Patent Application
20230002614
Cell transplantation holds significant potential for regenerating and repairing tissues and treating disease and disorders. Cells transplanted in suspension at or near their intended target may adhere and integrate into the target tissue. However, many factors can affect their rate of incorporation into the target tissue, and the cells may not effectively adhere to the intended target. Biopolymer scaffolds can provide a means for retaining cells at their intended target, increasing the rate of incorporation of the transplanted cells into the target tissue.
In vitro cell culture on polymer scaffolds provides a strategy for tissue creation and cell transplantation. A specific cell line is seeded onto the polymer scaffold. The polymer scaffold should provide good biocompatibility, controllable biodegradability, appropriate tensile strength, and flexibility. While collagen has often been used as a biopolymer scaffold, extensive cross-linking of the collagen is often required to provide the biopolymer with the requisite tensile strength which in turn reduces its flexibility, making it difficult to manipulate during both cell culture and transplantation. Furthermore, such biopolymer scaffolds are generally hazy and unsuitable for implantation in the eye.
According to one example (“Example 1”), provided herein is a reinforced biopolymer comprising i) a synthetic support membrane and ii) a biopolymer, the reinforced biopolymer having a) a measured optical transparency of at least 85%; b) a thickness of about 100 μm or less; and c) a toughness of at least 30 KJ/m3.
According to another example (“Example 2”), further to Example 1, the biopolymer includes a material selected from the group consisting of collagen, gelatin, laminin, fibronectin, fibrinogen, elastin, thrombospondin, heparan sulfate, chondroitin sulfate, polysaccharides, alginate, chitosan, glycosaminoglycan, hyaluronic acid, and combinations thereof.
According to another example (“Example 3”), further to Example 1 or 2, the biopolymer is functionalized to support cellular growth, cellular adhesion, or both cellular growth and adhesion.
According to another example (“Example 4”), further to any one of Examples 1-3, the biopolymer is configured to support a cellular monolayer on a surface distal to the synthetic support membrane.
According to another example (“Example 5”), further to any one of Examples 1-4, a layer of the biopolymer occurs on each side of the support membrane and the support membrane is approximately centrally located between two layers of the biopolymer.
According to another example (“Example 6”), further to Example 5, a layer of the biopolymer on only one side of the support membrane is configured to support a cellular monolayer.
According to another example (“Example 7”), further to any one of Examples 1-6, wherein the biopolymer is imbibed into the support membrane.
According to another example (“Example 8”), further to any one of Examples 1-7, the synthetic support membrane is biologically inert.
According to another example (“Example 9”), further to any one of Examples 1-8, the synthetic support membrane includes at least one of: expanded polytetrafluoroethylene (ePTFE); and expanded polyethylene.
According to another example (“Example 10”), further to any one of Examples 1-9, the synthetic support membrane is a biaxially oriented ePTFE membrane having a crystallinity index of at least 94% and a matrix tensile strength in both the longitudinal and transverse directions of at least about 600 MPa.
According to another example (“Example 11”), further to any one of Examples 1-10, the synthetic support membrane is configured to sustain mass transfer across the thickness of the synthetic support membrane.
According to another example (“Example 12”), further to any one of Examples 1-11, the synthetic support membrane has a thickness of about 0.5 μm to about 10 μm.
According to another example (“Example 13”), further to any one of Examples 1-12, the reinforced biopolymer has a thickness of about 0.5 μm to about 100 μm.
According to another example (“Example 14”), provided herein is an implantable cell therapy device comprising the reinforced biopolymer of any one of Examples 1-13 and a monolayer of cells on a surface of the biopolymer distal to the support membrane.
According to another example (“Example 15”), further to Example 14, the monolayer of cells includes cells selected from the group consisting of corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, photoreceptor cells, Müller glial cells, ganglion cells, endothelial cells, epithelial cells, pericytes, and combinations thereof.
According to another example (“Example 16”), provided herein is method of treating a disease or condition of or affecting a cellular layer in a subject, the method comprising implanting the implantable cell therapy device of Example 14 or Example 15 on or near the cellular layer in the subject.
