Patent Application
20070110962
The field of microfabrication generally concerns the manufacture and use of structures with dimensions on the order of micrometers to millimeters. The ability to make structures with micrometer resolution offers significant potential for applications in bioengineering and medicine, especially in tissue engineering. For example, in vitro models of vascular or other biological tissues can be developed based on such structures. Other applications of microfabricated structures or networks include their use as artificial tissues, medical devices, biosensors, drug delivery models or matrices for separations.
Hydrogels have garnered considerable interest as the chemical constituent of microstructures for biological applications. A hydrogel is a three-dimensional polymer, or array of polymers, that is hydrated by water or an aqueous solution. Tanaka, “Gels,” Sci. Am., 244, 124-138 (1981). Typical polymers that comprise hydrogels include proteins and/or sugars. Protein- or sugar-based hydrogels may exhibit properties that resemble those of various biological materials including extracellular matrices, particularly when the protein or sugar is a naturally occurring biological macromolecule.
The microfabrication of structures to date has primarily focused on non-hydrated materials, such as metals, ceramics and hard polymers, instead of hydrogels. These types of microstructures do not contain water as-fabricated. These structures are usually manufactured by pattern transfer methods such as photolithography using light or soft lithography using a micropatterned mold. In soft lithography, the surface of the mold contains a micropatterned topology. Xia et al., “Soft lithography,” Angew. Chem. Int. Ed., 37, 551-575 (1998). The inverse of this micropatterned topology is transferred to the structure during, for example, microtransfer molding, micromolding in capillaries or replica molding. Although polymeric and metal structures are convenient to fabricate by either photolithography or soft lithography, these microstructures poorly represent in vitro models of vascular or other biological tissues. These structures are also generally incapable of encapsulating a suspension of biological materials.
Pattern transfer methods have also been used to fabricate protein or cell layers on a micropatterned mold such as, for example, poly(dimethylsiloxane) (PDMS). Borenstein et al., “Microfabrication technology for vascularized tissue engineering,” Biomed. Microdevices, 4, 167-175 (2002). Although these layers consist of biological materials, they are too thin to represent in vitro models of three-dimensional biological gels or extracellular matrices. In addition, protein and cell layers are too thin to support a suspension of biological materials. These layers also cannot be interconnected or bonded together to yield more complex structures, such as a three-dimensional network.
Although bulk hydrogels of various compositions have been developed for many applications such as electrophoresis and chromatography, patterned microstructures or networks that have microscale inner and outer surface geometries cannot be easily fabricated in hydrogels. Photopolymerization of monomer and macromer solutions are suitable for forming simple hydrogel structures. Beebe et al., “Functional hydrogel structures for autonomous flow control inside microfluidic channels,” Nature, 404, 588-590 (2000). These methods, however, cannot consistently and accurately manufacture complicated hydrogel structures with a micropatterned surface topology. Moreover, these methods are poorly suited for fabricating hydrogels that consist of materials that cannot be photopolymerized, such as collagen, proteoglycans or living cells. A photopolymerization method is also not developed for manufacturing three-dimensional hydrogels that have open network topologies.
Methods based on cellular self-assembly have related shortcomings to those described above for fabricating micropatterned hydrogel structures. Cellular self-assembly involves interactions among cells and extracellular matrices that work to naturally form a microarray. Jakab et al., “Engineering biological structures of prescribed shape using self-assembling multicellular systems,” Proc. Natl. Acad. Sci. USA, 101, 2864-2869 (2004). Histogenesis and/or organogenesis are examples of such methods. Self-assembly is inconvenient for fabricating hydrogel structures comprising a suspension of biological materials or those that are networked together.
Based on the limitation of the methods described above, a need exists for a method to manufacture three-dimensional hydrogels that have microscale features. There is also a need for microfabricating hydrogels that can be interconnected to yield a network that can, for example, represent in vitro models of vascular or other biological tissues. A need also exists for bonding hydrogel structures together to yield more complex three-dimensional structures. There is also a need for hydrogel structures and networks that can support a suspension of biological or other materials.
