WO2017070392A1 - Appareil et procédés de production de tissus acellulaires pour la régénération d'organes - Google Patents

Appareil et procédés de production de tissus acellulaires pour la régénération d'organes Download PDF

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WO2017070392A1
WO2017070392A1 PCT/US2016/057977 US2016057977W WO2017070392A1 WO 2017070392 A1 WO2017070392 A1 WO 2017070392A1 US 2016057977 W US2016057977 W US 2016057977W WO 2017070392 A1 WO2017070392 A1 WO 2017070392A1
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organ
lung
cells
cell
sequence
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PCT/US2016/057977
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English (en)
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Jason Haruo SAKAMOTO
Filippo BEGARANI
Joan Elizabeth NICHOLS
Joaquin Cortiella
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The Methodist Hospital
Board Of Regents, University Of Texas System
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Publication of WO2017070392A1 publication Critical patent/WO2017070392A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/42—Respiratory system, e.g. lungs, bronchi or lung cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0688—Cells from the lungs or the respiratory tract
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention generally relates to the field of medicine, and in particular, to organ regeneration and transplantation.
  • apparatus and methods for improving decellularization of organ tissues while maintaining the extracellular matrix and scaffold are disclosed.
  • a novel method is provided for the decellularization of whole organs, which improves the preservation of the acellular scaffold.
  • the controlled release of one or more active agents may be accomplished to promote proper cellular differentiation, proliferation, and/or growth for the purpose of regenerating a functional organ.
  • Organ transplantation represents an important way of restoring function when an organ is irreparably damaged; the surgical process has saved tens of thousands of lives worldwide.
  • Organ rejection is a significant risk associated with
  • Immunosuppressive drugs such as cyclosporin and FK506 are usually given to the patient to prevent rejection. These immunosuppressive drugs however, have a narrow therapeutic window between adequate immunosuppression and toxicity. Prolonged immunosuppression can weaken the immune system, which can lead to a threat of
  • the present invention overcomes these and other inherent limitations in the art by providing, in a general sense, methods for the decellularization of a whole organ, which improves the preservation of the acellular scaffold.
  • the present disclosure also provides compositions and methods for integrating nano-based delivery strategies to provide the controlled release of active agents to promote proper cellular differentiation, proliferation, and growth for the purpose of regenerating a functional organ.
  • the present disclosure provides methods that reduce exposure of the tissues to detergents for preferably less than 3 or 4 hours. Furthermore, methods for controlling the delivery of one or more active agents (for example, and without limitation, such as growth factors, antibiotics, etc.) to the acellular scaffold have been employed post-decellularization to improve the acellular construct to better support organ regeneration.
  • active agents for example, and without limitation, such as growth factors, antibiotics, etc.
  • pluralities of nanovector delivery particles may also infused within the scaffold matrix, which can be pre-selected to deliver one or more of the active agents in timed-release and/or in sequential manner to promote proper cell growth and function of the resulting tissue scaffold and development of the functional organ.
  • the present invention overcomes this prior-art limitation by providing an apparatus and method for producing a superior decellularized ECM scaffold organ tissue.
  • an aeration mechanism in a conventional bioreactor apparatus, the surface tension of the detergent bubbles can be exploited to quickly remove the detergent from the bioreactor AND the acellular lung tissues.
  • This bubbling action not only mechanically agitates the tissue to further facilitate "washing" of detergent from the acellular tissue, but it also effectively removes the detergent from the perfusate solution itself.
  • a bioreactor apparatus that integrates a "bubbling bar" (i.e., an aeration tube, stone, fritted or scintered glass element, etc.) operably connected to a suitable air source, which effectively and simultaneously removes detergent from both the acellularized organ, and from the bioreactor itself through the creation of a bubbling or foaming effect that quickly diffuses excess detergent from the surface of the tissue or organ being prepared for acellular scaffolding.
  • a "bubbling bar” i.e., an aeration tube, stone, fritted or scintered glass element, etc.
  • the invention pertains to methods of producing decellularized organs, using an isolated organ or a part of an organ and a series of extractions that removes the cell membrane surrounding the organ, or part of an organ, and the cytoplasmic and nuclear components of the isolated organ, or part of an organ.
  • the invention provides a method for producing a decellularized organ comprising:
  • washing the isolated organ in a washing fluid to remove cellular debris without removing the interstitial structure of the organ until the isolated organ is substantially free of cellular material, to thereby produce a decellularized organ.
  • the method preferably also includes providing an aeration source in the organ decellurization chamber (as seen schematically in FIG. 1 and FIG. 12) to facilitate the controlled removal of the detergent-containing washing fluid from the decellularized organ in an expedited fashion to minimize untoward degradation of the resulting acellular scaffold.
  • the aeration source is a "bubbler" or aeration stone that facilitates agitation of the detergent fluid to form miscelles, which are then removed from the treatment chamber, thereby reducing the concentration of residual detergent in the washing medium.
  • the method can further comprise drying the decellularized organ, which can the optionally be stored at a suitable temperature, or equilibrated in a physiological buffer prior to use.
  • mechanical agitation of the solution comprising the isolated may be employed, which further comprises placing the isolated organ in a stirring vessel having one or more stirring devices (i.e. paddles) which can rotate at a speed ranging from about 25 to 250 revolutions per minute (rpm).
  • stirring devices i.e. paddles
  • rpm revolutions per minute
  • the step of mechanically agitating the isolated organ occurs in a fluid selected from the group consisting of distilled water, physiological buffer and culture medium.
  • the step of treating the isolated organ in the solubilizing fluid also occurs in a stirring vessel.
  • the solubilizing fluid is an alkaline solution having a detergent.
  • the alkaline solution is selected from the group consisting of sulphates, acetates, carbonates, bicarbonates and hydroxides
  • a detergent is selected from the group consisting of Triton X-100, Triton N-101, Triton X- 114, Triton X-405, Triton X-705, and Triton DF-16, monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), polyoxyethylene-23-lauryl ether (Brij 35), polyoxyethylene ether W-l (Polyox), sodium cholate, deoxycholates, CHAPS, saponin, n-Decyl ⁇ -D-glucopuranoside, n-heptyl ⁇ -D glucopyranoside, n-Octyl a-D- glucopyran
  • the step of washing the isolated organ also occurs in an aeration vessel.
  • the washing fluid can be selected from the group consisting of distilled water, physiological buffer and culture medium.
  • the invention features a method for producing a decellularized organ comprising:
  • the decellularized organ is a mammalian lung, and preferably, a human lung.
  • FIG. 1 shows a schematic of a bioreactor used to decellularized pig lung, notice that the parts of the organ, such as the pulmonary artery (PA) and the trachea, as well as the lung itself, are represented by simple shapes;
  • PA pulmonary artery
  • FIG. 2 and FIG. 3 show an exemplary bioreactor with tubes disposed as described in the prior schematic (FIG. 1);
  • FIG. 2 shows the bioreactor is connected to a porcine lung;
  • FIG. 3 is the empty bioreactor chamber itself;
  • FIG. 4A and FIG. 4B show screenshots of a Hamilton-C2 ventilator while operating in constant volume mode; APV cmv (FIG. 4A) or in constant pressure mode; PCV+ (FIG. 4B);
  • FIG. 5 shows exemplary embodiments in accordance with one aspect of the present invention involving lungs #4 and #5 after the decellularization process. Notice the numerous delaminations present on the surface. The presence of these delaminations did not constitute a significant issue, since it was noticed that few of those were present also just after lung grafts, indicating that they may be a minor concern also for healthy organs. Although delamination could be tolerated, lung 4 showed too many of them, thus leading to a further improvement of the final protocol; Lungs 5, 6 and 7 were treated with different protocols, but based on the same one employed for lung 4.
  • FIG. 6 shows an exemplary embodiment in accordance with one aspect of the present invention illustrating lung #5 after the decellularization. It is evident, in this case, how SDS damaged the ECM too much; it appeared almost transparent in certain areas;
  • FIG. 7 shows an exemplary embodiment in accordance with one aspect of the present invention illustrating lung #6 after the decellularization. This situation instead it really differs from the previous one; here the lung was still pink after the treatment, meaning that the SDS treatment was inefficient;
  • FIG. 8 shows an exemplary embodiment in accordance with one aspect of the present invention illustrating lung #7 after the decellularization.