According to another example (“Example 17”), further to Example 16, the disease or condition of or affecting the cellular layer is a corneal endothelial dystrophy, the implantable cell therapy device comprises a monolayer of corneal endothelial cells, and the cell therapy device is implanted at a posterior surface of the cornea.
According to another example (“Example 18”), further to Example 16, the disease or condition of or affecting the cellular layer is a disease or condition of or affecting retinal pigment epithelium, the implantable cell therapy device comprises a monolayer of retinal pigment epithelial cells, and the cell therapy device is implanted at the retina.
According to another example (“Example 19”), further to Example 16, the disease or condition of or affecting the cellular layer is a disease or condition of or affecting photoreceptors, the implantable cell therapy device comprises a monolayer of photoreceptor cells, and the cell therapy device is implanted at the retina.
The foregoing Examples are just that, and should not be read to limit or otherwise narrow the scope of any of the inventive concepts otherwise provided by the instant disclosure. While multiple examples are disclosed, still other embodiments will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative examples. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature rather than restrictive in nature.
The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description serve to explain the principles of the disclosure.
This disclosure is not meant to be read in a restrictive manner. For example, the terminology used in the application should be read broadly in the context of the meaning those in the field would attribute such terminology.
With respect to terminology of inexactitude, the terms “about” and “approximately” may be used, interchangeably, to refer to a measurement that includes the stated measurement and that also includes any measurements that are reasonably close to the stated measurement. Measurements that are reasonably close to the stated measurement deviate from the stated measurement by a reasonably small amount as understood and readily ascertained by individuals having ordinary skill in the relevant arts. Such deviations may be attributable to measurement error, differences in measurement and/or manufacturing equipment calibration, human error in reading and/or setting measurements, minor adjustments made to optimize performance and/or structural parameters in view of differences in measurements associated with other components, particular implementation scenarios, imprecise adjustment and/or manipulation of objects by a person or machine, and/or the like, for example. In the event it is determined that individuals having ordinary skill in the relevant arts would not readily ascertain values for such reasonably small differences, the terms “about” and “approximately” can be understood to mean plus or minus 10% of the stated value.
Persons skilled in the art will readily appreciate that various aspects of the present disclosure can be realized by any number of methods and apparatuses configured to perform the intended functions. It should also be noted that the accompanying drawing figures referred to herein are not necessarily drawn to scale, but may be exaggerated to illustrate various aspects of the present disclosure, and in that regard, the drawing figures should not be construed as limiting.
Various aspects of the present disclosure are directed toward reinforced biopolymers. The reinforced biopolymers may be configured to support and maintain cells on a surface thereof. The reinforced biopolymer may, for example, be used to support a monolayer of cells on a surface of the reinforced biopolymer. The cells may be, for example, stem cells, epithelial cells, endothelial cells, or retinal pigment epithelial cells. The reinforced biopolymers, as discussed in detail below, allow for mass transport across the reinforced biopolymers, have a high optical transparency, and have a sufficient toughness to endure manipulation, including bending and/or folding.
The reinforced biopolymers described herein may be used as a device or in a device, in methods, and in systems and may be configured to deliver a monolayer of cells to a target site. In certain instances, and as discussed in further detail below, the mechanical properties of the reinforced biopolymers are designed to avoid tearing or ripping during implantation and once implanted in vivo.
Although the synthetic support membrane 102 is positioned approximately centrally between two layers of biopolymer in each of reinforced biopolymer 100 of
In certain embodiments, the synthetic support membrane 102 is biologically inert. The synthetic support membrane 102 can include at least one of expanded polytetrafluoroethylene (ePTFE) and expanded polyethylene. In some embodiments, the synthetic support membrane 102 includes ePTFE.
