected biasing electrodes E.sub.ij are dynamically assigned as either driving electrodes or grounding (or reference) electrodes. To effect droplet actuation, the assignment of a given electrode as a drive electrode requires that an adjacent electrode be assigned as a ground or reference electrode to create a circuit inclusive with droplet D and thereby enable the application of an actuation voltage. In
In
Referring now to
It will be noted that a microactuator mechanism provided with the alternative single-sided electrode configurations illustrated in
The present invention also provides an apparatus adapted for electrostatically actuating a droplet, and preferably an array of droplets, out from one plane to another plane (i.e., z-axis actuation). The apparatus generally comprises a first plane on which droplets are initially supplied, an elongate intermediate element spaced from the first plane, and a second plane spaced from the intermediate element that serves as the destination for actuated droplets. The first and second planes and the elongate intermediate element are rendered conductive to bring about and control the actuation of the droplet. Thus, the main structural portion of the intermediate element can be composed of a conductive (or semiconductive) material. Alternatively, the main structural portion of the intermediate element can be plated, coated, or otherwise treated with a conductive layer or film by a conventional process such as thin film deposition, plating, spin-coating, metallization, or the like. The elongate intermediate element is employed primarily to electrically ground the droplet. Thus, depending on droplet size, the axial distance between the first plane and the intermediate element is small enough to ensure that a droplet residing on the first plane contacts the intermediate element as well.
In operation, the droplet is initially placed between the first plane and the elongate intermediate element. A voltage is then applied between the intermediate element and the second plane. The droplet becomes charged and attracted to the second plane. As a result, the droplet moves from the first plane, through or around the elongate intermediate element, and into contact with the second plane. In some embodiments, the first plane is a lower plane, the second plane is an upper plane, and the intermediate element is disposed between the upper and lower plane in terms of elevation. The actuation provided by the invention is strong enough to cause the droplet to move upwards into contact with the upper plane against the opposing gravitational force. An excessively high voltage is not needed to drive the actuation because, in the microscale context in which the invention is preferably implemented, the dominant physical factor is surface tension rather than other factors such as acceleration and gravity.
In one embodiment, the intermediate element comprises an elongate element such as a wire, or a plurality of such elongate elements. Droplets move around the elongate element during actuation.
Droplets can be supplied to the apparatus by different methods. In one embodiment, the first plane includes an array of electrodes. Using an electrowetting technique, selected electrodes can be sequentially energized and de-energized to cause droplets proximate to such electrodes to move to intended positions on the array prior to being actuated. To cause movement of the droplets by electrowetting, a voltage potential is applied between the elongate intermediate element (which is typically grounded) and one or more selected electrodes of the first plane. As an alternative to electrowetting-based droplet movement, droplets can be positioned on the first plane by more conventional dosing or dispensing methods, and the first plane then moved into position underneath the intermediate element and the second plane.
According to one embodiment of the present invention, an apparatus for actuating a droplet comprises a first conductive layer, a second conductive layer, a conductive elongate medial element, and a voltage source. The first conductive layer comprises a first hydrophobic surface. The second conductive layer comprises a hydrophilic surface facing the first hydrophobic surface. The second conductive layer is axially spaced from the first conductive layer to define a gap therebetween. The conductive elongate medial element is disposed in the gap between the first and second conductive layers, and comprises a second hydrophobic surface. The voltage source communicates with the second conductive layer and the elongate medial element.
The present invention also provides a method for electrostatically actuating a droplet. A droplet is placed on a first conductive layer. The droplet is grounded by contacting the droplet with an elongate grounding element that is axially spaced from the first conductive layer. The droplet is actuated by applying a voltage potential between the grounding element and a second conductive layer axially spaced from the grounding element. The droplet becomes charged and attracted to the second conductive layer. Accordingly, the droplet moves off the first conductive layer into contact with a hydrophilic surface of the second conductive layer.
