Patent Application
20220204911
It has been well established that cells, especially mesenchymal stem cells (MSCs), can sense their mechanical environment via focal adhesion molecules. Such sensation transmits the mechanical signal to the actin cytoskeleton, in turn, can drive lineage specification of MSCs. The discoveries of how the mechanical environment cells sense shape their behavior has led to the development of dynamic culturing systems that look at controlling stem cell fate for tissue engineering and regenerative applications along with looking at creating physiological systems to study disease progression and high throughput drug screening. However, in vitro studies look at multiple interactions of mechanical stimuli with microenvironmental stimuli have been limited due to the low experimental throughput of current bioreactor systems. Current researchers have looked at developing microlevel dynamic culture systems that could overcome the limited capabilities of bioreactors given their bulky and complicated systems. These microlevel systems have consisted of piezo array systems and pneumatic pressure systems that provide an actuation force that is translated through a cell culture substrate but is limited by potential disturbances in force or vibration translation through the substrate. Therefore, it would be advantageous to develop materials that could be directly interfaced with the cell culture and deliver the desired stimulation. The compositions and methods disclosed herein address these and other needs.
Magnetic stimuli responsive materials are sensitive transformable materials that can provide programmable, predictive, and highly controllable dynamic ranges that have not been attainable via other field responsive mechanical metamaterials. Recent advancements using magnetic metamaterials for dynamic biological system platforms include magnetic substrate and magnetic pillar configurations to induce dynamic mechanical stimuli on 2D cultures or single cell analysis. These platforms shown promise in the potential of studying in vitro mechanotransduction with high spatial-temporal control with both complex and localized stimuli. The next frontier in the study of mechanobiology is developing novel 3D platforms that have ability to provide highly specific mechanical microenvironments that could be interfaced with 3D cell cultures to study cellular activity in 3D dynamic in vivo mimicking systems. The largest hurdle is creating a system that has both a high-fidelity 3D structure at the micron level and fast, tunable transformation potential.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.
Like reference symbols in the various drawings indicate like elements.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
As used in this specification and the following claims, the terms “comprise” (as well as forms, derivatives, or variations thereof, such as “comprising” and “comprises”) and “include” (as well as forms, derivatives, or variations thereof, such as “including” and “includes”) are inclusive (i.e., open-ended) and do not exclude additional elements or steps. For example, the terms “comprise” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Accordingly, these terms are intended to not only cover the recited element(s) or step(s), but may also include other elements or steps not expressly recited. Furthermore, as used herein, the use of the terms “a”, “an”, and “the” when used in conjunction with an element may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Therefore, an element preceded by “a” or “an” does not, without more constraints, preclude the existence of additional identical elements.
The use of the term “about” applies to all numeric values, whether or not explicitly indicated. This term generally refers to a range of numbers that one of ordinary skill in the art would consider as a reasonable amount of deviation to the recited numeric values (i.e., having the equivalent function or result). For example, this term can be construed as including a deviation of ±10 percent of the given numeric value provided such a deviation does not alter the end function or result of the value. Therefore, a value of about 1% can be construed to be a range from 0.9% to 1.1%. Furthermore, a range may be construed to include the start and the end of the range. For example, a range of 10% to 20% (i.e., range of 10%-20%) can includes 10% and also includes 20%, and includes percentages in between 10% and 20%, unless explicitly stated otherwise herein.
It is understood that when combinations, subsets, groups, etc. of elements are disclosed (e.g., combinations of components in a composition, or combinations of steps in a method), that while specific reference of each of the various individual and collective combinations and permutations of these elements may not be explicitly disclosed, each is specifically contemplated and described herein.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.
System
Described herein are systems for cell culture. In some embodiments, as shown in
In some embodiments, the system (1000) can further include a 3D cell culture matrix (500) sized to be positioned within the vessel (100). In some embodiments, the system (1000) can include a 3D cell culture matrix (500) sized dimensioned to be positioned within the vessel (100) and disposed in contact with the porous, magnetic, elastomeric construct (200). In some embodiments, the system can include a 3D cell culture matrix (500) sized dimensioned to be positioned within the vessel (100) and disposed in contact with the porous, non-magnetic, construct (400). In some embodiments, the 3D cell culture matrix (500) can include a population of cells seeded (600) within a degradable polymer matrix.
In some embodiments, the polymer matrix can include, but is not limited to alginate, chitosan, agarose, fibrin, collagen, hyaluronic acid, a polyhydroxyalkanoate, a polyester, a polyalkylene oxide, a copolymer thereof, or a blend thereof. In some embodiments, the polymer matrix can include fibrin. In some embodiments, the polymer matrix can include alginate.
In some embodiments, as shown in
For example, in some embodiments, as shown in
In some embodiments, as shown in
For example, in some embodiments, as shown in
In some embodiments, as shown in
In some embodiments, the system can further include a force sensor, a microcontroller, a power supply, or any combination thereof. In some embodiments, the system can further include a force sensor. In some embodiments, the system can further include a microcontroller. In some embodiments, the system can further include a power supply.
Described herein are also methods for remote responsive control of dynamic cell culture. The method can include operatively positioning a porous, magnetic, elastomeric construct in proximity to a 3D cell culture matrix; and applying a magnetic field to the porous, magnetic, elastomeric construct.
Porous, Magnetic, Elastomeric Constructs The systems described herein can include a porous, magnetic, elastomeric construct (200). The porous, magnetic, elastomeric construct can be formed from a composite including a biocompatible elastomer and a population of magnetic particles dispersed within the biocompatible elastomer. In some embodiments, the construct can have a substantially cylindrical shape.
In some embodiments, the construct can have a diameter of at least 60% of the inner diameter of the well, (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). In some embodiments, the construct can have a diameter of 99% or less of the inner diameter of the well, (e.g., 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, or 65% or less).
The construct can have a diameter ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the construct can have a diameter of from 60% to 99% of the inner diameter of the well (e.g., from 60% to 90%, from 60% to 80%, from 60% to 70%, from 70% to 90%, from 70% to 80%, from 80% to 90%, or from 85% to 95%).
In some embodiments, the porous, magnetic, elastomeric construct can have a porosity of at least 10%, (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45%). In some embodiments, the porous, magnetic, elastomeric construct can have a porosity of 50% or less, (e.g., 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 15% or less).
The porous, magnetic, elastomeric construct can have a porosity ranging from any of the minimum values described above to any of the maximum values described above. In some embodiments, the porous, magnetic, elastomeric construct can have a porosity of from 10% to 50%, (e.g., from 15% to 50%, from 20% to 50%, from 25% to 50%, from 30% to 50%, from 35% to 50%, from 40% to 50%, from 45% to 50%, from 15% to 40%, from 20% to 40%, from 25% to 40%, from 30% to 40%, from 35% to 40%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 15% to 20%, from 10% to 20%, from 10% to 30%, or from 10% to 40%).
In some embodiments, the porous, magnetic, elastomeric construct can be operatively configured to apply nanovibration, strain, or a combination thereof to a population of cells within the vessel. In some embodiments, the porous, magnetic, elastomeric construct can be operatively configured to apply nanovibration to a population of cells within the vessel. In some embodiments, the porous, magnetic, elastomeric construct can be operatively configured to apply strain, or a combination thereof to a population of cells within the vessel. In some embodiments, the porous, magnetic, elastomeric construct can be operatively configured to apply nanovibration and strain to a population of cells within the vessel.
In some embodiments, the population of cells can include but is not limited to cell able to convert mechanical cues into intracellular biochemical signals for driving cellular growth, differentiation, morphology, homeostasis, and disease. In some embodiments, the cells can include, but are not limited to induced pluripotent stem cells, adipose stem cells, bone marrow stem cells, synovium stem cells, dental pulp stem cells, neural stem cells, umbilical cord mesenchymal stem cells, chondrocytes, osteoblasts, myoblasts, fibroblasts, and myeloid cells.
Magnetic Particles
The magnetic particles can comprise any suitable population of magnetic particles. In some embodiments, the magnetic particles can comprise nanoparticles. The term “nanoparticle,” as used herein, generally refers to a particle of any shape having one or more dimensions ranging from 1 nm up to, but not including, 1 micron. In some embodiments, the nanoparticles can comprise a particle of any shape having one or more dimensions ranging from 1 nm up to, but not including, 1 micron; and one or more dimensions of 1 micron or more (e.g., from 1 micron to 10 microns, from 1 micron to 20 microns, from 1 micron to 25 microns, or from 1 micron to 50 microns).