The present invention provides three-dimensional hydrogel structures patterned by a treated micropatterned mold. The treated mold is capable of transferring the inverse of its micropattern to a hydrogel by contact during formation or polymerization of the hydrogel from a precursor. The surface treatment of the micropatterned mold is designed to eliminate nonspecific binding between the hydrogel and mold. The hydrogel and mold can be separated from each other without collapsing the structure of the hydrogel or irreparably damaging its micropattern. The micropattern that is transferred may yield individual and/or interconnected features such as, for example, channels in the hydrogel that can sustain the flow of liquids. A hydrogel structure of the invention comprises a polymer array surrounded by a fluid, such as, for example, water or an aqueous solution, that hydrates the array.
The hydrogel precursor may be supported by a substrate, such as, for example, glass or a polystyrene dish, when it is contacted by the micropatterned mold. The substrate may also support the formed or polymerized hydrogel structure during and/or after patterning and separation from the mold. Alternatively, a hydrogel precursor can be formed or polymerized on top of the micropatterned mold. The polymer array of a micropatterned hydrogel structure may, for example, be protein and/or sugar-based. Such hydrogel structures can be embedded with biological, organic, metallic, and/or inorganic materials, such as drugs, macromolecules, micro- or nanoparticles, or cells.
The hydrogel structures of the invention can also be formed or polymerized to encapsulate other hydrogels of the same or different type. An encapsulated hydrogel that is a different type may be perturbed, for example, by an enzyme that specifically digests the encapsulated hydrogel, to form cavities in the encapsulating structure. These cavities can be micropatterned provided that the perturbed hydrogel was micropatterned. Hydrogel structures can also be interfaced with other hydrogels to yield more complicated structures. For example, a first hydrogel structure can be bonded or entangled with a second hydrogel structure.
The invention also provides a hydrogel network fabricated by interfacing at least two hydrogels in which one or more of the hydrogels may be a micropatterned structure. The hydrogel structures of a network may be patterned by a treated micropatterned mold such as described above. For example, the mold may transfer the inverse of its micropattern to one or more of the hydrogel structures. The micropattern that is transferred may yield individual features and/or interconnected channels in the hydrogel that are operable for microfluidic flow. Micropatterned hydrogel structures can also be specifically aligned to interconnect their patterns. These interconnected micropatterns can comprise a microfluidic network. Similarly, a micropatterned hydrogel structure featuring channels may be flushly contacted by a material, for example, a petri dish, such that the hydrogel and material form a microfluidic network. The interfaced hydrogels may also be combined into a multilayer structure.
Structures or networks of the invention comprise hydrogels that can adhere together by chemically bonding and/or mechanically entangling. The structures or networks may also not be adhered together. This bonding and/or entanglement can be facilitated by exposing the hydrogels to a controlled concentration of destabilizers at the area of interface. The diffusion of one or more hydrogel precursors into a hydrogel structure or network and then forming or polymerizing the precursors can also be used to bond and/or entangle hydrogels together. Hydrogel structures or networks can also be supported by one or more substrates such as, for example, glass or a polystyrene wafer. The invention also provides stabilizers, often kosmotropes, and destabilizers, often chaotropes, that can be used to affect the conformation of the hydrogels and facilitate bonding and/or entanglement. A hydrogel structure or network of the invention can be used to represent models of in vitro vascular or other biological tissues.
A treated micropatterned mold used to pattern one or more hydrogels can be a poly(dimethylsiloxane) (PDMS) mold. As described above, hydrogel precursors can be formed or polymerized while in contact with the treated surface of a micropatterned mold, such that the mold transfers the inverse of its pattern to the hydrogel structure. This pattern transfer can be accomplished by methods that include, for example, microtransfer molding, micromalding in capillaries and replica molding. These methods may involve the hydrogels being formed or polymerized while in contact with a substrate. These methods can allow individual and/or interconnected features, such as, for example, channels, to micropattern a hydrogel structure with a repeatable resolution that is less than about 5 μm. This resolution corresponds to the size of the features on the surface of the treated micropatterned mold.
The micropatterned surface of the mold is treated prior to contact with the hydrogel precursor, in order for the formed or polymerized structure to be separated from the mold without collapsing or irreparably deforming the micropattern on the hydrogel. The micropatterned mold can be treated by a release agent absorbed or layered on the surface of the mold. These release agents include, for example, bovine serum albumin (BSA), immunoglobulins, copolymers of ethylene oxide and propylene oxide, and/or oligo(ethylene glycol)-terminated self-assembled monolayers. The release agents can also be used to treat a substrate, such as, for example, glass or a polystyrene wafer. The type of release agent used can depend on the composition of the micropatterned mold or substrate being treated.