  • the lung appeared incompletely decellularized
  • FIG. 9 is a photograph of lung #8 after the decellularization. It has a homogeneous color and it showed all the structures typical of the organ when dissected;
  • FIG. 10 is a photograph of lung #11 before the decellularization while it was still attached to the ventilator
  • FIG. 11 shows a photograph of lung #11 just after the ventilation step that follows the end of the decellularization process
  • FIG. 12 shows a schematic representation of the bubbling technique starting from the scheme shown above. Bubbles coming from the bottom of the bioreactor, passing through the red tube system, should cause SDS to self-assemble, and go on the surface of the rinsing solution; there, thanks to a built-in pressure gradient, they leave the reactor throughout the yellow tube system;
  • FIG. 13 shows a plot of average SDS concentration of the five lungs at different moments of the bubbling rinse
  • FIG. 14A, FIG. 14B, and FIG. 14C show the results of various analyses of the lungs shown above.
  • FIG. 14A a plot of average SDS concentration of the five lungs at different moments of the bubbling rinse is compared to the concentration used during the decellularization.
  • FIG. 14B average SDS concentration of samples from rinsing solution is shown. Notice how the SDS concentration of drained bubbles and overflowed solution (2 columns on the right) are the highest, while the last sample collected (i.e., 27 hrs) is the lowest.
  • FIG. 14C is a plot that shows the ratio between the SDS (mg) found in samples and the mass (mg) of the samples for all the five lungs treated with the good protocol. Each column shows the average value and the standard error of the samples coming from the five different lungs but from the same lobes;
  • FIG. 15 shows the ratios between SDS (mg) present in the sample and the mass (mg) of the samples it is pretty straightforward that lungs washed with bubbling technique (i.e. lungs 8, 9, 10, and 11) (see also FIG. 10 and FIG. 11) have really low ratio values compared with the ones of lungs rinsed with different techniques (i.e., lungs 4 and 7 were the best ones among the others).
  • the legend on the right simply indicates from which zone lobe of the lung samples were collected;
  • FIG. 16 the curved shows the amount of protein during the decellularization process when 0.002% was used; it shows the average result evaluated for the 5 lungs treated with the final protocol. Notice the plateau that tends to from after a couple of hours since the beginning of the process or change of 0.002% Dextrose solution;
  • FIG. 17A and FIG. 17B show the average ratio between the protein amount and the weight of the relative tissue sample and the standard error; and the average ratio between the protein amount and the weight of the relative tissue sample including the control lungs, respectively;
  • FIG. 18A and FIG. 18B show physical loading of the growth factor inside the cavities of the mesoporous silicon particle, yellow arrows (FIG. 18A); and the chemical attachment of growth factor throughout functionalization of the silicon particles; blue arrows (FIG. 18B);
  • FIG. 19A, FIG. 19B, and FIG. 19C show IVIS images of a control porcine lung not perfused with particles (FIG. 19A); a normal porcine lung perfused with rhodamine-loaded MSVs (FIG. 19B); and a decellularized porcine lung not perfused with particles (FIG. 19C);
  • FIG. 20 shows the number of particles per mg of tissue found using the ICP technique: starting from the left, it reports the value for the experiments done, respectively with discoidal perfused in Hespan®, discoidal in Hespan® and blood solution, spherical in Hespan® solution and spherical in Hespan® and blood solution. It was straightforward to see how the discoidal particles in both cases exceeded the values of the spherical ones;
  • FIG. 21A, FIG. 21B, and FIG. 21C show SEM images of an illustrative sample.
  • the area delimited in green is the part of the sample where the tissue was found to be torn;
  • FIG. 21B shows a higher magnification SEM image of the area delimited in green in FIG. 21A, while FIG. 21C shows a still higher magnification SEM image of the area delimited in green in FIG. 21 A; and
  • FIG. 22 shows the curve obtained while loading different masses (pg) of fluorescent albumin in 5 million MSVs having a diameter of 2.6 pm.
  • End-stage lung disease is still today a cause of death and the only way to survive is throughout lung transplantation.
  • lung transplantation is a surgical operation that became quite common it still presents several related issues: the significant shortage of lungs for transplantation is the main one, this is then related with the need for immunosuppression and thus with transplanted lung rejection by the host organism.
  • end-stage lung disease is the fourth cause of death (Xu et al, 2007).
  • Even after transplantation the chance for surviving and keeping the graft function are not so high: in fact one third of the patients who received a lung transplant undergo a severe rejection episode, and only the 50% of the patients that survives the first here will be able to preserve graft function after 5 years (Christie et al, 2010).
  • Bioartificial lung creation characterized the first attempt to the goal, and it is still on going in many research laboratories, but, at the moment, the major efforts are focused on the creation of scaffolds starting from dead-animal organs throughout a decellularization process, i.e., a procedure aimed to remove all cells from an organ, or a piece of that, and leave the ECM (Song and Ott, 2012).
  • Literature shows several efforts made in accomplishing this goal and the importance of having a good scaffolds is of primary importance especially when dealing with recellularization; as Joaquin Cortiella and his group has proved on decellularized lung scaffolds (Nichols et al, 2012), preserving all the typical anatomical, chemical and morphological features of a real lung helps the differentiation and the three dimensional proliferation of stem cells seeded on the scaffold more than artificial scaffolds (Song and Ott, 2012; Nichols et al, 2009).
  • the matrix chemical composition and morphology play a crucial role in guiding seeded stem cells in their differentiation and proliferation and since lung is a complex organ, arranged by different type of cells (such as type I AEC and type II AEC and others), and have a complex, branched morphology it is difficult to reproduce these characteristic in an artificial one, for not even mentioning the bio-compatibility of those.
  • Tissue engineering and transplant surgery are increasingly focused on decellularized matrix scaffold. End-organ failure is an healthcare challenge still unresolved; indeed the only way to overcome this kind of diseases is a successful organ transplant, but the shortage of organs suitable for a transplant and the rejection response started by the host body are crucial factors of transplant failure.
  • Decellularization means the process by which, throughout the employment of specific solvents and solutions, the removal of all cells present in the organ and major histocompatibility complex (MHC) antigens is carried out.
  • MHC major histocompatibility complex
  • organ decellularization is a relatively new object of study and development, there are already many reports of different techniques and different materials employed to accomplish that.
  • Badylak et al. (2011) gave a detailed review on the most common decellularization protocol developed up to the moment (Crapo et al, 2011).
  • the preservation of the ECM is of vital importance for obtaining proper reconstruction of the whole organ, so it is really important to obtain a state of decellularization in which all original cells and immunogens are removed but where the protein and other materials, such as glycosaminoglycan (GAG), are preserved properly.
  • Decellularization agents can consist of the most different set of materials and solutions as well as physical approach.
  • Exemplary therapeutic agents which may be administered to a subject in need thereof, by incorporation of the agent(s) within one or more populations of the mesoporous silicon particles described herein, include, without limitation, one or more drugs, small molecules, proteins, lipids, nucleic acids, diagnostic markers, and such like.
  • mesoporous silicon nano or micro-particles may preferably be configured into a shape selected from the group consisting of discoidal, spheroid, non-spheroid, oblate spheroid, and combinations thereof.
  • the porous particle is fabricated of a porous or mesoporous silicon material that is discoidal in shape.
  • the biological barrier may be, for example, an epithelial or endothelial barrier, such as the blood-brain barrier, that is based on tight junctions that prevent or limit para-cellular transport of an active agent.
  • Cells of the reticulo-endothelial system may also act as a biological barrier against an active agent.
  • the biological barrier may also be represented by a cell membrane or a nuclear membrane of a target cell.
  • mesoporous silicon particles employed herein for delivery of one or more agents to the tissues or organs being prepared for transplantation are able to overcome at least one biological barrier, including one or more biological barriers selected from the group consisting of a hemo-rheology barrier, a reticuloendothelial barrier, a blood-brain barrier, a tumor-associated osmotic interstitial pressure barrier, an ionic- or a molecular-pump barrier, a cell-membrane barrier, an enzymatic - degradation barrier, a nuclear membrane barrier, and combinations thereof.
  • biological barriers selected from the group consisting of a hemo-rheology barrier, a reticuloendothelial barrier, a blood-brain barrier, a tumor-associated osmotic interstitial pressure barrier, an ionic- or a molecular-pump barrier, a cell-membrane barrier, an enzymatic - degradation barrier, a nuclear membrane barrier, and combinations thereof.
  • these mesoporous silicon particles may have at least one targeting moiety on its surface specifically directed against a target cell.
  • the at least one targeting moiety is selected from the group consisting of ligands, antibodies, antibody fragments, peptides, ap tamers, small molecules, and combinations thereof.
  • ligands can be chemically linked to appropriate reactive groups on the surface of the particle.
  • Protein ligands can be linked to amino- and thiol-reactive groups under conditions effective to form thioether or amide bonds respectively. Methods of attaching antibody or other polymer-binding agents to an inorganic or polymeric support are detailed elsewhere (see, e.g., Taylor, 1991).
