WO2010068728A2

WO2010068728A2 - Engineering functional tissue from cultured cells

Info

Publication number: WO2010068728A2
Application number: PCT/US2009/067453
Authority: WO
Inventors: Charles A. Vacanti
Original assignee: The Brigham And Women's Hospital, Inc.
Priority date: 2008-12-11
Filing date: 2009-12-10
Publication date: 2010-06-17
Also published as: WO2010068728A3

Abstract

Described herein is a method for islet cell engineering or regeneration in a subject, wherein a combination of pancreatic islet cells are cultured on a scaffold to form a cell aggregate, and subsequently suspended in a hydrogel and cultured under growth conditions sufficient to form a pancreatic islet. After sufficient growth and/or differentiation of the cells, an engineered islet is isolated from the scaffold and the hydrogel. Engineered islets produced in this manner are functional and can be used as in organ replacement, or augmentation of function. For example, described herein are methods and compositions suitable for producing a functional pancreatic islet for implantation into a subject having Type 1 diabetes.

Description

ENGINEERING FUNCTIONAL TISSUE FROM CULTURED CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U. S. C. § 119(e) of U.S. Provisional Application No. 61/121,835 filed on December 11, 2008, the contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for pancreatic islet cell engineering.

BACKGROUND

[0003] Currently, there are 18.2 million people in the United States who suffer from diabetes. Since type 1 diabetes occurs as a result of a lack of insulin secretion, pancreatic islet transplantation is a logical therapeutic option for these patients. Unfortunately, islet transplantation is only partly successful, due in part to the fact that Type 1 diabetes is an autoimmune disease, often resulting in widespread pancreatic cell death. Pancreatic islets (comprising insulin-secreting beta cells) are destroyed by lymphocyte infiltration and the recognition of antigens on the pancreatic cell, which drives the innate immune system to target and attack the beta cells, resulting in a loss of both beta cells and somatic insulin response.

[0004] Traditionally, the problem of allo rejection (i.e., rejection of a donor's islets by a recipient's body) following pancreatic islet transplantation, is overcome with immunosuppressive agents. However, when islets are transplanted into type 1 patients, both allo and autoimmune graft rejection are encountered. Although graft survival was dramatically improved with the publication of the Edmonton protocol (Shapiro, A. M., et al., N Engl J Med (2000) 343, 230-238), the existence of these immune barriers is one of the reasons why islet transplantation is so difficult in comparison to other organs. In addition, another obstacle to pancreatic islet transplantation is a lack of donors.

[0005] Thus, some trials (Knight, K. R.,et al., Faseb / (2006) 20, 565-567; Brown, D. L., Cell Transplant (2006)15, 319-324; Lechner, A., et al., Biochem Biophys Res Commun (2005) 327, 581-588) have reported attempts to establish tissue-engineered islets (TE islets). Although these reports show evidence of engineering islets in vivo using isolated islet cells, the functional reconstruction of islets from dissociated islets cells has not been demonstrated (Knight, K. R.,et al., Faseb / (2006) 20, 565-567; Brown, D. L., Cell Transplant (2006)15, 319-324). Lechner, et al. succeeded in reconstructing islets from dissociated islets, but were unable to confirm in vivo function after transplantation into diabetic animals (Lechner, A., et al., Biochem Biophys Res Commun (2005) 327, 581-588).

[0006] Some biomaterials have been explored for tissue-engineering islets, including naturally degrading polymers such as polyglycolic acid (PGA) and polylactide-co-glycolide acid (PLGA) that form a scaffolding necessary for engineering islets. However, tissue engineered islets were not established using the scaffolding materials alone (Pollok, J. M., et al., Dig Surg (2001) 18, 204-210; Linn, T., et al., Cell Transplant (2003) 12, 769-778), but rather hydrogels were also utilized in an attempt to keep the cells in a three-dimensional culture. In a three-dimensional cell culture system, the target cells have maximum surface area exposure that permits the cells to form spheroids while still allowing them to receive nutrition from contact with the medium. Despite all of the effort to generate TE islets using hydrogels, formation of an islet with normal insulin secreting function in vivo has not been confirmed (Lechner, A., et al., Biochem Biophys Res Commun (2005) 327, 581-588; Falorni, A., et al., Pancreas (1996) 12, 221-229; Tatarkiewicz, K., et al., Transplantation (2001) 71, 1518-1526).

SUMMARY OF THE INVENTION

[0007] Described herein are methods and compositions for engineering islets ex vivo by growing cells on a polymeric scaffold suspended in a hydrogel. Upon completion of cell culture based islet engineering, the tissues are isolated from the scaffold and hydrogel prior to implantation, reducing potential inflammatory reactions caused by the polymeric components following transplantation.

[0008] One aspect described herein relates to a method for producing functional pancreatic islets ex vivo, the method comprising (a) culturing cells of a dissociated islet on a scaffold to form a spheroid aggregate of the cells; (b) immersing the spheroid aggregate on the scaffold in a hydrogel under growth conditions sufficient to form an engineered islet; (c) isolating the engineered islet from the scaffold and the hydrogel, wherein the engineered islet is functional. [0009] In one embodiment of this aspect and all other aspect described herein, the method further comprises an expansion step of the cells prior to culturing the cells on a scaffold. [0010] In another embodiment of this aspect and all other aspects described herein, the scaffold is biodegradable. [0011] In another embodiment of this aspect and all other aspects described herein, the scaffold biodegrades.

[0012] In another embodiment of this aspect and all other aspects described herein, the scaffold comprises polyglycolic acid, or poly(lactic-co-glycolic acid).

[0013] In another embodiment of this aspect and all other aspects described herein, the hydrogel is thermoreversible.

[0014] In another embodiment of this aspect and all other aspects described herein, the isolating step comprises cooling the hydrogel and removing the engineered islet from the cooled hydrogel.