The present invention further provides a microarray structure synthesized according to the method just described. The resulting structure comprises a surface and a plurality of sample-containing spots disposed on the surface. It is therefore an object of the present invention to provide a method arid apparatus for performing non-contact electrostatic actuation of droplets from one plane to another plane. It is another object of the present invention to perform such actuation in the context of synthesizing a microarray, in which the contents of actuated droplets are stamped or printed onto a surface of the microarray.
Referring now to
First plane 20 comprises a first planar body 22 such as a plate or substrate. First planar body 22 can be composed of a non-conductive material, such as a glass or polymer, or can be a semiconductor. Typically, first planar body 22 is a dielectric material such as a glass coverslip that is rendered conductive by forming one or more control electrodes E (e.g., E.sub.1, E.sub.2, and E.sub.3 as illustrated in
A droplet D is shown residing on control electrode E.sub.2 prior to actuation. Droplet D is electrolytic, polarizable, or otherwise capable of conducting current or being electrically charged. Typically, droplet D ranges in size between approximately 10 μm to approximately 2 mm in diameter.
In the example illustrated in
Medial plate 32 of intermediate element 30 can be composed of a conductive material, a semi-conductive material, or a dielectric material. Preferably, medial plate 32 is a dielectric material such as a glass coverslip that is rendered conductive by applying a conductive layer 34 such as sputtered indium tin oxide (ITO). ITO is also preferred for its transparent property. During actuation as described herein below, each droplet D must move through its corresponding aperture A to reach second plane 40. Therefore, medial plate 32 is preferably hydrophobized as described above in connection with first plane 20, and thus
Second plane 40 comprises a second planar body 42 such as a plate or substrate. Second planar body 42 can be composed of a conductive material, a semi-conductive material, or a dielectric material. Preferably, second planar body 42 comprises a derivativized glass plate. As known in the art, particularly in the field of microarray fabrication, glass plates can be derivatized by applying, for example, a poly-(L)-lysine coating. Thus, the surface of second plane 40 facing first plane, surface 40A, is hydrophilic. Second plane 40 is rendered conductive by applying a conductive layer 44 such as sputtered ITO. Second plane 40 at least conceptually comprises an array of target sites T (e.g., T.sub.1, T.sub.2, and T.sub.3 as illustrated in
Control electrodes E.sub.1, E.sub.2 and E.sub.3 are useful for controlling the movement and positioning of droplets D prior to or after actuation. Thus, control electrodes E.sub.1, E.sub.2 and E.sub.3 can be employed to transport one or more droplets D from another portion of the structure of apparatus 10, such as a sample reservoir or injection site, to predetermined positions on the array of first plane 20 in alignment with corresponding apertures A.sub.1, A.sub.2, and A.sub.3 of intermediate element 30 and target sites T.sub.1, T.sub.2, and T.sub.3 of second plane 40. In a case where the stamping of droplets D results in unneeded, residual liquid masses, the residual material can returned to first plane 20 and transported away from apparatus 10 by control electrodes E.sub.1, E.sub.2 and E.sub.3 across first plane 20 to an appropriate waste location. Preferably, control electrodes E.sub.1, E.sub.2 and E.sub.3 operate on the principle of electrowetting, which is described in detail by Pollack et al., “Electrowetting-based actuation of liquid droplets for microfluidic applications”, Appl. Phys. Lett., Vol. 77, p. 1725 (September 2000). Additional disclosures of electrowetting techniques and applications therefor are provided in U.S. Pat. Nos. 6,911,132 and 6,989,234, the contents of which are incorporated herein in their entirety.
Briefly, the electrowetting technique involves controlling the surface tension on droplet D, and hence the contact angles droplet D makes with the surfaces it contacts, through application of a voltage potential between intermediate element 30 (serving as the ground plane) and first plane 20. The size of droplet D and the distance of gap g.sub.1 are such that the footprint of droplet D overlaps the electrodes (e.g., control electrodes E.sub.1 and E.sub.3) adjacent to the electrode (e.g., control electrode E.sub.2), thereby allowing droplet D to be moved electrode-by-electrode to any point on the array of first plane 20. Referring to
Referring now to
The principle underlying the droplet-based actuation process just described can be explained as follows. From Gauss' law, it is known that:
∇E=ρ/ε or,
∇2∇=−ρ/ε.
