WO2007025233A1 - Decellularization and recellularization of organs and tissues - Google Patents
Decellularization and recellularization of organs and tissues Download PDFInfo
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- WO2007025233A1 WO2007025233A1 PCT/US2006/033415 US2006033415W WO2007025233A1 WO 2007025233 A1 WO2007025233 A1 WO 2007025233A1 US 2006033415 W US2006033415 W US 2006033415W WO 2007025233 A1 WO2007025233 A1 WO 2007025233A1
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Definitions
- This invention relates to organs and tissues, and more particularly to methods and materials for decellularizing and recellularizing organs and tissues.
- Biologically derived matrices have been developed for tissue engineering and regeneration.
- the matrices developed to date generally have a compromised matrix structure and/or do not exhibit a vascular bed that allows for effective reconstitution of the organ or tissue.
- This disclosure describes methods for decellularization and recellularization of organs and tissues.
- the invention provides for methods and materials to decellularize an organ or tissue as well as methods and materials to recellularize a decellularized organ or tissue.
- the invention provides for a decellularized mammalian heart.
- a decellularized mammalian heart includes a decellularized extracellular matrix of the heart that has an exterior surface.
- the extracellular matrix of a decellularized heart substantially retains the morphology of the extracellular matrix prior to decellularization, and the exterior surface of the extracellular matrix is substantially intact.
- Representative hearts include but are not limited to rodent hearts, pig hearts, rabbit hearts, bovine hearts, sheep hearts, or canine hearts.
- Another representative heart is a human heart.
- the decellularized heart can be cadaveric, hi some embodiment, the decellularized heart is a portion of an entire heart.
- a portion of an entire heart can include, without limitation, a cardiac patch, an aortic valve, a mitral valve, a pulmonary valve, a tricuspid valve, a right atrium, a left atrium, a right ventricle, a left ventricle, septum, coronary vasculature, a pulmonary artery, or a pulmonary vein.
- the invention provides for a solid organ.
- a solid organ as described herein includes the decellularized heart described above and a population of regenerative cells attached thereto.
- the regenerative cells are pluripotent cells.
- the regenerative cells are embryonic stem cells, umbilical cord cells, adult-derived stem or progenitor cells, bone marrow-derived cells, blood-derived cells, mesenchymal stem cells (MSC), skeletal muscle-derived cells, multipotent adult progenitor cells (MAPC), cardiac stem cells (CSC), or multipotent adult cardiac-derived stem cells, hi some embodiments, the regenerative cells are cardiac fibroblasts, cardiac micro vasculature cells, or aortic endothelial cells.
- the number of the regenerative cells attached to the decellularized heart is at least about 1,000. In some embodiments, the number of the regenerative cells attached to the decellularized heart is about 1,000 cells/mg tissue (wet weight; i.e., pre-decellularized weight) to about 10,000,000 cells/mg tissue (wet weight). In some embodiments, the regenerative cells are heterologous to the decellularized heart. Also in some embodiments, the solid organ is to be transplanted into a patient and the regenerative cells are autologous to the patient.
- the invention provides a method of making a solid organ.
- Such a method generally includes providing a decellularized heart as described herein, and contacting the decellularized heart with a population of regenerative cells under conditions in which the regenerative cells engraft, multiply and/or differentiate within and on the decellularized heart.
- the regenerative cells are injected or perfused into the decellularized heart.
- the invention provides for a method of decellularizing a heart.
- Such a method includes providing a heart, cannulating the heart at one or more than one cavity, vessel, and/or duct to produce a cannulated heart, and perfusing the cannulated heart with a first cellular disruption medium via the one or more than one cannulations.
- the perfusion can be multi-directional from each cannulated cavity, vessel, and/or duct.
- the cellular disruption medium comprises at least one detergent such as SDS, PEG, or Triton X.
- Such a method also can include perfusing the cannulated heart with a second cellular disruption medium via the more than one cannulations.
- the first cellular disruption medium can be an anionic detergent such as SDS and the second cellular disruption medium can be an ionic detergent such as Triton X.
- the perfusing can be for about 2 to 12 hours per gram (wet weight) of heart tissue.
- Figure 1 is a schematic showing the initial preparation for the decellularization of a heart.
- the aorta, pulmonary artery, and superior caval vein are cannulated (A, B, C, respectively), and the inferior caval vein, brachiocephalic artery, left common carotid artery, and left subclavian artery are ligated. Arrows indicate the direction of perfusion in antegrade and retrograde.
- Figure 2 is a schematic of one embodiment of a decellularization / recellularization apparatus.
- Solid organs generally have three main components, the extracellular matrix (ECM), cells embedded therein, and a vasculature bed.
- ECM extracellular matrix
- a decellularized solid organ then can be used as a scaffold for recellularization.
- Mammals from which solid organs can be obtained include, without limitation, rodents, pigs, rabbits, cattle, sheep, dogs, and humans.
- Organs and tissues used in the methods described herein can be cadaveric.
- Solid organs as referred to herein include, without limitation, heart, liver, lungs, skeletal muscles, brain, pancreas, spleen, kidneys, uterus, and bladder.
- a solid organ as used herein refers to an organ that has a "substantially closed” vasculature system.
- a “substantially closed” vasculature system with respect to an organ means that, upon perfusion with a liquid, the majority of the liquid is contained within the solid organ and does not leak out of the solid organ, assuming the major vessels are cannulated, ligated, or otherwise restricted.
- many of the solid organs listed above have defined “entrance” and “exit” vessels which are useful for introducing and moving the liquid throughout the organ during perfusion.
- vascularized organs or tissues such as, for example, all or portions of joints (e.g., knees, shoulders, or hips), trachea, or spinal cord can be decellularized using the methods disclosed herein. Further, the methods disclosed herein also can be used to decellularize avascular tissues such as, for example, cartilage or cornea.
