Patent Application
20150352229
Some applications of the present invention relate generally to implantable sensors for detecting an analyte in a body and specifically to methods and apparatus for providing nutrients to cells in an implantable medical device.
The monitoring of various medical conditions often requires measuring the levels of various components within the blood. In order to avoid invasive repeated blood drawing, implantable sensors aimed at detecting various components of blood in the body have been developed. More specifically, in the field of endocrinology, in order to avoid repeated “finger-sticks” for drawing blood to assess the levels of glucose in the blood in patients with diabetes mellitus, implantable glucose sensors have been discussed.
One method for sensing the concentration of an analyte such as glucose relies on Fluorescence Resonance Energy Transfer (FRET). FRET involves the transfer of non-photonic energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when the donor and acceptor molecules are in close proximity to each other. FRET enables the determination of the relative proximity of the molecules for investigating, for example, the concentration of an analyte such as glucose.
PCT Patent Application Publication WO 2006/006166 to Gross et al., which is incorporated herein by reference, describes a protein which includes a glucose binding site, cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP). The protein is configured such that binding of glucose to the glucose binding site causes a reduction in a distance between the CFP and the YFP. Apparatus is described for detecting a concentration of a substance in a subject, the apparatus comprising a housing adapted to be implanted in the subject. The housing comprises a fluorescence resonance energy transfer (FRET) measurement device and cells genetically engineered to produce, in situ, a FRET protein having a FRET complex comprising a fluorescent protein donor, a fluorescent protein acceptor, and a binding site for the substance.
One of the challenges in the design of a cell-based implantable device is the maintenance of a significant population of cells over the long term, e.g., over a year or longer. In accordance with some applications of the present invention, techniques are provided for maintaining a desired cell population size over time, including both:
In order to balance the above-mentioned conflicting goals and preserve a generally constant cell population over a long period of time, e.g., at least one year, a three-layer cell encapsulation structure is provided, which comprises a substantially non-degradable three-dimensional scaffold having surfaces to which cells are attached, and a hydrogel, which is applied to the cells.
The scaffold, cells, and hydrogel are arranged such that the cells are sandwiched in spaces between the hydrogel and the surfaces of the scaffold. The cells are arranged in monolayers on at least 50% of an aggregate surface area of the surfaces of the scaffold. This arrangement allows mobility and proliferation of the cells in the spaces between the hydrogel and the surfaces of the scaffold, and prevents the mobility and the proliferation of the cells to locations outside of the spaces between the hydrogel and the surfaces of the scaffold. Cells within the spaces between the hydrogel and the surfaces of the scaffold that die leave a space upon disintegration. The structure provided by the surface of the scaffold on one side and the hydrogel on the other side maintain the patency of this space until one or more neighboring cells proliferate into the space.
Thus, in any local microscopic environment the encapsulation structure comprises a three-layer stack of (a) the surface of the solid scaffold, (b) the cells, and (c) the hydrogel, in this order. The cells at any location are thus generally limited to a monolayer, allowing free mobility and proliferation of the cells within the narrow space between the scaffold and the hydrogel, but preventing any proliferation into the rest of the volume and creation of three-dimensional cell structures.
The scaffold provides a three-dimensional structure with a high aggregate surface area, and high surface-to-volume ratio, which makes efficient use of the three-dimensional volume of the chamber. The surfaces of the scaffold, although often not flat, serve effectively as a two-dimensional substrate for seeding, growth, and attachment of the cells. If the hydrogel were not provided over the monolayer of the cells, the cells typically grow in three dimensions, away from the surfaces to which they are attached. Such three-dimensional growth would generally result in undesirable over-population, as described above. In addition, for many cell types, cell viability and protein expression, including expression of the sensor protein, are significantly enhanced when cells are attached and spread. Thus cells in this configuration will survive longer and function better than suspended cells, e.g., cells suspended in a hydrogel scaffold.
For some applications, the scaffold comprises microcarrier beads, fibers, a rigid structure, or a sponge structure having a plurality of interconnected internal pores.
The encapsulation structure may combine at least three benefits: (a) good cell attachment, leading to better cell viability and expression, lacking in simpler systems that for example use hydrogel as a scaffold, (b) prevention of over-population which often leads to a necrotic core, because of a limited number of cells and open diffusion channels to the cells via the hydrogel, and (c) enablement of cell mobility and proliferation within a two-dimensional culture, thereby enabling long-term steady state population.
Some applications of the present invention provide a multi-layer immunoisolation system. The viability of cells within a cell-based device strongly depends on an ample supply of oxygen. Generally, the foreign body response following device implantation creates dense fibrotic tissue that encapsulates the device, substantially reducing oxygen diffusion to the device from the blood circulation. Therefore, the viability of cells inside a cell-based device is enhanced by substantial vascularization of the tissue as close as possible to the implanted device, which increases oxygen levels at the device surface. More specifically, for a glucose measurement device, the creation of a dense fibrotic tissue is a potential diffusion barrier for glucose, leading to a time delay between glucose levels in the tissue and glucose levels measured by the device. Such dense fibrotic tissue should thus be avoided in order to maintain the accuracy of the glucose measurement.
The multi-layer immunoisolation system is configured to enhance long-term function of an implanted cell-based device. The multi-layer immunoisolation system comprises at least the following three layers: (a) a lower (inner) membrane layer, which is disposed at an external surface of the device, (b) an upper (outer) neovascularization layer, and (c) a middle protective layer, disposed between the lower membrane layer and the upper neovascularization layer. The multi-layer immunoisolation system comprises a biodegradable scaffold. Before biodegrading, the biodegradable scaffold spans both the upper neovascularization layer and the middle protective layer, such that the upper neovascularization layer comprises a first upper portion of the biodegradable scaffold, and the middle protective layer comprises a second lower portion of the biodegradable scaffold. In addition, the middle protective layer further comprises a non-biodegradable hydrogel that impregnates the second lower portion of the biodegradable scaffold. The upper neovascularization layer, which comprises the first upper portion of the biodegradable scaffold, is not impregnated with the hydrogel.
The biodegradable scaffold serves at least two functions: (a) during implantation of the device, the biodegradable scaffold protects the soft hydrogel of the middle protective layer from strong shear forces which might otherwise pull off the soft hydrogel; and (b) after implantation of the device, the biodegradable scaffold promotes vascularization of the tissue that grows into the upper neovascularization layer, until the biodegradable scaffold eventually degrades and is totally absorbed.
Upon biodegradation of the biodegradable scaffold, the middle protective layer (now comprising primarily the hydrogel) remains attached to the lower membrane layer. The middle protective layer typically serves to (a) prevent attachment of proteins to the lower membrane layer, thereby minimizing the creation of a fibrotic tissue, and/or (b) repel large proteins, thereby minimizing the fouling of the lower membrane layer. The high water content of the hydrogel of the middle protective layer prevents the attachment of various proteins, so that immune system cells are less likely to attach to the tissue-hydrogel interface, thereby minimizing the overall immune response. As a result of this triple-layer protection, the tissue surrounding the device is characterized by high vascularization and minimal fibrosis.
Some applications of the present invention provide another multi-layer immunoisolation system, which comprises at least the following three layers: (a) a lower (inner) membrane layer, which is disposed at an external surface of the device, (b) an upper (outer) protective layer, and (c) a middle attachment layer, which is disposed between the lower membrane layer and the upper protective layer, and which tightly fixes the upper protective layer to the lower membrane layer. The middle attachment layer comprises a non-biodegradable scaffold, which is tightly fixed to the lower membrane layer, such as by being deposited directly on the membrane using electrospinning.
The multi-layer immunoisolation system comprises a non-biodegradable hydrogel, which spans both the upper protective layer and the middle attachment layer. In other words, the middle attachment layer comprises a first portion of the hydrogel, and the upper protective layer comprises a second portion of the hydrogel. The hydrogel is impregnated in the scaffold of the middle attachment layer, and extends above the scaffold, i.e., in a direction away from the lower membrane, so as to provide the upper protective layer. The upper protective layer does not comprise the scaffold. As a result, the scaffold is not exposed to tissue, thereby reducing the likelihood that the multi-layer immunoisolation system generates an immune response.