The ePTFE synthetic support membrane of some embodiments is biaxially oriented and has high intrinsic strength, and can be prepared according to the general methodology described in U.S. Pat. No. 3,953,566 to Gore, which is hereby incorporated by reference in its entirety. The biaxially oriented ePTFE synthetic support membrane is highly crystalline (i.e., a crystallinity index of at least 94%) and has a matrix tensile strength in both the longitudinal and transverse directions of at least 600 MPa. The ePTFE synthetic support membrane may be comprised of a plurality of stacked ePTFE layers, wherein each layer has an areal density of less than 100 mg/m2.
In some instances, the ePTFE synthetic support membrane is prepared according to the methodology describe in U.S. Pat. No. 7,306,729 to Gore, which is hereby incorporated by reference in its entirety. In other instances, the ePTFE synthetic support membrane is prepared according to the methodology describe in U.S. Pub. No. 2012/0065649, which is hereby incorporated by reference in its entirety. In other instances, the ePTFE synthetic support membrane is prepared according to the methodology described in U.S. Pat. No. 5,814,405 to Gore, which is hereby incorporated by reference in its entirety.
In some embodiments, the synthetic support membrane is configured to permit mass transport across the synthetic support membrane when included in a reinforced biopolymer. By permitting mass transport across the synthetic support membrane, nutrients can diffuse across the membrane to reach cells on the surface of the biopolymer, and cellular waste byproducts can diffuse away from the cells and across the membrane. The mass transfer coefficient of the synthetic support membrane can be adapted to a desired use, increasing or decreasing mass transport potential across the synthetic support membrane. The mass transfer coefficient can be controlled by adjusting membrane pore size and/or membrane layering.
The synthetic support membrane of the reinforced biopolymer can have a thickness of about 0.5 μm to about 10 μm. In certain embodiments, the synthetic support membrane has a thickness of about 0.5 μm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. As described in detail below, in certain instances, the wetting out process can cause the synthetic support membrane to “collapse,” with a decrease in membrane thickness. The thicknesses provided above refer to the “collapsed” synthetic support membrane following wetting out and as it appears in the composite reinforced biopolymer.
In certain embodiments, the biopolymer includes collagen, gelatin, laminin, fibronectin, fibrinogen, elastin, thrombospondin, heparan sulfate, chondroitin sulfate, polysaccharides, alginate, chitosan, glycosaminoglycan, hyaluronic acid, or a combination of any two or more of these. In particular embodiments, the biopolymer includes a collagen. Many of these components may exist in different forms or as different types. For example, there are many different types of collagen (e.g., Types I, II, III, etc.) and gelatin (e.g., Type A, Type B). Further, these components may be derived from a variety of sources. It is contemplated that any type or form, derived from any source, may be included in a biopolymer described herein.
In some embodiments, the biopolymer also includes one or more additives, such as, for example, a coating or imbibing aid, a viscosity modifier, a density modifier, or a biopolymer cross-linker.
A single biopolymer layer occurring on one side of the synthetic support membrane can have a thickness of about 0 μm to about 95 μm. In some embodiments, the biopolymer is fully imbibed into the synthetic support membrane on one side of the synthetic support, the biopolymer layer thus having a thickness of about 0 μm.
In certain instances, the biopolymer of the reinforced biopolymer is cross-linked. Cross-linking the biopolymer can increase its tensile strength and thermostability. Methods for cross-linking biopolymer elements contemplated herein such as collagen, gelatin, laminin, fibronectin and the like are known in the art. Cross-linking, while increasing tensile strength, can have the negative effect of reducing the ‘stickiness’ of the biopolymer and it ability to retain cells and/or adhere to tissue in vivo. An advantage of the reinforced biopolymers disclosed herein is that because of the synthetic support membrane, the biopolymer does not need to be crosslinked to increase its tensile strength; the strength and toughness of the reinforced biopolymer is provided by the synthetic support membrane. Although the biopolymer may still be cross-linked, it does not need to be cross-linked as extensively as it would need to be if the synthetic support membrane were not present. This permits the biopolymer to retain its stickiness and ability to retain cells and/or adhere to tissues in vivo. Cross-linking the biopolymer may be done to, for example, increase the thermostability of the biopolymer so that it may be implanted in vivo without liquefying. The extent or level of cross-linking necessary to achieve the desired thermostability is generally lower than that required to achieve significant gains in tensile strength. Where the reinforced biopolymer includes a biopolymer layer on both sides of the synthetic support membrane, as in reinforced biopolymers 100 and 200 depicted by
In certain embodiments, a surface of a biopolymer layer is configured to support cellular growth, cellular adhesion, or both cellular growth and adhesion. In certain embodiments, a layer of cells are grown directly on the biopolymer layer's surface. In other embodiments, a suspension of cells is seeded onto the biopolymer layer's surface and the cells allowed to adhere to the biopolymer layer's surface. In certain instances, the cells grown on the biopolymer layer's surface or the cells adhered to the biopolymer layer's surface form a cellular monolayer (i.e., a layer of cells that is one-cell-thick).