- a decellularized organ or tissue as described herein e.g., heart or liver
- any portion thereof e.g., an aortic valve, a mitral valve, a pulmonary valve, a tricuspid valve, a pulmonary vein, a pulmonary artery, coronary vasculature, septum, a right atrium, a left atrium, a right ventricle, or a left ventricle
- a recellularized organ or tissue as described herein can be used to examine, for example, cells undergoing differentiation and/or the cellular organization of an organ or tissue.
- the invention provides for methods and materials to decellularize a mammalian organ or tissue.
- the initial step in decellularizing an organ or tissue is to cannulate the organ or tissue, if possible.
- the vessels, ducts, and/or cavities of an organ or tissue can be cannulated using methods and materials known in the art.
- the next step in decellularizing an organ or tissue is to perfuse the cannulated organ or tissue with a cellular disruption medium.
- Perfusion through an organ can be multi-directional (e.g., antegrade and retrograde). Langendorff perfusion of a heart is routine in the art, as is physiological perfusion (also known as four chamber working mode perfusion).
- aorta is cannulated and attached to a reservoir containing cellular disruption medium.
- a cellular disruption medium can be delivered in a retrograde direction down the aorta either at a constant flow rate delivered, for example, by an infusion or roller pump or by a constant hydrostatic pressure.
- the aortic valves are forced shut and the perfusion fluid is directed into the coronary ostia (thereby perfusing the entire ventricular mass of the heart), which then drains into the right atrium via the coronary sinus.
- a second cannula is connected to the left atrium and perfusion can be changed from retrograde to antegrade.
- Methods are known in the art for perfusing other organ or tissues. By way of example, the following references describe the perfusion of lung, liver, kidney, brain, and limbs. Van Putte et al., 2002, Ann. Thorac. Surg., 74(3):893- 8; den Butter et al., 1995, Transpl.
- a cellular disruption medium generally includes at least one detergent such as SDS, PEG, or Triton X.
- a cellular disruption medium can include water such that the medium is osmotically incompatible with the cells.
- a cellular disruption medium can include a buffer (e.g., PBS) for osmotic compatibility with the cells.
- Cellular disruption media also can include enzymes such as, without limitation, one or more collagenases, one or more dispases, one or more DNases, or a protease such as trypsin.
- cellular disruption media also or alternatively can include inhibitors of one or more enzymes (e.g., protease inhibitors, nuclease inhibitors, and/or collegenase inhibitors).
- a cannulated organ or tissue can be perfused sequentially with two different cellular disruption media.
- the first cellular disruption medium can include an anionic detergent such as SDS and the second cellular disruption medium can include an ionic detergent such as Triton X.
- a cannulated organ or tissue can be perfused, for example, with wash solutions and/or solutions containing one or more enzymes such as those disclosed herein. Alternating the direction of perfusion (e.g., antegrade and retrograde) can help to effectively decellularize the entire organ or tissue. Decellularization as described herein essentially decellularizes the organ from the inside out, resulting in very little damage to the ECM.
- An organ or tissue can be decellularized at a suitable temperature between 4 and 40°C.
- an organ or tissue generally is perfused from about 2 to about 12 hours per gram of solid organ or tissue with cellular disruption medium. Including washes, an organ may be perfused for up to about 12 to about 72 hours per gram of tissue. Perfusion generally is adjusted to physiologic conditions including pulsatile flow, rate and pressure.
- a decellularized organ or tissue consists essentially of the extracellular matrix (ECM) component of all or most regions of the organ or tissue, including ECM components of the vascular tree.
- ECM components can include any or all of the following: fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, ground substance, reticular fibers and thrombospondin, which can remain organized as defined structures such as the basal lamina.
- Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in histologic sections using standard histological staining procedures.
- residual cell debris also has been removed from the decellularized organ or tissue.
- morphology and the architecture of the ECM be maintained (i.e., remain substantially intact) during and following the process of decellularization.
- “Morphology” as used herein refers to the overall shape of the organ or tissue or of the ECM, while “architecture” as used herein refers to the exterior surface, the interior surface, and the ECM therebetween.
- the morphology and architecture of the ECM can be examined visually and/or histologically.
- the basal lamina on the exterior surface of a solid organ or within the vasculature of an organ or tissue should not be removed or significantly damaged due to decellularization.
- the fibrils of the ECM should be similar to or significantly unchanged from that of an organ or tissue that has not been decellularized.
- One or more compounds can be applied in or on a decellularized organ or tissue to, for example, preserve the decellularized organ, or to prepare the decellularized organ or tissue for recellularization and/or to assist or stimulate cells during the recellularization process.
- Such compounds include, but are not limited to, one or more growth factors (e.g., VEGF, DKK-I, FGF, BMP-I, BMP-4, SDF-I, IGF, and HGF), immune modulating agents (e.g., cytokines, glucocorticoids, IL2R antagonist, leucotriene antagonists), and/or factors that modify the coagulation cascade (e.g., aspirin, heparin-binding proteins, and heparin).
- growth factors e.g., VEGF, DKK-I, FGF, BMP-I, BMP-4, SDF-I, IGF, and HGF
- immune modulating agents e.g., cytokines, glucocortic
- a decellularized organ or tissue can be further treated with, for example, irradiation (e.g., UV, gamma) to reduce or eliminate the presence of any type of microorganism remaining on or in a decellularized organ or tissue.
- irradiation e.g., UV, gamma
- the invention provides for materials and methods for generating an organ or tissue.
- An organ or tissue can be generated by contacting a decellularized organ or tissue as described herein with a population of regenerative cells.
- Regenerative cells as used herein are any cells used to recellularize a decellularized organ or tissue.
- Regenerative cells can be totipotent cells, pluripotent cells, or multipotent cells, and can be uncommitted or committed.
- Regenerative cells also can be single-lineage cells.
- regenerative cells can be undifferentiated cells, partially differentiated cells, or fully differentiated cells.