  • any active agent a small molecule drug or a biomolecular drug, may be delivered using the mesoporous silicon particles employed herein for delivery of one or more agents to the tissues or organs being prepared for transplantation.
  • the at least one active agent is a biologically active compound selected from the group consisting of peptides, proteins, therapeutic agents, diagnostic agents, non-biological materials, and combinations thereof.
  • the therapeutic agent may be any physiologically or pharmacologically active substance that can produce a desired biological effect.
  • the therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, an anti-rejection agent, a cytokine, a cytotoxic agent, a nucleolytic compound, an anti-inflammatory compound, or a pro-drug enzyme, which may be naturally occurring, or produced by synthetic or recombinant methods, or by a combination thereof.
  • the therapeutic agent(s) may be a hydrophobic drug or a hydrophilic drug.
  • Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • a cancer chemotherapy agent may be a preferred therapeutic agent.
  • anticancer agents and other therapeutic agents those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician 's Desk Reference and to the reference by Goodman and Gilman (2001).
  • the therapeutic agent may be selected from the group consisting of genes, nucleic acids, shRNAs, siRNAs, microRNAs, DNA fragments, RNA fragments, plasmids, and combinations thereof.
  • the therapeutic agent is a siRNA or a microRNA that silences one or more genes expressed by the cancer cells or the tumor.
  • the therapeutic agent can also be applied to engineer the genome of cancer cells and/or stromal cells in the tumor, such as a CRISPR/Cas9 system.
  • composition of the disclosure may be designed, formulated and processed so as to be suitable for a variety of therapeutic and diagnostic uses and modes of administration.
  • composition of the disclosure may be administered to a subject, such as a human, via any suitable administration method in order to treat, prevent, and/or monitor a physiological condition, such as a disease.
  • Embodiments of the composition may be particularly useful for organ transplantation.
  • the mesoporous silicon particles employed herein may be employed as a single treatment modality, or alternatively may be combined with one or more additional therapeutic, diagnostic, and/or prophylactic agents, including, without limitation, one or more proteins, peptides, polypeptides (including, without limitation, enzymes, antibodies, antigens, antigen binding fragments etc.); RNA molecules (including, without limitation, siRNAs, microRNAs, iRNAs, mRNAs, tRNAs, or catalytic RNAs, such as ribozymes, and the like), DNA molecules (including, without limitation, oligonucleotides, polynucleotides, genes, coding sequences (CDS), introns, exons, plasmids, cosmids, phagemids, baculovirus, vectors [including, without limitation, viral vectors, virions, viral particles and such like]); peptide nucleic acids, detection agents, imaging agents, contrast agents, detectable gas, radio
  • Mesoporous silicon particles may also further optionally include one or more additional active ingredients, including, without limitation, one or more transcription factors, immunomodulating agents, immunostimulating agents, neuroactive agents, antiinflammatory agents, chemotherapeutic agents, hormones, so called “trophic factors,” cytokines, chemokines, receptor agonists or antagonists, or such like, or any combination thereof.
  • additional active ingredients including, without limitation, one or more transcription factors, immunomodulating agents, immunostimulating agents, neuroactive agents, antiinflammatory agents, chemotherapeutic agents, hormones, so called “trophic factors,” cytokines, chemokines, receptor agonists or antagonists, or such like, or any combination thereof.
  • the mesoporous silicon drug delivery formulations may also further optionally include one or more additional components to aid, facilitate, or improve delivery of a pro-drug and/or an active metabolite contained therein, including, without limitation, one or more liposomes, lipid particles, lipid complexes, and may further optionally include one or more binding agents, cell surface active agents, surfactants, lipid complexes, niosomes, ethosomes, transferosomes, phospholipids, sphingolipids, sphingosomes, or any combination thereof, and may optionally be provided within a pharmaceutical formulation that includes one or more additional nanoparticles, microparticles, nanocapsules, microcapsules, nanospheres, microspheres, or any combination thereof.
  • the polycation-functionalized nanoporous silicon carriers of the present disclosure will generally be formulated for systemic and/or localized administration to an animal, or to one or more cells or tissues thereof, and in particular, will be formulated for localized administration to a mammal, prior to, during, or following transplantation of one or more tissues, such as a lung.
  • drug-delivery formulations of the active compounds disclosed herein will be at least substantially stable at a pH from about 4.2 to about 8.2, and more preferably, will be substantially stable at a pH of from about 5 to about 7.5.
  • the active ingredient(s) and targeted drugs will be substantially active at physiological conditions of the animal into which they are being administered.
  • the present disclosure also provides for the use of one or more mesoporous silicon carriers in the manufacture of a medicament for therapy and/or for the amelioration of one or more symptoms of a disease, disorder, dysfunction, or condition, and particularly for use in the manufacture of a medicament for treating, one or more diseases, dysfunctions, or disorders involving, or arising from transplantation of one or more tissues or organs in a mammal, and, in a human, in particular.
  • the present disclosure also provides for the use of one or more of the disclosed polycation-functionalized nanoporous silicon drug delivery systems in the manufacture of a medicament for the transplantation of a mammalian organ, and in particular, in the transplantation of a human lung.
  • the invention also includes diagnostic and/or targeting compounds that may be optionally included in or on the surface of the silicon nanoparticle carriers to facilitate improvements in the treatment or prognosis of the organ transplantation.
  • Another important aspect of this disclosure concerns methods for using the polycation-functionalized nanoporous silicon carriers to facilitate treatment or the amelioration of one or more symptoms of the disease in a mammal having, suspected of having, or at risk for developing such a condition, and in particular for those mammals undergoing organ or tissue transplant.
  • Such methods generally involve administering to a mammal (and in particular, to a human in need thereof), one or more of the disclosed polycation-functionalized nanoporous silicon carriers formulated to contain one or more therapeutic or diagnostic agents, in an amount and for a time sufficient to diagnosis, monitor, treat (or, alternatively, to ameliorate one or more symptoms of) a condition in a mammal to which the composition has been administered.
  • the therapeutic formulations described herein may be provided to the animal as a single treatment modality, as a single administration, or alternatively provided to the patient in multiple administrations over a period of from several hours to several days, from several days to several weeks, or even over a period of several weeks to several months or longer, as needed following organ transplantation as may be needed.
  • kits that include one or more of the disclosed therapeutic drug compositions (and instructions for using the kit) also represent an important aspect of the present disclosure. Such kits may further optionally include one or more diagnostic agents, one or more therapeutic agents, or any combination thereof, either alone or further in combination with one or more additional compounds, pharmaceuticals, or such like.
  • kits of the present disclosure may be packaged for commercial distribution, and may further optionally include one or more delivery devices adapted to deliver one or more therapeutic composition(s) to an animal (e.g., syringes, injectables, and the like).
  • delivery devices adapted to deliver one or more therapeutic composition(s) to an animal (e.g., syringes, injectables, and the like).
  • kits typically include at least one vial, test tube, flask, bottle, syringe or other container, into which the mesoporous silicon carrier-based therapeutic composition(s) may be placed, and preferably suitably aliquotted.
  • the kit may also contain a second distinct container into which this second composition may be placed.
  • a plurality of mesoporous silicon carrier-based compositions as disclosed herein may be prepared in a single mixture, including those prepared as a suspension or in solution, and may be packaged in a single container, such as a vial, flask, syringe, catheter, cannula, bottle, or other suitable containment.
  • kits of the present disclosure may also typically include a retention mechanism adapted to contain or retain the vial(s) or other container(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) or other container(s) may be retained to minimize or prevent breakage, exposure to sunlight, or other undesirable factors, or to permit ready use of the composition(s) included within the kit.
  • a retention mechanism adapted to contain or retain the vial(s) or other container(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) or other container(s) may be retained to minimize or prevent breakage, exposure to sunlight, or other undesirable factors, or to permit ready use of the composition(s) included within the kit.
  • Another important aspect of the present disclosure concerns methods for using the disclosed mesoporous carriers as agents for delivering therapeutic or diagnostic compounds to selected cells or tissues or organs of a vertebrate mammal, and particularly in a mammal undergoing a tissue or an organ transplant, such as a human undergoing a lung transplant.
  • Such use generally involves administration to an animal in need thereof one or more of the disclosed therapeutic delivery vehicles, in an amount and for a time sufficient to prevent, treat, lessen, or cure the disease, disorder, dysfunction, condition, or deficiency in the affected animal, and/or to ameliorate one or more symptoms thereof.
  • the therapeutic compositions may be formulated to contain one or more anti -rejection, anti-inflammatory, or anti-microbial agents, or any combination thereof.