The invention provides a convenient method for fabricating the hydrogel structures and networks described above. For example, the invention provides a method for fabricating a micropatterned hydrogel structure by using a treated micropatterned mold. The method is capable of reproducibly transferring the inverse of the micropattern of the mold to a hydrogel structure at a resolution that is less than about 5 μm. A method of the invention generally comprises forming or polymerizing a hydrogel precursor while the precursor is in contact with the treated surface of the micropatterned mold. The method can also comprise the micropatterned hydrogel being formed or polymerized on a substrate. The micropatterned hydrogel can be separated from the mold, without being collapsed or irreparably deformed, by any suitable process. These processes include, for example, vibration, mechanical separation, application of air bubbles or application of buoyant forces from a fluid acting on the mold.
The method of the invention can be used to embed and/or suspend biological, organic, metallic, and/or inorganic materials within a hydrogel structure. The present method can also be used to interface a plurality of hydrogel structures. This interfacing may allow the structures to be combined into a microfluidic network or a multilayered structure. The interfacing may also comprise bonding and/or entangling hydrogels into a more complex structure or network. According to the invention, this bonding and/or entanglement can be aided by destabilizers acting alone, or with a stabilizer to conform the polymer array of a hydrogel. The diffusion of one or more hydrogel precursors into a hydrogel structure or network and then forming or polymerizing the precursors can also be used to bond and/or entangle the hydrogels. The method also describes the formation of a hydrogel structure that encapsulates other hydrogels of a different formulation. An encapsulated hydrogel that is a different type may be perturbed, for example, by an enzyme that specifically digests the encapsulated hydrogel, to form cavities in the encapsulating structure. These cavities can be micropatterned provided that the perturbed hydrogel was micropatterned.
Other features and advantages of the present invention will be apparent from the following detailed description of the invention, taken in conjunction with the accompanying drawings of which:
The present invention provides three-dimensional hydrogel structures and networks patterned by a treated micropatterned mold. The structures or networks of the invention can be used, for example, to represent in vitro models of vascular or other biological tissues. The treated mold is capable of transferring the inverse of its micropattern to a hydrogel by contact during formation or polymerization of the structure from a precursor. The micropattern that is transferred may yield individual and/or interconnected features such as, for example, channels in the hydrogel that are operable for microfluidic flow. The micropatterned features of the hydrogel structures can have a resolution that is less than about 5 μm.
The micropatterned hydrogel structures of the invention can also be interfaced so as to adhere together by chemically bonding and/or mechanically entangling. The diffusion of one or more hydrogel precursors into a hydrogel structure or network and then forming or polymerizing the precursors can also be used to bond and/or entangle the hydrogels. A network of hydrogels may also be fabricated by interfacing individual hydrogel structures. Micropatterned hydrogel structures can also be specifically aligned to interconnect their patterns. These interconnected micropatterns can comprise a microfluidic network. A hydrogel structure or network of the invention can be used, for example, as a component of artificial tissues, medical devices, biosensors, drug delivery models or separation processes.
Hydrogel structures are patterned by a treated micropatterned mold that contacts the precursor of the hydrogel during formation or polymerization. The transferred micropattern is the inverse of the pattern on the treated surface of the mold. The transferred micropattern defines the surface topology of the fabricated hydrogel structure. The treated micropatterned molds can, for example, be poly(dimethylsiloxane) (PDMS) molds, silicon wafers patterned with crosslinked photoresist or glass. The micropattern of the mold can be transferred by methods that include, for example, microtransfer molding, micromolding in capillaries and replica molding. The surface of a micropatterned mold employed in these methods is treated with a release agent, which is absorbed or layered on the mold. These release agents include, for example, bovine serum albumin (BSA), immunoglobulins, copolymers of ethylene oxide and propylene oxide, and/or oligo(ethylene glycol)-terminated self-assembled monolayers.
