Patent Application
20170049941
There is a critical shortage of organs for transplantation, with the organ waiting list currently at over 110,000 requests and increasing by 5% every year. Approximately 30,000 deaths are registered annually in the US due to liver disease. At this time, the only definitive treatment of hepatic failure is orthotopic transplantation. This form of transplantation will always be limited due to a paucity of available organs, and the delivery of cells directly is inefficient due to low engraftment. Thus, the generation of a transplantable tissue engineered liver graft could dramatically change this equation (e.g. organ engineering using natural organs scaffolds). However, the major challenge in tissue/organ engineering (including liver) has so far been limited graft survival after transplantation. The main gap that prevents advancement of the field is the lack of strategies to prevent acute thrombosis after graft transplantation.
Tissue engineering has so far had limited success in many tissues, including liver. The main gap that prevents advancement of the field is the lack of an ideal transplantable scaffold that has all the necessary microstructure and extracellular cues for cell attachment, differentiation, functioning, as well as vascularization, which has so far proven to be difficult to manufacture in vitro. In recent publications in Nature Medicine (Uygun et al. “Organ reengineering through development of a transplantable re-cellularized liver graft using decellularized liver matrix.” (2010) Nat Med. 16(7):814-20) and Tissue Engineering (Soto-Gutierrez et al. “A whole-organ regenerative medicine approach for liver replacement” (2011) Tissue Eng. Part C Methods, 17(6):677-686), cadaveric liver decellularization protocols to create a whole-liver scaffold for engineering hepatic grafts have been demonstrated. The decellularization process preserves intact the native microvascular network of the organ. Adult hepatocytes can be seeded into these scaffolds, remaining viable and providing essential liver functions for up to 10 days. Moreover, engineered livers could be implanted in the rats using the recipient left renal artery and vein. Liver graft function was documented for up to 8 hours after implantation. However, long-term transplantation of engineered livers remains a challenge.
Methods of preparing engineered organs with anti-thrombotic activity are provided to achieve long-term survival after transplantation using optimized vascular re-cellularization and/or polymer-based vascular surface modification to block acute thrombosis. The methods provide protocols to mitigate acute thrombosis with reendothelialization and protein-reactive polymers, such as N-hydroxysuccinimide-polyethylene glycol (NHS-PEG) and similar other molecules, and engineered organs for transplantation in patients with impaired organ functionality. Such engineered organs retain vasculature and are suitable for long-term survival following implantation. The organs described herein are based on extracellular matrix (ECM), and can be completely reendothelialized so as to not induce coagulation when exposed to blood (i.e., organs that are not at risk of acute thrombosis).
In one aspect, provided herein is a method of preparing a whole or partial organ extracellular matrix (ECM) construct including the steps of decellularizing a whole organ or partial organ by contacting the whole organ or partial organ with a decellularization solution and coating the decellularized whole organ or partial organ with an anticoagulant protein-associating composition. In some aspects the whole organ or partial organ is a whole liver or partial liver.
In some aspects, the step of decellularizing the whole or partial organ ECM construct includes contacting the whole organ or partial organ with a solution comprising about 0.02% trypsin and then contacting the whole organ or partial organ with a solution comprising about 0.1% Triton X-100.
In some aspects, the whole organ or partial organ is also disinfected. In some aspects, the whole organ or partial organ is disinfected with peracetic acid.
In some aspects of the decellularization solution further includes a chelating agent. In some aspects, the chelating agent is EGTA.
In some aspects of the method of preparing a whole or partial organ ECM construct, the whole organ or partial organ is frozen before decellularization.
In some aspects of the method, the anticoagulant protein-associating composition is a polyether polymer, copolymer, or block copolymer, such as a poly(C1-C6 alkylene oxide) moiety, such as a polyoxyethylene, a polyoxypropylene, or a polyoxytetramethylene linked to an amine or ECM-reactive group. In some aspects, the anticoagulant protein-associating composition includes an N-hydroxysuccinimide (NHS) moiety covalently linked to a non-reactive, hydrophilic, biocompatible polymer moiety. In some aspects of the method of preparing a whole or partial organ ECM construct the biocompatible polymer moiety comprises a polyether polymer, copolymer, or block copolymer, such as a poly(C1-C6 alkylene oxide) moiety, such as a polyoxyethylene, a polyoxypropylene, or a polyoxytetramethylene linked to an amine-reactive group.
In some aspects of the invention, the anticoagulant protein-associating composition includes poly(ethylene glycol) covalently linked to an NHS moiety.
In some aspects of the invention, the protein-associating polymer composition includes a phosphorylcholine (PC), sulfobetaine (SB), or carboxybetaine (CB) moiety.
In some aspects, the anticoagulant protein-associating composition includes an amine or ECM-reactive group. In some aspects the amine or ECM-reactive group is NHS, isocyanate (NCO), or carboxyl (COOH).
In some aspects of the method the anticoagulant protein-associating composition includes one or more of PEG-NHS, PEG-NCO, PC-NHS, PC-NCO, SB-PEG-NHS, PC-COOH, SB-COOH, or poly[N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide]-co-valine (PVLA-co-VAL).