[0015] In another embodiment of this aspect and all other aspects described herein, the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.

[0016] In another embodiment of this aspect and all other aspects described herein, the engineered islet secretes at least one of insulin, glucagon, or somatostatin.

[0017] In another embodiment of this aspect and all other aspects described herein, the method further comprises over-expressing Serpine 1 in the cells.

[0018] Another aspect described herein relates to a method for treating a subject having Type 1

Diabetes, the method comprising (a) culturing a cell of a dissociated islet on a scaffold to form a spheroid aggregate, (b) suspending the spheroid aggregate on the scaffold in a hydrogel under growth conditions sufficient to form an engineered islet; (c) isolating the engineered islet from the scaffold and the hydrogel, and (d) implanting the engineered islet into a subject having Type

1 Diabetes, wherein the engineered islet is functional and comprises a treatment for a subject having Type 1 Diabetes.

[0019] Another aspect described herein relates to a composition comprising a spheroid aggregate of cells on a scaffold immersed in a hydrogel.

[0020] In one embodiment of this aspect and all other aspects described herein, the cells comprise cells of a dissociated islet.

[0021] In another embodiment of this aspect and all other aspects described herein, the cells comprise stem cells.

[0022] In another embodiment of this aspect and all other aspects described herein, the scaffold is biodegradable.

[0023] In another embodiment of this aspect and all other aspects described herein, the scaffold comprises polyglycolic acid, or poly(lactic-co-glycolic acid).

[0024] In another embodiment of this aspect and all other aspects described herein, the hydrogel is thermoreversible. [0025] In another embodiment of this aspect and all other aspects described herein, the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.

Definitions

[0026] As used herein, the terms "isolated" and "isolating" when used in reference to e.g., a donor islet are used to describe the process of segregating a selected cell type or cell group (e.g., an islet) from a biological sample from a mammal. The term requires that an islet is removed from its native environment and preferably the cells are enriched in concentration relative to its native environment. It is specifically contemplated herein that a homogeneous population of cells or a heterogeneous (e.g., a plurality of cell types) population of cells can be used for the practice of the methods and compositions described herein. [0027] As used herein, the term "functional islet" refers to a cluster or aggregate of cells that together mimic the secretory action of a normally functioning (i.e., non-diabetic) endogenous pancreatic islet, in that a "functional islet" can secrete insulin, somatostatin and/or glucagon in response to one or more extracellular stimuli. For example, a functional islet secretes insulin in response to high concentrations of glucose. A functional islet can be distinguished from a non-insulin secreting islet clinically by using the dye dithizone or by measuring increased expression of the marker Serpine-1. In addition, it is contemplated herein that forced over- expression of Serpine-1 in cells of a dissociated islet will induce formation of a functional islet.

[0028] As used herein, the phrase "cells of a dissociated islet" refers to cell types that are typically present in an endogenous islet and can include e.g., beta cells, alpha cells, delta cells, epsilon cells, PP cells, gamma cells, or any combination thereof. Typically, an endogenous pancreatic islet comprises 15-20% alpha cells, 65-80% beta cells, 3-10% delta cells, 3-5% PP cells, and <1% epsilon cells, thus the ratio of the "cells of a dissociated islet" will be similar to that of an endogenous pancreatic islet. In some cases one or more cell types may be excluded from the culture of "cells of a dissociated islet", however it is preferred that the culture includes at least alpha cells, beta cells and delta cells. One of skill in the art can determine the appropriate ratio of each cell type relative to another in order to engineer a functional islet. As used herein the term "dissociated islet" refers to an Islet of Langerhans isolated from a donor pancreas.

[0029] As used herein, the term "spheroid aggregate" refers to a rounded cluster of "cells of a dissociated islet" as that term is used herein, that spontaneously arises when the cells are seeded on a scaffolding material. A "spheroid aggregate" comprises at least 50 cells, at least 100 cells, at least 200 cells, at least 500 cells, at least 1000 cells, at least 10,000 cells or more. [0030] The phrase "growth conditions sufficient to form" refers to the presence of cell- specific media, growth factors, temperature, pH, etc. necessary for the optimal growth of that cell-type or tissue-type in culture (i.e., pancreatic islet cells). It is to be expected that different combinations of cells will each have unique growth factor requirements, metabolic substrates and supplemental media beyond that of a basic cell culture growth media such as e.g., DMEM. It is well within the abilities of one skilled in the art to determine the specific cell culture needs of a certain cell combination from data reported in the scientific literature in order to achieve "growth conditions suitable for" e.g., forming an engineered islet. For example, media conditioned by insulin secreting cells such as INS-I cells or primary beta cells can be used as an enhanced medium for growth of an engineered islet. Cell-specific growth media and/or media supplements can also be purchased from a variety of commercial sources, such as INVITROGEN™, among others.

[0031] The term "biodegradable", as used herein refers to materials which are enzymatically or chemically degraded into simpler chemical species at least in a cell culture setting. It is also contemplated that a material is "biodegradable" if it is degraded into simpler chemical species in vivo.

[0032] The term "hydrogel" refers to a broad class of polymeric materials which are swollen extensively in water but which do not dissolve in water. Generally, hydrogels are formed by polymerizing a hydrophilic monomer in an aqueous solution under conditions where the polymer becomes cross-linked so that a three-dimensional polymer network sufficient to gel the solution is formed. Hydrogels are described in more detail in Hoffman, A. S., "Polymers in Medicine and Surgery," Plenum Press, New York, pp 33-44 (1974). [0033] As used herein, the term "thermoreversible" means that a hydrogel can be converted from a gel to a liquid by altering the temperature. Preferably, "thermoreversible" refers to a gel that forms a liquid by cooling the hydrogel.