These release agents may prevent or substantially minimize nonspecific binding between the micropatterned mold and the hydrogel precursor and the resulting hydrogel structure. The release agent that is selected to treat the micropatterned mold surface can also depend on the composition of the mold. A BSA solution, for example, may be used to treat a PDMS micropatterned mold. This solution may contain about one percent by weight of BSA in a phosphate buffered saline (PBS) solution. The BSA solution can be applied to the micropatterned mold at about 4° C. for about an hour or more. The solution that is not absorbed onto the surface of the mold can be rinsed away, if desired. A treated micropatterned mold can be separated from the hydrogel without collapsing the structure or irreparably deforming the micropattern on the hydrogel. The concentrations, temperatures and/or times identified above are provided merely as examples as any other suitable ranges of the same could be used according to the invention.
The treated mold can be separated from the micropatterned hydrogel structure by methods that include, for example, vibration, mechanical separation, application of air bubbles or application of buoyant forces from a fluid acting on the mold. A pneumatic or chemical separation of the micropatterned mold is also suitable in some embodiments. As an example, a PDMS micropatterned mold can be separated by application of buoyant forces from a fluid acting on the mold. In this separation method, PBS, a hydrophilic solution, may be applied around the PDMS mold, which is hydrophobic. The buoyant forces on the mold separate the mold from the hydrogel structure. It can also be useful to moisten the micropatterned mold surface prior to application of the release agent so as to reduce surface energy and prevent air babbles. The micropatterned mold may be moistened, for example, by a solvent, aqueous solution or alcohol, such as, ethanol.
A hydrogel structure of the invention comprises an interconnected array of polymers surrounded by a fluid that hydrates the array. The fluid may also comprise, for example, solvated particles such as ions and/or salts. The fluid acts in equilibrium with the polymer array and ions and/or salts that may be present, to help support the integrity of the hydrogel such that it does not collapse or deform. The present hydrogels may, for example, be protein and/or sugar-based. These hydrogels can be useful for embedding and/or suspending biological, organic, metallic, or inorganic materials, such as drugs, macromolecules, or cells, in the hydrogel structure. These hydrogel structures can also be supported by a substrate such as, for example, glass or a polystyrene wafer.
The hydrogel structures of the invention can also be formed or polymerized to encapsulate other hydrogels of the same or a different type. An encapsulated hydrogel that is a different type may be perturbed, for example, by an enzyme that specifically digests the encapsulated hydrogel, to form cavities in the encapsulating structure. These cavities can be micropatterned provided that the perturbed hydrogel was micropatterned. Hydrogel structures can also be interfaced with other hydrogels to yield more complicated structures. For example, a first hydrogel structure can be bonded or entangled with a second hydrogel structure.
In one embodiment, a three-dimensional hydrogel structure is micropatterned by contact with the surface of a treated micropatterned mold during formation or polymerization of a hydrogel precursor. This embodiment employs pattern transfer methods that include, for example, microtransfer molding, micromolding in capillaries or replica molding. The micropatterned hydrogel structure is capable of maintaining its integrity for an extended length of time that can include at least several months. The hydrogel generally comprises a polymer array of material surrounded by a fluid, such as, for example, water or an aqueous solution. The fluid hydrates the hydrogel and helps maintain the three-dimensional structure along with the polymer array and any solvated ions and/or salts that may also be present. The hydrogel structures may be protein and/or sugar-based. Examples of protein and/or sugar-based hydrogels include those that comprise an array of collagen, MATRIGEL, gelatin, agarose or combinations of these proteins and/or salts. As described above, these hydrogels can be formed or polymerized from a hydrogel precursor. The precursor may be prepared by any suitable method, which can depend on the composition of the hydrogel.
A hydrogel precursor can be formed or polymerized by a variety of methods. These methods can include, for example, photopolymerization, free radical polymerization, cationic polymerization or anionic polymerization. The formation and polymerization of a hydrogel structure can be influenced by parameters that include temperature, humidity, total ionic concentration, concentration of particular ions, time and pH. A suitable method for collagen-based hydrogel polymerization, for example, involves neutralizing the collagen-based precursor at about 4° C. such that polymerization occurs after the temperature is changed, for example, to about 37° C. The formation or polymerization of the precursor into a hydrogel is performed as the hydrogel precursor is contacted by the surface of the treated micropatterned mold. The temperatures identified above are provided merely as examples as any other suitable ranges of the same could be used according to the invention.