The fundamental implication of Gauss' law is that the charge density inside of any closed Gaussian surface containing a conductor can be increased by increasing the divergence of the electric field lines. An increase in surface charge density, ρ, will cause an increase in the force exerted on the surface. Hence, for a given electric field, higher forces can be achieved if the field divergence is larger. This means that if electric flux concentration is increased, a higher ρ can be obtained for the same voltage.
The example illustrated in
The problems attending devices such as apparatus 10 are removed with the wire traction system provided by the present invention. Referring now to
As a principal difference from apparatus 10 illustrated in
By comparison to the mid-plate configuration of apparatus 10, however, in the wire traction configuration of apparatus 100 there is a marked increase in the electric field density (and as a consequence, by Gauss' law, higher charge densities) on the surface of droplet D. The electric field strength near the surface of droplet D is nearly an order of magnitude greater than the electric field strength at a similar point in the mid-plate configuration of apparatus 10. Also, the surface area of droplet D directly under the influence of the electric field is greatly increased. The higher surface charge densities result in a higher electrostatic pressure on the droplet surface which, when integrated over the entire surface of droplet D, results in a much larger force on droplet D as compared to that in the mid-plate system illustrated in
It will be noted that charge density could also be increased on the droplet surface by: (1) increasing the dielectric permittivity of the dielectric medium surrounding droplet D (e.g., the use of a silicone oil); (2) increasing the electrostatic drive voltage; or (3) changing the geometry of the configuration of apparatus 10 or 100 in such a way that the .gradient.sup.2.gradient. term in the equation hereinabove increases even though the magnitude of the voltage itself is constant. For apparatus 100, however, the charge density on the droplet surface has been increased without increasing the voltage. The result is a much higher charge density for the same voltage in the wire traction system of apparatus 100 as compared to the mid-plate system of apparatus 10.
Referring now to
As shown in
An array-based print-head can be used to eliminate bottlenecks in scaffold fabrication by allowing for high-density rapid array printing, in seconds or minutes, rather than hours, and low and at high densities (e.g., 104 spots/cm2, or up to 105-106 spots/cm2 assuming spot sizes of 7-18 μm). Theoretically, array densities achievable with picoliter droplet printing are in the range of 1×105/cm2 can be greater of 5×105/cm2. Nanoliter droplet transfer rates have been performed in the range of about 1000 transfers/sec at linear velocities in the range of about 10 cm/sec. At such rates one could populate a 700×700 array, 10 columns at a time, followed by vertical printing, all in about 1 minute. Optionally, no intermediate cleaning or preprinting steps are required. These rates represent significantly greater throughput than any existing automated arraying system.
The EWOD printing system of the invention is based on self-aligned, non-contact, nanoliter or sub-nanoliter array printing using droplet-based microfluidics. One embodiment of the microfluidic printing array system of the invention is depicted in
Nanoliter-sized droplets have been dispensed using EWOD chips and the size of these droplets was on the order of 100 μm (Kolar and Fair, 2001, SmallTalk, pp. 139). Droplets (about 50-200/minute) can be delivered at a single location, so the anticipated build rate can be around 0.5-2 cm/minute for a nanoliter-volume droplet. The total size of the structure that can be built will depend on the size of microfluidic array. For picoliter-volume droplets, diameter is on the order of 10 μm and the build rate is approximately 0.05-0.2 mm/minute.