  • formulations of one or more of the drug delivery nanoparticles described herein will contain at least a chemotherapeutically-effective amount of a first active agent
  • the formulation may contain at least about 0.001% of each active ingredient, preferably at least about 0.01% of the active ingredient, although the percentage of the active ingredient(s) may, of course, be varied, and may conveniently be present in amounts from about 0.01 to about 90 weight % or volume %, or from about 0.1 to about 80 weight % or volume %, or more preferably, from about 0.2 to about 60 weight % or volume %, based upon the total formulation.
  • the amount of active compound(s) in each composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological t , route of administration, product shelf-life, as well as other pharmacological considerations will be contemplated by one of ordinary skill in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • compositions adapted for injectable administration include, but are not limited to, sterile aqueous solutions, dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions including, without limitation, one or more of those described in U.S. Patent No. 5,466,468 (specifically incorporated herein in its entirety by express reference thereto).
  • the form is preferably sterile, and is preferably fluid to the extent that easy syringability and/or ready administration to the patient is achievable. It is also preferably at least sufficiently stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as viruses, bacteria, fungi, and such like.
  • the carrier(s) can be a solvent or dispersion medium including, without limitation, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like, or a combination thereof), one or more vegetable oils, or any combination thereof, although additional pharmaceutically-acceptable components may be included.
  • a solvent or dispersion medium including, without limitation, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like, or a combination thereof), one or more vegetable oils, or any combination thereof, although additional pharmaceutically-acceptable components may be included.
  • Proper fluidity of the pharmaceutical formulations disclosed herein may be maintained, for example, by the use of a coating, such as, e.g., a lecithin, by the maintenance of the required particle size in the case of dispersion, by the use of a surfactant, or any combination of these techniques.
  • the inhibition or prevention of the action of microorganisms can be brought about by one or more antibacterial or antifungal agents, for example, without limitation, a paraben, chlorobutanol, phenol, sorbic acid, thimerosal, or the like.
  • an isotonic agent for example, without limitation, one or more sugars or sodium chloride, or any combination thereof.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example without limitation, aluminum monostearate, gelatin, or a combination thereof.
  • formulations of the disclosed drug delivery compositions may be suitable for direct injection into one or more organs, tissues, or cell types in the body. Administration of the disclosed compositions may be conducted using suitable means, including those known to the one of ordinary skill in the relevant medical arts.
  • compositions disclosed herein are not in any way limited to use only in humans, or even to primates, or mammals.
  • the methods and compositions disclosed herein may be employed during organ transplantation in avian, amphibian, reptilian, or other animal species.
  • the compositions disclosed herein are preferably formulated for administration to a mammal, and in particular, to humans, undergoing a transplantation procedure, such as a lung transplant.
  • compositions disclosed herein may also be provided in formulations that are acceptable for veterinary administration, including, without limitation, to selected livestock, exotic or domesticated animals, companion animals (including pets and such like), non-human primates, as well as zoological or otherwise captive specimens, and such like.
  • polynucleotides, nucleic acid segments, nucleic acid sequences, and the like include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
  • DNAs including and not limited to genomic or extragenomic DNAs
  • genes include peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
  • PNAs peptide nucleic acids
  • buffer includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.
  • carrier is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert (s), or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable.
  • the term "decellularized organ” as used herein refers to an organ, or part of an organ from which the entire cellular and tissue content has been removed leaving behind a complex interstitial structure.
  • Organs are composed of various specialized tissues.
  • the specialized tissue structures of an organ are the parenchyma tissue, and they provide the specific function associated with the organ.
  • Most organs also have a framework composed of unspecialized connective tissue which supports the parenchyma tissue.
  • the process of decellularization removes the parenchyma tissue, leaving behind the three- dimensional interstitial structure of connective tissue, primarily composed of collagen.
  • the interstitial structure has the same shape and size as the native organ, providing the supportive framework that allows cells to attach to, and grow on it.
  • Decellularized organs can be rigid, or semi-rigid, having an ability to alter their shapes.
  • Examples of decellularized organs include, but are not limited to the heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term “DNA segment,” are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • an effective amount refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.
  • engineered and recombinant cells are intended to refer to a cell into which an exogenous polynucleotide segment (such as DNA segment that leads to the transcription of a biologically active molecule) has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced exogenous DNA segment. Engineered cells are, therefore, cells that comprise at least one or more heterologous polynucleotide segments introduced through the hand of man.
  • epitope refers to that portion of a given immunogenic substance that is the target of (i.e. , is bound by), an antibody or cell- surface receptor of a host immune system that has mounted an immune response to the given immunogenic substance as determined by any method known in the art. Further, an epitope may be defined as a portion of an immunogenic substance that elicits an antibody response or induces a T-cell response in an animal, as determined by any method available in the art (see, e.g., Geysen et al, 1984).
  • An epitope can be a portion of any immunogenic substance, such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof.
  • immunogenic substance such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof.
  • epitope may also be used interchangeably with “antigenic determinant” or “antigenic determinant site.”
  • heterologous is defined in relation to a predetermined referenced DNA or amino acid sequence.
  • a heterologous promoter is defined as a promoter that does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation.
  • a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.
  • homologous means, when referring to polypeptides or polynucleotides, sequences that have the same essential structure, despite arising from different origins.
  • homologous proteins are derived from closely related genetic sequences, or genes.
  • an "analogous" polypeptide is one that shares the same function with a polypeptide from a different species or organism, but has a significantly different form to accomplish that function.
  • Analogous proteins typically derive from genes that are not closely related.
  • the term “homology” refers to a degree of complementarity between two polynucleotide or polypeptide sequences.
  • the word “identity” may substitute for the word “homology” when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence.
  • Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.
  • identity in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • the phrase "in need of treatment” refers to a judgment made by a caregiver such as a physician or veterinarian that a patient requires (or will benefit in one or more ways) from treatment. Such judgment may made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state that is treatable by one or more compound or pharmaceutical compositions such as those set forth herein.
  • isolated or “biologically pure” refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state.
  • isolated polynucleotides in accordance with the invention preferably do not contain materials normally associated with those polynucleotides in their natural, or in situ, environment.
  • isolated organ refers to an organ that has been removed from a mammal. Suitable mammals include humans, primates, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep. The term “isolated organ” also includes an organ removed from the subject requiring an artificial reconstructed organ. Suitable organs can be any organ, or part of organ, required for replacement in a subject. Examples include but are not limited to the heart, lung, kidney, liver, pancreas, spleen, bladder, ureter and urethra.
  • An organ, or a part of an organ can be isolated from the subject requiring an artificial reconstructed organ.
  • a diseased organ in a subject can be removed and decellularized, as long as the disease effects the parenchyma tissue of the organ, but does not harm the connective tissue, e.g., tissue necrosis.
  • the diseased organ can be removed from the subject and decellularized.
  • the decellularized organ, or a part of the organ can be used as a three-dimensional scaffold to reconstruct an artificial organ.
  • An allogenic artificial organ can be reconstructed using the subject's own decellularized organ as a scaffold and using a population of cells derived from the subject's own tissue. For example, cells populations derived from the subject's skin, liver, pancreas, arteries, veins, umbilical cord, and placental tissues.
  • a xenogenic artificial organ can be reconstructed using the subject's own decellularized organ as a scaffold, and using cell populations derived from a mammalian species that are different from the subject.
  • the different cell populations can be derived from mammals such as primates, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep.
  • An organ, or part of an organ can also be derived from a human cadaver, or from mammalian species that are different from the subject, such as organs from primates, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep. Standard methods for isolation of a target organ are well known to the skilled artisan and can be used to isolate the organ.
  • kit may be used to describe variations of the portable, self-contained enclosure that includes at least one set of reagents, components, or pharmaceutically-formulated compositions to conduct one or more of the assay methods of the present disclosure.
  • kit may include one or more sets of instructions for use of the enclosed reagents, such as, for example, in a laboratory or clinical application.
  • Link refers to any method known in the art for functionally connecting one or more proteins, peptides, nucleic acids, or polynucleotides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like.
  • local administration or “local delivery,” in reference to delivery of a composition, formulation, or device of the invention, refer to delivery that does not rely upon transport of the agent to its intended target tissue via the vascular or lymphatic system from a site of administration that is remote from the intended target tissue. The agent is delivered directly to its intended target tissue or in the vicinity thereof, e.g. by injection or implantation. It will be appreciated that a small amount of the delivered agent may enter the vascular system and may ultimately reach the target tissue via the vascular system.
  • mammal refers to the class of warm-blooded vertebrate animals that have, in the female, milk- secreting organs for feeding the young. Mammals include without limitation humans, apes, many four-legged animals, whales, dolphins, and bats. A human is a preferred mammal for purposes of the invention.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring.
  • laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally-occurring animals.
  • nucleic acid includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites).
  • nucleic acid also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like.