[0034] As used herein, an engineered tissue is considered "substantially free" of a polymer or hydrogel when little to no immune response or inflammatory reaction is detected in a subject following implantation with the engineered tissue. In addition, an engineered tissue is considered "substantially free" of a polymer or hydrogel when there is less than 20% of the original scaffold/hydrogel present following isolation of the engineered tissue; preferably less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or even absent (or below detectable limits). [0035] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0036] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. [0037] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. [0038] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. (A). Native rat islets are shown. (B). The islets were dissociated to single β cells one day after isolation of islets. (C). Islet cells were seeded on PGA fiber on the same day of dissociation. The upper two panels show dissociated cells before aggregation seeded on PGF. Thirty- six hours later, islet cells started to form aggregates in the lower two panels. (D). After five days in culture, the aggregated cells formed spheroids and proliferated to large sizes under magnification. (E). The reconstructed islets after being cultured in TPG (Mebiol gel) for 40 days. The massive proliferated TE islet cells were confirmed in gel at different magnifications. (F). After TPG was released, individual TE islets were collected manually. The average size of the TE islet was somewhat larger than native islets.

Figure 2. (A). Blood glucose levels before and after transplantation of TE islets (n=6). All recipients showed blood glucose elevation after the injection of streptozotocin (STZ). The blood glucose levels reversed to normoglycemia after transplantation of TE islets. This was maintained for 120 days. (B). Nude mice treated with STZ alone failed to demonstrate normogycemia (right). In addition, xenografts using native islet cells recovered normoglycemia to the same degree as animals treated with TE islets (left, n=5). Figure 3. An oral glucose tolerance test (OGTT) was performed in nude mice. (A). The upper panel indicates post-transplantation of TE islet grafts in the left. The peak of serum glucose levels was shown within 30 min. Serum glucose levels returned to a normal level during 60 to 120 min. In contrast, hyperglycemic blood glucose levels were displayed after the TE grafts were removed (right). In the lower panel, control xenografts treated with normal xenograft islets (left) and control untreated diabetic nude mice induced by STZ are shown (right). The control xenograft showed the same pattern as TE islet grafts (left). Furthermore, hyperglycemia continued after 120 min in the diabetic model (right). (B). Insulin ELISA was displayed and insulin secretions were inversely related to the glucose level. TE islets, xenograft controls, and diabetic controls are shown left, middle, and right. (C). C-peptide ELISA showed the selective secretion of C-peptide in rats. TE islets generated from rats and xenograft islets were both positively shown in the left and middle.

Figure 4. (A). Control and TE islet grafts stained using dithizone (DTZ) before and after are shown in the right or left panels, respectively. (B). HE staining showed the morphology under the kidney capsule where these were the graft sites of the islets. (C). Insulin and glucagon were stained in control and TE islet grafts. (D). Insulin and somatostatin were stained in control and TE islet grafts. High magnification is displayed in the right lower corner.

DETAILED DESCRIPTION

[0039] Described herein are methods and compositions useful for engineering a pancreatic islet ex vivo by culturing cells on a scaffold suspended in a hydrogel. These engineered islets can be isolated from the scaffold and hydrogel of the culture conditions prior to implantation into a subject. A variety of diseases requiring islet regeneration, such as e.g., Type 1 diabetes, can be treated by transplanting engineered tissues made by the methods described herein.

Cells

[0040] Essentially any pancreatic islet cell or cell combination can be used with the methods and compositions described herein. However, it is preferred that the cell is a human cell. The cell can be a primary cell e.g., a primary beta cell, primary alpha cell, or it may be a cell of an established cell line (e.g., INS-I cell). Where the cell is maintained under in vitro conditions, conventional tissue culture conditions and methods can be used, and are known to those of skill in the art. Methods for cell isolation are well known to those of skill in the art, and generally involve an enzymatic reaction (e.g., collagenase to dissociate cells from a desired tissue or biological sample (e.g., pancreatic tissue)), centrifugation, and/or plating of cells in tissue culture dishes. Methods suitable for isolating cells for the methods and compositions described herein can be found in, for example US Patent Nos. 6,475,764; 5,424,208; 7,217,568; 6,991,897; or 6,627,759, which are incorporated herein in their entirety. [0041] Cells can be obtained directly from a donor, from cell culture of cells from a donor, or from established cell culture lines. Cells are cultured using techniques known to those skilled in the art of tissue culture. Cells can either be autologous, or heterologous to the subject to which they are transplanted.

[0042] In one embodiment, the cells are comprised of "cells of a dissociated islet", including e.g., alpha cells, beta cells, delta cells, PP cells, and epsilon cells, or a combination or combinations, thereof.

[0043] Cell viability can be assessed using standard techniques including visual observation with a light or scanning electron microscope, histology, dye exclusion, or quantitative assessment with radioisotopes. The biological function of the cells delivered to the support structure can be determined using a combination of the above techniques and standard functional assays.

Scaffolds

[0044] Biocompatible synthetic, natural, as well as semi- synthetic polymers, can be used for synthesizing polymeric particles that can be used as a scaffold material. In general, for the practice of the methods described herein, it is preferable that a scaffold biodegrades such that an engineered islet can be isolated from the polymer prior to implantation. Thus, the scaffold provides a temporary structure for the spheroid aggregate until complete formation of a functional islet is achieved. Isolation of an engineered islet permits residual polymer to be removed from the islet before engraftment and reduces the risk of an inflammatory response or allo rejection caused by the polymer once the islet is transplanted into a recipient. [0045] Examples of polymers which can be used include natural and synthetic polymers, although synthetic polymers are preferred for reproducibility and controlled release kinetics. Synthetic polymers that can be used include biodegradable polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other polyhydroxyacids, poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyphosphazene, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and biodegradable polyurethanes; non-biodegradable polymers such as polyacrylates, ethylene- vinyl acetate polymers and other acyl-substituted cellulose acetates and derivatives thereof; polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefins, and polyethylene oxide. Examples of biodegradable natural polymers include proteins such as albumin, collagen, fibrin, synthetic polyamino acids and prolamines; polysaccharides such as alginate, heparin; and other naturally occurring biodegradable polymers of sugar units. Alternately, combinations of the aforementioned polymers can be used.