The middle attachment layer holds the hydrogel of the upper protective layer in place on the lower membrane layer. The upper protective layer has a smooth upper (outer) surface, which results in low biofouling of the lower membrane layer, allowing the membrane to efficiently diffuse nutrients into the device even after a long implantation period. In addition, the upper protective layer protects the device by presenting a highly biocompatible surface to the tissue.
In some applications of the present invention, a fully-implantable or partially-implantable sensor device comprises apparatus for facilitating cell growth. For some applications, the apparatus comprises a chamber and a membrane that surrounds the chamber at least in part and is permeable to nutrients. (“Nutrients,” in the context of the specification and in the claims, includes oxygen, glucose, and other molecules important for cell survival.) Typically, a scaffold comprising a hydrogel or other suitable material is disposed within the chamber, and a plurality of cells is disposed therein. Additionally, at least one nutrient supply compartment is typically disposed within the chamber, and interspersed with the scaffold such that at least 80% of the cells within the cell-growth medium are disposed within 100 um (microns) of the nutrient supply compartment. In this manner, the nutrient supply compartment is positioned within the chamber such that a diffusion path for nutrients is provided, by the nutrient supply compartment, between the membrane and the at least 80% of the cells.
In the context of the present application and in the claims, a membrane which is described as “surrounding” an element is to be understood as surrounding the element at least in part. Thus, for example, a membrane that surrounds a chamber may entirely surround the chamber, or may surround the chamber in part (while another portion of the chamber may be covered with something other than the membrane).
In some applications of the present invention, the apparatus comprises a chamber having isolated cells disposed (e.g., encapsulated) therein, and a membrane structure that surrounds the chamber at least in part. The membrane structure in a first state thereof has a first molecular weight cut off (MWCO), and transitions to a second state thereof, in which the membrane structure has a second MWCO, the second MWCO being higher than the first MWCO. One of the goals of the apparatus is to maintain a constant flow of nutrients into the chamber by increasing membrane permeability, thus reducing the adverse effect on nutrient flow due to membrane fouling (which otherwise may limit nutrient flow to the cells). The membrane structure is generally impermeable to white blood cells, for many months or even the entire time that the apparatus is implanted in the subject. Permeability to large molecules such as transferrin or even IgG increases over time.
It is noted that even though this application is described hereinabove and below as having the second MWCO being higher than the first MWCO, the scope of the present invention includes applications in which the first MWCO is the same or even larger than the second MWCO. A benefit in such an application case is enhanced total membrane thickness, which reduces the effect of some of the immune system components, especially reactive oxygen species (ROS). Additionally, a soft biodegradable membrane as provided by some applications of the present invention may recruit a weaker immune response compared to a rigid surface.
In some applications of the present invention, the membrane structure comprises (a) a first layer comprising a biodegradable material, and (b) a second layer that is non-biodegradable. In some applications, the membrane structure comprises a non-biodegradable material impregnated with a biodegradable material. Over time, the biodegradable material biodegrades, thereby leaving spaces in the non-biodegradable material, thereby increasing the permeability of the membrane structure. As a result the membrane structure initially has a low MWCO, which is effective for blocking cytokines, while after the biodegradable material has degraded, the non-biodegradable material having the larger MWCO remains. Thus, even if there has been fouling of the membrane, nutrients can still pass through the membrane due to the higher permeability of the membrane caused by degradation of the biodegradable material.
In some applications of the present invention, the apparatus comprises an optically-transparent scaffold; an optical waveguide, coupled to the scaffold; a plurality of cells on the scaffold; and a membrane structure surrounding the scaffold. The transparency of the scaffold enables light to pass through the optical waveguide to the scaffold, and through the scaffold to where the sensor protein secreted from the cells or produced within the cells are disposed. Similarly, fluorescent light emitted by the sensor protein in response to the excitation light is transmitted through the transparent scaffold to the optical waveguide.
In some applications of the present invention, apparatus for detecting a concentration of an analyte in a subject comprises an optical waveguide having a proximal end and a distal end. A sensing unit is disposed at the distal end of the optical waveguide and detects the analyte (e.g., by the binding of the analyte to a protein). The sensing unit comprises a first chamber. A second chamber is disposed around at least a distal end portion of the first chamber. Live cells that are genetically engineered to produce, in the body of the subject, a sensor protein having a binding site for the analyte, are disposed (e.g., encapsulated) within either the first chamber or the second chamber.
In some applications of the present invention, apparatus for detecting a concentration of an analyte in a subject comprises an optical waveguide having a first, distal, end and a second, proximal, end. A sensing unit for detecting analyte is disposed at the first end of the optical waveguide. The sensing unit comprises at least an inner axial portion without cells, disposed adjacent to the first end of the optical waveguide. A second chamber is adjacent to the inner axial portion, and is coaxial with the optical waveguide and the inner axial portion. Live cells that are genetically engineered to produce, in the subject's body, a sensor protein having a binding site for the analyte, are disposed in the second chamber and secrete a sensor protein. In this configuration, a relatively large surface area is provided for allowing transfer of analyte and nutrients between the subject's body and the second chamber.
In some applications of the present invention, the apparatus for detecting a concentration of an analyte in a subject comprises an optical waveguide; a chamber surrounding a distal portion of the optical waveguide, the distal portion of the optical waveguide extending along at least 75% of a length of the chamber; and live cells that are genetically engineered to produce, in a body of the subject, a sensor protein having a binding site for the analyte. The live cells are disposed (e.g., encapsulated) within the chamber.
In some applications of the present invention the apparatus for detecting a concentration of an analyte in a subject comprises an optical waveguide that transmits excitation light, and a chamber comprising (i.e., containing) live cells that are genetically engineered to produce, in a body of the subject, a fluorescent sensor protein having a binding site for the analyte. The fluorescent sensor protein is configured to emit fluorescent light in response to the excitation light. The chamber is disposed coaxially with respect to the optical waveguide. A lens is disposed between the optical waveguide and the chamber, the lens configured to focus light from the optical waveguide to the chamber and from the chamber to the optical waveguide. A first mirror is coupled to the chamber, and is disposed between a proximal end of the chamber and the lens. The first mirror reflects the excitation light within the chamber and transmits the fluorescent light from within the chamber toward the lens and the optical waveguide. The first mirror is shaped to define a pinhole that allows passage of the excitation light from the lens into the chamber. A second mirror is coupled to the chamber and disposed at a distal end of the chamber.
Applications of the present invention also include a method for facilitating the measurement of a concentration of an analyte in a body of a subject, from a subcutaneous location of the subject. This is accomplished by measuring a temperature of the subcutaneous location in conjunction with the facilitating of the measuring of the concentration of the analyte; and calibrating the measurement of the concentration of the analyte in response to the measured temperature.
There is therefore provided, in accordance with an application of the present invention, apparatus for detecting a concentration of an analyte in a subject, the apparatus configured to be implanted in a body of the subject and including:
an optical waveguide having a proximal end and a distal end;
a sensing unit disposed at the distal end of the optical waveguide and configured to detect the analyte, the sensing unit including:
For some applications, the analyte is glucose.
For some applications, the second chamber completely surrounds the first chamber.
For some applications, the optical waveguide includes an optical fiber. For some applications, the optical waveguide includes a planar optical waveguide.
For some applications, the live cells are disposed within the first chamber. For some applications, a first distal longitudinal segment of the second chamber is disposed around the first chamber at least at the proximal end portion of the first chamber, and a second proximal longitudinal segment of the second chamber does not surround the first chamber. For some applications, at least 60% of a volume of the second chamber is disposed along the second proximal longitudinal segment. For some applications, the first longitudinal segment of the second chamber completely surrounds the first chamber. For some applications, a diameter of the optical waveguide is equal to a diameter of the second chamber.