The treated micropatterned mold 4 in
The topology of the hydrogel structure may be micropatterned as individual and/or interconnected features. These features and/or channels can have a repeatable resolution that is less than about 5 μm. The interconnected channels can also be operable for microfluidic flow. The hydrogel structure may also be separated from the substrate in an embodiment of the invention and/or can be suspended in a solution.
An alternate scheme for micropatterning a hydrogel structure involves a microtransfer molding method. This scheme is represented in
The pattern transferred from the micropatterned mold to the hydrogel may be aided by pressure applied to the mold by a suitable weight. The weight used for micropatterning is dependent on, for example, the type of hydrogel and the surface area of the pattern. By adding a sufficient amount of pressure, the hydrogel structure may be micropatterned with holes so that, for example, an interconnection of channels can be fabricated between multiple bonded and/or entangled hydrogel structures. The release agent may be absorbed or layered on the mold. These release agents include, for example, bovine serum albumin (BSA), immunoglobulins, copolymers of ethylene oxide and propylene oxide, and/or oligo(ethylene glycol)-terminated self-assembled monolayers.
The release agent allows the micropatterned mold to be separated from the hydrogel without collapsing or irreparably deforming the structure or its micropattern. As shown, the topology of the hydrogel structure is micropatterned such that its features correspond to the inverse of those for the treated micropatterned mold. The features of
After the channels of the mold are moistened, a release agent 6, such as, for example, BSA, can be vacuum drawn by a device 30 through the channels to completely treat the micropatterned mold. As described above, treatment of the mold prevents or substantially minimizes nonspecific binding between the mold and the hydrogel precursor and the resulting hydrogel structure. With the mold and its channels treated, vacuum pressure is again provided to draw the precursor into the channels of the micropatterned mold. The hydrogel precursor is then formed or polymerized into the micropatterned hydrogel structure.
The treated surface of the micropatterned mold can pattern the hydrogel structure while it is in contact with the substrate. The micropattern that is transferred may yield individual and/or interconnected features such as, for example, channels in the hydrogel that are operable for microfluidic flow. The micropatterned features of the hydrogel structure can have al resolution that is less than about 5 μm. A typical micropatterned hydrogel structure prepared by this scheme and supported on a glass substrate is shown in the optical micrograph of
In another embodiment, the micropatterned hydrogel structures described above can be interfaced with other hydrogels to yield more complicated structures. For example,
The precursor 34 in this scheme and the precursor of the formed or polymerized micropatterned hydrogel 32 may be mixed with a suspension of biological materials, drugs, organics, metals, inorganics, macromolecules, cells or synthetic materials. The suspension can also comprise any combination of such materials. A suspension of fibroblasts and/or endothelial cells, for example, may be prepared in a media by growing cells on a substrate, and providing trypsin to allow the cells to be introduced into solution. This solution can also be centrifuged to increase the concentration of the cells. In the scheme of
The scheme also allows for the micropatterned hydrogel structure to comprise a suspension of mammalian fibroblast cells. This suspension may be labeled with a specific fluorescent marker, which can be used to distinguish, for example, the fibroblast cells of the micropatterned hydrogel from those cells suspended in the precursor. The micropatterned structure may also be fabricated by a microtransfer molding method employing a treated micropatterned mold. As described above, microtransfer molding can be used to fabricate individual and/or interconnected micropatterned features such as, for example, channels in the hydrogel that are operable for microfluidic flow. The micropatterned features of the hydrogel structure can have a resolution that is less than about 5 μm.
After mixing the precursor 34 with fibroblast cells and dispersing it to surround the micropatterned hydrogel structure 32, the precursor and hydrogel structure may be contacted by, for example, a glass layer 38 treated with a BSA solution 40. For example, the glass can be treated by an individual droplet of BSA solution containing about 5 μl. The treated glass can aid the interfacing of the precursor and micropatterned hydrogel. The precursor can then disperse by capillary forces into, for example, the channels of the micropattern hydrogel. The precursor is subsequently formed or polymerized with the treated glass in place to yield a hydrogel. The treated glass can also be separated from the hydrogels without collapsing or irreparably deforming the resulting coplanar structure 42. This coplanar structure may comprise two individual sets of fibroblast cells, each suspended within their respective hydrogel.