- Regenerative cells as used herein include embryonic stem cells (as defined by the National Institute of Health (NIH); see, for example, the Glossary at stemcells.nih.gov on the World Wide Web). Regenerative cells also include progenitor cells, precursor cells, and "adult"- derived stem cells including umbilical cord cells and fetal stem cells. Examples of regenerative cells that can be used to recellularize an organ or tissue include, without limitation, embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow-derived step or progenitor cells, blood-derived stem or progenitor cells, mesenchymal stem cells (MSC), skeletal muscle-derived cells, or multipotent adult progentitor cells (MAPC).
- NASH National Institute of Health
- MSC mesenchymal stem cells
- skeletal muscle-derived cells or multipotent adult progentitor cells
- Additional regenerative cells that can be used include cardiac stem cells (CSC), multipotent adult cardiac-derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, or aortic endothelial cells.
- CSC cardiac stem cells
- BM-MNC bone marrow mononuclear cells
- EPC endothelial progenitor cells
- BM-MNC bone marrow mononuclear cells
- EPC endothelial progenitor cells
- the number of regenerative cells that is introduced into and onto a decellularized organ in order to generate an organ or tissue is dependent on both the organ (e.g., which organ, the size and weight of the organ) or tissue and the type and developmental stage of the regenerative cells. Different types of cells may have different tendencies as to the population density those cells will reach. Similarly, different organ or tissues may be cellularized at different densities.
- a decellularized organ or tissue can be "seeded" with at least about 1,000 (e.g., at least 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000) regenerative cells; or can have from about 1,000 cells/mg tissue (wet weight, i.e., prior to decellularization) to about 10,000,000 cells/mg tissue (wet weight) attached thereto.
- Regenerative cells can be introduced ("seeded") into a decellularized organ or tissue by injection into one or more locations.
- more than one type of cell i.e., a cocktail of cells
- a cocktail of cells can be introduced at multiple positions in a decellularized organ or tissue or different cell types can be injected into different portions of a decellularized organ or tissue.
- regenerative cells or a cocktail of cells can be introduced by perfusion into a cannulated decellularized organ or tissue.
- regenerative cells can be perfused into a decellularized organ using a perfusion medium, which can then be changed to an expansion and/or differentiation medium to induce growth and/or differentiation of the regenerative cells.
- an organ or tissue is maintained under conditions in which at least some of the regenerative cells can multiply and/or differentiate within and on the decellularized organ or tissue. Those conditions include, without limitation, the appropriate temperature and/or pressure, electrical and/or mechanical activity, force, the appropriate amounts of O 2 and/or CO 2 , an appropriate amount of humidity, and sterile or near-sterile conditions.
- the decellularized organ or tissue and the regenerative cells attached thereto are maintained in a suitable environment.
- the regenerative cells may require a nutritional supplement (e.g., nutrients and/or a carbon source such as glucose), exogenous hormones or growth factors, and/or a particular pH.
- Regenerative cells can be allogeneic to a decellularized organ or tissue (e.g., a human decellularized organ or tissue seeded with human regenerative cells), or regenerative cells can be xenogeneic to a decellularized organ or tissue (e.g., a pig decellularized organ or tissue seeded with human regenerative cells).
- Allogeneic refers to cells obtained from the same species as that from which the organ or tissue originated (e.g., related or unrelated individuals), while “xenogeneic” as used herein refers to cells obtained from a species different than that from which the organ or tissue originated.
- an organ or tissue generated by the methods described herein is to be transplanted into a patient.
- the regenerative cells used to recellularize a decellularized organ or tissue can be obtained from the patient such that the regenerative cells are "autologous" to the patient.
- Regenerative cells from a patient can be obtained from, for example, blood, bone marrow, tissues, or organs at different stages of life (e.g., prenatally, neonatally or perinatally, during adolescence, or as an adult) using methods known in the art.
- regenerative cells used to recellularize a decellularized organ or tissue can be syngeneic (i.e., from an identical twin) to the patient, regenerative cells can be human lymphocyte antigen (HLA)-matched cells from, for example, a relative of the patient or an HLA-matched individual unrelated to the patient, or regenerative cells can be allogeneic to the patient from, for example, a non-HLA-matched donor.
- HLA human lymphocyte antigen
- the decellularized solid organ can be autologous, allogeneic or xenogeneic to a patient.
- a decellularized organ may be recellularized with cells in vivo (e.g., after the organ or tissue has been transplanted into an individual).
- In vivo recellularization may be performed as described above (e.g., injection and/or perfusion) with, for example, any of the regenerative cells described herein.
- in vivo seeding of a decellularized organ or tissue with endogenous cells may occur naturally or be mediated by factors delivered to the recellularized tissue.
- the progress of regenerative cells can be monitored during recellularization. For example, the number of cells on or in an organ or tissue can be evaluated by taking a biopsy at one or more time points during recellularization.
- the amount of differentiation that regenerative cells have undergone can be monitored by determining whether or not various markers are present in a cell or a population of cells. Markers associated with different cells types and different stages of differentiation for those cell types are known in the art, and can be readily detected using antibodies and standard immunoassays. See, for example, Current Protocols in Immunology, 2005, Coligan et al, Eds., John Wiley & Sons, Chapters 3 and 11. Nucleic acid assays as well as morphological and/or histological evaluation can be used to monitor recellularization.
- the invention also provides for a system (e.g., a bioreactor) for decellularizing and/or recellularizing an organ or tissue.
- a system e.g., a bioreactor
- Such a system generally includes at least one cannulation device for cannulating an organ or tissue, a perfusion apparatus for perfusing the organ or tissue through the cannula(s), and means (e.g., a containment system) to maintain a sterile environment for the organ or tissue.
- Cannulation and perfusion are well-known techniques in the art.
- a cannulation device generally includes size-appropriate hollow tubing for introducing into a vessel, duct, and/or cavity of an organ or tissue. Typically, one or more vessels, ducts, and/or cavities are cannulated in an organ.
- a perfusion apparatus can include a holding container for the liquid (e.g., a cellular disruption medium) and a mechanism for moving the liquid through the organ (e.g., a pump, air pressure, gravity) via the one or more cannulae.