  • Nucleic acids include single- and double- stranded DNA, as well as single- and double-stranded RNA.
  • nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.
  • operably linked refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
  • the term "patient” refers to any host that can serve as a recipient for one or more of the vascular access devices as discussed herein.
  • the recipient will be a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being).
  • a "patient” refers to any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.
  • phrases "pharmaceutically acceptable” refers to molecular entities and compositions that preferably do not produce an allergic or similar untoward reaction when administered to a mammal, and in particular, when administered to a human.
  • pharmaceutically acceptable salt refers to a salt that preferably retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects.
  • salts include, without limitation, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like); and salts formed with organic acids including, without limitation, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; salts formed with an organic cation formed from ⁇ , ⁇ ' dibenzylethylenediamine
  • salts refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects.
  • examples of such salts include, but are not limited to, acid-addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc,
  • plasmid refers to a genetic construct that is composed of genetic material (i.e., nucleic acids).
  • a plasmid or a vector contains an origin of replication that is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells including the plasmid.
  • Plasmids and vectors of the present invention may include one or more genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in a suitable expression cells.
  • the plasmid or vector may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and includes any chain or chains of two or more amino acids.
  • terms including, but not limited to “peptide,” “dipeptide,” “tripeptide,” “protein,” “enzyme,” “amino acid chain,” and “contiguous amino acid sequence” are all encompassed within the definition of a “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with, any of these terms.
  • polypeptides that have undergone one or more post- translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post- translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • post- translational modification(s) including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post- translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.
  • amino acids Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; lie), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys).
  • Amino acid residues described herein are preferred to be in the "L” isomeric form. However, residues in the "D" isomeric form may be substituted for L- amino acid residues provided the desired properties of
  • the terms "prevent,” “preventing,” “prevention,” “suppress,” “suppressing,” and “suppression” as used herein refer to administering a compound either alone or as contained in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state so as to prevent any symptom, aspect or characteristic of the disease state. Such preventing and suppressing need not be absolute to be deemed medically useful.
  • promoter refers to a region or regions of a nucleic acid sequence that regulates transcription.
  • Protein is used herein interchangeably with “peptide” and “polypeptide,” and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject.
  • polypeptide is preferably intended to refer to any amino acid chain length, including those of short peptides from about two to about 20 amino acid residues in length, oligopeptides from about 10 to about 100 amino acid residues in length, and longer polypeptides including from about 100 amino acid residues or more in length.
  • polypeptides and proteins of the present invention also include polypeptides and proteins that are or have been post-translationally-modified, and include any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.
  • a compound or entity may be partially purified, substantially purified, or pure.
  • a compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e. , is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.
  • a partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.
  • a compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e., is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.
  • a partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found( ⁇ ?.g., cellular material such as cellular proteins, peptides, nucleic acids, etc.).
  • the term "recombinant” indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment, or native state. Specifically, e.g., a promoter sequence is "recombinant” when it is produced by the expression of a nucleic acid segment engineered by the hand of man.
  • a "recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis
  • a "recombinant polypeptide” or “recombinant protein” is a polypeptide or protein which is produced by expression of a recombinant nucleic acid
  • a "recombinant virus,” e.g., a recombinant AAV virus is produced by the expression of a recombinant nucleic acid.
  • regulatory element refers to a region or regions of a nucleic acid sequence that regulates transcription.
  • exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.
  • RNA segment refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term “RNA segment,” are RNA segments and smaller fragments of such segments.
  • sequence when referring to amino acids, relates to all or a portion of the linear N-terminal-to-C-terminal order of amino acids within a given amino acid chain, e.g. , polypeptide or protein; "subsequence” means any consecutive stretch of amino acids within a sequence, e.g., at least 3 consecutive amino acids within a given protein or polypeptide sequence.
  • sequence and “subsequence” have similar meanings relating to the 5'-to-3' order of nucleotides.
  • biologically functional equivalent is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the invention.
  • the percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence.
  • the reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.
  • the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides.
  • the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).
  • Standard administration of two or more agents refers to administration of two or more agents to a subject such that the agents are not present together in the subject's body at greater than de minimis concentrations. Administration of the agents may, but need not, alternate. Each agent may be administered multiple times.
  • sequence homology as applied to an amino acid sequence means that the sequence displays at least approximately 20% identical or conservatively replaced amino acids, preferably at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60% identical or conservatively replaced amino acids, desirably at least approximately 70% identical or conservatively replaced amino acids, more desirably at least approximately 80% identical or conservatively replaced amino acids, and most desirably at least approximately 90% amino acid identical or conservatively replaced amino acids relative to a reference sequence. When two or more sequences are compared, any of them may be considered the reference sequence. % identity can be calculated using a FASTA or BLASTP algorithm, using default parameters. A PAM250 or BLOSUM62 matrix may be used.
  • a conservatively replaced residue is considered identical to the residue it replaces.
  • Conservative replacements may be defined in accordance with Stryer, L, Biochemistry, 3rd ed., 1988, according to which amino acids in the following groups possess similar features with respect to side chain properties such as charge, hydrophobicity, aromaticity, etc.
  • Aliphatic hydroxyl side chains S, T;
  • Basic side chains K, R, H;
  • Acidic amino acids D, E, N, Q; and (7) Cyclic aliphatic side chain: P.
  • subject describes an organism, including mammals such as primates, to which treatment with the compositions according to the present disclosure can be provided.
  • Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.
  • Substantial sequence homology as applied to a sequence means that the sequence displays at least approximately 60% identity, desirably at least approximately 70% identity, more desirably at least approximately 80% identity, and most desirably at least approximately 90% identity relative to a reference sequence. When two or more sequences are compared, any of them may be considered the reference sequence. % identity can be calculated using a FASTA, BLASTN, or BLASTP algorithm, depending on whether amino acid or nucleotide sequences are being compared. Default parameters may be used, and in exemplary embodiments, a PAM250 and/or BLOSUM62 matrix or such like may be employed in the practice of the invention.
  • a "sustained release formulation” is a composition of matter that comprises a therapeutic agent as one of its components and further comprises one or more additional components, elements, or structures effective to provide sustained release of the therapeutic agent, optionally in part because of the physical structure of the formulation.
  • Sustained release is release or delivery that occurs either continuously or intermittently over an extended period, e.g., at least several days, at least several weeks, at least several months, at least several years, or even longer, depending upon the particular formulation employed.
  • Suitable standard hybridization conditions include, for example, hybridization in 50% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 ⁇ g/ml of denatured salmon sperm DNA at 42°C for 16 hr followed by 1 hr sequential washes with O.lx SSC, 0.1% SDS solution at 60°C to remove the desired amount of background signal.
  • Lower stringency hybridization conditions for the present disclosure include, for example, hybridization in 35% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 ⁇ g/ml denatured salmon sperm DNA or E. coli DNA at 42°C for 16 h followed by sequential washes with 0.8x SSC, 0.1% SDS at 55°C.
  • Those of skill in the art will recognize that conditions can be readily adjusted to obtain the desired level of stringency.
  • structural gene is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.
  • nucleic acid segments that are complementary, essentially complementary, and/or substantially complementary to at least one or more of the specific nucleotide sequences specifically set forth herein.
  • Nucleic acid sequences that are “complementary” are those that are capable of base- pairing according to the standard Watson-Crick complementarity rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions such as those described immediately above.
  • the probes and primers of the present disclosure may be of any length.
  • an algorithm defining all probes or primers contained within a given sequence can be proposed:
  • n is an integer from 1 to the last number of the sequence
  • y is the length of the probe or primer minus one, where n + y does not exceed the last number of the sequence.
  • exemplary primer or probe sequence include, without limitation, sequences corresponding to bases 1 to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the entire length of the sequence.
  • probes or primers may correspond to the nucleotides from the first basepair to bp 40, from the second bp of the sequence to bp 41, from the third bp to bp 42, and so forth
  • probes or primers may correspond to a nucleotide sequence extending from bp 1 to bp 50, from bp 2 to bp 51, from bp 3 to bp 52, from bp 4 to bp 53, and so forth.
  • substantially complementary when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an mRNA encoding the selected sequence.
  • sequences will be highly complementary to the mRNA "target" sequence, and will have no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or so base mismatches throughout the complementary portion of the sequence.
  • sequences may be exact matches, i.e., be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch.
  • highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
  • Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or "% exact-match") to a corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds.
  • nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including about 96%, about 97%, about 98%, about 99%, and even about 100% exact match complementary to all or a portion of the target sequence to which the designed nucleic acid specifically binds.
  • Percent similarity or percent complementary of any of the disclosed nucleic acid sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • terapéutica period means the period of time that is necessary for one or more active agents to be therapeutically effective.