In other embodiments, the magnetic particles can comprise microparticles. The microparticles can be of any shape, and have one or more dimensions ranging from 1 micron to 100 microns. In some embodiments, all dimensions can range from 1 micron to 100 microns.
In some embodiments, the population of magnetic particles is a monodisperse population of magnetic particles. In other embodiments, the population of magnetic particles is a polydisperse population of anisotropic magnetic particles. In some instances where the population of magnetic particles is monodisperse, greater than 50% of the particle size distribution, more preferably 60% of the particle size distribution, most preferably 75% of the particle size distribution lies within 10% of the median particle size.
The magnetic particles can comprise any suitable magnetic material, such as ferromagnetic alloys comprising iron, cobalt, nickel, or combinations thereof. some embodiments, the magnetic particles can include iron, cobalt, zinc, cadmium, nickel, gadolinium, chromium, copper, gold, silver, platinum, manganese, metal oxide, or an alloy thereof. In certain embodiments, the magnetic particles can include nickel particles.
In some embodiments, the magnetic particles can comprise anisotropic magnetic particles. Such particles can be formed using methods known in the art, including synthesis driven by appropriate shaping ligands, template-assisted synthesis, template-assisted electrodeposition, and magnetically directed assembly. Examples of such materials are described, for example, in Lisjak et al. “Anisotropic Magnetic Nanoparticles: A Review of their Properties, Synthesis, and Potential Applications,” Progress in Materials Science, 2018, 95; 286-328 (which is hereby incorporated by reference in its entirety for its description of anisotropic magnetic particles, and which is attached to this filing).
The magnetic particles can be essentially homogeneous throughout, meaning that the composition does not vary throughout the particle cross-section (from the particle surface to the particle center). Alternatively, the anisotropic particles can possess a non-homogeneous structure. For example, the particles may possess a core-shell structure, or a multilayer structure (e.g., a magnetic core coated by a non-magnetic shell material).
The magnetic particles may have any desired shape. In certain embodiments, the particles can have a non-spherical shape. As generally used herein, “non-spherical” is used to describe particles having at least one dimension differing from another dimension by a ratio of at least 1:1.10. In one embodiment, the non-spherical particles have at least one dimension which differs from another dimension by a ratio of at least 1:1.25. A wide variety of shapes are considered “non-spherical” shapes. For example, non-spherical particles may be in the shape of rectangular disks, high aspect ratio rectangular disks, rods, high aspect ratio rods, worms, oblate ellipses, prolate ellipses, elliptical disks, UFOs, circular disks, barrels, bullets, pills, pulleys, bi-convex lenses, ribbons, ravioli, flat pill, bicones, diamond disks, emarginated disks, elongated hexagonal disks, tacos, wrinkled prolate ellipsoids, wrinkled oblate ellipsoids, or porous elliptical disks.
In some embodiments, the magnetic particles can comprise rod-shaped particles. “Rod-shaped,” as used herein, refers to a particle which has an elongated spherical or cylindrical shape (e.g., the shape of a pill) or a flattened rod-shape, such as the shape of a green bean. The rod-shaped particles can have an aspect ratio of at least 1.25 (e.g., at least 1.5, at least 2, at least 2.5, or at least 5). “Aspect ratio,” as used herein, refers to the length divided by the diameter of a particle. In some embodiments, the rod-shaped particles can have an aspect ratio ranging from 1.25 to 95.
In some embodiments, the magnetic particles can be present in an amount of 20% by weight or less, based on the total weight of the construct, (e.g., 15% by weight or less, 10% by weight or less, 5% by weight or less, 1% by weight or less, or 0.5% by weight or less). In some embodiments, the magnetic particles can be present in an amount of 0.1% by weight or more, based on the total weight of the construct, (e.g., 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, or 15% by weight or more).
The magnetic particles can be present in an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the magnetic particles can be present in an amount of from 0.1% by weight to 20% by weight, based on the total weight of the construct, (e.g., from 0.1% by weight to 15% by weight, from 0.1% by weight to 10% by weight, from 0.1% by weight to 5% by weight, from 0.1% by weight to 2.5% by weight, from 0.1% by weight to 2% by weight, from 0.1% by weight to 1.5% by weight, from 0.1% by weight to 1% by weight, from 0.5% by weight to 15% by weight, from 0.5% by weight to 10% by weight, from 0.5% by weight to 5% by weight, from 0.5% by weight to 2.5% by weight, from 0.5% by weight to 2% by weight, from 0.5% by weight to 1.5% by weight, from 0.5% by weight to 1% by weight, from 1% by weight to 15% by weight, from 1% by weight to 10% by weight, from 1% by weight to 5% by weight, from 1% by weight to 2.5% by weight, from 1% by weight to 2% by weight, from 1% by weight to 1.5% by weight, from 2% by weight to 15% by weight, from 2% by weight to 10% by weight, from 2% by weight to 5% by weight, from 2% by weight to 2.5% by weight, from 5% by weight to 15% by weight, from 5% by weight to 10% by weight, or from 10% by weight to 15% by weight).
The magnetic particles can be present in the composition in an amount of from 0.01% by volume to 2.0% by volume (e.g., from 0.01% by volume to 1.5% by volume, from 0.01% by volume to 1.0% by volume, from 0.01% by volume to 0.75% by volume, from 0.01% by volume to 0.5% by volume, from 0.01% by volume to 0.2% by volume, or from 0.01% by volume to 0.15% by volume), based on the total volume of the composition.
In some embodiments, the magnetic particles can be uniformly dispersed throughout the elastomer. In other embodiments, the magnetic particles can by non-homogenously dispersed throughout the elastomer. For example, the magnetic particles can be at varying concentrations throughout the elastomer (e.g., at a higher concentration at a region of the construct in proximity to a magnet and at a lower concentration at a region further away from a magnet). In some embodiments, a gradient of magnetic particles can be dispersed within the elastomer.
Elastomers
The elastomer can include any suitable biocompatible elastomer. For example, the biocompatible elastomer can comprise an acrylonitrile butadiene styrene (ABS), polyphenylene sulfide (PPS), poly(meth)acrylate, polyphenylsulfone (PPSU), cyclic olefin copolymer (COC), polyetheretherketone (PEEK), polyurethane (PU), polyetherimide (PEI), polyphenylene ether (PPE), polycarbonate (PC), poly(ethyleneterephthalate glycol) (PETG), polysiloxane, or any combination thereof. In some embodiments, the biocompatible elastomer comprises a polysiloxane, such as polydimethylsiloxane (PDMS).
In some embodiments, the elastomer can include a polymer formed by curing of an elastomeric resin. The elastomeric resin can comprise an elastomeric resin suitable for use in an additive manufacturing process. Such materials are well known in the art. In some examples, the elastomeric resin can comprise a thermoplastic polymer such as acrylonitrile butadiene styrene (ABS), polyphenylene sulfide (PPS), polyphenylsulfone (PPSU), polyetheretherketone (PEEK), polyurethane (PU), polyetherimide (PEI), polyphenylene ether (PPE), polycarbonate (PC), and combinations thereof. In some embodiments, the elastomeric resin can comprise a crosslinkable composition (e.g., a blend of monomers, oligomers, and/or polymers which can be crosslinked during the additive manufacturing process). Depending on the additive manufacturing process employed, the crosslinkable composition can be selected such that crosslinking can be induced thermally and/or by impinging electromagnetic radiation (e.g., UV and/or visible light). In certain embodiments, the elastomeric resin can comprise a crosslinkable silicone composition. For example, the elastomeric resin can comprise (A) a first organosilicon compound having at least two ethylenically unsaturated moieties per molecule; and optionally (B) one or more additional organosilicon compounds. Suitable silicone compositions are known in the art. See, for example, U.S. Pat. No. 10,155,884 to Dow Silicones Corp., U.S. Patent Application Publication No. 2017/0312981 to Wacker Chemie AG, U.S. Patent Application Publication No. 2018/0370141 to Wacker Chemie AG, U.S. Patent Application Publication No. 2018/0066115 to Wacker Chemie AG, U.S. Patent Application Publication No. 2018/0186076 to Dow Corning Corp., and U.S. Patent Application Publication No. 2019/0100626 to Lawrence Livermore National Security LLC, each of which is hereby incorporated by reference in its entirety. Other suitable elastomeric resins are described, for example, in U.S. Patent Application Publication No. 20160319150 to Cornell University.