In some aspects of the method of preparing a whole or partial organ ECM construct, each of the decellularization solution and the anticoagulant protein-associating composition are provided to the whole organ or partial organ by flushing vasculature of the whole organ or partial organ, thereby coating the vasculature with the anticoagulant protein-associating composition.
Also provided herein is a decellularized extracellular matrix (ECM) organ structure. The decellularized ECM organ structure includes a decellularized whole organ or partial organ comprising native ECM structure, and an anticoagulant protein-associating composition dispersed within the native ECM structure.
In some aspects, the anticoagulant protein-associating composition includes one or more of PEG-NHS, PEG-NCO, PC-NHS, PC-NCO, SB-PEG-NHS, PC-COOH, SB-COOH, or PVLA-co-VAL.
In some aspects of the invention, the anticoagulant protein-associating composition includes poly(ethylene glycol) covalently linked to an NHS moiety.
In some aspects of the invention, the organ structure further includes orthotopic, autologous, allogeneic or xenogeneic cells dispersed into the decellularized organ structure. In some aspects, the cells are primary cells, multipotent cells, or pluripotent cells.
The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values, as well as sub-ranges. For example, a range of temperatures of 4° C. to 37° C. includes 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 37° C., and sub-ranges such as 15° C. to 20° C. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases.
As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, are open ended and do not exclude the presence of other elements not identified. In contrast, the term “consisting of” and variations thereof is intended to be closed, and excludes additional elements in anything but trace amounts.
Provided herein are methods of preparing a whole or partial organ extracellular matrix (ECM) construct that is amenable to full re-endothelialization and suitable for long-term use in transplantation. The method comprises first decellularizing a whole or partial organ, for example a liver or partial liver, followed by providing an anti-thrombic coating to the whole or partial organ. As used herein, a whole or partial organ comprises macro- and micro-level structures such as vasculature, ducts and organ substructures, such as for example, in the case of liver, vasculature and bile ducts. The whole or partial organ is decellularized, leaving behind ECM, but retains organ native structure, meaning that three-dimensional organization of the structures of the organ are substantially retained in the ECM material left behind after decellularization.
One goal of the decellularization protocol is to provide an ECM construct that provides the lowest possibility of an unwanted host response. Parameters suitable for such constructs, such as amount of phospholipid and/or nucleic acid remaining following preparation of the construct, are disclosed, for example, in Keane et al. (“Consequences of ineffective decellularization of biologic scaffolds on the host response” Biomaterials (2012); 1771:1781).
In the experiments described herein, decellularized organ/organ constructs prepared as described herein are used as platforms for organ engineering. For example, although certain decellularization methods of whole or partial organs are known (e.g. as described in U.S. Pat. No. 8,470,520), such decellularized organs do not exhibit sufficient viability in vivo for use in long-term tissue transplantation. Rather, such organs must be re-endothelialized and provided with vascular surface modification proteins with polyethylene glycol (or other like molecules) to inhibit acute thrombosis and achieve long-term graft survival after transplantation. The methods described herein provide a feasible method of preserving the vascular perfusability to allow engineered tissues or organs complete to completely regenerate. As a result of these methods, and whole or partial organs prepared therefrom, long-term survival and regeneration of engineered tissues and organs is possible. The regenerated organs also allow for studying complex liver cell interactions.
For example, engineered liver grafts will enable performing much more aggressive hepatic resections in patients with malignancies, which is currently not possible due to the likelihood of developing hepatic failure as a consequence of insufficient hepatic mass. Moreover, these grafts could be sufficient to support patients with acute liver failure while their own liver recovers, without the risk of performing whole liver transplantation and the use of life-long immunosuppressant therapy. Scientifically, the system provides a feasible model to study liver development and hepatic maturation processes as well as a model to study the complex parenchymal and non-parenchymal liver cell interactions. Engineered organs as described herein could also be used as a tool to accurately predict the metabolism or toxicity of a compound in human liver grafts in vitro prior the exposure to the whole body, by providing a natural environment. This potentially translates into reduced costs and time in drug development, and less harmful patient exposure in clinical trials.
The organ to be decellularized and used as an ECM organ construct may be any organ amenable to decellularization and transplantation. In non-limiting embodiments the organ is a liver, kidney, spleen, gallbladder, lung, heart, muscle, and skin. The organs may be derived from humans, or may be porcine in origin. The organ is decellularized, for example and without limitation, by contacting the whole organ or partial organ by submersion or incubation in a decellularization solution. In a nonlimiting embodiment the decellularization solution is applied to the whole or partial organ by flushing the vasculature (e.g., perfusing) of the organ and/or ductwork of the organ. Decellularization solutions suitable for this use are known to those of skill in the art, but typically are aqueous solutions comprising a detergent or surfactant, and in one embodiment a non-ionic detergent, ionic or zwiterionic detergent, acid and base solutions, hypotonic and hypertonic solutions, alcohols, solvents, enzymes, chelating, physical and miscellaneous agents or any combination of any of the aforementioned solutions and agents. Examples of such detergents or surfactants include Triton X-100, however those of ordinary skill in the art will understand that any suitable decellularization solution may be utilized in the methods described herein.