For some applications, the optical waveguide has a diameter that is equal to a diameter of the first chamber.
For some applications, the apparatus further includes a first semi-permeable membrane between the first and second chambers, the live cells are genetically engineered to secrete the sensor protein, and the semi-permeable membrane is configured to facilitate passage of the sensor protein from the first chamber to the second chamber and restrict passage of the live cells therethrough. For some applications, the second chamber completely surrounds the first chamber. For some applications, the optical waveguide has a diameter that is equal to an outer diameter of the second chamber. For some applications, the apparatus further includes a second semi-permeable membrane surrounding the second chamber, the second semi-permeable membrane being configured to facilitate passage of nutrients into the second chamber and restrict cell passage therethrough.
For some applications, the first chamber has a proximal portion and a distal portion, and the proximal portion has a proximal-portion diameter that is smaller than a distal-portion diameter of the distal portion. For some applications, a diameter of the optical waveguide is equal to the distal-portion diameter.
For some applications, the second chamber surrounds the proximal portion of the first chamber, and the second chamber facilitates passage of nutrients to the live cells in the first chamber from fluid of the subject. For some applications, the apparatus further includes a semi-permeable membrane between the first and second chambers, the live cells are genetically engineered to secrete the sensor protein, and the semi-permeable membrane is configured to facilitate passage of the sensor protein from the first chamber to the second chamber. For some applications, the apparatus further includes a semi-permeable membrane surrounding at least one chamber selected from the group consisting of: the first and second chambers, the second semi-permeable membrane being configured to facilitate passage of nutrients into the selected chamber and restrict cell passage therethrough. For some applications, the semi-permeable membrane is configured to restrict cell passage into the second chamber. For some applications, the semi-permeable membrane is configured to restrict passage of the cells into the first chamber.
For some applications, the live cells are disposed within the second chamber. For some applications, a first proximal longitudinal segment of the second chamber completely surrounds the first chamber, and a second distal longitudinal segment of the second chamber does not surround the first chamber. For some applications, at least 60% of a volume of the second chamber is disposed along the second distal longitudinal segment. For some applications, a diameter of the optical waveguide is equal to a diameter of the first chamber.
For some applications, the apparatus further includes a first semi-permeable membrane between the first and second chambers, the live cells are genetically engineered to secrete the sensor protein, and the semi-permeable membrane is configured to facilitate passage of the sensor protein from the first chamber to the second chamber. For some applications, the optical waveguide has a diameter that is equal to a diameter of the first chamber.
For some applications, the optical waveguide has a diameter that is equal to a diameter of the second chamber.
For some applications, the first chamber includes optically-transparent material configured to transmit light through the first chamber. For some applications, the apparatus further includes a mirror disposed at a distal end of the first chamber and configured to reflect transmitted light through the first chamber.
For some applications, the first chamber includes optically-transparent material configured to transmit light through the first chamber. For some applications, the apparatus further includes a mirror disposed at a distal end of the first chamber and configured to reflect transmitted light through the first chamber.
There is further provided, in accordance with an application of the present invention, apparatus containing cells for implantation into a human subject, the apparatus including:
a substantially non-degradable three-dimensional scaffold having surfaces to which the cells are attached; and
a hydrogel, which is attached to the cells,
wherein the scaffold, the cells, and the hydrogel are arranged such that the cells are sandwiched in spaces between the hydrogel and the surfaces of the scaffold, and wherein the cells are arranged in monolayers on at least 50% of an aggregate surface area of the surfaces of the scaffold, thereby allowing mobility and proliferation of the cells in the spaces between the hydrogel and the surfaces of the scaffold, and preventing the mobility and the proliferation of the cells to locations outside of the spaces between the hydrogel and the surfaces of the scaffold.
For some applications, the cells are arranged in the monolayers on at least 70% of the aggregate surface area of the surfaces of the scaffold, such as on at least 90% of the aggregate surface area of the surfaces of the scaffold.
For some applications, the apparatus further includes a chamber, in which the scaffold, the cells, and the hydrogel are contained. For some applications, the apparatus further includes an external membrane, which surrounds at least a portion of the chamber.
For some applications, the cells are differentiated cells, such as terminally-differentiated cells, which are attached to the surfaces of the scaffold. Alternatively, for some applications, the cells are stem cells, which are attached to the surfaces of the scaffold.
For some applications, the cells are genetically engineered to produce a fluorescent sensor protein having a binding site for an analyte, the fluorescent sensor protein being configured to emit fluorescent light in response to excitation light. For some applications, the analyte is glucose.
For any of the applications described above, the scaffold may include microcarrier beads.
For any of the applications described above, the scaffold may include fibers.
For any of the applications described above, the scaffold may include a sponge structure having a plurality of interconnected internal pores.
For any of the applications described above, the scaffold may be rigid. For some applications, the rigid scaffold is shaped so as to define a plurality of wells.
There is still further provided, in accordance with an application of the present invention, a method for manufacturing a cell encapsulation structure, including:
providing a substantially non-degradable three-dimensional scaffold having surfaces suitable for cell attachment and growth;
seeding cells on the surfaces and allowing cell proliferation to reach at least 70% confluence; and
before the cells form three-dimensional structures on 50% of an aggregate surface area of the surfaces, filling, with a hydrogel, a volume of the cell encapsulation structure which is not already occupied by the cells or the scaffold, thereby preventing additional cell proliferation into the volume of the cell encapsulation structure which is not already occupied by the cells or the scaffold.
There is additionally provided, in accordance with an application of the present invention, apparatus including a multi-layer immunoisolation system for application to an implantable cell-based device, the multi-layer immunoisolation system including:
a lower membrane layer, which is disposed at an external surface of the device;
an upper neovascularization layer, which includes a first upper portion of the biodegradable scaffold; and
a middle protective layer, which (a) is disposed between the lower membrane layer and the upper neovascularization layer, and (b) includes:
wherein the upper neovascularization layer is not impregnated with the hydrogel.
For some applications, the lower membrane layer has a molecular weight cutoff (MWCO) of between 5 and 50 KDa.
For some applications, the biodegradable scaffold has a thickness of between 100 and 300 microns.
For some applications, the biodegradable scaffold includes a polymer.
For some applications, the lower membrane layer includes a material selected from the group consisting of: polysulfone (PS), polyethersulfone (PES), modified polyethersulfone (mPES), and polytetrafluoroethylene (PTFE).
For any of the applications described above, the biodegradable scaffold may be electrospun onto the lower membrane layer.
There is yet additionally provided, in accordance with an application of the present invention, apparatus including a multi-layer immunoisolation system for application to an implantable cell-based device, the multi-layer immunoisolation system including:
a non-biodegradable hydrogel;
a lower membrane layer, which is disposed at an external surface of the device;
an upper protective layer, which includes a first portion of the hydrogel; and
a middle attachment layer, which (a) is disposed between the lower membrane layer and the upper protective layer, and (b) includes:
For some applications, the lower membrane layer has a molecular weight cutoff (MWCO) of between 5 and 50 KDa.
For some applications, the lower membrane layer includes a material selected from the group consisting of: polysulfone (PS), polyethersulfone (PES), modified polyethersulfone (mPES), and polytetrafluoroethylene (PTFE).
For some applications, the non-biodegradable scaffold includes a polymer.
For some applications, the middle attachment layer has a thickness of between 50 and 150 microns.
For some applications, the upper protective attachment layer has a thickness of between 50 and 200 microns.
For any of the applications described above, the non-biodegradable scaffold may be electrospun onto the lower membrane layer.