[0046] PLA, PGA and PLA/PGA copolymers are particularly useful for forming the biodegradable scaffolds. PLA polymers are usually prepared from the cyclic esters of lactic acids. Both L(+) and D(-) forms of lactic acid can be used to prepare the PLA polymers, as well as the optically inactive DL-lactic acid mixture of D(-) and L(+) lactic acids. Methods of preparing polylactides are well documented in the patent literature. The following U.S. Patents, the teachings of which are hereby incorporated by reference, describe in detail suitable polylactides, their properties and their preparation: U.S. Pat. No. 1,995,970 to Dorough; U.S. Pat. No. 2,703,316 to Schneider; U.S. Pat. No. 2,758,987 to Salzberg; U.S. Pat. No. 2,951,828 to Zeile; U.S. Pat. No. 2,676,945 to Higgins; and U.S. Pat. Nos. 2,683,136; 3,531,561 to Trehu.

[0047] PGA is the homopolymer of glycolic acid (hydroxyacetic acid). In the conversion of glycolic acid to poly(glycolic acid), glycolic acid is initially reacted with itself to form the cyclic ester glycolide, which in the presence of heat and a catalyst is converted to a high molecular weight linear-chain polymer. PGA polymers and their properties are described in more detail in Cyanamid Research Develops World's First Synthetic Absorbable Suture", Chemistry and Industry, 905 (1970).

[0048] Fibers for a scaffold can be formed by melt- spinning, extrusion, casting, or other techniques well known in the polymer processing area. Preferred solvents, if used, are those which are completely removed by the processing or which are biocompatible in the amounts remaining after processing.

[0049] All polymers for use in the matrix must meet the mechanical and biochemical parameters necessary to provide adequate support for the cells with subsequent growth and proliferation. The polymers can be characterized with respect to mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy. [0050] Scaffolds can be of any desired shape and can comprise a wide range of geometries that are useful for the methods described herein. A non limiting list of shapes includes, for example hollow particles, tubes, sheets, cylinders, spheres, and fibers, among others. The shape of the scaffold should not impede cell growth, cell differentiation, cell proliferation or any other cellular process, nor should the scaffold induce cell death via e.g., apoptosis or necrosis. In addition, care should be taken to ensure that the scaffold shape permits appropriate surface area for delivery of nutrients from the culture media to the entire spheroid aggregate of cells, such that the spheroid aggregate develops into a functional islet. The scaffold porosity can also be varied as desired by one of skill in the art. [0051] In some embodiments, attachment of the cells to the polymer is enhanced by coating the polymers with compounds such as basement membrane components, agar, agarose, gelatin, gum arabic, collagens types I, II, III, IV, and V, fibronectin, laminin, glycosaminoglycans, polyvinyl alcohol, mixtures thereof, and other hydrophilic and peptide attachment materials known to those skilled in the art of cell culture. A preferred material for coating a polymeric scaffold is polyvinyl alcohol or collagen.

[0052] In some embodiments it may be desirable to add bioactive molecules to the scaffold. A variety of bioactive molecules can be delivered using the matrices described herein. These are referred to generically herein as "factors" or "bioactive factors". [0053] In a preferred embodiment, the bioactive factors are growth factors. Examples of growth factors include heparin binding growth factor (hbgf), transforming growth factor alpha or beta (TGFβ), alpha fibroblastic growth factor (FGF), epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), among others.

[0054] These factors are known to those skilled in the art and are available commercially or described in the literature.

Hydrogels

[0055] Any hydrogel can be used in the methods described herein as long as it is biocompatible with the cells necessary to engineer the desired tissue. It is preferred that the hydrogel used permits isolation of an engineered islet from the hydrogel. This is easily achieved if the hydrogel can be reversed in state from a gel to a liquid. For example, a preferred hydrogel for use in the practice of the methods described herein is Mebiol, a thermoreversible hydrogel that exists as a gel at high temperatures (>25°C) and a liquid at low temperatures (0-15°C). By simply cooling the gel comprising e.g., an engineered islet, the gel becomes a liquid and the engineered tissue, (e.g., the engineered islets) are easily removed from the hydrogel. The islets can be further washed to remove traces of the gel and then implanted in the absence of either hydrogel or scaffold material.

[0056] Hydrogels can be induced to favor the liquid state over a gel state by altering physical factors such as e.g., temperature, or pH, among others. It is important to note that a hydrogel should be chosen such that the manipulation necessary to induce the hydrogel to favor the liquid state will not affect the viability or function of the cells grown in the hydrogel (i.e., extreme temperature or pH shifts should be avoided).

[0057] Temperature-dependent, or thermosensitive hydrogels can be used in the methods described herein. These hydrogels have so-called "reverse gelation" properties, i.e., they are liquids at or below room temperature, and exist in the form of a gel when warmed to higher temperatures, e.g., body temperature. Thus, these hydrogels can be easily applied at or below room temperature as a liquid and automatically form a semi- solid gel when warmed to a temperature suitable for cell culture. Additional example of such temperature-dependent hydrogels include PLURONICS™ (BASF-Wyandotte), such as polyoxyethylene- polyoxypropylene F-108, F-68, and F-127, poly (N-isopropylacrylamide), and N- isopropylacrylamide copolymers.

[0058] These copolymers can be manipulated by standard techniques to affect their physical properties such as porosity, rate of degradation, transition temperature, and degree of rigidity. For example, the addition of low molecular weight saccharides in the presence and absence of salts affects the lower critical solution temperature (LCST) of typical thermosensitive polymers. In addition, when these gels are prepared at concentrations ranging between 5 and 25% (W/V) by dispersion at 4°C, the viscosity and the gel-sol transition temperature are affected, the gel-solid transition temperature being inversely related to the concentration. These gels have diffusion characteristics capable of allowing cells to survive and be nourished.