  • therapeutically-effective refers to reduction in severity and/or frequency of one or more symptoms, elimination of one or more symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and the improvement or a remediation of damage.
  • a “therapeutic agent” may be any physiologically or pharmacologically active substance that may produce a desired biological effect in a targeted site in a subject.
  • the therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioactive isotope, a receptor, and a pro-drug activating enzyme, which may be naturally-occurring, or produced by synthetic or recombinant methods, or any combination thereof.
  • Drugs that are affected by classical multidrug resistance such as the vinca alkaloids (e.g., vinblastine and vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel) may have particular utility as the therapeutic agent.
  • Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • a cancer chemotherapy agent may be a preferred therapeutic agent.
  • anticancer agents and other therapeutic agents those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician 's Desk Reference and to Goodman and Gilman's "Pharmacological Basis of Therapeutics" tenth edition, Hardman et al. (Eds.) (2001).
  • Transcriptional regulatory element refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences.
  • a transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.
  • transcription factor recognition site and a “transcription factor binding site” refer to a polynucleotide sequence(s) or sequence motif(s), which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding.
  • transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted based on known consensus sequence motifs, or by other methods known to those of ordinary skill in the art.
  • Transcriptional unit refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first ds-acting promoter sequence and optionally linked operably to one or more other cw-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.
  • the term "transformed cell” is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell.
  • transformation is intended to generally describe a process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell.
  • an exogenous polynucleotide sequence e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule
  • Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.
  • Treating refers to providing any type of medical or surgical management to a subject. Treating can include, but is not limited to, administering a composition comprising a therapeutic agent to a subject. "Treating” includes any administration or application of a compound or composition of the invention to a subject for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder, or condition. In certain aspects, the compositions of the present invention may also be administered prophylactically, i.e.
  • the subject will be one that has been diagnosed for being "at risk" of developing such a disease or disorder, either as a result of familial history, medical record, or the completion of one or more diagnostic or prognostic tests indicative of a propensity for subsequently developing such a disease or disorder.
  • vector refers to a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment.
  • a plasmid, cosmid, or a virus is an exemplary vector.
  • nucleic acid segments of the present disclosure in combination with an appropriate detectable marker (i.e., a "label,”), such as in the case of employing labeled polynucleotide probes in determining the presence of a given target sequence in a hybridization assay.
  • an appropriate detectable marker i.e., a "label”
  • a wide variety of appropriate indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay.
  • an enzyme tag such as urease, alkaline phosphatase or peroxidase
  • colorimetric, chromogenic, or fluorigenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences.
  • multiplexing assays where two or more labeled probes are detected either simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (for example, an emission and/or excitation spectral maximum), which also labeled a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discreet or discernible from the first label.
  • first detection property or parameter for example, an emission and/or excitation spectral maximum
  • FIG. 2 and FIG. 3 Photographs of an exemplary bioreactor are shown in FIG. 2 and FIG. 3, with and without an attached organ, respectively.
  • the techniques tested were all concerning a perfusion and immersion decellularization approach, since lungs were eased down in the bioreactor connected with the tubes as in the scheme, i.e., a tube connected to the pulmonary artery (PA) and one to the trachea, and then completely immersed in the proper solution until the bioreactor was filled up.
  • PA pulmonary artery
  • the standard configuration considers the presence of a tube having one end connected with the PA passing through a pump and the other end left free in the bioreactor, another tube connected in the same way of the one just described for the trachea and finally a tube with both the end terminating in the bioreactor and a pump connected to allow for recycle of the solution.
  • the techniques makes use of air bubbles pushed from the bottom of the bioreactor, filled with deionized water, to force the SDS molecules still present in the rinsing solution to self assembly and move towards the liquid/air surface; here they accumulate and form a layer of bubbles which created a pressure gradient which guides them towards the tube connected to the drain where they encounter the less resistance.
  • bubbles are force to leave the reactor as they form.
  • a first exemplary protocol utilized for the decellularization of organ tissues is reported below; it involves only the exploitation of an SDS solution in deionized water for the entire process:
  • PA pulmonary artery
  • PA pulmonary artery
  • PA pulmonary artery
  • step 8 If the bubbles are still there after too much time repeat step 8, otherwise follow step 10.
  • Ethanol was tried since it would also give a good method for disinfecting the organ: unfortunately for safety reason it was not possible to employ 70% concentration which is known to be the most effective one for disinfecting, but quick trails of piece of acellular tissue immersed in a special growing media for bacteria did not show any evidence of rapid growing of bacterial colony, proving that also 10% ethanol could be quite effective in eliminating bacteria in the organ. It must also be noticed that during the process of the dextrose contains antibacterials and antimicotics, and that SDS may help to lyse bacterial membranes.
  • bubbles are force to leave the reactor as they form.
  • the air compressor used to creates the bubbles is stopped and fresh deionized water is pushed in the bioreactor from the bottom of the reactor throughout the same tubes used for the air; the fresh water inserted pushed the liquid just beneath the liquid/air interface towards the drain port where it leaves the bioreactor.
  • the fresh water substitutes for the old one which is thought to contain more SDS. It is evident, also from the results presented below, that using deionized water to cover the lung helps to extract SDS form the inside of the organ by diffusion and it is also clear that this phenomenon is helped by the bubbles that gently it the lung anchored to the bioreactor.
  • Acellular parenchyma tissue samples were also homogenized and analyzed to look for the protein content left in the scaffold. This technique was also applied to control lung tissue so that it was possible to compare the results and see the difference between a normal tissue and an acellular one (FIG. 17A and FIG. 17B). As expected the amount of protein in the 5 lungs studied was much lower compared to the one of the controls, meaning that the decellularization may act not only on cells, which for sure contain protein, but also on the protein content of ECM.
  • a pro-inflammatory environment specifically with the presence of INF- ⁇ and TNF-k is needed to activate the immunosuppressive effects of MSCs (Macchiarini et al, 2008; Crapo et al , 2011).
  • Activated MSCs then exert immune-modulatory effects by the secretion of soluble factors and by cell-cell contact dependent interactions upon activation with pro-inflammatory cytokines.
  • TGF- ⁇ transforming growth factor ⁇
  • IL-10 interleukin 10
  • PGE2 prostaglandin E2
  • PDL1 programmed death ligand 1
  • MSCs play a role in immunomodulation and control of immune responses (Crapo et al, 2011; Song and Ott, 2011; Cortiella et al, 2010; Hopkinson et al, 2008; and Funamoto et al, 2010), and recently data has been generated that supports a role for MSCs in the regulation of tumor development in pancreatic cancer (Phillips et al, 2010).
  • Preliminary data indicates the ability to identify and isolate MSCs from lung or peripheral blood.
  • Human MSCs can be identified by the expression of a specific set of cell surface molecules such as CD105, CD90, CD29 and CXCR4 co-expression which can be used to evaluate the presence of MSCs in normal as well as injured lung environments.
  • lung scaffold may be used to determine if RA-primed MSCs have the capacity to replicate neuronal processes found in the lung and to determine if the lung scaffold influences differentiation of cells to neuronal, lung epithelium or endothelial cell lineages.
  • AC scaffolds are lungs whose original cells have been destroyed by exposure to detergents and physical methods of removing cells and cell debris (Fox et al, 2005). This creates a lung scaffold from the skeleton of the lungs themselves.
  • Stem cell transplantation is a therapeutic strategy which has the potential to replace damaged cells as well as modify the environment through production of trophic factors.
  • a variety of stem cell sources such as adult human umbilical cord blood, bone marrow or the brain itself have been considered for development of cell-therapy for a variety of diseases.
  • MSC-based therapies for lung, brain or other soft tissues there needs to be a consistent cell selection and development of differentiation strategies to produce cells of specific lineages in order to be able to realize their clinical benefits.
  • MSCs stem cells have the capacity for precise migration during embryogenesis and later in life in response to injury, using the CXCR4/SDF-1 (Sawada et al, 2008) pathway. Because of this, subpopulations of peripheral blood mononuclear cells (MNC) were examined for expression of stem cell markers of immaturity combined with expression of CXCR4 and examined neural lineage potential of these subpopulations after RA-priming. A population of CXCR4+ CD133+ ABCG2+ cells were identified that had a high degree of neuronal lineage differentiation efficiency and migrated using a CXCR4/SDF-1 mechanism. Implantation of RA-primed CXCR4+ CD133+ ABCG2+ cells into the lateral ventricle of uninjured or TBI rats resulted in survival of cells, migration to the injury site and differentiation of cells after implantation.
  • MSCs have an excellent candidate for use as an autologous stem cell therapy for the treatment of traumatic brain injury and neurodegenerative disorders.