There is also provided in accordance with an inventive concept 1, apparatus for facilitating cell growth, the apparatus configured to be implanted in a body of a subject and comprising:
a chamber;
a membrane that surrounds the chamber at least in part and is permeable to nutrients;
a scaffold within the chamber, the scaffold having at least 1000 cells coupled thereto; and
at least one nutrient supply compartment within the chamber, interspersed with the scaffold such that at least 80% of the cells coupled to the scaffold are disposed within 100 microns of the nutrient supply compartment, the nutrient supply compartment being positioned within the chamber such that a diffusion path for nutrients is provided, by the nutrient supply compartment, between the membrane and the at least 80% of the cells.
Inventive concept 2. The apparatus according to inventive concept 1, wherein a volume of the nutrient supply compartment is 25%-75% of a volume of the chamber.
Inventive concept 3. The apparatus according to inventive concept 1, wherein the scaffold has at least 2000 cells coupled thereto.
Inventive concept 4. The apparatus according to inventive concept 1, wherein the scaffold has fewer than 20,000 cells coupled thereto.
Inventive concept 5. The apparatus according to inventive concept 4, wherein the scaffold has fewer than 10,000 cells coupled thereto.
Inventive concept 6. The apparatus according to inventive concept 1, wherein at least 80% of the cells coupled to the scaffold are disposed within 50 microns of the nutrient supply compartment.
Inventive concept 7. The apparatus according to claim 1, wherein the scaffold comprises a hydrogel.
Inventive concept 8. The apparatus according to any one of inventive concepts 1-7, further comprising a nutrient permeable medium that is disposed within the nutrient supply compartment and that is not conducive to cell growth.
Inventive concept 9. The apparatus according to inventive concept 8, wherein the nutrient permeable medium comprises a material selected from the group consisting of: silicone rubber, fused glass powder, sintered glass powder, a hydrogel, and alginate.
Inventive concept 10. The apparatus according to inventive concept 8, wherein the nutrient permeable medium is shaped to define one or more spheres.
Inventive concept 11. The apparatus according to inventive concept 10, wherein the one or more spheres comprises 100-1000 spheres.
Inventive concept 12. The apparatus according to inventive concept 10, wherein a volume of the chamber is at least 20 times a volume of at least one of the spheres.
Inventive concept 13. The apparatus according to inventive concept 12, wherein the volume of the chamber is at least 100 times the volume of the at least one of the spheres.
Inventive concept 14. The apparatus according to inventive concept 13, wherein the volume of the chamber is 200-1000 times the volume of the at least one of the spheres.
Inventive concept 15. The apparatus according to inventive concept 10, wherein the spheres are disposed in the chamber in an efficient packing configuration.
There is further provided in accordance with an inventive concept 16, apparatus for facilitating cell growth, the apparatus configured to be implanted in a body of a subject and comprising:
a chamber having cells disposed therein; and
a membrane structure that surrounds the chamber at least in part, which membrane structure in a first state thereof has a first molecular weight cut off (MWCO), and which is configured to transition to a second state thereof, in which the membrane structure has a second MWCO, the second MWCO being higher than the first MWCO.
Inventive concept 17. The apparatus according to inventive concept 16, wherein the membrane structure is permeable to nutrients.
Inventive concept 18. The apparatus according to inventive concept 16, wherein the second molecular weight cutoff (MWCO) is at least three times higher than the first MWCO.
Inventive concept 19. The apparatus according to inventive concept 16, wherein the second MWCO is greater than 150 kilodaltons.
Inventive concept 20. The apparatus according to inventive concept 16, wherein the membrane structure in the first state is not permeable to IgG.
Inventive concept 21. The apparatus according to inventive concept 20, wherein the membrane structure in the second state is permeable to IgG.
Inventive concept 22. The apparatus according to inventive concept 16, wherein the membrane structure in the first state is permeable to glucose and not permeable to IgG.
Inventive concept 23. The apparatus according to inventive concept 22, wherein the membrane structure in the second state is permeable to glucose and permeable to IgG.
Inventive concept 24. The apparatus according to inventive concept 16, wherein the membrane structure in the first state is not permeable to transferrin.
Inventive concept 25. The apparatus according to inventive concept 24, wherein the membrane structure in the second state is permeable to transferrin.
Inventive concept 26. The apparatus according to inventive concept 16, wherein the membrane structure in the first and second states is not permeable to white blood cells.
Inventive concept 27. The apparatus according to inventive concept 16, wherein the first MWCO is less than 150 kilodaltons.
Inventive concept 28. The apparatus according to inventive concept 27, wherein the first MWCO is less than 100 kilodaltons.
Inventive concept 29. The apparatus according to inventive concept 28, wherein the first MWCO is less than 50 kilodaltons.
Inventive concept 30. The apparatus according to inventive concept 16, wherein the second MWCO is greater than two times the first MWCO, and wherein the first MWCO is less than 100 kilodaltons.
Inventive concept 31. The apparatus according to any one of inventive concepts 16-30, wherein the membrane structure comprises:
a first material that is biodegradable and has the first MWCO; and
a second material, which is non-biodegradable and has the second MWCO.
Inventive concept 32. The apparatus according to inventive concept 31, wherein the first material has a thickness of 50-500 microns.
Inventive concept 33. The apparatus according to inventive concept 31, wherein the second material is impregnated with the first material.
Inventive concept 34. The apparatus according to inventive concept 31, wherein the first material is configured to biodegrade in the presence of body fluids within a period of two weeks to six months.
Inventive concept 35. The apparatus according to inventive concept 31, wherein the second material is permeable to molecules that are 80-300 kilodaltons.
Inventive concept 36. The apparatus according to inventive concept 31, wherein the membrane structure comprises:
a first layer, comprising the first material; and
a second layer, comprising the second material.
Inventive concept 37. The apparatus according to inventive concept 36, wherein the second layer is disposed between the cells and the first layer.
Inventive concept 38. The apparatus according to inventive concept 36, wherein the non-biodegradable material comprises a material selected from the group consisting of: polysulfone (PSU), polytetrafluoroethylene (pTFE), and polyethersulfone (PES).
Inventive concept 39. The apparatus according to inventive concept 36, wherein the first layer has a thickness of 50-500 microns.
Inventive concept 40. The apparatus according to inventive concept 36, wherein the second layer has a thickness of 50-250 microns.
Inventive concept 41. The apparatus according to inventive concept 36, wherein the biodegradable material comprises a hydrogel.
Inventive concept 42. The apparatus according to any one of inventive concepts 16-30, further comprising:
a scaffold to which the cells are attached; and
a nutrient supply compartment disposed at least partially between the scaffold and the membrane structure, the nutrient supply compartment being permeable to nutrients and configured to inhibit growth of the cells into the nutrient supply compartment.
Inventive concept 43. The apparatus according to inventive concept 42, further comprising an optical waveguide, wherein the scaffold is: (a) coupled to the optical waveguide, and (b) configured to facilitate illumination of the chamber by the optical waveguide.
Inventive concept 44. The apparatus according to inventive concept 43, wherein the optical waveguide comprises an optical fiber.
Inventive concept 45. The apparatus according to inventive concept 43, wherein the optical waveguide comprises a planar optical waveguide.
Inventive concept 46. The apparatus according to inventive concept 42, wherein the scaffold mechanically supports the membrane structure.
Inventive concept 47. The apparatus according to inventive concept 42, wherein the scaffold is optically transparent.
Inventive concept 48. The apparatus according to inventive concept 42, wherein the scaffold comprises a material selected from the group consisting of: a hydrogel, molded plastic and polystyrene.
Inventive concept 49. The apparatus according to inventive concept 42, wherein the nutrient supply compartment is optically transparent.
There is still further provided in accordance with an inventive concept 50, apparatus for facilitating cell growth, the apparatus configured to be implanted in a body of a subject and comprising:
an optically-transparent rigid scaffold;
an optical waveguide, coupled to the scaffold;
a plurality of cells disposed on the scaffold; and
a membrane structure at least partially surrounding the scaffold.
Inventive concept 51. The apparatus according to inventive concept 50, wherein the optical waveguide comprises an optical fiber.