- a holding container for the liquid e.g., a cellular disruption medium
- a mechanism for moving the liquid through the organ e.g., a pump, air pressure, gravity
- the sterility of an organ or tissue during decellularization and/or recellularization can be maintained using a variety of techniques known in the art such as controlling and filtering the air flow and/or perfusing with, for example, antibiotics, antifungals or other anti-microbials to prevent the growth of unwanted microorganisms.
- a system to decellularize and recellularize organ or tissues as described herein can possess the ability to monitor certain perfusion characteristics (e.g., pressure, volume, flow pattern, temperature, gases, pH), mechanical forces (e.g., ventricular wall motion and stress), and electrical stimulation (e.g., pacing).
- perfusion characteristics e.g., pressure, volume, flow pattern, temperature, gases, pH
- mechanical forces e.g., ventricular wall motion and stress
- electrical stimulation e.g., pacing
- a pressure-regulated perfusion apparatus is advantageous to avoid large fluctuations.
- the effectiveness of perfusion can be evaluated in the effluent and in tissue sections. Perfusion volume, flow pattern, temperature, partial O 2 and CO 2 pressures and pH can be monitored using standard methods.
- Sensors can be used to monitor the system (e.g., bioreactor) and/or the organ or tissue.
- Sonomicromentry, micromanometry, and/or conductance measurements can be used to acquire pressure-volume or preload recruitable stroke work information relative to myocardial wall motion and performance.
- sensors can be used to monitor the pressure of a liquid moving through a cannulated organ or tissue; the ambient temperature in the system and/or the temperature of the organ or tissue; the pH and/or the rate of flow of a liquid moving through the cannulated organ or tissue; and/or the biological activity of a recellularizing organ or tissue, hi addition to having sensors for monitoring such features, a system for decellularizing and/or recellularizing an organ or tissue also can include means for maintaining or adjusting such features.
- Means for maintaining or adjusting such features can include components such as a thermometer, a thermostat, electrodes, pressure sensors, overflow valves, valves for changing the rate of flow of a liquid, valves for opening and closing fluid connections to solutions used for changing the pH of a solution, a balloon, an external pacemaker, and/or a compliance chamber.
- a thermometer e.g., a thermostat
- electrodes e.g., electrodes
- pressure sensors e.g., pressure sensors
- overflow valves e.g., valves for changing the rate of flow of a liquid
- valves for opening and closing fluid connections to solutions used for changing the pH of a solution e.g., a balloon, an external pacemaker, and/or a compliance chamber.
- the chambers, reservoirs and tubings can be water-jacketed.
- a balloon inserted into the left ventricle via the left atrium can be used to place mechanical stress on a heart.
- a piston pump that allows adjustment of volume and rate can be connected to the balloon to simulate left ventricular wall motion and stress.
- left ventricular wall motion and pressure can be measured using micromanometry and/or sonomicrometry.
- an external pacemaker can be connected to a piston pump to provide synchronized stimulation with each deflation of the ventricular balloon (which is equivalent to the systole).
- Peripheral ECG can be recorded from the
- Mechanical ventricular distention can also be achieved by attaching a peristaltic pump to a canula inserted into the left ventricle through the left
- ventricular distention achieved by periodic fluid movement (e.g., pulsatile flow) through the canula can be synchronized with electrical stimulation.
- a mammalian heart can be decellularized and recellularized and, when maintained under the
- a functional heart that undergoes contractile function and responds to pacing stimuli and/or pharmacologic agents can be generated.
- This recellularized functional heart can be transplanted into a mammal and function for a period of time.
- Figure 2 shows one embodiment of a system for decellularizing and/or
- the embodiment shown is a bioreactor for decellularizing and recellularizing a heart.
- This embodiment has an adjustable rate and volume peristaltic pump (A); an adjustable rate and volume piston pump connected to an intraventricular balloon (B); an adjustable voltage, frequency and amplitude external pacemaker (C); an ECG recorder (D); a pressure sensor in the 'arterial line' (which equals coronary artery pressure) (E); a pressure sensor in the 'venous' line (which equals coronary sinus pressure) (F); and synchronization between the pacemaker and the piston pump (G).
- a system for generating an organ or tissue can be controlled by a computer-readable storage medium in combination with a programmable processor (e.g., a computer-readable storage medium as used herein has instructions stored thereon for causing a programmable processor to perform particular steps).
- a storage medium in combination with a programmable processor, can receive and process information from one or more of the sensors.
- Such a storage medium in conjunction with a programmable processor also can transmit information and instructions back to the bioreactor and/or the organ or tissue.
- An organ or tissue undergoing recellularization can be monitored for biological activity.
- the biological activity can be that of the organ or tissue itself such as electrical activity, mechanical activity, mechanical pressure, contractility, and/or wall stress of the organ or tissue.
- the biological activity of the cells attached to the organ or tissue can be monitored, for example, for ion transport/exchange activity, cell division, and/or cell viability. See, for example, Laboratory Textbook of Anatomy and Physiology (2001, Wood, Prentice Hall) and Current Protocols in Cell Biology (2001, Bonifacino et al., Eds, John Wiley & Sons).
- a computer- readable storage medium of the invention in combination with a programmable processor, can be used to coordinate the components necessary to monitor and maintain an active load on an organ or tissue.
- the weight of an organ or tissue can be entered into a computer-readable storage medium as described herein, which, in combination with a programmable processor, can calculate exposure times and perfusion pressures for that particular organ or tissue.
- a storage medium can record preload and afterload (the pressure before and after perfusion, respectively) and the rate of flow.
- a computer-readable storage medium in combination with a programmable processor can adjust the perfusion pressure, the direction of perfusion, and/or the type of perfusion solution via one or more pumps and/or valve controls.
- a donor rat was systemically heparinized with 400 U of heparin/kg of donor. Following heparinization, the heart and the adjacent large vessels were carefully removed. The heart was placed in a physiologic saline solution (0.9%) containing heparin (2000 U/ml) and held at 5°C until further processing. Under sterile conditions, the connective tissue was removed from the heart and the large vessels. The inferior venae cava and the left and right pulmonary veins were ligated distal from the right and left atrium using monof ⁇ l, non-resorbable ligatures.