[0059] U.S. Pat. No. 4,188,373 describes using PLURONIC™ polyols in aqueous compositions to provide thermal gelling aqueous systems. U.S. Pat. Nos. 4,474,751, '752, 753, and 4,478,822 describe drug delivery systems which utilize thermosetting polyoxyalkylene gels; with these systems, both the gel transition temperature and/or the rigidity of the gel can be modified by adjustment of the pH and/or the ionic strength, as well as by the concentration of the polymer. In one example, a 23% PLURONIC gel can be prepared that is a viscous liquid at below 15°C and above 50° C, and is a hydrogel at temperatures between 15 and 50°C.

[0060] Other hydrogels suitable for use in the methods of the invention are pH-dependent. These hydrogels are liquids at, below, or above specific pH values, and gel when exposed to specific pHs, e.g., 7.35 to 7.45, the normal pH range of extracellular fluids within the human body. Thus, these hydrogels can be maintained as a hydrogel in cell culture conditions and manipulated to permit removal of an engineered tissue, without subjecting the engineered tissue to an extreme shift in pH. Examples of such pH-dependent hydrogels are TETRONICS™ (BASF-Wyandotte) polyoxyethylene-polyoxypropylene polymers of ethylene diamine, poly(diethyl aminoethyl methacrylate-g-ethylene glycol), and poly(2- hydroxymethyl methacrylate). These copolymers can be manipulated by standard techniques to affect their physical properties.

Transplantation

[0061] Methods for transplanting an engineered tissue into a recipient are known to those of skill in the art. In one embodiment of the methods described herein, the engineered islet cells are transplanted beneath the kidney capsule.

[0062] It is likely that islets engineered from a donor or from a cell line may still be subject to allograft rejection and may require the use of immunosuppressive agents such as e.g., glucocorticoids, cyclosporin, tacrolimus, or sirolimus, among others. These drugs can be added to the treatment regime as deemed necessary by one skilled in the art of medicine. [0063] For this reason, it is preferred that cells are isolated from the individual requiring treatment, expanded in culture, differentiated as required, cultured using the methods described herein to form an engineered tissue and then transplanted back into the same individual. This autologous mode of treatment would prevent allograft rejection responses from occurring in the individual. In one embodiment, the engineered tissue is transplanted underneath the kidney capsule to reduce the autoimmune rejection of the graft and is contemplated for use with the methods described herein.

Efficacy of Treatment

[0064] The efficacy of a given treatment for a disease (e.g., engineered islet transplantation for Type 1 diabetes) can be determined by the skilled clinician. However, a treatment is considered "effective treatment," as the term is used herein, if any one or all of the signs or symptoms of the disease are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, e.g., by at least 10% following transplant with an engineered islet. Efficacy can also be measured by a failure of an individual to develop further disease symptoms (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) preventing the disease from occurring in an individual which may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease; e.g., prevention of complete islet cell loss; (2) inhibiting the disease, e.g., arresting its development by preventing further islet cell deterioration; or (3) relieving the disease, e.g., causing regression of the symptoms, promoting regeneration of islets, reducing the need for insulin shots, reduced hospitalization frequency or length of stay, reduced incidence or severity of diabetic complications (e.g., impaired wound healing) and/or improving survival of an affected individual. An effective graft size for the treatment of a disease means that amount which, when transplanted to a mammal in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an engineered tissue (i.e., pancreatic islet) can be determined by assessing physical indicators of disease, where the disease is diabetes, for example, blood glucose level, serum insulin response, and basal insulin level provide indicators of disease status.

[0065] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents. The present invention may be as defined in any one of the following numbered paragraphs.

1. A method for producing functional pancreatic islets ex vivo, the method comprising:

(a) culturing cells of a dissociated islet on a scaffold to form a spheroid aggregate of the cells;

(b) immersing the spheroid aggregate on the scaffold in a hydrogel under growth conditions sufficient to form an engineered islet;

(c) isolating the engineered islet from the scaffold and the hydrogel, wherein the engineered islet is functional.

2. The method of paragraph 1, further comprising an expansion step of the cells prior to said culturing on said scaffold.

3. The method of paragraph 1, wherein the scaffold is biodegradable.

4. The method of paragraph 1, wherein the scaffold biodegrades during said culturing of step (b).

5. The method of paragraph 1, wherein the scaffold comprises polyglycolic acid, or poly(lactic- co-glycolic acid).

6. The method of paragraph 1, wherein the hydrogel is thermoreversible.

7. The method of paragraph 1, wherein the isolating step comprises cooling the hydrogel and removing the engineered islet from the cooled hydrogel.

8. The method of paragraph 1, wherein the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.

9. The method of paragraph 1, wherein the engineered islet secretes at least one of insulin, and/or glucagon, and/or somatostatin.

10. The method of paragraph 1, further comprising over-expressing Serpine 1 in the cells.

11. A method for treating a subject having Type 1 Diabetes, the method comprising:

(a) culturing cells of a dissociated islet on a scaffold to form a spheroid aggregate of said cells,

(b) suspending the spheroid aggregate on the scaffold in a hydrogel under growth conditions sufficient to form an engineered islet;

(c) isolating the engineered islet from the scaffold and the hydrogel, and

(d) implanting the engineered islet into a subject having Type 1 Diabetes, wherein the engineered islet is functional and comprises a treatment for a subject having Type 1 Diabetes.

12. The method of paragraph 11, further comprising an expansion step of the cell prior to said culturing on said scaffold.

13. The method of paragraph 11, wherein the scaffold is biodegradable. 14. The method of paragraph 11, wherein the scaffold biodegrades during said culturing of step (b).