Where the reinforced biopolymer includes a biopolymer layer on both sides of the synthetic support membrane, as in reinforced biopolymers 100 and 200 depicted by
Where the reinforced biopolymer includes a biopolymer layer only on one side of the synthetic support membrane, as in reinforced biopolymer 300 depicted by
The biopolymer can be selected to support growth of a desired cell type. In some embodiments, it may be desirable to grow corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, photoreceptor cells, Müller glial cells, ganglion cells, endothelial cells, epithelial cells, pericytes, or a combination thereof on a surface of the reinforced biopolymer. In certain instances, these cells form a monolayer of cells on a surface of the reinforced biopolymer. Biopolymer substrates suitable for supporting growth of these and other cell types are known in the art.
In some embodiments, the surface of the biopolymer layer configured to support cellular growth, cellular adhesion, or both cellular growth and cellular adhesion is functionalized. The surface may be functionalized by, for example, including cell adhesion molecules, antigens, epitopes, and/or stem cell differentiation factors at the surface of the biopolymer layer. It will be recognized that such functionalization factors are cell-specific, and can be selected according to the specific cell type.
In certain instances, the biopolymer layer(s) is configured to minimize or avoid an inflammatory response when implanted in vivo.
The thickness of the reinforced biopolymer can be from about 0.5 μm to about 150 μm. In some embodiments, the reinforced biopolymer can have a thickness of about 10 μm to about 50 μm. In other embodiments, the reinforced biopolymer can have a thickness of about 20 μm to about 30 μm. In a particular embodiment, the reinforced biopolymer can have a thickness of about 20 μm.
In some embodiments, a reinforced biopolymer described herein has a toughness of at least about 30 KJ/m3. As the toughness of the synthetic support membrane is a few orders of magnitude greater than that of the biopolymer layer(s)—even if crosslinked, the toughness of the synthetic support membrane approximates the toughness of the reinforced biopolymer. The contribution of the biopolymer to the toughness of the reinforced biopolymer can be neglected.
The synthetic support membrane-biopolymer composite construction of the reinforced biopolymers described herein results in the biopolymer having sufficient tensile strength and toughness to withstand handling and manipulation during both cell culture procedures and in vivo implantation. Unlike a substrate made up only of biopolymer, which requires extensive cross-linking to achieve sufficient tensile strength to withstand manipulation and implantation, the described reinforced biopolymers are flexible and tough, while minimizing the need to further manipulate (e.g., cross-link) the biopolymer layer(s).
In certain embodiments, a reinforced biopolymer described herein has a measured optical transparency of at least about 85%. In some embodiments, a reinforced biopolymer described herein has a measured optical transparency of at least about 90%. Optical transparency is measured in air with the biopolymer in a fully hydrated state. When measured in air, Fresnel reflection at the outside interfaces between the sample and its environment reduces transmission values compared to optical transparency of the reinforced biopolymer when in use in accordance to the present disclosure. It will be understood that the optical transparency of the reinforced biopolymer in use will be higher than the measured value in air.