  • This MSC cell population also has potential for use to treat traumatic brain injury for lung or other soft tissue injuries.
  • CD105+CD90+CD29+ MSCs and MSC subsets were isolated as previously described from peripheral blood buffy coats (Sawada et al, 2008) or from human bone marrow.
  • the mononuclear (MNC) fraction was isolated using Ficoll density gradient separation medium (Amersham-Biosciences, Piscataway, NJ, USA). Subpopulations of MNC were isolated by counter-current centrifugal elutriation using a Beckmann J6M elutriator (Beckman Instruments) in a Sanderson chamber. A Masterplex peristaltic pump (Cole Parmer Instruments) was used to provide the counter current flow.
  • RPMI 1640 medium supplemented with 2 mM glutamine, 100 U penicillin G, 100 ⁇ g/mL streptomycin, and 10% donor-derived autologous serum was used as the elutriation medium.
  • 3-6 x 10 6 cells were loaded at 3000 RPM, and cell fractions were isolated using a step-wise reduction of rotor speed and medium flow to allow for collection of subpopulations of MNC based on cell size and density.
  • CFDA CFDA
  • SE is a fixable-cell- permeant, fluorescein-based tracer for very long- term cell labeling.
  • CellTrace Far Red DDAO-SE is a fixable, far- red— fluorescent tracer for very long-term cell labeling.
  • the succinimidyl ester (SE) reactive group forms a strong covalent attachment to primary amines that occur in proteins and other biomolecules on the inside and outside of cells. Deposition of CFSE labeled MSCs, proliferation and dispersal of cells was determined in CFDA-labeled cell constructs as previously described (Sawada et al, 2008; Arand et al, 1992).
  • Antibodies for phenotyping were conjugated to fluorescein isothiocyanate (FITC), phycoerytherin (PE) or PerCP were purchased from commercial sources and were used as described by each manufacturer.
  • Corresponding immunoglobulin (IgG) matched isotype control antibodies were used to set baseline values for analysis markers. Staining for ABCG2 (Stem Cell Technologies, Vancouver, BC, CANADA), CD133 (Miltenyi Biotech, Auburn, CA, USA) or CXCR4 (BD Biosciences, San Jose, CA, USA) was done with PE conjugated antibodies.
  • MNCs were stained in PBS (Ca- and Mg-free) supplemented with 5% autologous serum.
  • BD Becton Dickenson
  • IL-2 Interleukin-2
  • IL-4 Interleukin-4
  • IL-5 Interleukin-5
  • IL-10 Interleukin-10
  • TNF Tumor Necrosis Factor
  • IFN- ⁇ Interferon- ⁇ protein levels produced by elutriated MNC pre and post RA- priming as described by the manufacturer.
  • Phagocytic capacity of elutriated cells was evaluated using uptake of fluorescent beads as evaluated by uptake of fluorescent beads.
  • Cell cultures were exposed to 3.5 ⁇ g/mL lipopoly saccharide (LPS), or 4 ⁇ g/mL phytohemagglutinin-M (PHAM).
  • LPS lipopoly saccharide
  • PHAM phytohemagglutinin-M
  • Cells may be cultured in DMEM-LG supplemented with 10% human AB serum, 10-3 M ⁇ -mercaptoethanol ( ⁇ - ⁇ ) (Sigma; St. Louis, MO, USA) + 5 x 10 "7 M all-irans-retinoic acid (RA) (Sigma) for 24 hrs (Sawada et al., 2008).
  • 3-6 ⁇ MSCs may be induced to differentiate using time release MultistageTM (Leonardo Biosystems, Inc., Houston, TX, USA) nanoparticles loaded with 2 mM L-glutamine, B-27 supplement (Invitrogen), 10 ⁇ g/mL epidermal growth factor (EGF) and 2.5 ⁇ g/mL fibroblast growth factor-beta (FGF- ⁇ ) and 10% autologous or human AB serum. Viability of cells may be determined using a Molecular Probes LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, San Jose, CA, USA).
  • Methods of MSC deposition may be done by infusion of the MSCs into the chambers containing lung scaffold using a catheter method for recellularization by insertion of a set of 2-3 catheters in the main stem bronchus or main bronchi of the acellular lung scaffold. This method has been used previously to place other cell types in the acellular scaffold (Arand et al, 1992). A single aliquot of MSCs may be injected into the acellular lung scaffold and the system may be cultured at 37°C for the duration of the project. When the experiment is to be terminated media and cell culture products may be removed from the bioreactor chambers.
  • the MSC- scaffold construct may be fixed using 2% paraformaldehyde which may be added to the bioreactor chambers. MSC cell supematants and fixed MSC cell constructs are then analyzed for levels of inflammatory mediators produced by MSCs or MSC subsets using ELISA as well as immunostaining for the presence of these factors as has been done previously (Sawada et al, 2008). Initially production of IL-4, IL-10 or IL-13 by MSCs will be examined. Proteomic screens using Mass spectrometry will be performed to determine neural, lung epithelial and lung endothelial phenotypes as previously described (Sawada et al, 2008; Arand et al, 1992).
  • sections may be stained with hematoxylin and eosin or may be stained with anti-CD 105, CD90 and CD29 (Pharmingen) primary antibodies followed by staining with anti-mouse FITC, PE, or Rhodamine conjugated secondary antibodies (Molecular Probes) as previously described.
  • Cells may be fixed with paraformaldehyde (PAF) before analysis using a FACSAria instrument (BD Biosciences), with acquisition and analysis using the FACSDiva program (BD Biosciences).
  • PAF paraformaldehyde
  • MSC may be seeded onto the scaffold in the bioreactor and adhered cells may be fixed using PAF.
  • the MSC-lung scaffold construct may be fixed with 2% (wt./vol.) paraformaldehyde for 30 min at 37°C and held at 4°C until assay. Following transfer, the construct may be washed in phosphate buffered saline (PBS) and then frozen sections may be cut. Sections may be permeabilized in 1% BD permeabilizing solution (BD Biosciences) for 10 min with a final wash in Tris-buffered saline (TBS). Nonspecific binding was blocked by a 1-hr treatment in TBS plus 0.1% w/v Tween containing defatted milk powder (30 mg/mL).
  • PBS phosphate buffered saline
  • TBS Tris-buffered saline
  • cells may be incubated in secondary antibodies conjugated to fluorescein isothiocyanate (FITC), rhodamine, or Cy5 anti-mouse, anti-rat, or anti-rabbit IgGs (1:500 dilution) for 1 hr at 37°C, then stored at 4°C until analyzed.
  • FITC fluorescein isothiocyanate
  • Cy5 anti-mouse, anti-rat, or anti-rabbit IgGs (1:500 dilution)
  • Use of isotype matched controls and omission of primary antibodies will serve as negative controls and resulted in no detectable staining in confocal analysis or less than 2% background staining for flow cytometry analysis of samples.
  • the preparations will then be mounted in Slow Fade GOLD with DAPI (Molecular Probes) and observed using an LSM 510 Meta advanced laser scanning confocal microscope (Zeiss, Thornwood, NY, USA).
  • MSCs may be evaluated for cell adhesion, migration in then scaffold, cytokine production and apoptosis induction using a conventional TUNEL assay. Sections of the fixed MSC-lung scaffold construct may be frozen sectioned and MSCs may be examined for production of immunomodulatory factors. Location and extent of fluorescent labels may be examined using a Nikon T300 Inverted Fluorescent microscope (Nikon Corp., Melville, NY, USA).
  • Statistical Analysis For cell phenotype analysis 10,000 cells were collected for each sample. Statistical analysis may be performed using GraphPad InSTAT software (version 2003). Mean values and standard deviation between data collected for MSCs may be determined. Analysis of variance (ANOVA) may be performed and data subjected to Tukey Kramer multiple comparison test. Mean differences in the values are considered significant when p is less than 0.05.
  • MSVs were designed, above all, for delivery of specific drugs during the treatment of cancer (Ferrari, 2005). Their particular structures and material composition allow for the attachment and encapsulation of different type of drugs.
  • the payload can be chemically attached on the particle surface through a previous chemical functionalization or they can be physically loaded in the pores of the particle; this approach offers the opportunity to deliver different types of payloads at the same time but also allows for the sequential delivery of different substances: a particle can be loaded with three or more types of drugs, the first , for instance, can be physically loaded in the pores of the vector, the second one can be encapsulated in liposomes that in turn are loaded in the pores of the particle, and finally a third drug can be chemically attached on the surface.
  • the different drugs can be released in different moment, allowing for a sequential and multiple drug release. Focusing on the example just given, it is straightforward to understand that the first physically loaded drug would be the last to be released finding itself in the bottom of the pores covered by the second physically loaded drug; moreover, the release of drug encapsulated in liposomes or micelles will depend of the degradation on the phospholipidic shell that surround it.