- the heart was mounted on a decellularization apparatus for perfusion (Figure 1).
- the descending thoracic artery was cannulated to allow retrograde coronary perfusion (Figure 1, Cannula A).
- the branches of the thoracic artery e.g., brachiocephalic trunc, left common carotid artery, left subclavian artery
- the pulmonary artery was cannulated before its division into the left and right pulmonary artery ( Figure 1, Cannula B).
- the superior vena cava was cannulated (Figure 1, Cannula C). This configuration allows for both retrograde and antegrade coronary perfusion.
- Example 3 Decellularization After the heart was mounted on the decellularization apparatus, antegrade perfusion was started with cold, heparinized, calcium-free phosphate buffered solution containing 1-5 mmol adenosine per L perfusate to reestablish constant coronary flow. Coronary flow was assessed by measuring the coronary perfusion pressure and the flow, and calculating coronary resistance. After 15 minutes of stable coronary flow, the detergent-based decellularization process was initiated.
- a heart was perfused antegradely with a detergent. After perfusion, the heart can be flushed with a buffer (e.g., PBS) retrogradely. The heart then was perfused with PBS containing antibiotics and then PBS containing DNase I. The heart then was perfused with 1% benzalkonium chloride to reduce microbial contamination and to prevent future microbial contamination, and then perfused with PBS to wash the organ of any residual cellular components, enzymes, or detergent.
- PBS buffer
- Hearts were isolated from 8 male nude rats (250-30Og). Immediately after dissection, the aortic arch was cannulated and the hearts were retrogradely perfused with the indicated detergent. The four different detergent-based decellularization protocols (see below) were compared with respect to their feasibility and efficacy in (a) removing cellular components and (b) preserving vascular structures.
- Decellularization generally included the following steps: stabilization of the solid organ, decellularization of the solid organ, renaturation and/or neutralization of the solid organ, washing the solid organ, degradation of any DNA remaining on the organ, disinfection of the organ, and homeostasis of the organ.
- PEG Decellularization Protocol #1
- Hearts were washed in 200 ml PBS containing 100 U/ml penicillin, 0.1 nig/ml Streptomycin, and 0.25 ⁇ g/ml Amphotericin B with no recirculation. Hearts were then decellularized with 35 ml polyethyleneglycol (PEG; 1 g/ml) for up to 30 minutes with manual recirculation. The organ was then washed with 500 ml PBS for up to 24 hours using a pump for recirculation. The washing step was repeated at least twice for at least 24 hours each time. Hearts were exposed to 35 ml DNase I (70 U/ml) for at least 1 hour with manual recirculation. The organs were washed again with 500 ml PBS for at least 24 hours. B) Decellularisation Protocol #2 (Triton X and Trypsin)
- Hearts were washed in 200 ml PBS containing 100 U/ml Penicillin, 0.1 mg/ml Streptomycin, and 0.25 ⁇ g/ml Amphotericin B for at least about 20 minutes with no recirculation. Hearts were then decellularized with 0.05% Trypsin for 30 min followed by perfusion with 500 ml PBS containing 5% Triton-X and 0.1% ammonium-hydroxide for about 6 hours. Hearts were perfused with deionized water for about 1 hour, and then perfused with PBS for 12 h. Hearts were then washed 3 times for 24 hours each time in 500 ml PBS using a pump for recirculation. The hearts were perfused with 35 ml DNase I (70 U/ml) for 1 hour with manual recirculation and washed twice in 500 ml PBS for at least about 24 hours each time using a pump for recirculation.
- DNase I 70 U/ml
- Hearts were washed in 200 ml PBS containing 100 U/ml Penicillin, 0.1 mg/ml Streptomycin, and 0.25 ⁇ g/ml Amphotericin B for at least about 20 mins with no recirculation.
- the hearts were decellularized with 500 ml water containing 1% SDS for at least about 6 hours using a pump for recirculation.
- the hearts were then washed with deionized water for about 1 hour and washed with PBS for about 12 hours.
- the hearts were washed three times with 500 ml PBS for at least about 24 hours each time using a pump for recirculation.
- the heart was then perfused with 35 ml DNase I (70 U/ml) for about 1 hour using manual recirculation, and washed three times with 500 ml PBS for at least about 24 hours each time using a pump for recirculation.
- Hearts were washed with 200 ml PBS containing 100 U/ml Penicillin, 0.1 mg/ml Streptomycin, and 0.25 ⁇ g/ml Amphotericin B for at least about 20 mins with no recirculation. Hearts were then decellularized with 500 ml water containing 5%Triton X and 0.1% ammonium hydroxide for at least 6 hours using a pump for recirculation. Hearts were then perfused with deionized water for about 1 hour and then with PBS for about 12 hours. Hearts were washed by perfusing with 500 ml PBS 3 times for at least 24 hours each time using a pump for recirculation. Hearts were then perfused with 35 ml DNase I (70 U/ml) for about 1 hour using manual recirculation, and washed three times in 500 ml PBS for about 24 hours each time.
- the decellularisation apparatus was set up within a laminar flow hood. Hearts were perfused at a coronary perfusion pressure of 60 cm H 2 O. Although not required, the hearts described in the experiments above were mounted in a decellularisation chamber and completely submerged and perfused with PBS containing antibiotics for 72 hours in recirculation mode at a continuous flow of 5 ml/min to wash out as many cellular components and detergent as possible.
- Successful decellularization was defined as the lack of myofilaments and nuclei in histologic sections. Successful preservation of vascular structures was assessed by perfusion with 2% Evans Blue prior to embedding tissue sections. Highly efficient decellularization took place when a heart was first perfused antegradely with an ionic detergent (1% sodium-dodecyl-sulfate (SDS), approximately 0.03 M) dissolved in deionized H 2 O at a constant coronary perfusion pressure and then was perfused antegradely with a non-ionic detergent (1% Triton X-100) to remove the SDS and presumably to renature the extracellular matrix (ECM) proteins. Intermittently, the heart was perfused retrogradely with phosphate buffered solution to clear obstructed capillaries and small vessels.