In one embodiment, the invention is an apparatus for engineering a tissue by actuating droplets by electrowetting. In some embodiments the apparatus has at least two microarray print heads, but in other embodiments can have at least three, or four, or more microarray print heads. In other embodiments, the apparatus has at least four microarray print heads. Each of the print heads of the apparatus contains a first conductive layer having an array of control electrodes covered by a first hydrophobic insulator surface. Each of the print heads of the apparatus also contains a second conductive layer having a second conductive layer surface that faces the first hydrophobic surface. The second conductive layer is spaced from the first conductive layer to define a gap between them. The second conductive layer has an actuation voltage ranging from about 10 to about 100 volts or more. Each of the print heads of the apparatus also has a wire traction system having at least one conductive elongate wire element disposed in the gap between the first and second conductive layers, and a second hydrophobic surface, having a voltage that is less than the second conductive layer actuation voltage. Each of the print heads of the apparatus has a voltage source communicating with the second conductive layer, and with the elongate wire element, that provides an actuation voltage to the second conductive layer ranging from about 10 to about 100 volts or more. Upon application of the actuation voltage, a droplet is caused to move along a pathway extending around the conductive elongate wire element and from the first conductive layer towards the second conductive layer. In some embodiments having at least four microarray print heads, one of the print heads can be used to actuate droplets of a hydrogel, one of the print heads can be used to actuate droplets of a crosslinker, one of the print heads can be used to actuate droplets of a cell suspension, and one of the print heads can be used to actuate droplets of a bioactive material, such as, for example, a growth factor.
In another embodiment, each of the print heads of the apparatus contains a substrate having a substrate surface and an array of drive electrodes disposed on the substrate surface. Each of the print heads of the apparatus also contains a dedicated array of reference elements settable to a common reference potential and disposed in at least substantially co-planar relation to the electrode array, so that the array of reference elements is electrically and physically distinct from the drive electrode array, and so that each drive electrode is adjacent to at least one of the reference elements. Each of the print heads of the apparatus also contains a dielectric layer disposed on the substrate surface to cover the drive electrodes. Each of the print heads of the apparatus also contains an electrode selector for sequentially activating and de-activating one or more selected drive electrodes of the array to sequentially bias the selected drive electrodes to an actuation voltage, so that a droplet disposed on the substrate surface moves along a desired path defined by the selected drive electrodes. In some embodiments having at least four microarray print heads, one of the print heads can be used to actuate droplets of a hydrogel, one of the print heads can be used to actuate droplets of a crosslinker, one of the print heads can be used to actuate droplets of a cell suspension, and one of the print heads can be used to actuate droplets of a bioactive material, such as, for example, a growth factor.
In another embodiment, each of the print heads of the apparatus contains a substrate having a substrate surface and an array of electrodes disposed in at least substantially co-planar relation on the substrate surface, wherein the array of electrodes contains drive electrodes and dedicated reference electrodes. Each of the print heads of the apparatus also contains a dielectric layer disposed on the substrate surface and covering the array of electrodes. Each of the print heads of the apparatus contains an electrode selector for dynamically creating a sequence of electrode pairs, each electrode pair comprising a selected one of the drive electrodes biased to a first voltage and a selected one of the reference electrodes disposed adjacent to the selected drive electrode and biased to a second voltage less than the first voltage, so that a droplet disposed on the substrate surface moves along a desired path running between the electrode pairs created by the electrode selector, and so that manipulation of the droplet is accomplished by electrowetting actuation so that the droplet overlaps a selected one of the drive electrodes and a selected one of the reference electrodes continuously. In some embodiments having at least four microarray print heads, one of the print heads can be used to actuate droplets of a hydrogel, one of the print heads can be used to actuate droplets of a crosslinker, one of the print heads can be used to actuate droplets of a cell suspension, and one of the print heads can be used to actuate droplets of a bioactive material, such as, for example, a growth factor.