Optically transparent reinforced biopolymers can be used, for example, as substrates or vehicles for delivering cells to the eye without adversely affecting the eye's optics. For example, a monolayer of corneal endothelial cells grown on a surface of a biopolymer layer of a reinforced biopolymer can be implanted at the posterior surface of the cornea, providing a layer of healthy endothelial cells to the cornea (i.e., an endothelial keratoplasty). In another example, retinal pigment epithelial cells can be introduced directly to the retina as a monolayer formed on a surface of a reinforced biopolymer. In both of these examples, the reinforced biopolymers allow normal optical function of the eye, with good transparency and minimal scattering.
The transparency of ePTFE is largely governed by how much residual scattering is present in the bulk of the material. To obtain high specular (clear image-forming) transmission, diffuse (scattered) components in both transmission and reflection must be minimized. This is typically done by either thinning of the structure or incorporation of a substance with a near-matching refractive index (around 1.37 for ePTFE) into ePTFE's microstructure. In some embodiments, the ePTFE synthetic support membrane is prepared according to the methodology describe in U.S. Pat. No. 7,306,729 to Gore, which is hereby incorporated by reference in its entirety. Some members of this family of membranes possess high clarity while having high degrees of porosity, which normally reduces the amount of visible light passing through the membrane. Certain membranes can have a light transmission, or optical transparency, of at least 50% and a porosity of at least 50%, while light transmission values of 85% and higher are obtainable in membranes that are greater than 75% porous.
The amount of air trapped within the synthetic support membrane and the hydration index of imbibed substances are important determinants in the optical transparency of the support membrane, contributing to the optical transparency of the reinforced biopolymer. In some embodiments, all or substantially all air is expelled from the synthetic support membrane during a wetting out process that occurs prior to the synthetic support membrane being imbibed with a biopolymer. In some instances, the wetting out process results in the membrane “collapsing” on itself. For example, an ePTFE membrane having a thickness of about 5 μm prior to wetting out may collapse to a thickness of about 1 μm. The wetted out synthetic support membrane is then prepared and imbibed with the desired biopolymer to produce a reinforced biopolymer that is free or substantially free of air. In some instances, where the reinforced biopolymer is imbibed with biopolymer and is free or substantially free of air, it is considered to be “fully imbibed.”
In certain embodiments, the biopolymer has a refractive index that is the same as or substantially similar to the refractive index of the synthetic supporting membrane.
In another aspect, provided herein are implantable cell therapy devices. The implantable cell therapy device includes a reinforced biopolymer and a monolayer of cells on a surface of the biopolymer that is distal to the synthetic support membrane. The monolayer of cells can include, but is not limited to, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, photoreceptor cells, Müller glial cells, ganglion cells, endothelial cells, epithelial cells, pericytes, stem cells, and combinations thereof. In some embodiments, the stem cells are pluripotent stem cells. In other embodiments, the stem cells are multipotent stem cells.
In another aspect, methods of treating a disease or condition are provided. In some instances, the disease or condition affects a cellular layer in a subject. The disease or condition can be, for example, corneal endothelial dystrophy, a disease or condition affecting the retinal pigment epithelium, or a disease or condition affecting the photoreceptors. The methods generally include surgically implanting an implantable cell therapy device described herein on or near the diseased site or otherwise affected area, where the implantable cell therapy device includes a monolayer of cells selected to treat or otherwise improve the disease or condition. For example, corneal endothelial dystrophy can be treated by surgically implanting a cell therapy device including a monolayer of corneal endothelial cells at a posterior surface of the cornea. In some embodiments, the implantation procedure includes first removing some or all of the diseased or non-functional corneal endothelium. A disease or condition of or affecting retinal pigment epithelium can be treated by surgically implanting a cell therapy device including a monolayer of retinal pigment epithelial cells at the retina. A disease or condition of or affecting photoreceptors can be treated by surgically implanting a cell therapy device including a monolayer of photoreceptor cells at the retina.
It should be understood that although certain methods and equipment are described below, other methods or equipment determined suitable by one of ordinary skill in the art may be alternatively utilized.
Synthetic support membrane thickness is measured via a non-contact measurement using a Keyence LS-7010.