Other Components
Optionally, the porous, magnetic, elastomeric construct may include one or more additional components. For example, in some embodiments, the porous, magnetic, elastomeric construct may further include a non-magnetic filler. The non-magnetic filler may be, for example, an organic filler, an inorganic filler, a ceramic powder, or combinations thereof. The organic filler may be a polymer, such as, but not limited to, polystyrene, polyethylene, polypropylene, polysulfone, polyamide, polyimide, polyetheretherketone, etc. The organic filler can also be a smaller molecule either amorphous or crystalline in nature, and can be of in various shapes and sizes. The inorganic filler or ceramic powder can be any inorganic compounds that are compatible with the curing chemistry. Examples include, but are not limited to, silicon dioxide, titanium dioxide, zirconium dioxide, barium titanate, strontium titanate, etc. A mixture of more than one inorganic or organic with inorganic fillers are also suitable.
In some embodiments including the non-magnetic filler, the non-magnetic filler can be present as any suitable wt. % of the composition. In some embodiments, the non-magnetic filler can be present in an amount of 0.05 wt. % or more, 0.1 wt. % or more, 1 wt. % or more, 2 wt. % or more, 3 wt. % or more, 4 wt. % or more, 5 wt. % or more, 6 wt. % or more, 7 wt. % or more, 8 wt. % or more, 9 wt. % or more, 10 wt. % or more, 11 wt. % or more, 12 wt. % or more, 13 wt. % or more, 14 wt. % or more, 15 wt. % or more, 16 wt. % or more, 18 wt. % or more, 20 wt. % or more, 25 wt. % or more, 30 wt. % or more, 35 wt. % or more, 40 wt. % or more, 45 wt. % or more, 50 wt. % or more, 55 wt. % or more, 60 wt. % or more, 65 wt. % or more, 70 wt. % or more, 80 wt. % or more, or 85 wt. % or more. In some embodiments, the non-magnetic filler can be present in an amount of 90 wt. % or less, 85 wt. % or less, 80 wt. % or less, 75 wt. % or less, 70 wt. % or less, 65 wt. % or less, 60 wt. % or less, 55 wt. % or less, 50 wt. % or less, 45 wt. % or less, 40 wt. % or less, 35 wt. % or less, 30 wt. % or less, 25 wt. % or less, 20 wt. % or less, 15 wt. % or less, 10 wt. % or less, 5 wt. % or less, 1 wt. % or less, 0.5 wt. % or less, 0.1 wt. % or less, or 0.05 wt. % or less.
The non-magnetic filler can be present in an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the non-magnetic filler can be present in an amount of from 0.01 wt. % to 90 wt. % of the compositions, (e.g., from 1 wt. % to 90 wt. %, from 5 wt. % to 90 wt. %, from 10 wt. % to 90 wt. %, from 15 wt. % to 90 wt. %, from 25 wt. % to 90 wt. %, from 30 wt. % to 90 wt. %, from 40 wt. % to 90 wt. %, from 50 wt. % to 90 wt. %, from 60 wt. % to 90 wt. %, from 70 wt. % to 90 wt. %, from 80 wt. % to 90 wt. %, from 1 wt. % to 80 wt. %, from 5 wt. % to 80 wt. %, from 10 wt. % to 80 wt. %, from 15 wt. % to 80 wt. %, from 25 wt. % to 80 wt. %, from 30 wt. % to 80 wt. %, from 40 wt. % to 80 wt. %, from 50 wt. % to 80 wt. %, from 60 wt. % to 80 wt. %, from 70 wt. % to 80 wt. %, from 1 wt. % to 75 wt. %, from 5 wt. % to 75 wt. %, from 10 wt. % to 75 wt. %, from 15 wt. % to 75 wt. %, from 25 wt. % to 75 wt. %, from 30 wt. % to 75 wt. %, from 40 wt. % to 75 wt. %, from 50 wt. % to 75 wt. %, from 60 wt. % to 75 wt. %, from 70 wt. % to 75 wt. %, from 55 wt. % to about 75 wt. %, from 1 wt. % to 60 wt. %, from 5 wt. % to 60 wt. %, from 10 wt. % to 60 wt. %, from 15 wt. % to 60 wt. %, from 25 wt. % to 60 wt. %, from 30 wt. % to 60 wt. %, from 40 wt. % to 60 wt. %, from 50 wt. % to 60 wt. %, from 1 wt. % to 50 wt. %, from 5 wt. % to 50 wt. %, from 10 wt. % to 50 wt. %, from 15 wt. % to 50 wt. %, from 25 wt. % to 50 wt. %, from 30 wt. % to 50 wt. %, from 40 wt. % to 50 wt. %, from 1 wt. % to 40 wt. %, from 5 wt. % to 40 wt. %, from 10 wt. % to 40 wt. %, from 15 wt. % to 40 wt. %, from 25 wt. % to 40 wt. %, from 30 wt. % to 40 wt. %, from 1 wt. % to 30 wt. %, from 5 wt. % to 30 wt. %, from 10 wt. % to 30 wt. %, from 15 wt. % to 30 wt. %, from 25 wt. % to 30 wt. %, from 1 wt. % to 20 wt. %, from 5 wt. % to 20 wt. %, from 10 wt. % to 20 wt. %, from 15 wt. % to 20 wt. %, from 1 wt. % to 10 wt. %, from 5 wt. % to 10 wt. %, from 1 wt. % to 5 wt. %, from 0.5 wt. % to 1 wt. %, from 0.5 wt. % to 5 wt. %, from 0.5 wt. % to 10 wt. %, from 0.5 wt. % to 20 wt. %, from 0.5 wt. % to 30 wt. %, from 0.5 wt. % to 40 wt. %, from 0.5 wt. % to 50 wt. %, from 0.5 wt. % to 60 wt. %, from 0.5 wt. % to 70 wt. %, from 0.5 wt. % to 80 wt. %, from 0.5 wt. % to 90 wt. %, from 0.05 wt. % to 1 wt. %, from 0.05 wt. % to 5 wt. %, from 0.05 wt. % to 10 wt. %, from 0.05 wt. % to 20 wt. %, from 0.05 wt. % to 30 wt. %, from 0.05 wt. % to 40 wt. %, from 0.05 wt. % to 50 wt. %, from 0.05 wt. % to 60 wt. %, from 0.05 wt. % to 70 wt. %, from 0.05 wt. % to 80 wt. %, from 0.05 wt. % to 90 wt. %).
The non-magnetic filler can have any suitable particle size, e.g., the longest dimension of the particle, such as the average longest dimension. In some embodiments, the non-magnetic filler can have a primary particle size of 100 microns or less, (e.g., 90 microns or less, 80 microns or less, 70 microns or less, 60 microns or less, 50 microns or less, 40 microns or less, 30 microns or less, 20 microns or less, 10 microns or less. In some embodiments, the non-magnetic filler can have a primary particle size of 5 microns or more, (e.g., 10 microns or more, 20 microns or more, 30 microns or more, 40 microns or more, 50 microns or more, 60 microns or more, 70 microns or more, or 80 microns or more).
The non-magnetic filler can have a primary particle size ranging from any of the minimum values described above to any of the maximum values described above. For example, the non-magnetic filler can have a primary particle size of from 5 microns to 100 microns, (e.g., 5 microns to 100 microns, 10 microns to 100 microns, 20 microns to 100 microns, 30 microns to 100 microns, 40 microns to 100 microns, 50 microns to 100 microns, 60 microns to 100 microns, 70 microns to 100 microns, from 80 microns to 100 microns, 90 microns to 100 microns, from 5 microns to 90 microns, from 10 microns to 90 microns, from 20 microns to 90 microns, from 30 microns to 90 microns, from 40 microns to 90 microns, from 50 microns to 90 microns, from 60 microns to 90 microns, from 70 microns to 90 microns, from 80 microns to 90 microns, from 5 microns to 80 microns, from 10 microns to 80 microns, from 20 microns to 80 microns, from 30 microns to 80 microns, from 40 microns to 80 microns, from 50 microns to 80 microns, from 60 microns to 80 microns, from 70 microns to 80 microns, from 5 microns to 70 microns, from 10 microns to 70 microns, from 30 microns to 70 microns, from 30 microns to 70 microns, from 40 microns to 70 microns, from 50 microns to 70 microns, from 60 microns to 70 microns, from 5 microns to 60 microns, from 10 microns to 60 microns, from 20 microns to 60 microns, from 30 microns to 60 microns, from 40 microns to 60 microns, from 50 microns to 60 microns, from 5 microns to 50 microns, from 10 microns to 50 microns, from 20 microns to 50 microns, from 30 microns to 50 microns, from 40 microns to 50 microns, from 5 microns to 40 microns, from 10 microns to 40 microns, from 20 microns to 40 microns, from 30 microns to 40 microns, from 5 microns to 30 microns, or from 10 microns to 30 microns, from 20 microns to 30 microns, from 5 microns to 20 microns, from 10 microns to 20 microns, from 5 microns to 10 microns. In some embodiments, the non-magnetic filler can have a primary particle size of 90 microns or more. In some embodiments, the non-magnetic filler can have a primary particle size of 5 microns or less. As used herein, “primary” particle size refers to the actual particles in their un-conglomerated state, which can optionally conglomerate to form larger “secondary” particles.