15. The method of paragraph 11, wherein the scaffold comprises polyglycolic acid, or poly(lactic-co-glycolic acid).

16. The method of paragraph 11, wherein the hydrogel is thermoreversible.

17. The method of paragraph 11, wherein the isolating step comprises cooling the hydrogel and removing the engineered islet from the cooled hydrogel.

18. The method of paragraph 11, wherein the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.

19. The method of paragraph 11, wherein the islet secretes at least one of insulin, glucagon, and somatostatin.

20. The method of paragraph 11, wherein the implanting step comprises transplantation under the kidney capsule.

21. A composition comprising a spheroid aggregate of cells on a scaffold immersed in a hydrogel.

22. The composition of paragraph 21, wherein the cells comprise cells of a dissociated islet.

23. The composition of paragraph 21, wherein the scaffold is biodegradable.

25. The composition of paragraph 21, wherein the scaffold comprises polyglycolic acid, or poly(lactic-co-glycolic acid).

26. The composition of paragraph 21, wherein the hydrogel is thermoreversible.

27. The composition of paragraph 21, wherein the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.

EXAMPLES

[0066] Described below are methods for the generation of tissue-engineered islets. In this example, an exemplary scaffold material (e.g., PGA) was evaluated as a temporary biodegradable scaffold that was seeded with cells from a dissociated islet. The cells and the scaffold were then suspended in an exemplary hydrogel (e.g., Thermoreversible gelatin polymer (TPG, Mebiol)) for the formation of functional islet cells. Islet cell reconstruction

[0067] Islets isolated from rats were enzymatically dissociated using 0.05% Trypsin and EDTA (Invitrogen, Carlsbad, CA) followed by gentle pasteur pipetting the next day (Ono, J., et al., Endocrinol Jpn (1977) 24, 265-70). Islets were isolated using collagenase and separated by Ficoll-Conray gradients. Then the cells were selected manually. [0068] Yields of islets and cells from the pancreas of two rats were 1008 ± 155 and 1.99 x 10⁶, respectively (Fig 1A,B). The single cells were placed in a culture dish containing PGA fibers in CMRL1099 medium (INVITROGEN™) supplemented with EGF and NGF; FBS was not added (Figure 1C,D). Cells exposed to PGA and growth factors formed clusters (Figure 1C), while cells that were not exposed to PGA did not form clusters (data not shown) 24 hours later. The cell aggregates were cultured for five days in CMRLl 099 medium with EGF, NGF, and IGF (Figure ID). The clustered cells on PGA were then suspended in TPG (Mebiol gel). The cell and gel combination was then cultured for 40 days (Figure IE). Finally, the temperature reactive gels were removed via extraction below 24°C. The tissue- engineered (TE) islets were washed repeatedly using DMEM with FBS. TE islets were then collected manually and used for transplantation (Figure IF).

Transplantation of TE islet cells

[0069] Streptozotocin (STZ) was administered to nude mice for one week to establish a diabetic phenotype. Five hundred native islets or tissue engineered islets were transplanted under the kidney capsule of diabetic nude mice as a control group and an experimental group, respectively. As a negative control, materials composed with PGA in gel but devoid of cells were used. Mice were monitored for blood glucose, body weight, and urine glucose levels three times per week for one month, then once per week for 120 days. After four months, the recipient mice received nephrectomies to remove the grafts containing the source of exogenous insulin. Hyperglycemia reappeared in all mice following nephrectomy in the TE islets graft group (Figure 2A). The positive control indicated a similar pattern with TE islets (Figure 2B). The negative control group remained hyperglycemic following nephrectomy and did not return to normoglycemia (Figure 2C).

Analysis of functional TE islet cells in vivo

[0070] To confirm that the TE islets were functional (i.e., responsive to glucose stimulation) in vivo, oral glucose tolerance tests were evaluated before and after nephrectomy in mice (Figure 3 A, upper column). Age- and gender- matched mice in the same strain were used as normal controls (Figure 3 A, lower left). In addition, animals with a moderate level of diabetes, which ranged from 200 to 300mg/dl, were used as a diabetic control (Figure 3 A, lower right). The recipients of TE islet graft indicated responses identical to the normal controls (Figure 3 A, upper left).

[0071] Insulin secretion and C-peptide were confirmed in recipient mice before and after administration of STZ, transplantation, and nephrectomy. Insulin secretion levels were found to be inversely proportional to blood glucose levels (Figure 3B). Since rat islets were transplanted as xenografts into nude mice, mouse C-peptide was discriminated as a species- specific peptide from rat C-peptide. Therefore, an increased level of C-peptide was found in the TE islet and xenografts control, as C-peptide selectively reacted with rat C-peptide but not mouse C-peptide (Figure 3C).

Functional TE islets in vitro

[0072] Dithizone (DTZ; Sigma, St. Louis, MO) is a widely accepted dye to distinguish insulin-positive cells in isolated islets in a clinical setting (Baharvand, H., et al., Develop Growth Differ (2006) 48, 323-332, (2006)). TE islets and rat islets were stained positively for DTZ before transplantation (Figure 4A). The morphology of both transplanted controls and TE islets was evaluated using H & E (Figure 4B). Multi-lineage cells having endocrine functions were identified using three-color staining. Insulin and glucagon were confirmed positively in graft sites of both the control and TE islets (Figure 4C) as well as somatostatin (Figure 4D). Although gamma cell function was confirmed in the control islets graft using pancreatic polypeptide antibody, there was no expression seen in the TE islet grafts (data not shown).