In some embodiments, the invention is a method of tissue engineering by actuating droplets by electrowetting. In one embodiment, the steps of the method include the horizontal actuation of each droplet of a group of hydrogel droplets, to position the hydrogel droplets at discrete locations on a microfluidic chip, followed by the vertical actuation of the group of hydrogel droplets so that the group of hydrogel droplets is deposited on a tissue growth surface. In another embodiment, the steps of the method include the horizontal actuation of each droplet of a group of crosslinker droplets, to position the crosslinker droplets at discrete locations on a microfluidic chip, followed by the vertical actuation of the group of crosslinker droplets so that the group of crosslinker droplets is deposited on a tissue growth surface. In another embodiment, the steps of the method include the horizontal actuation of each droplet of a group of cell suspension droplets, to position the cell suspension droplets at discrete locations on a microfluidic chip, followed by the vertical actuation of the group of cell suspension droplets so that the group of cell suspension droplets is deposited on a tissue growth surface. In another embodiment, the steps of the method include the horizontal actuation of each droplet of a group of growth factor droplets, to position the growth factor droplets at discrete locations on a microfluidic chip, followed by the vertical actuation of the group of growth factor droplets so that the group of growth factor droplets is deposited on a tissue growth surface. In various embodiments, the steps of horizontally and then vertically actuating droplets of hydrogel, crosslinker, cell suspension, and growth factor are repeated to deposit a second layer, or a third layer, or a fourth layer, or additional layers up to a hundred, or up to a thousand, or up to a million or more layers of droplets onto the tissue growth surface.
In the EWOD multi-microarray printing system of the invention, at least four different kinds of materials can be combined to make soft tissue constructs: hydrogels, crosslinkers, cells and bioactive materials, such as growth factors. This multiple material scaffold can be treated as a heterogeneous object in the modeling process.
To represent a heterogeneous tissue construct, tissue geometry, as well as material distributions of the object, can be designed. The CAD model can thus be enriched with useful material information. The continuously varying material composition produces gradation in material properties, sometimes referred to as functionally gradient materials (FGM) (see Cheng and Lin, 2005, Computer-Aided Design 37:1115-1126). In the scheme of heterogeneous tissue construct modeling, in some embodiments, voxel-based volumetric datasets to represent the material variations can be used.
The core issue of heterogeneous tissue representation is to design a scheme to represent the geometry and material information in order to integrate with the CAD system and material design. First, a geometric space, S, having the macroscopic shape of the tissue, is defined. A heterogeneous solid, H, on geometric space S, is defined, such that H consists of different kinds of materials having different material composition functions within the interior, specified by the material designer. For material composition, the material space as a vector space, and the components as the material primitives, are designed. A FGM of the heterogeneous class can thus be represented; and a distance field is referred to as a distance map of all inner points to the selected features. The FGM takes the distance from inner object point to the selected feature(s) as arguments, and it must satisfy the requirement of 0<FGM<1 in the material gradient range. In various embodiments, the user interface for this EWOD microarray system includes various support modules: a voxelization module, a material evaluation module (material designer), and a 2D slicing module.
The hydrogel printing system of the invention, for freeform fabrication of tissue constructs, can deposit living cells and other biological and bioactive compounds in a biological friendly environment, for example, at temperatures ranging from about 4° C. to about 37° C. A key component of the inventive system is a multi-microarray (e.g., 2 array units, 3 array units, 4 array units, 5 array units, 6 array units, or more) printing-head that is designed to make accurate micro-deposits of various viscosity fluids, suspensions and solutions, with exceptional control (
The EWOD-based print head is fixed on the moving planar arm and moves in X and Y directions (i.e., horizontally) based on the scaffold CAD model (
Under the control of a computer, the moving table moves up one layer height in the Z direction (i.e., vertically) after finishing each layer. In this manner, the hydrogel scaffold is stacked up layer by layer and a three-dimensional hydrogel scaffold can be fabricated. The multiple microarray system allows for the near-in-time deposition of cells, growth factors, and scaffold materials, thus enabling the fabrication of heterogeneous tissue scaffolds that contain living cells and bioactive compounds.