The incubation, submersion, or flushing of the whole or partial organ in decellularization solution may be performed for durations of, for example and without limitation, 30 minutes to 24 hours, and may be performed at temperatures ranging from 0° C. to 37° C.
Prior to contacting the whole organ or partial organ, for example by flushing the organ with decellularization solution, the whole organ or partial organ is optionally digested with a protease-containing solution, such as a solution comprising an acid protease. As used herein, a protease is an enzyme that breaks down proteins or polypeptides into smaller polypeptides or amino acids. Those of skill in the art are aware of suitable proteases for use in decellularization protocols. However, in non-limiting embodiments, the protease is pepsin or trypsin. In some embodiments, the protease solution is included in the decellularization solution.
In one embodiment, the protease of the protease-containing solution is an acid protease, for example trypsin or pepsin. In a non-limiting example, the organ or partial organ is decellularized by flushing and digestion with a protease-containing solution comprising from 0.005% wt. (percent by weight) to 0.1% trypsin, followed by flushing and treatment with a detergent solution comprising from 0.01% to 5% Triton X-100. In a preferred embodiment, the protease solution comprises 0.02% (by weight) trypsin and the decellularization solution includes 0.1% (by weight) Triton X-100.
In any embodiment, the detergent solution may further comprise a chelating agent, such as 0.001 mM to 10 mM EDTA or EGTA, or, by weight of the decellularization solution, 0.01% to 5% EDTA or EGTA. Prior to decellularization, the whole organ or partial organ may optionally frozen, for example by flash freezing, and thawed, or the organ surface may be cross-linked by exposure to formaldehyde or any other fixative agents.
In an exemplary embodiment, the whole or partial organ is digested with a protease solution for durations ranging from 30 minutes to 24 hours, and digestion occurs at temperatures ranging from 4° C. to 37° C. Following digestion, the digested whole or partial organ is washed, for example by rinsing or flushing, with a wash solution, such as those known to those of skill in the art. Examples of such wash solutions include water, deionized water, cell-free culture medium, phosphate buffered saline (PBS), and combinations thereof. Rinsing/washing may also be performed anytime a step of the decellularization method is completed. Thus, for example, following flushing or other incubation/submersion/immersion in the protease solution, the whole or partial organ may be washed/rinsed with any suitable wash solution and then immersed in or otherwise flushed with the decellularization solution.
According to one embodiment, the organ or partial organ is decellularized by flushing and digestion with a protease-containing solution followed by flushing and treatment with a decellularization solution, followed by disinfecting the ECM construct, again optionally with washing/rinsing steps between the digestion, decellularization, and disinfecting steps. The decellularization also optionally comprises a disinfecting step, e.g., by flushing or otherwise contacting the ECM construct with a solution comprising an appropriate amount of peracetic acid at concentrations ranging from 0.1% to 3% from 10 minutes to 6 hours. In addition, other disinfecting agents may be used, for example and without limitation, antibiotics such as penicillin (1,000-10,000 Units/ml), streptomycin (50-100 μg/m1), gentamycin (1-100 μg/ml) diluted in buffer saline solution (PBS). The whole or partial organ construct cane be exposed to these disinfecting agents for from 30 minutes to 24 hours at temperatures ranging from 4° C. to 25° C. Those of skill in the art will understand that any suitable disinfecting solution or protocol may be used within the spirit of the invention.
One goal for improving host response outcomes is to reduce the formation of thrombi. Accordingly, in an exemplary embodiment, the method of preparing a whole organ or partial organ ECM construct comprises digestion and decellularizing the whole or partial organ as described above, and providing, for example by submersion, immersion, incubation, or flushing the vasculature with, an anticoagulant such as a protein-associating composition (e.g. N-hydroxysucinnimide (NHS)-heparin), such as a polymer-based composition (e.g. NHS-poly(ethylene glycol) (PEG) or equivalent compositions).
As used herein, the term “polymer” includes copolymers, block copolymers, homopolymers, and modified polymers. The composition comprises a non-reactive moiety (e.g. PEG) attached to an amine- or ECM-reactive group (e.g. NHS). A polymer is prepared by polymerization of one or more monomers by any useful polymerization method, such as radical polymerization, such as controlled-radical polymerization, living polymerization, e.g., atom-transfer radical polymerization, though poly(C1-6 alkylene oxide) polymer are typically produced by ionic mechanisms—both cationic or anionic mechanisms—such as in the case of polymerization of ethylene oxide in water. A “residue” is an incorporated monomer. By “attached”, unless indicated otherwise, it is meant linked or covalently bonded. The non-reactive moiety is biocompatible—that is, it does not substantially inhibit cell growth and differentiation and implementation of the methods of producing a whole or partial organ ECM construct as described herein. By “non-reactive”, it is meant that a moiety essentially does not covalently bind, react or link to the whole or partial organ ECM construct under physiological conditions, such as in water, cell culture medium, blood, serum, plasma, PBS, and/or saline.