A critical role of bio-absorbable material, PGA, to aggregation of cells

[0073] To reconstruct functional islets, it was essential that a scaffold was used, such that single cells were cultured on PGA to form spheres. Gene expression in the cells was examined using a microarray, whether cultured alone or in the presence of PGA. Some specific extra-cellular matrix mRNA was up-regulated in cells on PGA. Although MMP12 and MMP3 were slightly increased when cells were cultured with PGA, Serpinel was dramatically up-regulated five-fold higher in cells cultured to PGA versus controls cultured in the absence of PGA (Table 1). [0074] Many approaches have been examined to expand islets. However, these approaches do not permit islets to grow beyond a constant size using some hydro gels. Consequently, while the dissociated cells have been used to expand the cell yield, this has not been confirmed in vivo (Lechner, A., et al., Biochem Biophys Res Commun (2005) 327, 581-588). [0075] Thermoreversible gelatin polymer (TPG) is a relatively new material used in tissue engineering and has been previously reported as a scaffold for chondrocytes (Yasuda, A., et al. Tissue Eng (2006) 12, 1237-1245). TPG (Mebiol gel) changes from gel to liquid at approximately 20°C. Hence, TPG has an advantage in that it can be extracted from cells and tissues prior to transplantation. Although some hydrogels have been used for islet reconstruction, they have not enabled reaggregation of cells into functional islets. Described herein is a combination of materials capable of enhancing aggregate formation and spheroids that are both structurally and functionally similar to islets.

[0076] Polyglycolic acid (PGA) is widely used clinically. For our purpose, PGA was custom-manufactured to form a fiber scaffold (Cortiella, J., et al., Tissue Eng (2006) 12, 1213-25). When single cells from islets were seeded to PGA, the cells started to aggregate. As the aggregation progressed over a few days, the aggregated cells were investigated using microarrays to focus on mechanisms of molecular regulation. In particular, genes encoding extra-cellular matrix molecules and adhesion molecules were examined. [0077] Matrix metalloproteinases (MMPs) play a leading role in the resolution of extracellular matrix proteins. Currently, up-regulation of MMPs has been reported not only for extra-cellular matrix suppression but also for acceleration of angiogenesis and oncogenesis (Noel, A., et al., Semin Cell Dev Biol (2008) 19, 52-60). Serpinel, also known as serine protease inhibitor or plasminogen activator inhibitor- 1, down-regulates the production of plasmin that activates MMPs indirectly and acts as an antagonist of MMP activity. In addition, increased levels of Serpinel have been reported in various disease states, including e.g., in numerous types of cancer as a prognostic factor (Gils, A., and Declerck, P. J. Curr Med Chem (2004) 11, 2323-34).

[0078] In this study, the expression of MMP3 and MMP 12 was slightly increased in the cells seeded on a PGA scaffold. However, their inhibitor, Serpinel, was further up-regulated five times higher than controls. Up-regulation of MMPs and Serpinel has been reported in the active oncogenesis phase (Gils, A., and Declerck, P. J. Curr Med Chem (2004) 11, 2323-34). Alternatively, aggregation of the β cells may have prevented the interruption of cell growth caused by Serpine-1. [0079] The insulin secreting function of TE islets was studied using various functional assays, including the oral glucose tolerance test (OGTT). TE islets showed the potential to secrete exogenous insulin following transplantation in mice. TE islets were able to secrete adequate insulin to maintain glucose levels in a normal range for up to 120 days after transplantation. In addition, blood glucose levels were reversed after graft removal. Furthermore, OGTT indicated a completely normal pattern in recipients before nephrectomy. Secretion of insulin was monitored with C-peptide. C-peptide transitions reflected the presence of TE islet grafts. [0080] In addition to the secretion of insulin, other endocrine functions were confirmed in the tissue engineered islets using fluorescent immunohistochemistry. The engineered islets showed multi-lineage reconstruction of endocrine tissue as evidenced by the co-expression of insulin with glucagon or somatostatin.

[0081] The pancreatic islet cells automatically functioned to secrete insulin in response to high glucose levels. The tissue engineered islets demonstrate autoregulatory properties, requiring only the formation of the islet without the need for further vasculogenesis and/or neurogenesis in theTE islet cell graft. [0082] Described herein is a successful method for regenerating functional TE islets.

METHODS

Animals and cells.

[0083] Female Fisher rats (Taconic Farms, Germantown, NY) and female nude mice (Jackson Laboratory, Bar Harbor, ME) were used as donors and recipients, respectively. Blood glucose and urine glucose were monitored using Elite (Bayer Corporation, Elkhart, IN) and Diastix (Bayer), respectively. Nude mice, age adjusted to eight weeks old, were administered 220mg/kg STZ via a single tail vein injection each day for seven days before transplantation (Kemp, CB. , et al., Nature (1973) 244:447). Mice with blood glucose levels that exceeded 400mg/dl twice continuously per week were used as diabetic transplantation recipients. Pancreatic islet cells were isolated from Fisher rats, processed in collagenase digestion and purified using a Ficoll gradient method as previously described (Sutton, R., et al., Transplantation (1986) 42, 689-691; Okeda, T., et al., Endocrinol Jpn (1979) 26, 495-499; Ikehara, Y., et al., / Clin Invest (2000) 105, 1761-7). Isolated islets were used for transplantation or dissociation to single cells. Enzyme ELISA.

[0084] Serum was collected four times: before injection of STZ, after the onset of diabetes before transplantation, before nephrectomy in normoglycemia, and after nephrectomy with reversal to diabetes. Insulin (Linco Research, St. Charles, MO) and C-peptide (WAKO, Richmond, VA) concentrations were measured using sandwich enzymatic ELISA kits.

OGTT.

[0085] OGTT was performed before and after removal of the TE islet graft. Controls including untreated or treated STZ-induced diabetic mice were also used. Blood was drawn after a 6-hour fast. Baseline (0 min) blood glucose measurements were recorded. Mice were then challenged with 1.5mg/g body weight of D-glucose (SIGMA™) dissolved in phosphate- based saline (PBS),(INVITROGEN™) passed through a stomach tube (FISHER SCIENTIFIC™, Pittsburgh, PA). Blood glucose levels were measured at 15, 30, 60, and 120 minutes after the glucose challenge (Rink, C, et al., Physiol Genomics (2006) 27, 370-379).

Histological Examination.