Generally, SFF systems provide some kind of a support structure build mechanism to deal with overhang features on the built part. Although not specifically excluded from the system of the invention, the tissue fabrication system of the invention need not include this support structure build mechanism for at least three reasons. First, most scaffolds have self-supported internal tissue matrix and can be oriented to eliminate support structures. Second, generally, the deposited hydrogel is sticky, and thus easily sustained and interconnected. Third, hydrogel and cell densities are similar to water, and a buffer solution may be used to support the weight of the deposited hydrogel and cells. Optional elimination of the support structure can serve to simplify the system and reduce build time.
Generally, the creation of a 3D CAD model is the first step in SFF. The 3D CAD model can be created directly using CAD software (commercially available examples include, Pro/E, Ideas, Solidworks, or AutoCAD) or can be created using reverse engineering, which can reconstruct the 3D model from surface coordinates or multi-planar images of cross-sections of the model. The 3D model of the imaged parts is reconstructed from these high-resolution multi-planar images through biomimetic model software. Several commercially available biomimetic software packages, such as Mimics (Materialize) and AMIRA (TGS), can be used to reconstruct a 3D model from CT and MRI images and generate an STL file, which is a commonly used data format in SFF. By way of a non-limiting example, these processes can be used to create a CAD-based flow and manufacturing model of a child's airway based on MR volume imaging. By way of a further non-limiting example, these processes can be used to create a CAD-based flow and manufacturing model of a trabecular bone model from MicroCT for SFF manufacturing of bone tissue scaffold. By way of further non-limiting example, these processes can be used to create a CAD-based flow and manufacturing model of a biomimetic myocardial model from imaging studies of rat hearts. By way of further example, myocardial ultrastructure can be mapped using serial sectioning, staining with hematoxilin and eosin, imaging by brightfield microscopy, and 3D reconstruction in AMIRA.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used.
The term “layer” is used herein to denote a structure or body that is typically but not necessarily planar or substantially planar, and is typically deposited on, formed on, coats, treats, or is otherwise disposed on another structure.
The term “communicate” (e.g., a first component “communicates with” or “is in communication with” a second component) is used herein to indicate a structural, functional, mechanical, electrical, optical, or fluidic relationship, or any combination thereof, between two or more components or elements. As such, the fact that one component is said to communicate with a second component is not intended to exclude the possibility that additional components may be present between, and/or operatively associated or engaged with, the first and second components.
For purposes of the present disclosure, it is understood that when a given component such as a layer, region or substrate is referred to herein as being disposed or formed “on”, “in”, or “at” another component, that given component can be directly on the other component or, alternatively, intervening components (for example, one or more buffer layers, interlayers, electrodes or contacts) can also be present. It will be further understood that the terms “disposed on” and “formed on” are used interchangeably to describe how a given component is positioned or situated in relation to another component. Hence, the terms “disposed on” and “formed on” are not intended to introduce any limitations relating to particular methods of material transport, deposition, or fabrication.
For purposes of the present disclosure, it will be understood that when a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being “on”, “at”, or “over” an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
The materials and methods employed in the experiments disclosed herein are now described.
The capability of EWOD to dispense hydrogels has been assessed as follows. Several solutions were mixed, filtered, and then used as described below: a 1% w/v sodium alginate, a 2% w/v sodium alginate with a viscosity of 250 cP at 25° C., and a 1% w/v calcium chloride solution. The experiments described herein were performed on a glass chip with patterned chrome electrodes, having a pitch of 0.75 mm and a gasket to maintain top-plate height above the electrodes. The chip was first coated with Parylene C, which functions as a dielectric and chemical insulator, and then was coated with a thin layer of Teflon AF for hydrophobicity. The top plate consisted of a sputtered indium-tin oxide (ITO)-coated film, which was later coated with Teflon AF for hydrophobicity. ITO is a transparent conductor, allowing the top plate to remain grounded during operation. All of the experiments were performed at room temperature.