Examples of additional ingredients include, but are not limited to, adhesion promoters; dyes; pigments; anti-oxidants; initiators for crosslinking; carrier vehicles; heat stabilizers; flame retardants; thixotropic agents; flow control additives; inhibitors; extending and reinforcing fillers; and cross-linking agents.
One or more of the additives can be present as any suitable wt. % of the composition. In some embodiments, the additives can be present in an amount of 15 wt. % or less of the composition, (e.g., 14 wt. % or less, 13 wt. % or less, 12 wt. % or less, 11 wt. % or less, 10 wt. % or less, 9 wt. % or less, 8 wt. % or less, 7 wt. % or less, 6 wt. % or less, 5 wt. % or less, 4 wt. % or less, 3 wt. % or less, 2 wt. % or less, 1 wt. % or less, or 0.5 wt. % or less). In some embodiments, the additives can be present in an amount of 0.1 wt. % or more of the composition, (e.g., 0.5 wt. % or more, 1 wt. % or more, 2 wt. % or more, 3 wt. % or more, 4 wt. or more, 5 wt. % or more, 6 wt. % or more, 7 wt. % or more, 8 wt. % or more, 9 wt. % or more, 10 wt. % or more, 11 wt. % or more, 12 wt. % or more, 13 wt. % or more, or 14 wt. % or more).
The additives can be present in an amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the additives can be present in an amount ranging from 0.1 wt. % to 15 wt. % of the composition, (e.g., from 0.1 wt. % to 5 wt. %, from 0.1 wt. % to 10 wt. %, from 0.5 wt. % to 5 wt. %, from 0.5 wt. % to 10 wt. %, from 0.5 wt. % to 15 wt. %, from 1 wt. % to 5 wt. %, from 1 wt. % to 10 wt. %, from 1 wt. % to 15 wt. %, from 2 wt. % to 5 wt. %, from 2 wt. % to 10 wt. %, from 2 wt. % to 15 wt. %, from 5 wt. % to 10 wt. %, from 5 wt. % to 15 wt. %, from 8 wt. % to 10 wt. %, from 8 wt. % to 15 wt. %, from 10 wt. % to 15 wt. %).
Construct Fabrication
The porous, magnetic, elastomeric constructs described herein can be formed using an additive manufacturing process, such as fused deposition modeling (FDM), fused filament fabrication (FFF), fused pellet fabrication, fused particle fabrication, composite filament fabrication (CFF), direct ink writing (DIW), stereolithography (SLA), digital light processing, continuous liquid interface production, selective heat sintering, or selective laser sintering.
The constructs can be interfaced with a magnet (e.g., an electromagnet) configured to apply a magnetic field within the construct. By applying a magnetic field of varying strength, the mechanical properties of the construct (e.g., the Young's modulus of the material) can be varied and/or the construct can be deformed.
Magnet
The systems described herein can include a magnet (300). The magnet can include a permanent magnet (302) as shown in
The permanent magnet can generate an external magnetic field ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the permanent magnet can generate an external magnetic field of from 200 mT to 450 mT, (e.g., from 200 mT to 400 mT, from 250 mT to 300 mT, from 250 mT to 350 mT, from or from 250 mT to 400 mT, from 300 mT to 350 mT, from 300 mT to 400 mT, or from 350 mT to 400 mT).
In some embodiments, the magnet (300) comprises an electromagnet (301) as shown in
The electromagnet can generate an external magnetic field ranging from any of the minimum values described above to any of the maximum values described above. In some embodiments, the electromagnet can generate an external magnetic field of from 15 mT to 60 mT, (e.g, from 15 mT to 30 mT, from 15 mT to 40 mT, from 15 mT to 50 mT, from 30 mT to 40 mT, or from 40 mT to 60 mT).
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.
Mechanical tissue environment is crucial in developing novel therapy concepts and tissue regenerative medicine. However, the understanding of tissue mechanical responsive mechanisms is substantially lacking, due to limited tissue models and resources available for well-controlled ex vivo study. Ferromagnetic soft composite materials offer controlled and untethered fast transformation of complex 3D shapes which is needed for such biomedical manipulation applications. Therefore, we introduce a smart, responsive 3D porous ferromagnetic interface that can reconstruct in vivo tissue microvibrational environment with desired full control of tissue dynamics. The objectives of these examples were to develop precise and controllable ferromagnetic porous composites; characterize the mechanical properties of smart responsive composites; distinguish the deformation capabilities of the responsive porous composites; verify the ability of the interface design to be suited for 3D cell culture with efficient waste and nutrient transport; establish the interface is compatible with 3D stem cell growth; and utilize 3D printing and particulate leaching techniques to develop high-fidelity microporous magnetic composite constructs with various micro patterns and shapes.
Cells respond to mechanical signals provided by the local extracellular matrix (ECM) and can control cell fate via cellular mechanotransduction. Therefore, it is important to be able to control and characterize the interface micromechanical environment. Utilizing 3D printing and particulate leaching techniques can be used to develop high-fidelity microporous magnetic composite constructs with various micro patterns and shapes (
Ferromagnetic composites can be controlled via external magnetic fields and the correlation between magnetic field strength and deformation and pressure can be used to dictate the mechanical cues provided to a cellular network. The increased complexity allows for the generation of relevant dynamic stimuli that is experienced under biological conditions. This could be used to direct gene expression and cell fate through mechanosensitive cell receptors or channels.
Described is a fabrication process that yields a well-ordered microporous magnetic elastomer composite that has controllable 4D transformation, or the ability to change the 3D conformation of the composites with time. The microporous magnetic elastomer has the capacity to induce tunable dynamic mechanical stimulation to a 3D cell culture.
Magnetic PDMS (Sylgard 184) composites are strong candidates for dynamic culture systems but are limited in their potential as a high-fidelity 3D platform given the highly viscous liquid precursor properties of PDMS that render these materials unsuitable for 3D printing. Therefore, we evaluated an indirect 3D printing fabrication technique to construct high-fidelity, remote-control microporous magnetic composites utilizing a 3D printed water-soluble sacrificial template for the fabrication for 3D PDMS tissue engineering scaffolds8. This process has not been utilized for the development of high-fidelity magnetic composites with low viscosity PDMS.
This fabrication technique allows for the assembly of magnetic porous constructs with controlled porosity and micropatterns, controllable dynamic mechanical manipulation, cell viability, and waste and nutrient transport for full interface culture conditions (
The flexibility in design allows for the integration of the magnetic composites into readily available cell culture platforms, such as well plates for high-throughput screening capabilities. Well plate compartmental devices can be designed to control different modes of magnetic actuation to the magnetic porous composites with the potential for both electromagnetic and permanent magnetically driven actuation. Electromagnetic actuation can lead to the development of a nanovibration environment that has been shown to have strong implications on the control of mesenchymal stem cell fate in both 2D and 3D cell cultures3,9,10. Permanent magnet movable platforms can provide cyclic permanent magnetic actuation profiles that can lead to macroscopic deformation of the magnetic composites shown in
Construct design. In a first example, a construct including a magnetic composite is inserted at the bottom of 96-well plate. A 3D cell-laden gel can then be placed on top of the construct (see
In a second example, a non-magnetic composite (e.g., PDMS) can be inserted at the bottom of 96-well plate. The 3D cell-laden gel can then be placed on top of the non-magnetic composite. Then, a non-constrained magnetic composite can be placed above the cell laden gel (see
The development of the smart responsive ferromagnetic porous composites for each sample is illustrated in
The printing infill percentage and infill patterns were varied to tune the mechanical properties, porosity, and topography of the interface design. The infill percentage refers to the amount of filament printed per layer with 100% infill being a filled structure with no pores. Given the PVA 3D print is a sacrificial template, the infill printed represents the pores of the final construct. A 70% infill percentage was chosen as this was the highest consistent attainable porosity for the final porous composite. The infill pattern indicates the micropattern printed to form the 3D geometric structure and both the rectangular grid with 90 degree offset every layer (
The PVA printed template, shown in
Actuation device designs. We developed two design iterations to encompass a 96-well plate with design flexibility for spatial control of a magnetic actuation. First iteration is a 3D Printed container that could enclose an electromagnet, microcontroller, and power supply with designed edges for the placement of a well plate (see
The second iteration was used in the assays due to ease of implantation given the power supply and microprocessor being outside of the incubator; only requires two wires for power supply to the electromagnet; more control during the actuation process; and closer proximity of magnet to the 96-well plate given no lid compartment.