Optical micrographs in
The micropatterned hydrogel structures described above can also be interfaced with other hydrogels. These interfaced hydrogels may yield a multilayered hydrogel structure. The interfaced hydrogels can also adhere together by chemically bonding and/or mechanically entangling. This bonding and/or entanglement also allows hydrogel structures to be combined and fabricated into more complex hydrogel structures. Bonding and/or entanglement can be facilitated by destabilizers acting alone, or with a stabilizer to conform the polymer array of a hydrogel. For example, when a hydrogel is exposed to a controlled concentration of destabilizers, its polymers can change conformation so that they may substantially interact with an interfaced hydrogel.
The hydrogel that is conformed is then stabilized while in contact with the interfaced hydrogel. During stabilization, the polymers of each hydrogel are conjectured to adhere together by chemically bonding and/or mechanically entangling. The degree to which the hydrogels can be bonded and/or entangled together may be manipulated by varying the parameters under which the destabilizer and stabilizer, if any, are used. These parameters can include, for example, temperature, humidity, total ionic concentration, concentration of a destabilizer and/or stabilizer, time and pH. The type of hydrogels that are interfaced together can also affect the degree of chemical bonding and/or mechanical entanglement.
A suitable destabilizer and stabilizer can depend on the composition of the hydrogel's polymer array. In general, these destabilizers and stabilizers can be chaotropes and kosmotropes, respectively. Common destabilizers in water include, for example, SCN−, H2PO4−, guanidinium, HSO4−, HCO3−, urea, I−, Cl−, NO3−, tetramethylammonium, NH4+, Cs+ or K+. Examples of stabilizers in water can include SO42−, HPO42−, Mg2+, Ca2+, Li+, Na+, H+, OH− or HPO42−. More specifically, destabilizers of collagen, MATRIGEL and/or agarose polymers can include, for example, NaSCN, urea, LiCl, glucose, NaClO4, MgCl2, glycerol, CON2H4 or guanidinium HCl. Stabilizers of collagen polymers can include NaF, NaSO4, betaine, NH4Cl or trimethylamine N-oxide. The destabilizers and stabilizers identified above can also be used in combination with each other or different solutions, such as, for example, a PBS solution, to affect the conformation of a particular hydrogel. A hydrogel may also be stabilized by a chaotrope and destabilized by a kosomotrope.
There are several approaches to using destabilizers and stabilizers for chemically bonding and/or mechanically entangling interfaced hydrogels. One approach is to controllably diffuse or flow a destabilizer into a first hydrogel, which is interfaced with a second hydrogel, at a concentration sufficient to change the conformation of the first hydrogel's polymer array. The destabilizers can affect the conformation of the polymers at the area of interface between the hydrogels by diffusing or flowing through the hydrogel structure and/or channels micropatterned on the surface of a hydrogel. A destabilizer may also be applied to the surface of one or both of the hydrogels before they are interfaced.
The destabilizer is conjectured to allow the polymers of the first hydrogel to substantially interact with the interfaced hydrogel, whose polymers may also be affected by the destabilizer. A solution is then introduced, for example, by controlled diffusion or flow, to lower the relative concentration of the destabilizer so that the first hydrogel's polymers stabilize and can chemically bond and/or mechanically entangle with the polymers of the interfaced hydrogel. This solution may, for example, be a PBS solution, which is relatively neutral in terms of destabilizing and/or stabilizing hydrogels.
Another approach is to employ a destabilizer and a stabilizer, which can act together on the polymer array of a hydrogel. In this approach, the concentrations of the stabilizer and destabilizer can be regulated to control the conformation of the hydrogel's polymers. As described above, the destabilizers and stabilizers can affect the conformation of polymers at the area of interface between the hydrogels by diffusing or flowing through the hydrogel structure and/or channels micropatterned on the surface of a hydrogel. The first hydrogel is initially destabilized to affect the conformation of its polymers. The relative concentration of the destabilizer is then lowered by increasing the concentration of the stabilizer so that the first hydrogel stabilizes and can chemically bond and/or mechanically entangle with the polymers of the interfaced hydrogel.
After the precursor 46 forms or polymerizes, the treated micropatterned mold 56 and a treated substrate 58 are separated from the second micropatterned hydrogel structure 48. The mold and substrate are preferably treated by a release agent 6. The second structure can remain in contact with the PDMS frame 50. The second hydrogel structure can then be interfaced with the first micropatterned hydrogel structure 52. This interfacing can be aided by immersing the structures in a solution, such as, for example, saline, so that when the solution is removed the hydrogels contact each other. The hydrogels may also be interfaced in a nonbinding manner. When the hydrogels are interfaced, the resulting structure 44 comprises multilayered micropattern hydrogels. This multilayered micropatterned hydrogel structure can be supported on the substrate 54.