First, experiments were conducted to determine whether the alginate and crosslinking solutions could be actuated, dispensed, combined, and split. For each test, the glass chip and top plate were washed and dried, then the top plate was aligned and placed on top of the glass chip and the space between them was filled with 2 cSt silicone oil, which serves to prevent evaporation and reduce the actuation voltage. The first test was performed by filling a reservoir with 650 nl of 1% alginate. A 55V actuation voltage was chosen, ensuring a reasonable velocity without causing dielectric breakdown. The solution was dispensed by extending a ‘finger’ of the solution from the reservoir through electrowetting, then by applying voltage only to the last electrode in the ‘finger’ and the reservoir, causing the finger to narrow and eventually split between the two electrodes (
Second, experiments were conducted to determine whether the alginate solution and a crosslinking solution could be dispensed, actuated, and then combined to create the hydrogel on chip. Approximately 650 nl of the 1% alginate and the 1% Genipin crosslinking solution were inserted into their own adjacent reservoirs. The crosslinker was dispensed first, moved to a holding electrode, and then the alginate was dispensed and moved toward the crosslinker. When the droplets moved together, they rapidly became unable to be actuated, indicating a change in the surface tension or the viscosity or both the surface tension and the viscosity. To determine whether the alginate had gelled, the top plate was removed and the droplet was ‘squashed’ using a pipette tip (
The capability of the EWOD process for handling, dispensing and actuating cell suspensions was examined. Further, the voltage that can be applied to cells without damaging them was examined.
Tests were conducted on the EWOD chip using the human fetal osteoblast cell line hFOBs 1.19 (obtained from ATCC between passage 11 and 13). Cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) containing 10% FBS and 1% Penicillin-Streptomycin prior to the experiment. Cultured cells were trypsinised, suspended in PBS and separated by centrifuging. The separated cells were treated with a Live Dead Assay (Molecular Probes) reagent solution (6 μM ethidium homodimer-1 and 2 μM Calcein in PBS) according to the manufacturer's instructions. The cell suspension was loaded on chips, which were actuated with voltages ranging from 40-60 V. After actuation the EWOD chips were observed under fluorescent microscope to quantify live and dead cells. The fraction of live cells after actuation was about 94% (
Vertical actuation of droplets ranging in size between nanoliter-sized and microliter-sized was accomplished (
The performance of the printing system depicted in part in
H9c2 myoblasts are used. H9c2 myoblasts are maintained in DMEM containing 10% FBS and 1% Penicillin-Streptomycin. Upon reaching confluence, and in the presence of low serum concentrations and differentiating growth factors (e.g., bFGF, IGF-1, all-trans-retinoic acid, etc.), H9c2 myoblasts differentiate into cardiac myocytes and eventually fuse to form myotubes. While not contracting spontaneously, the differentiated/fused cells can contract in response to pacing and express numerous cardiomyocyte-like electrophysiological properties, thus demonstrating their histiotypic phenotype.
During the EWOD printing process (
Initially, cell proliferation is assessed every other day using the Alamar blue assay. In select scaffolds, the viability of the cells in the scaffolds is evaluated by confocal fluorescence microscopy using the Live-Dead Assay. Upon reaching confluence (as verified by phase contrast microscopy), differentiation of the myoblasts into myocytes and fusion into myotubes is induced, for example, by reducing the serum contents to 1% and addition of IGF-1 (10 ng/ml) and/or all-trans-retinoic acid (10 nM). Constructs containing differentiated myocytes/myotubes are stimulated electrically. One of skill in the art will be able to empirically determine the optimal pulse parameters (e.g., duration, height, pacing frequency). Electrical activity is monitored using the Microelectrode Array System from Multi Channel Systems (Reutlingen, Germany). Contraction is monitored optically using video graphic analysis software.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.
While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This invention was made, in part, using funds obtained from the U.S. Government (National Science Foundation Award No. 0700139) and the U.S. Government therefore has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/36371 | 3/6/2009 | WO | 00 | 11/29/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61068501 | Mar 2008 | US |