All magnetic constructs were designed to fit within a cell culture well plate. Well plate compartmental devices will be constructed to have control of the dynamic external magnetic environment placed on the magnetic constructs. The defined magnetic environment will control the strain level, frequency, and duration of actuation placed on the magnetic constructs that will be translated to the 3D cell culture. Two different modes of magnetic actuation will be investigated (nanovibration and strain) with the development of two different magnetic actuation devices.
Nanovibration will be the first mode of actuation investigated with the usage of electromagnets to generate external magnetic fields of 15-60 mT. These magnetic fields are unable to induce macrolevel actuation to the magnetic composites, however; they create micro and nanolevel vibrational environments due to the low external magnetic fields. The electromagnet could be modulated in a way to induce low frequency nano vibrations directly to a 3D stem cell culture. This is interesting for preconditioning 3D cell laden gels for potential tissue grafts. This has been studied by using piezo actuators to translate nanolevel deformations to the bottom of a well plate. The force developed by the magnetic composites will be assessed by studying the response of the materials at different electromagnetic fields with a low force sensor.
The nanovibration system can include a 3D printed housing (
A permanent magnet system to induce macrolevel constraints can include a micro linear actuator driven stage that can move permanent magnets in the vertical direction. The movement of the stage can create a dynamic magnetic field profile that can induce macrolevel strain, as shown in
The 3D printed device for the permanent magnet system can include a housing that has two inner pillars [16] that are designed to fit the curvature of two micro linear actuators (
By way of example, for the induction of strain, 200-400 mT applied magnetic fields can be used to deform the magnetic material (e.g., an elastomer such as PDMS containing NdFeB microparticles dispersed therein). Permanent magnets can be used to create a pulsated magnetic field via moving the stage up and down. The linear actuators can perform a 1 Hz pulsated field with 7-8 mm translation (speed of linear actuators=14-15 mm/s).
In the case of Samples 1 and 2, a porous PDMS/magnetic composite and PDMS porous construct can be interfaced at the top and bottom of a 3D cell culture. An external magnetic field can actuate the PDMS/magnetic composite to allow for control of the pressure placed on a 3D cell culture. Variability in composition of the magnetic composite can allow for adjustment of the initial mechanical properties of the soft composites and force potential to mimic in vivo specific mechanical environments as shown by
In the case of Sample 3, a PDMS/magnetic composite could provide an interface system that could be in full contact of a 3D cell culture. The cell culture could be inserted into a magnetic/PDMS porous reservoir and an external magnetic field could be used to control the mechanical stimuli placed on the cell culture with defined micropatterns. This system is viable due to the interconnected porosity of magnetic reservoir system allowing for waste and nutrient transport (
Sample 4 includes a 3D magnetic porous composite scaffold that can be seeded with cells. Then the magnetic composite interface could directly transmit mechanical stimuli to the adherent cells and computational modeling and experimental techniques to develop well controlled and defined micromechanical environments and study cellular behavior. The development of these models could be obtained by utilizing the works described by Mokhtari-Jafari, F. et al., Zhao, R., et al., and Malvè, M., Bergstrom, D. J. & Chen, X. B. 15-17 This can allow for the study of the structural and fluid mechanics at the microscale level within the scaffold to best simulate the forces being applied at the structural and fluid mechanic interface. To our knowledge, this has never been studied with a bioinert actuating scaffold.
Sample 1
Sample 1 includes a 3-D PDMS magnetic porous composite that is fabricated to establish a dynamic interface for a 3D cell culture. The magnetic composite is designed to be inserted into the bottom of well plate and provide a surface contact for a 3D cell culture gel that can placed on top. Modifications can be made to the surface of the magnetic composite to increase absorption of the gel to the top surface of the magnetic composite. This can permit the most ideal dynamic interaction translation between the magnetic composite and the gel. A microcontroller is used to control the magnetic field strength, waveform, frequency, and duration. The external magnetic field created will influence the dynamic tensile strain or vibration environment placed on the 3D cell culture.
Sample 2
Sample 2 includes a non-magnetic (PDMS) porous construct and a 3-D PDMS magnetic porous composite (
Sample 3
Sample 3 has the same set-up as the Sample 2 but the surfaces of both porous constructs are functionalized. The interface of the constructs includes a full infill or nanopatterned thin membrane that can be developed using photolithography (
Sample 4
Sample 4 includes a magnetic composite reservoir construct that can provide 360 degrees 3D dynamic actuation by being in full contact with a cell culture. The 360 degrees actuation system is described in
Sample 5
Sample 4 includes a dynamic bioinert magnetic porous scaffold system that can provide a programmable microenvironment to cells. The cells are seeded on the scaffold material and the microporous pores allow for the cells to migrate throughout the 3D construct (
Additionally,
Magnetic Field Modeling
Magnetic Flux Density Modeling can be used to calculate the magnetic flux density at certain locations from a magnet. This can help with size constraints of the porous PDMS and magnetic composite constructs along with the height of the 3D gel. Increased or varying location of the magnetic composite away from the magnetic will change the magnetic field applied and the sequential response. These models can help to ensure that the proper magnetic field is applied given the location of the magnetic composites in respect to the magnet.
Force Response
Assessment of Force Translation of Magnetic Composites. A 0-11b small force sensor can be used to detect the amount of force the magnetic composites generate at different flux densities. The force readings can be measured via both an electromagnet and permanent magnets at different distances from the magnetic composite.
A Magnetic Flux Modeling and a Gauss Meter can be used to access the magnetic fields at different distances. This is needed to access the nano vibration actuation potential and the driving force of the piston-like set-up for the magnetic composites.
Abstract
Mechanical stimuli have been shown to play a large role in cellular behavior, including cellular growth, differentiation, morphology, homeostasis, and disease. Therefore, developing bioreactor systems that can create complex mechanical environments for both tissue engineering and disease modeling drug screening is appealing. However, many of existing systems are restricted due to their bulky size with external force generators, destructive microenvironment control, and low throughput. These shortcomings have preceded to the utilization of magnetic stimuli responsive materials, given their untethered, fast, and tunable actuation potential at both the microscale and macroscale level, for seamless integration into cell culture wells and microfluidic systems. Nevertheless, magnetic soft materials for cell culture have been limited due to the inability to develop well-defined 3D structures for more complex and physiological relevant mechanical actuation. Herein, we introduce a facile fabrication process to develop magnetic-PDMS (polydimethylsiloxane) porous composite designs with both well-defined and controllable microlevel and macrolevel features to dynamically manipulate 3D cell-laden gel at the scale. The intrinsic stiffness of the magnetic-PDMS porous composites is also modulated to control the deformation potential to mimic physiological relevant strain levels, with 2.89 to 11% observed in magnetic actuation studies. High cell viability was achieved with the culturing of both human adipose stem cells (hADMSCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in 3D cell-laden gel interfaced with the magnetic-PDMS porous composite. Also, the highly interconnected porous network of the magnetic-PDMS composites facilitated free diffusion throughout the porous structure showcasing the potential of a multi-surface contact 3D porous magnetic structure in both reservoir and 96-well plate insert designs for more complex dynamic mechanical actuation. In conclusion, these studies provide a means for establishing a biocompatible, tunable magnetic-PDMS porous composite with fast and programmable dynamic
Introduction
Cells have been shown, especially mesenchymal stem cells (MSCs), to both sense and act upon their mechanical environment via a mechanism called mechanotransduction1-7. This process consists of the cell being able to convert mechanical cues into intracellular biochemical signals for driving cellular growth, differentiation, morphology, homeostasis, and disease. Mechanical stimuli experienced by cells can come in both intrinsic and extrinsic forms. Current studies have looked at primarily one dependent force variable at a time to understand how each stimuli affects cellular behavior1-2. Most of these investigations utilized extrinsic forces to manipulate the intrinsic behavior of the cells through actomyosin contractility and cytoskeleton rearrangement, which can trigger biochemical signals depending on the dynamic nature of the force3-5. There are many different platforms to mimic in vivo physiological relevant forces experienced on cell cultures including substrate stiffness6-7, micro/nanopattering8-10, hydrostatic pressure11-12, perfusion flow13-14, shear forces15-16, compressive forces17-20, tensile forces21-23, and vibration24-26. The findings have led to the development of bioreactor systems for providing specific and complex mechanical environment used in tissue engineering and disease modeling27-28.Some developed microscale systems can overcome the complications involved with conventional bioreactor systems which are bulky, lack microenvironment control, and have low experimental throughput19, 25, 30-31 The mechanical stimuli to 3D cell cultures also can be achieved via pneumatic actuation of a PDMS membranes19,31, dielectric elastomer actuators32-33, and magnetic elastomer actuation34-37 in high-throughput microfluidic, micro array, or cell culture well-plate systems. However, the current existing microscale systems to date are still lacking the capacity for micro level spatial control of mechanical actuation with most needing external forces to act upon an interface substrate.