Inventive concept 52. The apparatus according to inventive concept 50, wherein the optical waveguide comprises a planar optical waveguide.
Inventive concept 53. The apparatus according to inventive concept 50, wherein the scaffold is shaped to define one or more wells in which cell-growth medium is disposed, and on which the cells are disposed.
Inventive concept 54. The apparatus according to inventive concept 53, wherein a total surface area of the scaffold upon which the cells are disposed is at least 60% of a total surface area of the scaffold which is illuminated when light passes through the optical waveguide.
Inventive concept 55. The apparatus according to inventive concept 50, wherein the cells form a monolayer on the scaffold.
Inventive concept 56. The apparatus according to inventive concept 50, wherein a length of the scaffold is 2-4 mm.
Inventive concept 57. The apparatus according to inventive concept 50, wherein a volume of the scaffold is 0.5-2 microliter.
Inventive concept 58. The apparatus according to inventive concept 50, wherein a total surface area of the scaffold upon which the cells are disposed is 2.5-3.5 mm̂2.
Inventive concept 59. The apparatus according to inventive concept 50, wherein (a) within an exit cone of twenty-two degrees from a tip of the optical waveguide, and (b) within a distance from the tip, the distance being four times a diameter of the waveguide, (c) there is a scaffold surface area for cell growth that is at least four times the surface area of a distal tip of the waveguide.
Inventive concept 60. The apparatus according to any one of inventive concepts 50-59, wherein the scaffold has a plurality of surfaces perpendicular to the optical waveguide.
Inventive concept 61. The apparatus according to inventive concept 60, wherein the plurality of cells do not secrete a sensor protein.
Inventive concept 62. The apparatus according to inventive concept 60, wherein the plurality of cells secrete a sensor protein.
Inventive concept 63. The apparatus according to any one of inventive concepts 50-59, further comprising a mirror coupled to the scaffold and configured to reflect light, transmitted from the optical waveguide, back to the optical waveguide.
Inventive concept 64. The apparatus according to inventive concept 63, wherein the mirror is not flat.
Inventive concept 65. The apparatus according to inventive concept 64, wherein the non-flat mirror is concave.
There is additionally provided in accordance with an inventive concept 66, apparatus for detecting a concentration of an analyte in a subject, the apparatus configured to be implanted in a body of the subject and comprising:
an optical waveguide having a first end and a second end;
a sensing unit disposed at the first end of the optical waveguide and configured to detect the analyte, the sensing unit comprising:
There is yet additionally provided in accordance with an inventive concept 70, apparatus for detecting a concentration of an analyte in a subject, the apparatus configured to be implanted in a body of the subject and comprising:
an optical waveguide;
a chamber surrounding a distal portion of the optical waveguide, the distal portion of the optical waveguide extending along at least 75% of a length of the chamber; and
live cells that are genetically engineered to produce, in a body of the subject, a sensor protein having a binding site for the analyte, the live cells being disposed within the chamber.
Inventive concept 71. The apparatus according to inventive concept 70, wherein the analyte is glucose.
Inventive concept 72. The apparatus according to inventive concept 70, wherein the distal portion of the optical waveguide has a distal-portion diameter that is smaller than a proximal-portion diameter of a proximal portion of the optical waveguide.
Inventive concept 73. The apparatus according to inventive concept 72, wherein the proximal portion diameter is equal to a combined diameter of the chamber and the distal portion of the optical waveguide.
Inventive concept 74. The apparatus according to inventive concept 70, wherein the optical waveguide comprises an optical fiber.
Inventive concept 75. The apparatus according to inventive concept 70, wherein the optical waveguide comprises a planar optical waveguide.
There is further provided in accordance with an inventive concept 76, apparatus for detecting a concentration of an analyte in a subject, the apparatus configured to be implanted in a body of the subject and comprising:
an optical waveguide configured to transmit excitation light;
a chamber comprising live cells that are genetically engineered to produce, in a body of the subject, a fluorescent sensor protein having a binding site for the analyte, the fluorescent sensor protein being configured to transmit fluorescent light in response to the excitation light, the chamber being disposed coaxially with respect to the optical waveguide;
a lens disposed between the optical waveguide and the chamber, the lens being configured to focus light from the optical waveguide to the chamber and light from the chamber to the optical waveguide;
a first mirror, optically coupled to the chamber and disposed between a proximal end of the chamber and the lens, the first mirror configured to reflect the excitation light within the chamber and transmit the fluorescent light from within the chamber toward the lens and the optical waveguide, the first mirror being shaped to define a pinhole configured to allow passage of the excitation light from the lens into the chamber; and
a second mirror, optically coupled to the chamber and disposed at a distal end of the chamber.
Inventive concept 77. The apparatus according to inventive concept 76, wherein the analyte is glucose.
Inventive concept 78. The apparatus according to inventive concept 76, wherein the first mirror comprises a dichroic mirror.
Inventive concept 79. The apparatus according to inventive concept 76, wherein the optical waveguide comprises an optical fiber.
Inventive concept 80. The apparatus according to inventive concept 76, wherein the optical waveguide comprises a planar optical waveguide.
There is also provided in accordance with an inventive concept 81, a method, comprising:
facilitating measuring of a concentration of an analyte in a body of a subject, from a subcutaneous location of the subject;
measuring a temperature of the subcutaneous location in conjunction with the facilitating of the measuring of the concentration of the analyte; and calibrating the measurement of the concentration of the analyte in response to the measured temperature.
Inventive concept 82. The method according to inventive concept 81, wherein facilitating the measuring comprises subcutaneously implanting a device configured to measure the analyte, and wherein the method further comprises calibrating the device prior to the measuring of the concentration of the analyte.
Inventive concept 83. The apparatus according to inventive concept 81, wherein the analyte is glucose.
The present invention will be more fully understood from the following detailed description of some applications thereof, taken together with the drawings, in which:
A membrane structure 22 permeable to nutrients surrounds scaffold material 28 at least in part and is mechanically supported by the scaffold material. Membrane structure 22 may be a simple membrane (e.g., a homogeneous membrane), or a membrane having multiple components, such as a spatially non-homogeneous membrane structure (e.g., as described hereinbelow with reference to
For some applications, an optical system (e.g., optical system 59 as indicated in
In general, in applications described herein with reference to all of the figures, the cells secrete a sensor protein. Alternatively, the cells express but do not secrete a sensor protein.
In accordance with some applications of the present invention, the diameter of a part of the scaffold material 28 in which optical waveguide 48 is inserted is about 300-600 microns (e.g., 500 microns), and the waveguide itself typically has a diameter of 300-600 microns (e.g., 500 microns). The cells are typically disposed only on a portion 29 of scaffold material 28 that is shorter than the entire length of the scaffold material. Portion 29 is typically 1-10 mm (e.g., 2-4 mm) in length. Although four rings 33 of scaffold material 28 defining wells 30 are shown in
In accordance with some applications of the present invention, cells 26 are genetically engineered to produce, in situ, sensor protein (not shown) comprising a fluorescent protein donor (e.g., cyan fluorescent protein (CFP)), a fluorescent protein acceptor (e.g., yellow fluorescent protein (YFP)), and a binding site (e.g., glucose-galactose binding site) for an analyte. When the protein binds an analyte such as glucose, binding of the glucose causes a conformational change in the sensor protein and a corresponding changing in the distance between respective donors and acceptors. Fluorescence resonance energy transfer (FRET) involves the transfer of energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when the donor and acceptor molecules are in close proximity to each other. FRET enables the determination of the relative proximity of the molecules for investigating, for example, the binding of analyte, and thus the concentration of the analyte. All of the apparatus and methods described herein, with reference to each of the figures, may be combined with techniques described in the above-referenced PCT Patent Application Publication WO 2006/006166 to Gross et al. and U.S. Pat. No. 7,951,357 filed in the national stage thereof, and in US 2010/0202966 to Gross et al., which are incorporated herein by reference.