Non-limiting examples of a non-reactive moiety include polyethers, such as a polyoxyalkylene polymer, such as poly(C1-6 alkylene oxide) polymers or copolymers where two or more different C1-6 alkylene oxide monomer residues are incorporated into the poly(C1-6 alkylene oxide) polymer. “Alkylene” refers to a saturated bivalent, linear or branched, aliphatic hydrocarbon radical, such as methylene (—CH2—), ethylene (e.g., —CH2—CH2—), propylene (e.g., —CH2—CH2—CH2—), tetramethylene (e.g., —CH2—CH2—CH2—CH2— or —CH2—CH2—CH2(CH3)—), etc. An exemplary polyether or poly(C1-6 alkylene oxide) polymer is polyoxyethylene (PEG), having the structure —(O—CH2—CH2)n—OH, in which n is an integer greater than or equal to 2. In one non-limiting embodiment, n is 2-50. Other examples of the poly(C1-6 alkylene oxide) polymer moiety include polypropylene glycol (PPG; H—(O—CH2—CH2—CH2—CH2—)n—OH) or polytetramethylene glycol (PTMEG; H—(O—CH2—CH2—CH2(CH3)—)n—OH), in which n is an integer greater than or equal to 2. In one non-limiting embodiment, n is 2-50. In addition, polyether-containing block polymers, comprising blocks of different polyether, polyoxyalkylene or poly(C1-6 alkylene oxide) blocks, such as PEG-PPG-PEG block copolymers may be used as the non-reactive moiety. The non-reactive moiety, such as a polyether, is modified with an amine-(or ECM) reactive moiety, such as NHS, isocyanate (NCO), carboxyl (COOH), aldehyde (C═O), or chloride (Cl) groups. Suitable block copolymers can be formed using living radical polymerization techniques as well as click chemistry techniques, as are known to those of skill in the art.
Other compositions suitable for use in the protein-associating composition include, without limitation: zwitterionic moieties (e.g., phosphorylcholine (PC), sulfobetaine (SB), carboxybetaine (CB)), macromolecules or polymers with amine reactive groups (N-hydroxysucinnimide (NHS), isocyanate (NCO), carboxyl (COOH)) (e.g., PC-NHS, PC-NCO, SB-PEG-NHS, PC-COOH, SB-COOH, PEG-PPG-PEG-NHS, PEG-SB-NHS compositions), or poly[N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide]-co-valine (PVLA-co-VAL), or PVLA-co-VAL-PEG-NHS to inhibit acute thrombosis in damaged vascular and biomaterial surfaces. These compositions can be additionally bound to biotin, for example for detection. An exemplary polymer including biotin is poly(ethylene glycol) (N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane.
In addition to the above-identified compounds that may be used to reduce and/or eliminate formation of thrombi in the whole or partial organ ECM construct, additional suitable compounds may be found in U.S. Pat. No. 5,977,252, including, for example and without limitation, compounds including ester, anhydride (including N-carboxy anhydride), isocyanate (as described above), aldehyde, tosylate, tresylate or epoxide groups/moieties. Reactive end groups that will not release small molecules or toxic molecules upon the covalent attachment of the polymer are preferred. To this end, cyclo-esters, cyclo-anhydrides and isocyanates are also suitable reactive groups to attach to the end of the polymer and effectuate the covalent modification.
In another embodiment, the protein associating composition comprises an NHS moiety covalently linked to a non-reactive, biocompatible polymer moiety. In yet another embodiment, the NHS moiety is linked (covalently bonded) to a PEG moiety. In an exemplary embodiment, the protein-associating polymer comprises N-hydroxysuccinimide (NHS)-modified poly(ethylene glycol) (PEG).
The compositions described above for use as anticoagulants in decellularized organs produce more than 80% of adequate re-endothelization of all vessels in the whole or partial organ, and re-epithelialization up to 30-40% of the whole or partial organ. These levels are possible because the compositions reduce the formation of thrombi.
The step of immersion/submersion/incubation/flushing of the whole organ or partial organ with the above-described protein-associating compositions may be conducted prior to ex vivo population of the whole organ or partial organ with cells, or after ex vivo population of the organ or partial organ with cells, or, for example, immediately before implantation of the cell-populated organ into a patient. Exposure of the whole or partial organ to the polymer may be for durations ranging from 30 minutes to 24 hours, and may occur at temperatures ranging from 4° C. to 37° C. and can be done under flow conditions ranging from 1 ml/min to 100 ml/min, or in static conditions. Those of skill in the art will appreciate that reaction times will vary based on the protein-associating polymer that is used.