The precursors in this scheme can be mixed with a suspension of biological materials, drugs, organics, metals, inorganics, macromolecules, cells or synthetic materials. The suspension can also comprise any combination of such materials. A suspension of fibroblasts and/or endothelial cells, for example, may be prepared in a media by growing cells on a substrate. This solution can also be centrifuged to increase the concentration of the cells. In the scheme of
Optical micrographs in
The hydrogel structures of the invention can also be formed or polymerized to encapsulate other hydrogels of the same or different type. An encapsulated hydrogel that is a different type may be perturbed, for example, by an enzyme that specifically digests the encapsulated hydrogel, to form cavities in the encapsulating structure. Alternatively, hydrogels can be heated to perturb the encapsulated hydrogel, such as, for example, gelatin, and form cavities in the encapsulating hydrogel. These cavities can be micropatterned provided that the perturbed hydrogel was micropatterned. A scheme for fabricating a micropatterned hydrogel comprising cavities is represented in
In this scheme, a MATRIGEL-based structure 60 is formed or polymerized while in contact with a flat collagen-based precursor 62. The collagen is formed or polymerized over the MATRIGEL structure, which will be the perturbed hydrogel. The perturbed hydrogel may be MATRIGEL-based comprising a solution of basement membrane proteins derived from mouse sarcoma. The MATRIGEL-based hydrogel is heated and molded on the collagen-based layer at about 37° C. The collagen-based precursor encapsulates the MATRIGEL-based structure. The collagen-based precursor is slowly added at about 4° C. and heated until about 37° C. Temperature gradients are avoided to prevent deformation of the structures. The MATRIGEL-based hydrogel is digested by an enzyme dispase under mild conditions of about 2 U per ml for one to three hours. The digestion rapidly degrades the MATRIGEL-based hydrogel and minimally digests the collagen-based structure. The resulting hydrogel structure 64 comprises cavities, where the MATRIGEL-based hydrogel was. The parameters identified above are provided merely as examples as any other suitable ranges of the same could be used according to the invention.
A typical hydrogel structure comprising cavities is also shown in the optical micrographs of
One embodiment of the invention comprises fabricating a hydrogel network that can be unsupported or can include a supporting substrate(s). The network is capable of maintaining its integrity for an extended length of time that can include several months. The hydrogel network comprises a plurality of interfaced individually patterned structures of the invention. These networks may be fabricated to comprise two hydrogel structures. The interfaced hydrogels can, for example, be entangled and/or bonded together. Hydrogels in a network may be interfaced as stacked multilayers or configured structures. This embodiment also provides stabilizers, often kosmotropes, and destabilizers, often chaotropes, that can be used to modify the conformation of the hydrogel and facilitate entanglement and/or bonding among the hydrogel structures.
Hydrogel networks of the invention can also comprise a suspension of biological materials, drugs, organics, metals, inorganics, macromolecules, cells and/or synthetic materials. These materials may be suspended in some or all of the individual hydrogels. This hydrogel network can also be formed or polymerized to encapsulate other hydrogels of a different type. The other hydrogels can be perturbed or digested, for example, by an enzyme or suitable perturbant to form cavities in the structure. The cavities can include biological materials, drugs, organics, metals, inorganics, macromolecules, cells or synthetic materials. The type of enzyme used for digestion depends on the hydrogel that is to be perturbed. A MATRIGEL-based hydrogel, for example, can be digested by enzyme dispase. Other suitable enzymes for digesting collagen and agarose include collagenase and agarase, respectively.
The examples herein are presented to illustrate other advantages of the present invention and to assist in preparing the hydrogel structures or networks of the invention. These examples can include or incorporate any of the variations or embodiments of the invention described above. Moreover, the embodiments described above may each include or incorporate the variations of all other embodiments. The following examples are not intended in any way to otherwise limit the scope of the disclosure.