Reinforced biopolymer thickness and biopolymer layer thickness (in both hydrated and dehydrated conditions) is determined by laser scanning confocal microscopy (LSCM) cross-sectioning. The reinforced biopolymer can be stained (e.g., with Rhodamine) to offset the biopolymer layer from the synthetic support membrane. Thickness of the synthetic support membrane, whether alone or when incorporated into the reinforced biopolymer, can also be determined by LSCM. LSCM is used as the composite thickness (and layers thereof) is measured in a fully hydrated state. The thickness of the reinforced biopolymer in a partially hydrated or dry state is significantly thinner (e.g., about 200%-500%).
Tensile properties of the synthetic support membrane, which approximate the tensile properties of the reinforced biopolymer, are determined in accordance with ASTM D412-Dogbone F. Peak tensile load, tensile load, and toughness (i.e., area under the curve up to peak tensile load) can each be calculated from the standard test data.
The spectral transmission and haze of a specimen (membrane, film, etc.) is measured in air using a Shimadzu UV-2700 spectrophotometer. The instrument contains an integrating sphere and has a range of measurement from 185 to 900 nm. Both haze and transmission measurements are described in ASTM standard D1003-13 and implemented accordingly.
A 10% gelatin solution was prepared with Type A gelatin. A 3 mil (76.2 μm thick) ePTFE membrane was placed and fixated onto a table. The membrane was then hydrophilically treated and dried. The dry, hydrophilically treated membrane was then mounted onto a hoop. An excess of the gelatin solution was pipetted onto the membrane, and a drawdown procedure using RDS15 Mayer bar was carried out to form a layer of gelatin on the membrane. This procedure involved putting a line of gelatin solution (in excess) across a top edge of the membrane and drawing it down with the Mayer bar. Care was taken not to move the membrane. The membrane immediately wetted out with the gelatin. The formed reinforced biopolymer composite was then allowed to dry. The composite was optionally rewetted to allow for easier peel off. Gelatin layer thickness can be controlled by using different drawdown bars. The membrane with gelatin layer was dried and hydrophilically treated. The dry, treated membrane was then
Gelatin of the gelatin layer was crosslinked using a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-N-hydroxysuccinimide (EDC-NHS) crosslinking procedure (see, e.g., Kuijpers et al. (2000), Journal of Biomaterials Science, Polymer Edition, 11(3):225-243). Concentrations of EDC/NHS were altered to generate cross linking from 5% to ‘100%’ (functional groups crosslinked to their maximum in view of steric hinderance). The gelatin weight of the reinforced biopolymer was determined, and the amount of gelatin was used to calculated how much EDC would be required to activate carboxylic acid residues. A 2.5% w/v EDC solution with NHS (molar ratio NHS/EDC=0.2) was prepared. MES buffer (0.05M, pH 5.3) was used to immerse the ePTFE-gelatin composite so the concentration of gelatin was 1 g/50 ml. The required quantity of EDC/NHS solution was subtracted from this volume, and the required quantity of EDC/NHS solution was added. The following formula illustrates the calculation:
where x is the degree of carboxylic acid residue activation desired. Note that 79 mml COO− per 100 g gelatin is only true for gelatin type A, based on an average of 78-80 mmol.
Reinforced biopolymers were incubated in the refrigerator for 16 hours before quenching in quenching solution for 2 hours. The reinforced biopolymers were washed in deionized water for 4 hours, including replacement of solution and agitation to assure removal of reaction byproducts.
The reinforced biopolymers were then mounted on a PTFE ring, which was placed in a 24 well plate. The reinforce biopolymers were kept hydrated until cell culture was seeded onto the samples.
The invention of this application has been described above both generically and with regard to specific embodiments. It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments without departing from the scope of the disclosure. Thus, it is intended that the embodiments cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
This application is a national phase application of PCT Application No. PCT/US2020/064561, internationally filed on Dec. 11, 2020, which claims the benefit of Provisional Application No. 62/947,933, filed Dec. 13, 2019, which are incorporated herein by reference in their entireties for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/064561 | 12/11/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62947933 | Dec 2019 | US |