Also provided herein is an extracellular matrix (ECM) organ structure, comprising a decellularized whole organ or partial organ substantially comprising native three-dimensional ECM structure, and an anticoagulant, such as a protein-associating composition, such those described above, dispersed within and/or coating the native ECM structure (e.g., comprising essentially all macro-structural elements of the organ or partial organ from which the organ structure is prepared). The organ structure optionally comprises cells. For example, one embodiment is a commercial product comprising a decellularized organ structure comprising the anticoagulant. In another embodiment, the commercial product is the organ structure populated with cells, such as a patient's autologous cells for transplantation into the patient, and comprising the anticoagulant, which is applied to the organ structure after population of the organ structure with cells and prior to implantation thus coating exposed ECM material in the organ structure.
In one embodiment, the ECM organ structure is prepared according to any method described herein, or any suitable method known to those of skill in the art to provide a whole or partial organ ECM construct with low immunogenicity and suitable for implantation, and provided with (for example coated with) an anticoagulant polymer as described herein.
Also provided is a method of producing an artificial organ, comprising, prior to or after administration of the anticoagulant, perfusing the ECM whole or partial organ structure, as described herein, with one or more cells, such as, for example, primary cells (e.g., hepatocytes), multipotent cells and/or pluripotent cells, for example progenitor cells or stem cells, as are broadly known in the field. The cells may be, according to certain embodiments, orthotopic, autologous, allogeneic and/or xenogeneic. The artificial organ (organ structure) is implanted in a patient in need thereof, for example and without limitation, a liver ECM structure as described herein is perfused with hepatocytes and incubated, for example as described below, flushing the organ structure with the anticoagulant prior to, for example immediately prior to, implantation of the organ structure in a patient.
Quality Assessment Protocols to Evaluate Whole Organ Liver Decellularization
Protocols for whole liver decellularization. Different detergents (SDS, trypsin and Triton X-100) were evaluated for their effect on the organ ECM. System criteria for evaluation were based on the preservation of structural and extracellular proteins, DNA remnants, the presence of growth factors, and integrity of the collagenous capsule covering the external surface of the liver (i.e. Glisson's capsule). For instance; to quantitate the desired features of an extracellular matrix after liver decellularization using multiphoton imaging 5 75-90 μm (depth) beyond the liver surface (Glisson's capsule), it was found that 0.02% trypsin and 0.1% Triton X-100 maintained the structure, orientation and density of the collagen better than use of 0.02% trypsin and 3% Triton X-100 as decellularization. In normal livers, collagen fibers were long and widely separated by the cellular content. Moreover, scanning electron microscopy (SEM) images confirmed the presence and higher collagen density preserved when 0.02% trypsin and 0.1% Triton X-100 protocol was used. A meticulous analysis of the surface of the Glisson's capsule showed complete integrity of the Glisson's capsule in both decellularization protocols. It was found that 100% of the fibrillar collagen of native liver was retained after 0.02% trypsin and 0.1% Triton X-100 decellularization. It was also found that residual DNA content in both decellularization protocols was less than 10%. Based on these results, 0.02% trypsin/0.1% triton was selected as being most optimized and, as expected, fibronectin and laminin components of the basement membrane were preserved. The criteria that define a successful organ decellularization process are poorly understood. From empirical experience it is known that the goal is to maintain the quality and quantity of the collagen content and the preservation of the basement membrane components as close to normal liver as possible. Under these circumstances, an organ scaffold can be produced with no leakage in flow culture conditions.
A new methodology to easily check the quality of the decellularization process based on the previous criteria was developed. The results show that an optimized decellarization protocol consists of a combination of 0.02% trypsin and 0.1% Triton X-100/0.05% EGTA.
The liver decellularization protocol described herein preserves the structure and alignment of the collagen fibers as shown and analyzed by multiphoton fluorescence microscopy, this technique allows imaging several hundreds of micrometers deep into biological samples as scattering of red-shifted light for collagen fibers (
The method and condition with which the liver tissue is decellularized will have profound impact on both the structure and biological composition of extracellular tissue matrices. Differential scanning calorimeter (DSC) analysis is a useful tool for assessing the extent of decellularization and effects on structure/composition.
As the results in
Liver Graft Re-Cellularization and Culture-Perfusion System
Two different systems useful for re-cellularization are shown in
Optimization and evaluation system of vascular re-endothelialization and bile duct re-epithelialization. Ultimately reconstruction of liver grafts in vitro also requires the addition of liver non-parenchymal cells. Previous work has demonstrated intact vasculature using corrosion cast technique.