Microscale actuator systems are critically needed in evaluating and developing physiological relevant actuation to cell cultures with multiple modes and ranges of actuation. Herein, we introduced a magnetic stimuli responsive materials to bridge such technology gap. Although magnetic materials have shown to provide highly tunable, predictive, complex transformation potential at both the macroscopic and microscopic level38-40, developing magnetic elastomer actuators with defined 3D structures and porosity using PDMS (polydimethylsiloxane) for dynamic cell culture is emerging6-10, 19, 34-36, 41. Investigators have explored the development of magnetic porous composites as devices for on-demand drug delivery by the usage of sacrificial molds of salt or sugar microparticles to define the porous network42-43. Yet, there has been minimal investigation into porous magnetic composites as scaffold or porous interfaces with high-fidelity porous networks for cell culture considerations. Therefore, we developed a high-fidelity, tunable magnetic PDMS porous composite that can be actuated remotely via an external magnetic field. To our knowledge, this will be the first magnetic PDMS porous composite with well-defined 3D microstructures for full control over the porosity and micropatterns to study the topographical cues in 3D cell culture. Because of the ability to precisely manipulate the intrinsic stiffness and magnetic particle density in the porous magnetic PDMS composites, the resulted physiological relevant strain level enables the potential control of human umbilical cord mesenchymal stem cells (hUCMSCs) differentiation. Also, the porous magnetic PDMS showed to be a suitable 3D cell culture substrate with high cell viability for both adipose-derived mesenchymal stem cells (hADMSCs) and hUCMSCs in a 3D fibrin gel. The highly interconnected porous network of the magnetic PDMS porous composites enhances free diffusion through the material unlocking the potential to use as the multiple surface contact porous constructs for more complex dynamic mechanical actuation. With this platform using remotely controllable porous materials, small and complex cell culture magnetic interfaces can be implemented into standard cell culture 96 well plate as a potential high-throughput platform for advancing dynamic 3D cell culture systems.
Materials and Methods
FDM 3D Printing of Sacrificial Templates
A fused filament fabrication printer (N2, Raise 3D), or fused deposition modeling (FDM), was used to print the PVA sacrificial templates. The 3D template structure was designed using a computer aided design (CAD) 3D slicer software provided by Raise 3D (ideaMaker). The 3D CAD design file then could be exported to the printer by a .STL file to perform the 3D print. The method of printing the 3D templates include the PVA filament being fed through a heated extrusion nozzle that melts the thermoplastic and then solidifies on the heated stage bed after extrusion. The .STL computer slice file guides the nozzle to execute the print layer-by-layer by stacking x-y planes on top of each other. The printing infill percentage and infill patterns were varied to tune the mechanical properties, porosity, and topography of the interface design. The infill percentage refers to the amount of filament printed per layer with 100% infill being a filled structure with no pores. Given the PVA 3D print is a sacrificial template, the infill printed represents the pores of the final construct. A 70% infill percentage was chosen as this was the highest consistent attainable porosity for the final porous composite. The infill pattern indicates the micropattern printed to form the 3D geometric structure and both the rectangular grid and gyroid patterns were investigated. The printing parameters are shown in Table 1.
3D Fabrication of Magnetic Porous Composites
The PVA printed templates were used to form the 3D magnetic porous composite structures. Carbonyl iron microparticles (Sigma-Aldrich) and PDMS (Sylgard 184, Dow Corning) were mixed in a 1:1 (w/w) ratio with varying base to curing agent ratios of PDMS (10:1, 15:1, 20:1). The carbonyl iron/PDMS precursor solution was mixed thoroughly and degassed via centrifugation at 1000×g for 1 minute. The physics of magnetic actuation and properties were characterized previously. The magnetic precursor was then poured in a well and the PVA template was placed on top of the magnetic precursor pool. The PVA template and carbonyl iron/PDMS precursor were placed in a vacuum chamber at 400 kPa vacuum pressure for 2 hours to allow for the magnetic precursor to completely penetrate the pores of the PVA template via capillary action. The PVA templates, with infiltrated carbonyl iron/PDMS precursor, were then wiped of any excess magnetic precursor and then baked at 65° C. for 4 hours to allow for the carbonyl iron/PDMS precursor to cure within the template. The PVA template with infiltrated cured PDMS was then placed in a hot stir plate (above 70° C.) for 3-4 hours to allow for the PVA to completely dissolve. Once the PVA is completely dissolved, the magnetic-PDMS porous composites could be removed and left out to dry.
Mechanical Analysis of Magnetic Porous Composites
Mechanical analysis of the magnetic-PDMS porous composites was tested by performing controlled axial compressions utilizing an ElectroForce 5500 (TA Instruments). The testing parameters including a controlled axial compression at 0.01 mm/s of cylinder composites of the gyroid infill pattern (8.5 mm×7.8 mm) and grid infill pattern with 90-degree layer offset (9 mm×7.8 mm). The Young's modulus of the composites were determined by the linear portion of the stress vs strain curve which was determined to be 2% to 10% strain.
Controlled Deformation Characterization of Magnetic Composites Controlled deformation studies were performed to assess the ability to induce strain on the magnetic-PDMS composites with varying magnetic fields. A 3D printed compartmental box was printed to hold both a phone and 96-well plate at a defined distance and location to have reproducible image collection for both qualitative and quantitative strain analysis. Grid (6.8×5 mm) and gyroid (7.2×5 mm) PVA templates were prepared to make magnetic composite structures to fit inside a 96-well plate. 15:1 (PDMS base: cure) grid and 20:1 (PDMS base: cure) gyroid magnetic composites were fabricated and used for controlled deformation studies. The 3D printed box was designed to have a permanent magnet insert location. 3D printed blocks were placed underneath the permanent magnet to adjust the applied magnetic field to the magnetic composites within the 96 well plate. Applied magnetic fields of 0, 240, 325, and 415 mT were performed on the magnetic-PDMS composites in a 96-well plate with DPBS media. Image collection was obtained via a 12-megapixel f/1.8 camera iPhone XR, Apple). Applied magnetic fields were measured via a gaussmeter (TD8620 Handheld Digital Gauss TeslaMeter, China),
Dynamic actuation studies of the magnetic composites were performed using a 3D printed box that was designed to develop a dynamic magnetic field profile to magnetic composites within a 96-well plate. The box was built to hold two micro linear actuators that drive a stage, encompassing permanent magnets, in the z-direction to control the frequency, amplitude, and duration of an applied magnetic field experienced by the magnetic porous composites. A cyclic, 325 mT magnetic field was used for all the magnetic actuation studies. Magnetic porous composite recovery studies were performed by imaging the height of both gyroid (20:1) and grid (15:1) under no applied magnetic field after different duration of dynamic magnetic field actuation cycles (0, 5, 25, 50, 100). On-demand precise actuation analysis of the magnetic porous composites was executed by inducing a pause function at the maximum applied magnetic field during the dynamic magnetic actuation cycle for imaging the strain levels of the gyroid (20:1) and grid (15:1) for the same actuation cycle durations studied for the recovery tests.