Typically, scaffold material 28 is optically transparent. Scaffold material 28 may comprise, for example, molded plastic or polystyrene. Excitation light generated by control unit 50 passes through optical waveguide 48, and enters each well 30 via transparent scaffold material 28. A signal of light of different wavelengths emitted by the sensor proteins is passed by the optical waveguide 48 to control unit 50. Control unit 50 interprets the different wavelengths in the received light signal as indicative of which portion of the sensor proteins have undergone the conformational change, and, therefore, of the concentration of the analyte (e.g., glucose). Typically, scaffold material 28 is rigid.
For some applications, the scaffold is fabricated using 3D printing, and may comprise a biocompatible material, such as MED610.
Reference is now made to
For example, the first MWCO may be less than 150 kilodaltons (e.g., less than 100 kilodaltons, e.g., less than 50 kilodaltons), while the second MWCO is typically greater than 150 kilodaltons. In a particular example, the first MWCO is less than 100 kilodaltons and the second MWCO is greater than two times the first MWCO.
Typically, membrane structure 22 in the first state is not permeable to IgG, while in the second state structure 22 is permeable to IgG. Alternatively or additionally, membrane structure 22 in the first state is not permeable to transferrin, while in the second state structure 22 is permeable to transferrin. In both states, membrane structure 22 is permeable to glucose, and not permeable to white blood cells.
The transition from the first state to the second state may be achieved (as described hereinabove with reference to
Reference is now made to
For some applications, a mirror 35 (e.g., a non-flat mirror, such as a concave mirror) is disposed at the distal end of apparatus 18, in order to reflect light back toward the sensor protein and toward optical waveguide 48. Use of such a mirror is shown in
At least one nutrient supply compartment comprising a nutrient permeable medium 42 that is arranged to not be conducive to cell growth therein is interspersed with scaffold 19, such that at least 80% of the cells within scaffold 19 are disposed within 100 um (e.g., within 50 um) of nutrient permeable medium 42. The nutrient permeable medium is positioned such that an easy diffusion path for nutrients is thus provided, by the nutrient permeable medium, between the subject's body and the at least 80% of the cells.
A volume of the nutrient supply compartment comprising nutrient permeable medium 42 is typically 25%-75% of a volume of chamber 155. Typically, nutrient permeable medium 42 comprises a hydrogel, but in general may comprise any material which suitably diffuses nutrients. The nutrient permeable medium may alternatively or additionally comprises one or more materials such as silicone rubber, fused glass powder, sintered glass powder, a hydrogel, and/or an alginate. This material may be shaped to define one or more spheres, e.g., at least 100 and/or less than 1000 spheres. The volume of chamber 155 is typically at least 20 times (e.g., at least 100 times, e.g., 200-1000 times) a volume of at least one of the spheres. For some applications, the spheres are disposed in the chamber in an efficient packing configuration.
It is noted that (as shown in
The glucose level and the time response to glucose changes that the sensor protein experiences while still inside the cells depends on the uptake dynamics of glucose into the cells. This dynamic adds complexity to the sensing mechanism. This complexity does not exist when the sensor protein is secreted from the cells and can react with the glucose as soon as the glucose enters the device. Accordingly, the two optical signals obtained by reading the fluorescence from cytosolic sensor protein and from free secreted sensor protein have different time responses and different calibration factors. For improved accuracy, some applications of the present invention reduce mixing of these two optical signals. Providing transparent sensor chamber 57 and non-optically-transparent cell chamber 55 enables the optical signal to be obtained primarily or exclusively from the free secreted sensor protein, rather than the cytosolic sensor protein. Alternatively or additionally, optical waveguide 48 is positioned so as to reduce the amount of light passing therethrough that includes fluorescence generated within cell chamber 55.
For some applications, cell chamber 55 and sensor protein chamber 57 are separated by internal membrane 60, which has a molecular weight cutoff sufficiently large to allow the sensor protein to freely diffuse between the two chambers while preventing cells from crossing between the chambers. Therefore, for typical applications in which the sensor protein has a size of 90 KDa and cells cannot pass through a membrane with pore size of 1 um or less, the inner membrane typically has a MWCO which is larger than 50 kDa (e.g., larger than 90 KDa) and a pore size smaller than about 1 um.
Alternatively, for other applications, internal membrane 60 is not provided, and separation between the cells of cell chamber 55 and the free sensor protein of sensor protein chamber 57 is provided by the two chambers comprising different materials. The cell chamber typically comprises a material that supports cell growth, and optionally also allows cell attachment. For example, the materials of the cell chamber may include sponge-like structures formed by dehydrated alginate; randomly-scattered fibers, e.g., created by electro-spinning, the fibers typically comprising plastic types which enable cell attachment and are optionally plasma-treated for enhanced surface charge; and/or solid structures comprising, for example, collagen, fibrinogen, or other proteins present in the external cellular matrix (ECM) of cells. The sensor protein chamber is filled with an optically-transparent material that does not allow cell proliferation but does allow free diffusion of the fluorescent biosensor, typically a hydrogel, e.g., alginate or Poly(ethylene glycol) (PEG). These separation techniques may be used instead of or in addition to internal membrane 60 in any of the configurations described hereinbelow with reference to
Typically, but not necessarily, sensing unit 62 further comprises an external membrane 58, which surrounds all or a part of the sensing unit, and thus provides an interface between the sensing unit and tissue 61 of the subject. External membrane 58 is configured to prevent the sensor protein from escaping the sensing unit (both from cell chamber 55 and sensor protein chamber 57), while maintaining ample diffusion of nutrients to the chambers. Typically, membranes effectively block the escape of molecules which are at least three times the rated MWCO. Thus for a sensor protein of a typical size of 90 KDa to be maintained long-term in the sensing unit, the MWCO of external membrane 58 should be no greater than 30 KDa. On the other hand, in order to maintain diffusion of nutrients into the sensing unit, the MWCO of the external membrane should be no less than a few KDa. Therefore, the typical MWCO of the outer membrane is in between 3 KDa and 30 KDa.
As appropriate for various applications of the present invention, membrane 58 may be a single membrane, surrounding both cell chamber 55 and sensor protein chamber 57. Alternatively, a membrane may surround cell chamber 55 while another membrane surrounds sensor protein chamber 57, each of these membranes separating the respective chambers 55 and 57 from tissue 61.
In the configurations described with reference to
Optical waveguide 48 is typically disposed parallel to a longitudinal axis of transparent inner axial portion 72 and outer cell chamber 76. Optical waveguide 48 for this application typically comprises an optical fiber. Inner axial portion 72 may also effectively be an optical fiber, however unlike many optical fibers, inner axial portion 72 for this application typically does not have a clad around a portion of the lateral surface thereof (as shown) from which it is desired that light escapes (and enters). Therefore, inner axial portion 72 releases light and receives light through its lateral surface, both to and from outer cell chamber 76. Alternatively or additionally, the lateral surface of inner axial portion 72 is roughened (or otherwise treated) in order to enhance the passage of light between inner axial portion 72 and outer cell chamber 76. Further alternatively or additionally, the refractive index of inner axial portion 72 is matched to that of chamber 76, in order to minimize reflections. For some applications (configuration not shown), inner axial portion 72 has a profile different than a simple cylinder, e.g. a cone, thereby increasing the angle between light rays and the normal to the surface.
Reference is made to
As shown in
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For some applications, protective layer 98 serves one or more of the following functions:
(a) to filter the molecules exchanged between the sensing unit and the tissue, allowing the passage of small molecules, e.g., glucose, but preventing larger molecules, e.g., molecules of the immune system (e.g. IgG); and/or
In order to achieve one or both of the above purposes, protective layer 98 may comprise, for example, a hydrogel (e.g., the hydrogel comprising either synthetic polymers (e.g. Poly(ethylene glycol) (PEG), Poly(ethylene oxide) (PEO), Poly(propylene oxide) (PPO), poly(hydroxyethyl methacrylate) (pHEMA)) or polymers based on proteins (e.g. fibrinogen, collagen), or a combination of both synthetic and protein-based polymers.