Introduction of a pressure difference can make a solution flow through, for example, the collagen-based hydrogel and the cavities. The flow enhances an exchange of materials between the cavities and the surrounding hydrogel. A fluorescently labeled immunoglobulin IgG of about 150 kDa is incorporated into the MATRIGEL-based hydrogel during formation or polymerization from a precursor, and before digestion of the hydrogel. A fluorescence microscopy method is used to track the release and transport of the antibody out of the cavities after digestion. The decay in fluorescence in the hexagonal cavities is slow without flow. The slow decay is contemplated to be due to nonspecific binding of the antibody with the fibrils in the MATRIGEL-based hydrogel. The binding is sufficient to immobilize a large percentage of the antibody in the hydrogel. The comparisons of inclusion of flow through the network and of non-inclusion of flow are shown in the optical micrographs in
During flow, the bound protein is transported out of the cavities. With flow for two hours, the intensity of fluorescence decreased by 50 percent. By contrast, without flow fluorescence was decreased by 10 percent. Six hours after the introduction of flow, the cavities lost nearly all of the fluorescence. At all times under flow, the initially uniform distribution of fluorescence of the hexagons develops an outward ring that surrounds the cavities. The ring is polarized by the direction of flow. Although flow enhances the transport of molecules into and out of the cavities, it does not alter the shapes of the cavities. Applications for this structure include, for example, the controlled transport of drugs or growth factor to and from through the cavities, and the release of drugs or growth factor in response to an external signal, such as, for example, the introduction of a specific enzyme. For example, the two-dimensional array of cell-filled cavities described above could be used to study cell behavior due to the introduction of drugs. Also, the two-dimensional array of hydrogels could be used to release an encapsulated drug according to a desired time schedule and geometry to enhance the pharmacokinetic profile of the drug in vivo.
The invention provides a convenient method for fabricating the hydrogel structures and networks described above. For example, the invention provides a method for fabricating a micropatterned hydrogel structure by using a treated micropatterned mold. The mold is preferably treated with a release agent as described above. The method is capable of reproducibly transferring the inverse of the micropattern of the mold to a hydrogel structure at a resolution that is less than about 5 μm. A method of the invention generally comprises forming or polymerizing a hydrogel precursor, while the precursor is in contact with the treated surface of the micropatterned mold. The method can also comprise the micropatterned hydrogel being formed or polymerized on a substrate. The micropatterned hydrogel can be separated from the mold, without being collapsed or irreparably deformed, by any suitable process. These processes include, for example, vibration, mechanical separation, application of air bubbles or application of buoyant forces from a fluid acting on the mold.
The method of the invention can be used to embed and/or suspend biological materials, drugs, organics, metals, inorganics, macromolecules, cells or synthetic materials within a hydrogel structure. The present method can also be used to interface a plurality of hydrogel structures. This interfacing may allow the structures to be combined into a multilayer structure or network that can be microfluidic. The interfacing may also comprise chemically bonding, and/or mechanically entangling hydrogels into a more complex structure or network. According to the invention, this bonding and/or entanglement can be aided by destabilizers acting alone, or with a stabilizer to alter the conformation of the polymer array of a hydrogel. The diffusion of one or more hydrogel precursors into a hydrogel structure or network and then forming or polymerizing the precursors can also be used to bond and/or entangle the hydrogels. The method also describes the formation or polymerization of a hydrogel structure that encapsulates other hydrogels that have the same or a different composition. An encapsulated hydrogel that is a different type may be perturbed, for example, by an enzyme that specifically digests the encapsulated hydrogel, to form cavities in the encapsulating structure. These cavities can be micropatterned provided that the perturbed hydrogel was micropatterned.
While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill in the art, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents and other alterations to the compositions, methods and/or articles set forth herein. It is therefore intended that the protection granted by Letter Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.
This application claims the priority of U.S. Provisional Application No. 60/505,155, filed Sep. 23, 2003, entitled METHODS TO MOLD THREE-DIMENSIONAL MICROSTRUCTURES OF GELS, and 60/592,717 filed on Jul. 30, 2004, entitled THREE-DIMENSIONAL GEL MICROSTRUCTURES, both of which are hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US04/31177 | 9/23/2004 | WO | 3/23/2006 |
| Number | Date | Country | |
|---|---|---|---|
| 60505155 | Sep 2003 | US | |
| 60592717 | Jul 2004 | US |