Cell Culture of hUCMSCs and hADMSCs
Patient derived human umbilical cord stem cells (hUCMSCs) and adipose stem cells (hADMSCs) were provided by the Midwest Stem Cell Therapy Center (MSCTC). Cells were maintained in Gibco™ DMEM, high glucose, pyruvate media supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin, all purchased from Gibco™, Dublin, Ireland. Cells were cultured in a humidified incubator at 37° C. and 5% CO2. Cells were detached by the incorporation of trypsin-EDTA (Gibco™) followed by the addition of cell culture media, and then centrifuged for 5 minutes at 1500 rpm. Passages 3-5 of hUCMSCs were used in 3D fibrin gel preparation.
Fibrin hUCMSC Laden 3D Gel Preparation
Stock solution aliquots of fibrinogen (Millipore Sigma, US) and thrombin at concentrations of 45 mg/mL and 10 U/mL were used in the preparation of the fibrin gels. The aliquots were heated up to 37° C. prior to preparation. The fibrinogen stock solution is then diluted with DPBS and mixed gently to give a final concentration of 22.5 mg/mL of fibrinogen. DPBS is also added to the thrombin stock solution to yield a final concentration of 5 U/mL thrombin solution. The pelleted cells were then suspended and mixed carefully into the thrombin solution. 50 μL of the fibrinogen preparation is then added to the top of the porous constructs followed by the addition of the 50 μL of the thrombin-cell suspension. The gel is then mixed with the pipette tip immediately given the rapid polymerization and placed in the cell incubator for 15 minutes to ensure full polymerization. Cell culture media is then added on top of the gel and placed back in the incubator. The final fibrinogen and thrombin concentrations of the gels are 11.25 mg/mL and 2.5 U/mL respectively with 12,000 cells per gel preparation.
3D Cell Culture for Cell Viability Testing
Grid (3×6.8 mm) and gyroid (3×7.2 mm) cylinder PVA structures were printed to fit at the bottom of a 96-well plate well. Porous composites were fabricated as previously described with 8 different groups for cell viability analysis. The groups were represented by the two different infill patterns (grid, gyroid), two PDMS crosslinking ratios (10:1, 15:1), and both magnetic and PDMS porous constructs. For cell viability studies, the porous magnetic and PDMS constructs were sterilized by a 70% ethanol bath followed by UV exposure. The constructs were thoroughly washed with DPBS before insertion into a cell culture plate well.
Fibrin hADMSC laden 3D Gels were prepared as described for the hUCMSC laden 3D gel, while using hADMSCs, with a fibrinogen and thrombin concentrations of 10 mg/mL and 2.5 U/mL. Fibrin gels were cultured in both magnetic and PDMS only 3D porous well constructs. The 3D porous well design consisted of a 4 mm diameter and 2 mm tall well formed in a cylinder with 3 mm thick walls. The fibrin gels were cultured for 5 days in 48 well tissue culture plates.
For qualitative and quantitative analysis of the cell viability of the different porous construct groups and the no construct control group, a live/dead-assay was performed using a live/dead imaging kit (LIVE/DEAD® Viability/Cytotoxicity Kit, Invitrogen™). The live/dead staining was performed on the cell cultures on the fourth day under cell culture conditions. The attached hUCMSC cell-laden fibrin gels attached to the porous constructs were removed from the 96 well-plate and flipped over and placed at the bottom of the wells for imaging. Given the high porosity and thin nature of the constructs, they provided sufficient transparency for imaging without removing the construct from the gel. As for the magnetic well cell culture studies, hADMSC cell-laden fibrin gels were removed from the well constructs with a spatula and placed in the bottom a 96-well plate for imaging.
For staining, 20 μL of 2 mM EthD-1 and 5 μL of 4 mM calcein AM from the LIVE/DEAD® reagents were added to 10 mL of DPBS. The resulting working dye was then vortexed to ensure thorough mixing and a final concentration of 4 μM EthD-1 and 2 μM calcein AM was achieved. Cell culture media was then aspirated from the well of each sample and 100 μL of the live-dead solution was added to each well. The well-plate was then covered with aluminum foil and incubated for 25 minutes. After incubation, the live/dead solution was aspirated from each well and each sample was washed with DPBS and imaged with a fluorescent microscope (EVOS, Invitrogen and Primovert, ZEISS). Qualitative and quantitative analysis was performed by using post-processing imaging software (Fiji, ImageJ).
Diffusivity Studies
To determine the viability of the ferromagnetic interface system to be used as a 360-degree interface system, the ability for waste and nutrients to diffuse through the interconnected micropores of the magnetic porous required investigation. Therefore, a diffusivity study of the composite interfaces was performed by developing a magnetic composite reservoir with a 5×5 mm cylinder well with 3 mm thick porous walls. The diffusivity would be measured by the absorbance of a 5% (w/w) alginate sol gel (Alginic acid, Fischer Scientific) with 15 mg/mL of aniline blue (Fischer Scientific) in the bulk fluid. The reservoirs were first submerged in 1 mL of PBS in 24 well plates and preconditioned via physical agitation to allow for PBS to enter the pores of magnetic porous composites. The alginate/aniline blue sol gel was then loaded in a 1 mL syringe with a 26 G needle and 100 μL of solution was injected into each magnetic composite reservoir, followed by the addition of another 800 μL of DPBS. The cumulative mass transport out of the magnetic reservoir composites was measured by removing 200 μL for absorbance analysis of the well plate bulk solution outside the composite interface at specified time points (20, 40, 60, 80, 100, 120, and 150 min), 200 μL of PBS was added after each time point. Studies were completed in triplicates for both grid and gyroid reservoirs magnetic composites.
Cumulative Release Profile
A calibration curve was found by performing seven 1:1 serial dilutions of 1.75 g/mL of aniline blue and then measuring the absorbance at 625 nm of 200 uL of each dilution with a plate reader (Synergy H1 Hybrid Multi-Mode Reader, BioTek). Absorbance levels for PBS were also measured to allow for the calculation of relative absorbance values from the diffusivity studies. Release profiles were found by measuring the absorbance at 625 nm of the aliquots taken at each time point with a plate reader. Relative increase in the absorbance profiles for each time point were then calculated by subtracting the measured value by the previous absorbance level accounting for the addition of 200 μL after each removed sample. Cumulative release profiles were then established by calculating the cumulative sum of the relative increase values.
Results
Facile and Versatile Fabrication of 3D Porous Ferromagnetic Interface
Combining 3D printing and particulate leaching methods, we were able to fabricate high-fidelity 3D porous magnetic composites. We used PDMS to develop a soft porous magnetic substrate actuator for micromechanical actuation of 3D dynamic cell cultures44. Both grid and gyroid infill patterns were investigated for precise control and high-fidelity fabrication of 3D magnetic-PDMS porous constructs. The grid pattern structure was chosen given it is a widely used pattern in many different extrusion based tissue engineering scaffolds. As for substrate interface considerations, rectangular grid patterns have been widely studied to induce anisotropic cell morphology in both 2D static10 and time dependent 3D cell culture surfaces45. The grid structure is widely used given there are many different tissue types in the mesoderm lineage that have anisotropic cellular network formations that align in a linear fashion. The gyroid infill pattern was also investigated as triply minimal surfaces have emerged as a 3D printed pattern for porous biomaterial fabrication. Gyorid structures have been shown to have good mechanical properties, and due to their minimal surface, they allow for high permeability and nutrient and waste transport46-47. The facile fabrication with simple three steps were illustrated in
A range of infill volume percentages (30%, 50%, 70%) were also studied to analyze the potential bounds in controlling the porosity, mechanical properties, interface dimensions, and geometries to influence cellular behavior. Microscopic images of 3D printed PVA sacrificial templates were shown in
Because of the increased distance between strands, the liquid-air surface tension and adhesive forces were not sufficient for inducing capillary action to infill the PVA template volume with magnetic-PDMS precursor at lower infill percentages (<50%). The inability to establish the full porosity range exhibited by Mohanty, S. et al. was likely due to the higher density of the carbonyl iron and PDMS precursor compared to the PDMS only precursor (980 vs. 4420 kg/m3). This result can be described by the equation for capillary action Eq. (1), as the height that the liquid precursor can travel (h) will decrease with both an increase in distance between the strands (r) and density (p) with the assumption that there is little or no change in the liquid-air surface tension (y) and contact angle (θ).