- SDS sodium-dodecyl-sulfate
- Triton X-100 1% Triton X-100
- a decellularized heart is stained via Langendorff perfusion with Evans Blue to stain vascular basement membrane and quantify macro- and micro-vascular density.
- polystyrene particles can be perfused into and through a heart to quantify coronary volume, the level of vessel leakage, and to assess the distribution of perfusion by analyzing coronary effluent and tissue sections.
- a combination of three criteria are assessed and compared to isolated non- decellularised heart: 1) an even distribution of polystyrene particles, 2) significant change in leakiness at some level 3) microvascular density.
- Fiber orientation is assessed by the polarized-light microscopy technique of Tower et al. (2002, Fiber alignment imaging during mechanical testing of soft tissues, Ann Biomed Eng., 30(10):1221-33), which can be applied in real-time to a sample subjected to uniaxial or biaxial stress. During Langendorff perfusion, basic mechanical properties of the decellularised ECM are recorded (compliance, elasticity, burst pressure) and compared to freshly isolated hearts.
- the caval and pulmonary veins, the pulmonary artery and the thoracic aorta were transsected and the heart was removed from the chest.
- a prefilled 1.8 mm aortic canula (Radnoti Glass, Monrovia, CA) was inserted into the ascending aorta to allow retrograde coronary perfusion (Langendorff).
- the hearts were perfused with heparinized PBS (Hyclone, Logan, UT) containing 10 ⁇ M adenosine at a coronary perfusion pressure of 75 cm H 2 O for 15 minutes followed by 1% sodium dodecyl sulfate (SDS) or 1% polyethylene glycol 1000 (PEG 1000) (EMD Biosciences, La Jolla, Germany) or 1% Triton-X 100 (Sigma, St. Louis, MO) in deionized water for 2 - 15 hours. This was followed by 15 minutes of deionized water perfusion and 30 minutes of perfusion with 1% Triton-X (Sigma, St. Louis, MO) in deionized water.
- SDS sodium dodecyl sulfate
- PEG 1000 polyethylene glycol 1000
- Triton-X 100 Sigma, St. Louis, MO
- the hearts were then continuously perfused with antibiotic-containing PBS (100 U/ml penicillin-G (Gibco, Carlsbad, CA), 100 U/ml streptomycin (Gibco, Carlsbad, CA) and 0.25 ⁇ g/ml Amphotericin B (Sigma, St. Louis, MO)) for 124 hours.
- antibiotic-containing PBS 100 U/ml penicillin-G (Gibco, Carlsbad, CA), 100 U/ml streptomycin (Gibco, Carlsbad, CA) and 0.25 ⁇ g/ml Amphotericin B (Sigma, St. Louis, MO)
- antibiotic-containing PBS 100 U/ml penicillin-G (Gibco, Carlsbad, CA)
- streptomycin Gabco, Carlsbad, CA
- Amphotericin B Sigma, St. Louis, MO
- kidney isolation the entire peritoneal content was wrapped in wet gauze and carefully mobilized to the side to expose the retroperitoneal space.
- the mesenteric vessels were ligated and transected.
- the abdominal aorta was ligated and transected below the take off of the renal arteries.
- the thoracic aorta was transected just above the diaphragm and canulated using a 1.8 mm aortic canula (Radnoti Glass, Monrovia, CA).
- the kidneys were carefully removed from the retroperitoneum and submerged in sterile PBS (Hyclone, Logan, UT) to minimize pulling force on the renal arteries.
- Characteristic structures such as Bowman's capsule delineating the glomerulus from surrounding proximal and distal tubules and glomerular capillary basement membranes within the glomeruli were preserved.
- SEM images of decellularized renal medulla showed intact medullary pyramids reaching into the renal pelvis with intact collecting duct basal membranes leading towards the papilla. Thus, all the major ultrastructures of the kidney were intact after decellularization.
- the lung (with the trachea) were carefully removed from the chest and submerged in sterile PBS (Hyclone, Logan, UT) to minimize pulling force on the pulmonary arteries. 15 minutes of heparinized PBS perfusion was followed by 2 - 12 hours of perfusion with 1% SDS (Invitrogen, Carlsbad, CA) in deionized water and 15 minutes of perfusion with 1% Triton-X (Sigma, St. Louis, MO) in deionized water.
- sterile PBS Hyclone, Logan, UT
- the lung was then continuously perfused with antibiotic containing PBS (100 U/ml penicillin-G (Gibco, Carlsbad, CA), 100 U/ml streptomycin (Gibco, Carlsbad, CA), 0.25 ⁇ g/ml Amphotericin B (Sigma, St. Louis, MO)) for 124 hours.
- antibiotic containing PBS 100 U/ml penicillin-G (Gibco, Carlsbad, CA), 100 U/ml streptomycin (Gibco, Carlsbad, CA), 0.25 ⁇ g/ml Amphotericin B (Sigma, St. Louis, MO)
- the caval vein was exposed through a median laparotomy, dissected and canulated using a mouse aortic canula (Radnoti Glass, Monrovia, CA).
- the hepatic artery and vein and the bile duct were transsected and the liver was carefully removed from the abdomen and submerged in sterile PBS (Hyclone, Logan, UT) to minimize pulling force on portal vein.
- 15 minutes of heparinized PBS perfusion was followed by 2 - 12 hours of perfusion with 1% SDS (Invitrogen, Carlsbad, CA) in deionized water and 15 minutes of 1% Triton-X (Sigma, St. Louis, MO) in deionized water.
- the liver was then continuously perfused with antibiotic containing PBS (100 U/ml penicillin-G (Gibco, Carlsbad, CA), 100 U/ml streptomycin (Gibco, Carlsbad, CA), 0.25 ⁇ g/ml Amphotericin B (Sigma, St. Louis, MO)) for 124 hours.