As described above, micro computed tomography (CT) was also utilized to characterize the architectural vasculature of the bile duct, portal vein and central vein (
Assembly of Whole-Organ Liver Vasculatures and Bile Duct
In order to corroborate the histological quantification of the whole organ re-cellularization protocols in a more systematic fashion, micro Magnetic Resonance Imaging (MRI) was used. The liver was divided in different segments (SRL/IRL; superior right lobe/inferior right lobe, RML; right median lobe, LML; left median lobe, LLL; left lateral lobe, AC/PC; anterior caudate lobe/posterior caudate lobe) and obtained 2D images of the intrahepatic biliary tree, portal and central vein vasculature of each segment (
The best perfusion protocol results in repopulation of 68±9% of the portal vein and 78.3±16% of the central vein, and microscopy analysis also confirmed that microvascular endothelial cells lined the interior of the portal and central veins. Histological quantification revealed that 86±3% of the portal vein system and 81±9% of the central venous system were repopulated (
The resulting evaluation was compatible with the histological evaluation previously developed. This novel, powerful imaging technique is capable of providing a systematic three-dimensional or two-dimensional quantitative analysis of the normal intrahepatic biliary tree, portal and central vein as well as the corresponding evaluation of the re-cellularization of whole liver scaffolds (
Functional evaluation of liver vascular re-endothelialization. Most importantly, to date the major challenge in tissue/organ engineering (including liver) has so far been limited graft survival after transplantation. That is, the main gap that prevents advancement of the field is the lack of strategies to prevent acute thrombosis after graft transplantation. Thus, the development of a functional liver vasculature is imperative to achieve long-term survival of engineered organs. It has previously been demonstrated that intact vasculature using corrosion cast technique. Additionally, micro computed tomography has been performed to characterize the architectural vasculature of the decellularized liver. In addition, systems to monitor liver re-cellularization of the entire vasculature and the bile duct based on imaging techniques (micro computed tomography and magnetic resonance imaging) have been developed. Based on these techniques. It was found that up to 80-90% of the vessels in the all liver were adequately re-cellularized with endothelial cells and about 60-80% of the bile ducts were adequately re-cellularized with cholangiocytes using the methods described herein. This data demonstrate that hepatocytes, endothelial and bile duct cells can be seeded in the re-cellularized grafts with great efficiency and limited damage. The functionality of the engineered liver vasculature in the organ culture system was analyzed by the evaluation of the intake of acetylated low-density lipoprotein (ac-LDL) using confocal microscopy, a characteristic of endothelial cells to use the “scavenger cell pathway” of LDL metabolism. Additionally, tissue plasminogen activator (tPA) secretory ability was measured in the culture medium (a protein involved in the breakdown of blood clots) after the exposure of vitamin D, and endothelial gene expression was also characterized.
Acetylated low-density lipoprotein (ac-LDL) is known to be incorporated into microvascular endothelial cells. Uptake of fluorescence-labeled ac-LDL was evaluated and, as expected, the newly engineered liver vasculature took up Dil-labeled (Dil is available commercially, for example from Life Technologies) acetylated low-density lipoprotein (Ac-LDL), a specific function of endothelial cells in vitro, and demonstrated the detailed three-dimensional structure of the portal and central venous system (
Analysis of the expression of endothelial cell-related genes via quantitative RT-PCR after whole liver vasculature engineering revealed that expression levels of genes related to angiogenesis (endothelial cell growth and remodeling) and coagulation in the re-cellularized liver vasculature were similar to those measured in 3D-fibronectin cultures (
Next, it was determined whether the biliary system could be re-assembled in the decellularized livers, a prerequisite for producing a functional liver graft. The matrix of the biliary system was repopulated with a total of 6×106 bile duct epithelial cells through the matrix of the main bile duct. For quantification, iron-fluorescent-microparticle-labeled cells were used. Optimization was based on the percentage of the bile duct area lined by infused cells. Micro-imaging revealed that 59±24% of the bile ducts could be repopulated (
Assembly and Function of Bioengineered Liver
To test hepatic function, the functional characteristics of the three engrafted cellular compartments (hepatocytes, bile duct cells, and microvascular endothelial cells) in the decellularized matrix were also analyzed.