However, this result is tolerable given higher percentage infill PVA templates lead to higher porous constructs. This is desired for interface and scaffolding in 3D cell culture systems, due to the ability for higher nutrient and waste transport from higher interconnected microchannels. The higher infill PVA templates also allow for casting smaller strand widths of the magnetic porous composites, which is advantageous when considering topographical contact points for controlled alignment of cells in particular tissue sets such as bones, muscles, and nerves. Such tissue sets have been extensively studied in vitro but is usually limited to a means for static control of 3D dynamic cell culture.8-10Our developed platform supplies suitable topographical contact points and mass transport for interfacing 3D cell culture with remote-control of dynamic conditions, which could advance deep understanding in a variety of tissue engineering models. Further developments can study the usage of smaller FDM nozzles to explore the ability to fabricate 3D structures with smaller strand gaps to yield both more porous and smaller strand width magnetic porous composites.
Owing to the power of 3D printing, many complicated macroscopic designs can be achieved to cast the 3D magnetic PDMS porous composites with well-defined microstructures and high fidelity. These structural examples were included in
3D Magnetic PDMS Porous Composites Enable the Remote-Controlled Actuation
The non-destructive, remotely controlled actuation in the field of tissue engineering is emerging, especially for inducing dynamic mechanical strain which could play a large role in the fate of mesenchymal stem cells35,36,48,49 Carbonyl iron microparticles were chosen to be embedded within PDMS given their high magnetization level with a low hysteresis which leads to a strong translational force in the direction of an applied field50. When this combinational force is greater than the intrinsic stiffness of the PDMS then this will lead to a deformation of the bulk PDMS, which is why we observed that actuation capacity of our developed 3D magnetic PDMS porous composites is directly correlated to their intrinsic mechanical properties. We were able to control such mechanical properties by altering the stiffness of PDMS composites casted from 70% infill grid and gyroid PVA templates which possessed the high-fidelity in small constructs with higher porosity to increase actuation potential. Three different base-to-cure cross linking ratios (10:1, 15:1, 20:1) were studied to yield less stiff PDMS composites at higher ratios. Strain-dependent uniaxial compression of the magnetic composites was performed to assess the Young's modulus from all the samples shown in
The ability to alter the intrinsic stiffness of the magnetic porous composites should allow for a wide mechanical strain range, with the less stiff composites having the largest strain potential. Thus, we investigated the remotely controlled deformation on the lowest stiffness attainable grid (15:1) and gyroid (20:1) magnetic composites, respectively in
Additional dynamic magnetic composite actuation studies were then evaluated to establish the on-demand and precise actuation capacity of the magnetic porous composites. A dynamic actuation compartmental box was developed to utilize two linear actuators to control the z-directional movement of a stage, encompassing an array of permanent magnetism to control the dynamic magnetic field experienced by a magnetic porous composite (seen in
Cyclic strain actuation of a gyroid construct at 325 mT can be observed across a range of different frequencies (0.25, 1, and 2 Hz). In addition, hUCMSC laden fibrin gels were polymerized on the top of gyroid (20:1) magnetic composites, that were the same height (3 mm) of the porous composites used for cell viability studies, and actuated with a 325 mT to show the ability for the composites to induce a strain that could be translated to the 3D fibrin gel placed above.
With the proposed magnetic porous composite bottom surface interface, the magnetic porous composite will induce a tensile strain utilizing similar design principles utilized by the commercially available Flexcell® Tissue Train® 3D Cell Culture System and other 3D tensile strain platforms51-52, which use fixed constraints to anchor the cell culture on select surfaces and have one or more stretchable interface substrates to induce a stretching event. For the particular system studied, the angular surface of the well plate acts as the anchor surface with the PDMS interface acting as the deformable surface that induces a uniaxial stretching event. The height of the 3D fibrin gel interfaced with the magnetic composite can also be modified to control the translatable strain experienced by the 3D gel as a thinner gel will result in a higher strain.
The higher actuation potential of the gyroid constructs could be correlated to its lower stiffness at a 20:1 ratio, although the magnetic volume fraction of embedded magnetic particles was kept constant in all magnetic-PDMS composite groups. The reproducible magnetic porous composite strain levels studied here are comparable with the compression and tensile mechanical stimuli used in controlling the differentiation of MSCs in 3D gels from reported actuation studies (5-15% strain)19,53,54, which indicates the feasibility for using our developed 3D magnetic PDMS composites as dynamic interface to induce physiological mechanical actuation.
3D Porous Ferromagnetic Interface Enables the 3D Dynamic Culture of hMSCs
Although micropatterning has been shown to control the alignment of hMSCs in 2D culture,45,55,56 the 3D patterned dynamic interfaces have not been well studied due to the lack of capable dynamic micropattern platforms which is critically needed as an improved 3D culture system in mimicking in vivo MSCs cellular behavior. Our developed 3D magnetic PDMS porous composites are desired as both scaffolding and interface materials for precise remote-control of the dynamic mechanical environment in a 3D cell culture system.
Patient derived human umbilical cord stem cells were chosen as the cell source owing to their sensitive response to various mechanical loading in culturing environment and be able to differentiate into adipose, bone, tendon, muscle, and cartilage linages depending on both the intrinsic and extrinsic mechanical environments provided.6,53,54 Fibrin gels with final concentrations of 2.5 U/mL thrombin and 11.25 mg/mL fibrinogen were polymerized on the top of magnetic composites and PDMS porous constructs. The live-dead two-color cell viability assay was performed after four days of cell culture. High cell viability (>84%) was observed across all the experimental interface groups and were comparable to the positive fibrin control group (
We also implemented the specific 3D microstructure designs which is imperative for improving the waste and nutrient mass transport and exchange through the interconnected networks of the 3D porous ferromagnetic constructs. We introduced a reservoir interface design which could provide a dynamic micromechanical environment to all surfaces of a 3D cell-laden gel for highly controlled actuation. Both grid and gyroid patterned reservoirs were designed with 3 mm thick, and porous walls surrounding a 5×5 mm reservoir space for housing an alginate sol gel (5%, w/w). Mass transport analysis was then determined by the ability for fluid to move from the gel through the exterior porous walls to the bulk fluid. The mass transport out of reservoir composite structures was quantified by incorporating aniline blue dye into the gels and measuring the absorbance of the bulk fluid at different time points shown in
A 3D cell culture and viability study were further performed in this 3D well construct using human derived adipose mesenchymal stem cells (hADMSCs) which is a different type of mesenchymal stem cell source and more available than hUCMSCs used in
Magnetic stimuli responsive materials have been emerging as the next generation of dynamic cell culture platforms. These materials allow for fast, highly tunable actuation at both the macroscopic and microscopic scale without any external pressure systems and tethered electronics, leading to the ease of integration into high-throughput systems such as 96 cell culture well plates and microfluidic chips. However, current existing bioreactor systems using such magnetic responsive materials are still lacking the precision in microenvironment control, or in low throughput and bulky size. In this research, we overcame such limitations by developing a novel magnetic elastomer cell culture platform and using a porous magnetic PDMS composite with the full tunability of microstructures. The fabrication of the 3D magnetic PDMS composites are simple and straightforward with high-fidelity control of microlevel and macrolevel structures, which can reproduce strain levels suited in 3D stem cell culture and differentiation. The porous magnetic PDMS composite also demonstrated biocompatibility given the high cell viability experienced for 3D culturing both hUCMSC and hADMSCs. The high interconnected porosity of the porous structure also supports the development of a multiple surface contact design for more complex mechanical actuation given improved fluid permeability and mass transport. Our results reveal a promising biocompatible, remotely controlled magnetic PDMS composite interface material with full control over the porosity, microlevel and macrolevel structure, mechanical properties, and dynamic strain for tunable dynamic cell culture studies at the scale. For future studies, multiple embodiment designs will be established to look at both tensile and compression actuation and the ability to control the differentiation of hUCMSCs. The magnetic actuation device will be developed to remotely control the strain levels of an array of porous magnetic-PDMS composites in a cell culture plate to induce a well-defined dynamic mechanical environment in high throughput employed in drug screening.
The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
The application claims the benefit of U.S. Provisional Application No. 63/130,596, filed Dec. 24, 2020; and U.S. Provisional Application No. 63/170,298, filed Apr. 2, 2021, which are hereby incorporated herein by reference in their entirety.
This disclosure was made with Government Support under Grant No. 1R35GM133794 awarded by the National Institutes of Health. The Government has certain rights to this disclosure.
| Number | Date | Country | |
|---|---|---|---|
| 63130596 | Dec 2020 | US | |
| 63170298 | Apr 2021 | US |