- antibiotic containing PBS 100 U/ml penicillin-G (Gibco, Carlsbad, CA), 100 U/ml streptomycin (Gibco, Carlsbad, CA), 0.25 ⁇ g/ml Amphotericin B (Sigma, St. Louis, MO)
- Movat Pentachrome staining was performed on paraffin embedded decellularized tissues following the manufacturers instructions (American Mastertech Scientific, Lodi, CA). Briefly, deparaffmized slides were stained using Verhoeff s elastic stain, rinsed, differentiated in 2% ferric chloride, rinsed, placed in 5% sodium thiosulfate, rinsed, blocked in 3% glacial acetic acid, stained in 1% alcian blue solution, rinsed, stained in crocein scarlet — acid fuchsin, rinsed, dipped in 1% glacial acetic acid, destained in 5% phosphotungstic acid, dipped in 1% glacial acetic acid, dehydrated, placed in alcoholic saffron solution, dehydrated, mounted and covered.
- Immunofluorescence staining was performed on decellularized tissues. Antigen retrieval was performed on paraffin-embedded tissue (recellularized tissue) but not on frozen sections (decellularized tissue) as follows: Paraffin sections were de- waxed and rehydrated by 2 changes of xylene for 5 minutes each, followed by sequential alcohol gradient and rinsing in cold running tap water. The slides were then placed in antigen retrieval solution (2.94 g tri- sodium citrate, 22 ml of 0.2 M hydrochloric acid solution, 978 ml ultra-pure water, and adjusted to a pH of 6.0) and boiled for 30 minutes. After rinsing under running cold tap water for 10 minutes, immunostaining was begun.
- Tissue samples were then dehydrated in increasing concentrations of EtOH (50% for 10 minutes, 70% for 10 minutes two times, 80% for 10 minutes, 95% for 10 minutes two times, 100% for 10 minutes two times). Tissue samples then underwent critical point drying in a Tousimis Samdri-780A (Tousimis, Rockville, MD). Coating was performed with 30 seconds of Gold/Palladium sputter coating in the Denton DV-502A Vacuum Evaporator (Denton Vacuum, Moorestown, NJ). Scanning electron microscopy images were taken using a Hitachi S4700 Field Emission Scanning Electron Microscope (Hitachi High Technologies America, Pleasanton, CA). Mechanical Testing.
- Fibrin gels were cast into cross-shaped molds with a final concentration of 6.6 mg of fibrin/ml. The average thickness of the fibrin gels was 165.2 ⁇ 67.3 ⁇ m. All samples were attached to a biaxial mechanical testing machine (Instron Corporation, Norwood, MA) via clamps, submerged in PBS, and stretched equibiaxially to 40% strain. In order to probe the static passive mechanical properties accurately, the samples were stretched in increments of 4% strain and allowed to relax at each strain value for at least 60 seconds. Forces were converted to engineering stress by normalizing the force values with the cross sectional area in the specific axis direction (5 mm x initial thickness). Engineering stress was calculated as the displacement normalized by the initial length. In order to compare the data between the two axes as well as between sample groups, a tangential modulus was calculated as follows:
- mESC mouse embryonic stem cells suspended in 1 cc of standard expansion media (Iscove's Modified Dulbecco's Medium (Gibco, Carlsbad, CA), 10% Fetal Bovine Serum
- the decellularized ECM was compatible with cell viability, attachment and proliferation. Seeded mESCs engrafted on the ECM scaffolds and began to invade the matrix within 72 h of cell seeding.
- Example 7 Evaluation of Decellularized Organs
- Aortic valve competence and integrity of the coronary vascular bed of SDS decellularized rat heart was assessed by Langendorff perfusion with 2% Evans blue dye. No left ventricular filling with dye was observed, indicating an intact aortic valve. Macroscopically, filling of the coronary arteries up to the fourth branching point was confirmed without signs of dye leakage. In tissue sections, perfusion of large (150 ⁇ m) and small (20 ⁇ m) arteries and veins was subsequently confirmed by red fluorescence of Evans blue-stained vascular basal membrane.
- SEM Scanning electron micrographs of SDS decellularized cardiac ECM demonstrated that fiber orientation and composition were preserved in aortic wall and aortic valve leaflet with an absence of cells throughout the entire tissue thickness.
- Decellularized left and right ventricular wall retained ECM fiber composition (weaves, struts, coils) and orientation, while myofibers were completely removed.
- intact vascular basal membranes of different diameters without endothelial or smooth muscle cells were observed.
- a thin layer of dense epicardial fibers underneath an intact epicardial basal lamina was retained.
- the stress at 40% strain varied between 5 and 14 kPa in the longitudinal direction and between 15 and 24 kPa in the circumferential direction, which is in agreement with previously published data.
- the circumferential direction was stiffer than the longitudinal direction, most likely due to muscle fiber orientation of the heart. While the fiber orientation changes through the thickness of the cardiac tissue, the majority of the fibers were oriented in the circumferential direction and thus, this direction would be expected to be stiffer.
- the decellularized tissue was significantly stiffer than the intact tissue. This also would be expected since the extracellular matrix is stiffer than the cells themselves, and the combination of ECM and cells would likely not be as stiff as just the ECM alone.
- Pig kidneys were isolated from heparinized male animals. To allow perfusion of the isolated organs, the renal artery was canulated and blood was washed out with PBS perfusion over 15 minutes. Perfusion with 27 L of 1% SDS in deionized water was performed for 35.5 hours at a pressure of 50-100 mmHg. Perfusion with 1% Triton-X in deionized water was initiated to remove SDS from the ECM scaffold. Washing and buffering of the decellularized kidneys was then performed by perfusion with antibiotic containing PBS for 120 hours to remove detergents and obtain a biocompatible pH.
- Organ clearing was observed within two hours of initiating perfusion. Clear white color predominated 12 hours into perfusion. Decellularization was terminated with the organ was white semi-transparent.