For some applications, protective layer 98 experiences minimal fouling while in the body, i.e., pores of layer 98 remain open and are not clogged with various molecules (e.g. proteins).
Reference is made to
For some applications, the second chamber is disposed around only a proximal end portion of the first chamber (as shown in
As noted, the second chamber may surround substantially the whole length of the first chamber (as shown in
For some applications, optical waveguide 48 has a diameter that is equal to a diameter of the first chamber (e.g., as shown in
Optional internal membrane 60 of sensing unit 62 is typically semi-permeable, configured to facilitate passage of the sensor protein from the chamber containing the cells (i.e., the first or the second chamber) to the other chamber (i.e., the second or the first chamber, respectively), while restricting passage of cells through membrane 60.
Typically, a first distal longitudinal segment 124 of external sensor protein chamber 122 surrounds (i.e., is disposed around) at least a portion of, e.g., at least at a proximal end portion of, such as all of, internal cell chamber 120, and a second proximal longitudinal segment 126 of external sensor protein chamber 122 does not surround (i.e., is not disposed around) internal cell chamber 120. Optical waveguide 48 is typically contiguous with second proximal longitudinal segment 126 of external sensor protein chamber 122. Typically, because internal cell chamber 120 occupies a portion of first distal longitudinal segment 124 of external sensor protein chamber 122, but not of second proximal longitudinal segment 126 of external sensor protein chamber 122, a cross-sectional area of external sensor protein chamber 122 is greater along second proximal longitudinal segment 126 than along first distal longitudinal segment 124. In addition, typically at least 60% of a volume of external sensor protein chamber 122 is disposed along second proximal longitudinal segment 126. As a result, the light transmitted by optical waveguide 48 interacts well with the sensor protein in external sensor protein chamber 122. For some applications, a diameter of optical waveguide 48 is equal to a diameter of sensing unit 62 and/or to a diameter of external sensor protein chamber 122.
Because first distal longitudinal segment 124 of external sensor protein chamber 122 surrounds at least a portion of, e.g., all of, internal cell chamber 120, a relatively large surface area is provided for allowing (a) transfer of analyte and nutrients between the subject's body and internal cell chamber 120, via external sensor protein chamber 122, and (b) transfer of the sensor protein from internal cell chamber 120 to external sensor protein chamber 122. In addition, because external sensor protein chamber 122 is typically disposed at the surface of sensing unit 62 along the entire sensing unit, a relatively large surface area is provided for allowing transfer of analyte and nutrients between the subject's body and external sensor protein chamber 122, via external membrane 58.
Typically, a first proximal longitudinal segment 224 of external cell chamber 220 surrounds (i.e., is disposed around) at least a portion of, e.g., at least at a proximal end portion of, such as all of, internal sensor protein chamber 222, and a second distal longitudinal segment 226 of external cell chamber 220 does not surround (i.e., is not disposed around) internal sensor protein chamber 222. Optical waveguide 48 is typically contiguous with first proximal longitudinal segment 224 of internal sensor protein chamber 222. Typically, because internal sensor protein chamber 222 occupies a portion of first proximal longitudinal segment 224 of external cell chamber 220, but not of second distal longitudinal segment 226 of external cell chamber 220, a cross-sectional area of external cell chamber 220 is greater along second distal longitudinal segment 226 than along first proximal longitudinal segment 224. In addition, typically at least 60% of a volume of external cell chamber 220 is disposed along second distal longitudinal segment 226. For some applications, a diameter of optical waveguide 48 is equal to a diameter of internal sensor protein chamber 222.
Because first proximal longitudinal segment 224 of external cell chamber 220 surrounds at least a portion of, e.g., all of, internal sensor protein chamber 222, a relatively large surface area is provided for allowing transfer of the sensor protein from external cell chamber 220 to internal sensor protein chamber 222. In addition, because external cell chamber 220 is typically disposed at the surface of sensing unit 62 along the entire sensing unit, a relatively large surface area is provided for allowing transfer of analyte and nutrients between the subject's body and external cell chamber 220, via external membrane 58.
Reference is made to both
Reference is still made to both
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In order to enable immediate testing and qualification of sensing unit 62, and make efficient use of device shelf life, there are benefits to manufacturing sensing unit 62 as close as reasonably possible to its typical operational state and to eliminate as much as possible any “maturation” gradients. In the case of a device such as sensing unit 62 that is based on secreted biosensor protein, the long-term steady state concentration of sensor protein in sensor protein chamber 57 is determined by the balance between (a) protein generation rate by the cells, and (b) protein loss because of catabolism of the proteins, caused by proteases and other factors and possibly protein leakage out of the device. This steady state may take a week or more to reach, thus preventing immediate testing of the device after manufacture and the need for a maturation period.
In some applications of the present invention, the device manufacturing process comprises the loading of purified biosensor protein, at the expected steady state concentration, into sensor protein chamber 57. The protein may be separately manufactured in the same cells, in other cells, or in bacteria and purified from the growth medium. An additional benefit of providing a steady-state level of sensor protein in the manufacturing process is that the number of cells required for the operation of the sensing unit is only the number necessary to support the steady state over the long term, thereby allowing a smaller number of cells and thus a smaller volume for cell chamber 55.
Reference made to
For any of the configurations of cell chamber 55 described hereinabove with reference to
High concentrations of the sensor protein may enhance the intensity of the optical signal. For some applications of the present invention, in order to achieve a high local concentration of sensor protein, the sensor protein is targeted to specific surfaces of the apparatus which enjoy a higher collection efficiency by the optics. Targeting may be achieved, for example, by creating a specific interaction between the protein and the surface, e.g., by the addition of a linker to the protein. The linker has enhanced binding to the specific surface either through a physical interaction (e.g., a hydrophobic or hydrophilic interaction) or through a specific biological interaction (e.g., a biotin-avidin interaction).
Reference is made to
Reference is now made to
As can be seen in the graphs of
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Scaffold 310, cells 314, and hydrogel 316 are arranged such that cells 314 are sandwiched in spaces between hydrogel 316 and surfaces 312 of scaffold 310. Cells 314 are arranged in monolayers on at least 50%, such as at least 70%, e.g., at least 90% (for example, 100%) of an aggregate surface area of surfaces 312 of scaffold 310 (the “aggregate surface area” is the sum of the surface areas of all of the surfaces of the scaffold). This arrangement allows mobility and proliferation of cells 314 in the spaces between hydrogel 316 and surfaces 312 of scaffold 310, and prevents the mobility and the proliferation of cells 314 to locations outside of the spaces between hydrogel 316 and surfaces 312 of scaffold 310. Typically, cells 314 occupy at least 75% of the aggregate volume of the spaces between hydrogel 316 and surfaces 312 of scaffold 310. Cells within the spaces between hydrogel 316 and surfaces 312 of scaffold 310 that die, such as because of stress or apoptosis, leave a space upon disintegration. The structure provided by the surface of the scaffold on one side and the hydrogel on the other side maintain the patency of this space until one or more neighboring cells proliferate into the space.
Thus, in any local microscopic environment encapsulation structure 300 comprises a three-layer stack of (a) surface 312 of solid scaffold 310, (b) cells 314, and (c) hydrogel 316, in this order. The cells at any location are thus generally limited to a monolayer, allowing free mobility and proliferation of the cells within the narrow space between the scaffold and the hydrogel, but preventing any proliferation into the rest of the volume and creation of three-dimensional cell structures.