As described above, hepatic function was analyzed via immunostaining of cytokeratin 19 (CK19) for bile duct cells, albumin for hepatocytes, and Von Willebrand factor for microvascular endothelial cells (
To assess the metabolic activity of engrafted hepatocytes, albumin urea synthesis, production, and total bile acid secretion were measured. The cumulative urea, albumin and total bile acids amounts in the re-cellularized liver system were not different within the experimental groups and not higher than hepatocyte sandwich culture during the 9 days culture period (
Long-Term Function and Regeneration Capacity Following Auxiliary Transplantation
Establishment of an auxiliary liver transplantation model in albumin-deficient mutant rats. Previously, the survival of bioengineered decellularized liver grafts has been limited to a few hours as a result of vascular thrombosis or bleeding following the use of systemic anticoagulation. To avoid these complications, liver grafts were bioengineered to incorporate anti-thrombotic activity (
The next step was to develop an auxiliary liver transplant model in order to investigate engraftment, long-term function and the regenerative capacity of the assembled liver grafts (
As described, Nagase analbuminemic rats (NARs) were preconditioned by retrorsine treatment before transplantation in some studies to impair host hepatocyte replication capacity, allowing a regenerative advantage to the donor liver graft. To assess function and an increase in the mass of donor hepatocytes in the transplants, serum albumin was serially measured after transplantation (
Prior to APLT, the recipient animal was injected with retrorsine and underwent a reduction of portal blood flow at the time of APLT, to create an environment where there was a selective growth advantage to transplanted grafts. The auxiliary partial graft was obtained by resection of the donor median and left lateral lobes, and was heterotopically transplanted into the recipient. Portal-portal anastomosis and infrahepatic-infrahepatic vena cava anastomosis were performed in an end-to-side manner and bile duct was implanted into the duodenum of the recipient. Graft survival was evaluated over time (up to 28 days) by graft weight, histological evaluation of proliferative markers and serum albumin levels in analbuminemic rats. FK506-based immunosuppression protocol effectively control graft rejection. Transplanted grafts revealed regenerative potential as evaluated by increase of liver mass weight of the donor graft. Serum albumin levels were maintained for the duration of the study. A novel auxiliary partial liver transplantation in rats for the future evaluation of engineered liver grafts was thus developed and standardized (
The regenerative effect of retrorsine preconditioning was not evident for the first days after auxiliary liver transplant. However, serum albumin levels increased continuously in retrorsine-conditioned recipient rats, reaching levels of 3.04±0.36 mg/mL on day 17 after transplantation, whereas, and levels in naive rats were 0.18±0.11 mg/mL. In contrast serum albumin levels were 16.71±0.60 mg/mL in retrorsine-conditioned recipient rats transplanted with normal liver grafts. Thus, the lower but parallel upward trend of serum albumin levels in assembled liver-transplanted conditioned-NAR recipients was approximately 18% that in animals transplanted with normal liver grafts. These results suggest that assembled liver grafts while functionally inferior to transplanted normal liver grafts (approximately one-fifth), demonstrated a proliferative/regenerative response when transplanted into animals preconditioned to deliver a regenerative stimulus to the graft (
The highest serum albumin levels in retrorsine-conditioned recipient rats transplanted with normal liver grafts were observed around 14-17 days after transplant, thus, further histological analysis was performed at this time. At seventeen days after transplantation into retrorsine conditioned recipients the diameter of assembled liver grafts measured from 2-3 cm and had the color and texture of a normal liver (
Modification of Whole Liver Vascular Surface to Prevent Acute Thrombosis
The objective here was to achieve interruption of acute thrombosis in polyethylene-glycol-modified vascular surface of engineered liver grafts after re-connection to portal vein blood flow. It was previously demonstrated that modifying an injured vascular surface with a protein-reactive polymer could block undesirable platelet deposition (J Biomed Mater Res. 1998,41(2):251-6; J Vasc Surg. 2012, 55(4):1087-95). For this purpose, the utility of surface modification using a protein-reactive polymer, Nhydroxysuccinimide-polyethylene glycol, NHS-PEG to block platelet activation, deposition and formation of thrombus were evaluated. The entire vascular surfaces of the decellularized livers were coated, as indicated below (
Additionally, thrombus formation was quantified. Briefly, as described above the ECM-surface was modified with N-hydroxysuccinimide-polyethylene glycol (NHS-PEG) and was conjugated with biotin for detection purposes. 50 mg/mL of NHS-PEG-biotin cover 73±8% of the decellularized liver surface area. The ability of the coating to limit thrombosis was then tested by perfusion of coated livers with blood for approximately 15 min directly through the portal vein. PEG-NHS coated decellularized livers were reconnected to the blood flow by portal-portal anastomosis. Platelet deposition and thrombus formation was analyzed at several early time points (t=0, t=5, t=10, t=15, t=20, t=30). Thrombus formation was evaluated by: i) immunohistochemical analysis of CD41, ii) scanning electron microscope for platelet deposition and iii) measurement of blood pressure of the portal-portal anastomosis. There was a significant reduction in thrombus formation in the perfused NHS-PEG-coated decellularized livers (
Assembling Liver Grafts for Transplantation
As described above, it was demonstrated that hepatocytes, endothelial and bile duct epithelial cells can be seeded into the whole-liver scaffolds and kept viable while providing essential liver functions. It was also demonstrated that acute thrombosis of decellularized whole livers after transplantation can be attenuated with re-endothelialization and vascular surface modification using protein-reactive polymers. Additionally, a clinically relevant rat model of auxiliary liver transplantation was described. Taken together, all this data demonstrated that re-cellularization protocols are compatible and can be performed efficiently while minimizing damage. Thus, the next step was to design the methods to engineer functional liver grafts and demonstrate long-term survival after transplantation.
Following the above protocols in 5 general steps produced transplantable liver grafts that survive for long-term (up to 17 days, at which time transplanted animals were sacrificed). The liver grafts transplanted in Retrorsine-treated Nagase rats demonstrated histological areas of liver sinusoidal tissue similar to normal liver. Histological tissue of the assembled and transplanted liver grafts was recovered after 3 and 17 days. H&E analysis demonstrated areas that showed liver tissue around the larger vessels, populating the surrounding parenchyma, and areas populated with inflammatory cells. These results demonstrate that the methods developed here are crucial to assembled liver grafts that achieve long term survival and function (17 days) compared to the previously published survival of assembled liver grafts (8 hours).