[0086] DTZ was prepared at a concentration of lmg/ml with dimethylsulfoxide (DMSO) as a stock solution. In vitro DTZ staining was performed by adding lOμL of the stock solution to ImI of culture medium. The DTZ solution was added to culture dishes and incubated at 37⁰C for 15 minutes. The stained cells were then examined.

[0087] After the transplantation of native or TE islets, the graft site of the kidney was removed at 120 days. The grafts were fixed in Bouin's solution (Sigma) overnight. Bouin's solution was then changed to 10% Formalin before embedding with paraffin. The tissues were cut at 5μm thickness, for immunohistochemistry and Hematoxylin & Eosin (Richard- Allen Scientific, Kalamazoo, MI) staining. After the de-paraffination, the continuous slides were co-stained with insulin and glucagon (Linco Research) or somatostatin (Chemicon, Temecula, CA) or pancreatic polypeptide (Linco Research) antibodies. The primary antibodies were diluted 1/50 to incubate overnight at 4°C. Anti-Guinea Pig IgG (Texas Red) and anti-rabbit IgG (FITC) (JACKSON IMMUNORESEARCH LABORATORIES™, Inc., West Grove, PA) secondary antibodies, diluted at 1/50, were used for 2 hours. Fluorescent intensity was analyzed using a fluorescence microscope (Nikon) and spot image software (Diagnostic Instruments Inc., Sterling Heights, MI). PCR array analysis.

[0088] Single cells from rat islets adjusted to a cell density of 2.0xl0⁶/ml were cultured on 35mm x 10mm treated polystyrene cell culture dishes (BD Biosciences, San Jose, CA) and exposed to medium containing growth factors (GFs), EGF (100ng), NGF (400ng), and IGF (200ng) with or without PGA. After exposure to GFs, cells were cultured for 36 hours. The dishes were gently rinsed with PBS. The cells were collected into 1.5ml Eppendorf tubes and supplied with ImI of PBS. After the tubes were centrifuged, pellets were collected. RNA was purified using the RNeasy Mini kit (Qiagen Inc., Valencia, CA). Samples were conjugated with lysis buffer and homogenized. RNA was trapped in the membrane of spin column included in the kit. After three washes, mRNA was extracted. Purified mRNA was processed by PCR array analysis (SuperArray Bioscience, Frederick, MD). Briefly, this was accomplished by converting the mRNA (10ng) samples into first strand cDNA, which was the template for the polymerase chain reaction (PCR) using a cDNA synthesis kit (SuperArray Bioscience). This was combined with a template with ready-to-use RT² Real-Time™ SYBR Green PCR master mix (SuperArray Bioscience). Equal aliquots of this mixture were added to each well of the same PCR (rat extracellular matrix and adhesional molecules) array plate (SuperArray Bioscience) containing the predispensed gene specific primer sets, and PCR was performed to collect real-time amplification data in AB 17500 (Applied Biosystems, Foster City, CA) with software. The expressions of genes were compared using the ΔΔC_t method (Livak K.J., and Schmittgen T.D. Method (2001) 25, 402-8). The final result was reported from SuperArray Bioscience as reference number #P2783.

Table 1. mRNA levels compared using microarray. Control expressions and PGA seeded expression were calculated as group 1 and group 2, respectively. The genes that expressed 1.5 times higher than control are shown, 2.0 times higher genes were defined as positive.

^Position of the microarray panel, **Gene over-expressed in Group 2 vs. Group 1

Claims

2. The method of claim 1, further comprising an expansion step of the cells prior to culturing on the scaffold.

3. The method of claim 1, wherein the scaffold is biodegradable.

4. The method of claim 1, wherein the scaffold biodegrades during the culturing of step (b).

5. The method of claim 1, wherein the scaffold comprises polyglycolic acid, or poly(lactic- co-glycolic acid).

6. The method of claim 1, wherein the hydrogel is thermoreversible.

7. The method of claim 1, wherein the isolating comprises cooling the hydrogel and removing the engineered islet from the cooled hydrogel.

8. The method of claim 1, wherein the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.

9. The method of claim 1, wherein the engineered islet secretes at least one of insulin, glucagon, or somatostatin.

10. The method of claim 1, further comprising over-expressing Serpine 1 in the cells.

(a) culturing cells of a dissociated islet on a scaffold to form a spheroid aggregate of the cells,

(c) isolating the engineered islet from the scaffold and the hydrogel, and (d) implanting the engineered islet into a subject having Type 1 Diabetes, wherein the engineered islet is functional and comprises a treatment for a subject having Type 1 Diabetes.

12. The method of claim 11 , further comprising an expansion step of the cell prior to the culturing on the scaffold.

13. The method of claim 11, wherein the scaffold is biodegradable.

14. The method of claim 11 , wherein the scaffold biodegrades during the culturing of step (b).

15. The method of claim 11 , wherein the scaffold comprises polyglycolic acid, or poly(lactic-co-glycolic acid).

16. The method of claim 11 , wherein the hydrogel is thermoreversible.

17. The method of claim 11 , wherein the isolating comprises cooling the hydrogel and removing the engineered islet from the cooled hydrogel.

18. The method of claim 11 , wherein the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.

19. The method of claim 11, wherein the islet secretes at least one of insulin, glucagon, or somatostatin.

20. The method of claim 11 , wherein the implanting comprises transplantation under the kidney capsule.

22. The composition of claim 21 , wherein the cells comprise cells of a dissociated islet

23. The composition of claim 21, wherein the scaffold is biodegradable.

24. The composition of claim 21, wherein the scaffold comprises polyglycolic acid, or poly(lactic-co-glycolic acid).

25. The composition of claim 21, wherein the hydrogel is thermoreversible.

26. The composition of claim 21 , wherein the hydrogel is selected from the group consisting of: thermoreversible gelatin, a blood clot, calcium/alginate and Mebiol.