Scaffold 310 provides a three-dimensional structure with a high aggregate surface area, and high surface-to-volume ratio, which makes efficient use of the three-dimensional volume of the chamber. The surfaces of the scaffold, although often not flat, serve effectively as a two-dimensional substrate for seeding, growth, and attachment of the cells. If hydrogel 316 were not provided over the monolayer of the cells, the cells typically grow in three dimensions, away from the surfaces to which they are attached. Such three-dimensional growth would generally result in undesirable over-population, as described above. In addition, for many cell types, cell viability and protein expression, including expression of the sensor protein, are significantly enhanced when cells are attached and spread. Thus cells in this configuration will survive longer and function better than dispersed cell, e.g., cells dispersed in a hydrogel scaffold.
For some applications, encapsulation structure 300 further comprises a chamber, such as sensor protein chamber 57 in any of the configurations described hereinabove with reference to
For some applications, scaffold 310 comprises:
In accordance with an application of the present invention, encapsulation structure 300 is manufactured by the following process:
Ideally, hydrogel 316 penetrates all spaces in encapsulation structure 300 that are not occupied by scaffold 310 or cells 314. Therefore, the minimum feature size of surfaces 312 is typically at least a few tens of micrometers.
Encapsulation structure 300 may combine at least three benefits: (a) good cell attachment, leading to better cell viability and expression, lacking in simpler systems that for example use hydrogel as a scaffold, (b) prevention of over-population which often leads to a necrotic core, because of a limited number of cells and open diffusion channels to the cells via the hydrogel, and (c) enablement of cell mobility and proliferation within a two-dimensional culture, thereby enabling long-term steady state population.
For some applications, cells 314 are differentiated cells, such as terminally-differentiated cells. For other applications, cells 314 are stem cells. For some applications, cells 314 are genetically engineered to produce a fluorescent sensor protein having a binding site for an analyte, such as glucose, the fluorescent sensor protein being configured to emit fluorescent light in response to excitation light, such as using the techniques described hereinabove.
Reference is now made to
The resulting dynamics of cell populations are shown in
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Multi-layer immunoisolation system 400 is configured to enhance long-term function of an implantable cell-based device 410. Multi-layer immunoisolation system 400 is disposed at an external surface of device 410. For example, multi-layer immunoisolation system 400 may be integrated into any of the sensing devices described herein instead of, or as an implementation of, external membrane 58.
Multi-layer immunoisolation system 400 comprises at least the following three layers:
Multi-layer immunoisolation system 400 comprises a biodegradable scaffold 418. Before biodegrading, biodegradable scaffold 418 spans both upper neovascularization layer 414 and middle protective layer 416, such that upper neovascularization layer 414 comprises a first upper portion of biodegradable scaffold 418, and middle protective layer 416 comprises a second lower portion of biodegradable scaffold 418.
In addition, middle protective layer 416 further comprises a non-biodegradable hydrogel that impregnates the second lower portion of biodegradable scaffold 418. Upper neovascularization layer 414, which comprises the first upper portion of biodegradable scaffold 418, is not impregnated with the hydrogel.
Biodegradable scaffold 418 serves at least two functions:
Upon biodegradation of biodegradable scaffold 418, middle protective layer 416 (now comprising primarily the hydrogel) remains attached to lower membrane layer 412. Middle protective layer 416 typically serves to (a) prevent attachment of proteins to lower membrane layer 412, thereby minimizing the creation of a fibrotic tissue, and/or (b) repel large proteins, thereby minimizing the fouling of lower membrane layer 412. The high water content of the hydrogel of middle protective layer 416 prevents the attachment of various proteins, so that immune system cells are less likely to attach to the tissue-hydrogel interface, thereby minimizing the overall immune response. Typically, the hydrogel of middle protective layer 416 has a thickness of at least 50 um, e.g., at least 100 um, such as in order to enable reactive oxygen species (ROS) decay between inflamed tissue and the device cells. Without the use of the techniques described herein, it is generally difficult to attach a hydrogel to a membrane, particularly with a thickness of more than a few um.
As a result of this triple-layer protection, the tissue surrounding device 410 is characterized by high vascularization and minimal fibrosis.
Typically, lower membrane layer 412 (and lower membrane 512, described hereinbelow with reference to
Typically, biodegradable scaffold 418 is highly porous, and has an average pore size of at least 5 um, no more than 50 um, and/or or between 5 and 50 um. For some applications, the scaffold comprises a mesh. Biodegradable scaffold 418 may comprise a polymer, such as polylactic acid (PLA), poly(DL-lactic-co-glycolic acid) (PLGA), poly(3-hydroxypropionic acid) (P(3-HP)), or 3-hydroxypropionic acid (3-HP). Biodegradable polymers and the products of their degradation are typically non-toxic, so as to not evoke a strong immune response. Additionally, biodegradable polymers typically maintain good mechanical integrity until degraded in order to evoke enhanced vascularization in its vicinity. Finally, biodegradable polymers typically have controlled degradation rates leading to complete disintegration in the body within a few weeks to a few months, which is enough time to evoke vascularization but not become a potential annoyance for the patient a long time after the device is explanted.
Biodegradable scaffold 418 (of upper neovascularization layer 414 and the middle protective layer 416 in combination) typically has a thickness of between 100 and 300 um and promotes neovascularization by virtue of the large pore size and the slow biodegradation effect. As mentioned above, the scaffold additionally holds the hydrogel layer in place. For some applications, biodegradable scaffold 418 is fixed to the upper (outer) surface of membrane layer 412 by gluing. Alternatively or additionally, for some applications, biodegradable scaffold 418 is fixed to the upper (outer) surface of membrane layer 412 by being directly deposited using electrospinning, i.e., the scaffold is electrospun onto the membrane.
The hydrogel (and hydrogel 520, described hereinbelow with reference to
Reference is now made to
Multi-layer immunoisolation system 500 is configured to enhance long-term function of an implanted cell-based device 510. Multi-layer immunoisolation system 500 is disposed at an external surface of device 510. For example, multi-layer immunoisolation system 500 may be integrated into any of the sensing devices described herein instead of, or as an implementation of, external membrane 58.
Multi-layer immunoisolation system 500 comprises at least the following three layers:
Middle attachment layer 516 comprises a non-biodegradable scaffold, which is tightly fixed to lower membrane layer 512, such as by being deposited directly on the membrane using electrospinning, i.e., the scaffold is electrospun onto the membrane. Typically, the scaffold is highly porous, and may comprise, for example, a polymer such as polyurethane, polyvinylidene fluoride (PVDF), or polyethylene terephthalate (PET). Middle attachment layer 516 typically has a thickness of between 50 and 100 um.
Multi-layer immunoisolation system 500 comprises a non-biodegradable hydrogel 520, which spans both upper protective layer 514 and middle attachment layer 516. In other words, middle attachment layer 516 comprises a first portion of hydrogel 520, and upper protective layer 514 comprise a second portion of hydrogel 520. Hydrogel 520 is impregnated in the scaffold of middle attachment layer 516, and extends above the scaffold, i.e., in a direction away from lower membrane 516, so as to provide upper protective layer 514. Upper protective layer 514 does not comprise the scaffold. As a result, the scaffold is not exposed to tissue, thereby reducing the likelihood that multi-layer immunoisolation system 500 generates an immune response.
Middle attachment layer 516 holds the hydrogel of upper protective layer 514 in place on lower membrane layer 512. Upper protective layer 514 has a smooth upper (outer) surface, which results in low biofouling of lower membrane layer 512, allowing the membrane to efficiently diffuse nutrients into device 510 even after a long implantation period. In addition, upper protective layer 514 protects device 510 by presenting a highly biocompatible surface to the tissue. Upper protective layer 514 typically has a thickness of between 50 and 200 um.
It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove. Rather, the scope of the present invention includes both combinations and subcombinations of the various features described hereinabove, as well as variations and modifications thereof that are not in the prior art, which would occur to persons skilled in the art upon reading the foregoing description.
The present application claims priority from U.S. Provisional Application 61/746,691, filed Dec. 28, 2012, which is assigned to the assignee of the present application and is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2013/061368 | 12/27/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61746691 | Dec 2012 | US |