Scalability of Organ Decellularization Protocol
Scalability of Organ Decellularization Protocol. Different protocols for whole rat liver decellularization have been developed. To determine if the decellularization protocol was feasible in large livers, native whole porcine livers, which are similar to human in size and anatomy, were utilized. The decellularization protocol consisted first of a freezing-thawing technique for at least 12 hours to induce cellular lysis. The whole organ decellularization was achieved then by portal perfusion with sodium dodecyl sulfate (SDS), which is an anionic detergent that simultaneously can lyse cells and solubilize cytoplasmic components. The protocol was based on the rat liver decellularization protocol that was previously described above. Decellularization was achieved by perfusing the liver with sodium dodecyl sulfate (SDS; Sigma, St. Louis, MO, USA) in deionized water for a total of 72-96 h starting with 0.01% SDS for 24 h followed by 0.1% SDS for another 24 h, which was followed by 1% SDS for 48 h or more. Subsequently, the liver was washed with deionized water 15 min and with 1% Triton X-100 (Sigma) for 30 min. The decellularized livers were washed with PBS for 1 h. The liver bioscaffold was sterilized in 0.1% peracetic acid (Sigma) in PBS for 3 h. The liver bioscaffold was washed extensively with sterile PBS and preserved in PBS supplemented with antibiotics and kept at 4° C. for up to 7 days. (Yagi et al. Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach. Cell Transplant. 2013;22(2):231-42.) The objective of the studies below was to establish an effective and minimally disruptive method for the decellularization of intact porcine whole liver and to demonstrate that reconstitution of liver parenchyma is possible using the methodology developed in the rat model. Moreover, the bioreactors used to assemble whole livers were upscaled, and the anti-thrombotic studies previously developed in rodent studies were translated to the porcine model. The methods and techniques established in these studies represent a significant step towards the decellularization, re-cellularization and transplantation procedures necessary for a successful regenerative medicine approach to liver bioengineering for transplantation at a human scale.
Immunological reaction of the remaining materials of the decellularized liver matrix has to be avoided if further clinical application is intended in order to elude any inflammatory reactions. As porcine livers have a much larger tissue density and area, the DNA content of the different areas and lobes was analyzed in order to measure the homogeneity of the decellularization process. Samples involved the right lateral, right median, left median and left lateral lobe of the decellularized whole liver (
A customized organ culture chamber, which was specifically constructed for a large-scale organ perfusion was developed; the perfusion system was designed based on previously developed system for rat liver that consisted of a peristaltic pump, bubble trap, and oxygenator. The system was placed in an incubator for temperature control, and the oxygenator was connected to atmospheric gas mixture. The graft was continuously perfused through the portal vein at 4 ml/min with continuous oxygenation that delivered an inflow partial oxygen tension of ˜300 mmHg.
The experiments and analyses above show:
i) Establishment of easy-to-use systems to monitor qualitatively the organ decellularization process based on a) multiphoton fluorescence microscopy, b) differential scanning calorimeter (DSC) analysis, c) DNA content and d) histological analysis of structural and basement membrane components (fibronectin and laminin);
ii) Optimized re-cellularization protocols for the vascular system (portal vein, central vein) and characterization of the functionality of the engineered liver vasculature based on a) histological evaluation, b) Ac-LDL incorporation, c) tPA reactive secretion and d) gene expression;
iii) Establishment of optimized re-cellularization protocols that combine three different compartments a) hepatocytes, b) microvascular endothelial cells and c) bile duct cells. Hepatic functionality using liver grafts re-cellularized with three different cell types is also reported;
iv) Design of protocols for the re-cellularization of the bile duct system and histological evaluation revealed that up to 60-70% of the bile ducts in the decellularized liver can be adequately re-cellularized with biliary epithelial cells;
v) Establishment and standardization of a clinically relevant model of Auxiliary Partial Liver Transplantation in the rat. This model represents a driving force of the laboratory as optimized protocols of liver engineering can easily be tested and validated. Immune-suppressed Nagase rats (analbuminemic rats) can be used, and serum albumin levels evaluated by ELISA to monitor the function of the transplanted graft show that the engineered tissue prepared according to the above provide such functionality;
vi) Development of an engineered liver graft with anti-thrombotic activity to achieve long-term survival after transplantation using optimized protocols to reconstitute the liver parenchyma and vascular endothelialization and polymer-based vascular surface modification to block acute thrombosis; and
vii) Establishment of scaled-up methods and techniques in porcine livers based on the systems developed in the rodent models (
The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.
This application claims priority to U.S. Provisional Patent Application No. 61/985,690, filed Apr. 29, 2014, the contents of which are incorporated herein by reference in their entirety.
This invention was made with government support under Grant No. DK083556 awarded by the National Institutes of Health. The government has certain rights in the invention. Financial support for this invention was also provided under the Research Center Network for Realization of Regenerative Medicine, provided by the Japan Agency for Medical Research and Development (AMED).
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/028238 | 4/29/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61985690 | Apr 2014 | US |