- Hearts from F344 rats were prepared by cannulating the aorta distal to the Ao valve and ligating all other great vessels and pulmonary vessels except the left branch of the pulmonary trunk (distal to its bifurcation) and the inferior vena cava (IVC). Decellularization was achieved using Langendorf retrograde coronary perfusion and 2 liters of 1% SDS over 12-16 hours. The hearts were then renatured with 35 mL of 1% Triton-X over 30-40 minutes, and then washed with antibiotic and antifungal-containing PBS for 72 hours. The IVC was ligated before the transplantation. A large (380 to 400 gram) RNU rat was prepared for reception of the decellularized heart.
- a blunt-angled mosquito clamp was applied to both the JVC and the abdominal Ao of the host animal to ensure isolation of areas of anastomosis.
- the aorta of the decellularized heart was anastomosed to the host abdominal aorta proximal and inferior to the renal branches using 8-0 silk suture.
- the left branch of the decellularized heart's pulmonary trunk was anastomosed to the closest region of the host IVC to minimize physical stress on pulmonary trunk.
- the clamp was released after both vessels were sewn into the host animal, and the decellularized heart filled with the host animal's blood.
- the recipient animal's abdominal aortic pressure was observed visually in the decellularized heart and aorta.
- the decellularized heart became distended and red, and bleeding was minimal at the site of anastomosis.
- Heparin was administered (3000 IU) by IP injection 3 minutes after clamp release (initiation of perfusion).
- the heart was photographed and positioned in the abdomen to minimize stress on the sites of anastomosis.
- the abdomen was closed in sterile fashion and the animal monitored for recovery.
- the animal was found dead from hemorrhage at approximately 48 hours after transplantation. Transplantation time is currently in the 55 to 70 minute range.
- a decellularized heart was transferred to an organ chamber and continuously perfused with oxygenised cell culture media under cell culture conditions (5% CO 2 , 60% humiditiy, 37°C).
- 120 x 10 6 PKH labelled HUVECs (suspended in 50 ml of endothelial cell growth media) were infused at 40 cm H 2 O coronary perfusion pressure.
- Coronary effluent was saved and cells were counted.
- the effluent was then recirculated and perfused again to deliver a maximum number of cells. Recirculation was repeated two times. After the third passage, approximately 9O x IO 6 cells were retained within the heart.
- the heart was continuously perfused with 500 ml of recirculating oxygenised endothelial cell culture media for 120 hours. The heart was then removed and embedded for cryosectioning. HUVECs were confined to arterial and venous residues throughout the heart, but were not yet completely dispersed throughout the extravascular ECM.
- the plates were removed from the cooler and placed in a sterile hood on ice. Tissue and trypsin-containing buffer were transferred to 50 ml conical tubes on ice using wide-mouth pipettes. Trypsin Inhibitor (Reagent #3) was reconstituted with 1 ml HBSS (Reagent #1) and added to the 50 ml conical tube and gently mixed. The tissue was oxygenated for 60-90 seconds by passing air over the surface of the liquid. The tissue was then warmed to 37 0 C and collagenase (300 units/ml) reconstituted with 5 ml Leibovitz L-15 was added slowly. The tissue was placed in a warm (37 0 C) shaker bath for 45 minutes.
- the tissue was titrated ten times using a 10 ml pipet to release the cells (3 mis per second) and then strained through a 0.22 ⁇ m filter.
- the tissue was washed with an 5 additional mis of L-15 media, titrated a second time, and collected in the same 50 ml conical tube.
- the solution of cells was then incubated at room temperature for 20 minutes, and spun at 50 xg for five minutes to pellet the cells. The supernatant was gently removed and the cells were resuspended in the desired volume using Neonatal-Cardiomyocyte Media.
- No. 12440-053 was supplemented with 10% Fetal Bovine Serum (HyClone), 100 U/ml penicillin-G (Gibco), 100 U/ml streptomycin (Gibco), 2 mmol/L L-glutamine (Invitrogen), and 0.1 mmol/L 2-mercaptoethanol (Gibco, Cat. No. 21985-023) and sterile filtered before use.
- Amphotericine-B was added as needed (0.25 ⁇ g/ml final concentration). This media was enhanced with 1.2 niM CaCl (Fisher Scientific, Cat. No. C614-500) and 0.8 mM MgCl (Sigma, Cat. No. M-0250).
- the isolated ECM was recellularized with neonatal heart-derived cells.
- Completely decellularized hearts (made as described herein) were injected with a combination of 50 x 10 6 freshly isolated rat neonatal cardiomyocytes, fibrocytes, endothelial and smooth muscle cells.
- the heart tissue was then sliced and the slices were cultured in vitro to test the biocompatibility of the decellularized ECM and the ability of the resulting constructs to develop into myocardium rings.
- these rings were mounted between two rods to measure contractile force under different preload conditions.
- the rings could be electrically paced up to a frequency of 4 Hz and created contractile force of up to 3 niN under a preload of up to 0.65 g.
- contractile tissue was obtained that generated an equally effective force as that generated by optimized engineered heart tissue rings using artificial ECM constructs.
- a more in-depth functional assessment was performed including insertion of a pressure probe into the left ventricle to record left ventricular pressure (LVP) and video recording of wall motion as the stimulation frequency was gradually increased from 0.1 Hz to 10 Hz and performed pharmacological stimulation with phenylephrine (PE).
- LVP left ventricular pressure
- PE phenylephrine
- the recellularized heart showed contractile response to single paces with spontaneous contractions following the paced contractions with corresponding increases in LVP. After a single pace, the heart showed three spontaneous contractions and then converted to a fibrillatory state. Similar to the stimulated contractions, spontaneous depolarizations caused a corresponding increase in LVP and a recordable QRS complex possibly indicating the formation of a developing stable conduction pattern.
- vWF vonWillebrand Factor
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ES06790024T ES2384721T3 (en) | 2005-08-26 | 2006-08-28 | Decellularization and recelularization of organs and tissues |
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