  • the types of drugs that can be loaded on MSVs can be of different nature, such as, chemical or physical; they can release substances able to kill the target cells throughout chemical reactions, enzimatic reactions or physical phenomena like thermal ablation,meaning a treatment that employs special metal nanoparticles that once excited with an opportune electromagnetic source they start increasing their temperature until they reach a level that damages and kills cells, and many other technique that exploit particle activation or photon emission to create an environment harmful for target cells.
  • MSVs can be sequestered from the blood flow in different ways: they can take and follow smaller and smaller capillaries until they are extravasated from them, they can be swallowed by phagocytic cells but that can also leave the blood vessels thanks to the presence of fenestration especially in the cancer region where they are usually more frequent and finally they can also adhere to blood vessel walls.
  • Another interesting strategy for targeting cancer cells consists in the coating of particles with different targeting moieties that allow the attachment of particles to specific target present on cancer cells, keeping the porous structure the drug to annihilate them once attached.
  • MSVs can be created in different shape, they can be spherical, cylindrical, hemispherical or discoidal depending on the fabrication process employed. It has been noticed that also the shape of the MSVs is crucial for their uptake by different tissues and organs (Gilbert et al, 2009). The study conducted to address and answer this aspect showed that spherical particles are the ones able to reach the majority of the organs with the lowest loss in the liver.
  • MSVs were designed initially for cancer treatment it turned out that they could have been used for other applications too. Studies are now conducted to use them in the imaging field (Ferrari, 2005). They can be loaded for example with contrast agents for magnetic resonance imaging (MRI) and other imaging techniques.
  • MRI magnetic resonance imaging
  • Growth factors are in general substances capable of triggering and guide cell proliferation, differentiation and growth; they are above all proteins but they could be represented by hormone too.
  • Nerve growth factor (NGF) discovered by Nobel Laureate Rita Levi-Montalcini ,was just the first one to be identified, today we can count many of them as, for instance, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) or hepatocyte growth factor and so on.
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • hepatocyte growth factor hepatocyte growth factor and so on.
  • Growth factor are molecules, usually proteins or steroid hormone, able to give the cells different instructions to allow for their proliferation or differentiation, depending on the situation and the needs.
  • Tissue engineering deals with techniques and methods to induce regeneration; using cells to repair damages that the body can not normally repair, or recreate biological functions thus to substitute the ones that an organs can no more support are the goals of tissue engineering.
  • the idea of using cells as mentioned above is brilliant and may constitute the solution to this issue, but cells alone are not able to proliferate and differentiate and the required rate and quantity thus, more often, the employment of growth factors is essential.
  • cell differentiation depends also on the environmental conditions that surround them, so not only growth factor presence and type but also ECM composition and morphology; this explains why we need a well-decellularized lung to achieve a good cell differentiation and proliferation.
  • growth factor should also be time- release adjustable, that means, to have the possibility to tune the time and the amount per time that these devices need to release the molecules.
  • more than one growth factor as to be employed requiring the possibility of simultaneous or sequential, depending on the situation, delivery of multiple growth factors.
  • biocompatible scaffolds is only one of the many existing: there are techniques that exploit the change in Ph or temperature to release growth factor, others that, thanks to a greater concentration of particular proteinase, in the site of interest, that cleaves the chemical bond keeping the growth factor linked to the delivery system, releases the signaling molecules just where needed. There are also different way of loading the proteins onto the delivery systems; it can be done throughout the exploitation of physical principles or chemical ones depending on the situation where they have to be used. This last observation is of great importance since it has been proven that the method f loading can denature the protein and make the growth factor complete useless for the purpose it was employed for.
  • MSVs mesoporous silicon particles
  • FIG. 18A and FIG. 18B The main idea underlying the delivery system designed for our purpose is the loading of the proper protein in the mesoporous silicon particles (MSVs) (FIG. 18A and FIG. 18B). It is required that the particles are perfused into the lung so that they can spread and can reach the inmost zones. To be sure that those particles can reach even the most difficult place inside the lung a brief study was conducted: MSVs particle were loaded with Rhodamine dye and perfused into an acellular lung, then images with an IVIS imaging system were taken and compared to the control images taken before the particle perfusion; then discoidal and spherical particles loaded with Rhodamine were used to see if effectively the discoidal ones could reach and accumulate better in the lung than the spherical ones.
  • ICP Inductive Coupled Plasma
  • SEM scanning electron microscopy
  • Particles used for these experiments were all discoidal; they were initially suspended in Isopropanol were disposed in small tubes and the tubes were eased down in a vacuum chamber to let the isopropanol evaporate; once dried, the particles were then suspended in different concentration of albumin, than sonicated to mix and help the particles to spread in the solution containing the protein and finally laid on a plate shaker for half an hour in order to help the loading of the protein into the MSVs.
  • kits were used: the Fibrin Gel In Vitro Angiogenesis Assay Kit (a product of EMD Millipore Corporation) and the Colorimetric (MTT) Kit for Cell Survival and Proliferation (EMD Millipore Corporation). These kits were employed with two types of human cells: the Human Lung Microvascular Endothelial Cells (HLMVEC) (purchased from Cell Applications, Inc.) and the Human Pulmonary Alveolar Epithelial Cells (HPAEpiC) (Science CellTM Research Laboratories).
  • HLMVEC Human Lung Microvascular Endothelial Cells
  • HPAEpiC Human Pulmonary Alveolar Epithelial Cells
  • Human lung microvascular endothelial cells are primary endothelial cells isolated from normal adult human lung capillaries; also human pulmonary alveolar epithelial cells, containing alveolar type I (AEC I) and alveolar type II (AEC II), responsible for lining more than 99% of the internal surface of the lung, were isolated from human lung tissue.
  • the kits were purchased with the intention to study cell behaviors in the presence of certain growth factors.
  • the fibrin gel in vitro angiogenesis assay kit was chosen to demonstrate whether cells were able to migrate and start to differentiate and thus showing, as the name suggests, tube and capillary formation; it exploits a fibrin gel since endothelial cells can rapidly align and make interconnections and finally display tube formation, a process that involves cell adhesion, differentiation, migration and proliferation; the colorimetric (MTT) kit for cell survival and proliferation instead is designed to study the proliferation of a cell culture under specific conditions; MTT is a yellow substrate that yields formazan product which has a dark blue color; this is due to the ability of only living cells to cleave MTT, explaining its purpose of studying the proliferation of a cell culture.
  • MTT colorimetric
  • FGFb Recombinant Human Fibroblast Growth Factor-basic
  • PDGF Human Recombinant Derived Growth Factor
  • VEGF Human Vascular Endothelial Growth Factor
  • AEC II cells may have an essential role in a future recellularization since they are believed to have self renewal ability and they seem to be able to reenter the cell cycle and differentiate in other type of cells such as AEC I.
  • each experiment was performed in triplicate, including the controls, which did not receive growth factors.
  • three different doses (or concentrations) were administrated to each type of cell in triplicate. That means that 48 wells were examined: having two different type of cells (HL1VIVEC and HPAEpiC), and, for each, a different concentration of a growth factor type administrated, more exactly three per growth factor type, plus the controls, everything done in triplicate, gives 24; times 2, since two different assay kits were used, gives 48.
  • the first study conducted for the growth factor distribution project was the perfusion of solution containing particles in the acellular lung scaffold (FIG. 19A, FIG. 19B, and FIG. 19C).
  • the IviSVs used for this study were discoidal since from preliminary results they ended up to be more dispersed in tissues than the others and they were suspended in a IX PBS solution. To see where the particles, loaded with rhodamine spread after their perfusion it was employed the IVIS imaging system mentioned above: a normal pig lung was used as control and a normal and a decellularized lung as the main samples. It was interesting to observe how the results were markedly different: the control lung appeared to be completely full of particle, while the acellular one was almost empty.
  • IVIS images gave unexpected results, to verify whether a significant amount of particles spread in the acellular organ, ICP mass spectroscopy was employed. In this case discoidal and spherical particles were perfused in different acellular pig lungs in solution of Hespan® and blood (FIG. 20).
  • ARAND M et al, "Colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins," Anal. Biochem., 207(1):73- 75 (Nov. 1992).
  • GHANNAM S et al, "Mesenchymal stem cells inhibit human Thl7 Cell Differentiation and Function and Induce a T regulatory cell phenotype," /. Immunol, 185(1):302-312 (May 2010).
  • GRIBSKOV, M, and BURGESS, RR "Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins," Nucleic Acids Res., 14(16):6745- 6763 (Aug. 1986).
  • HALES NW et al, "A countermeasure to ameliorate immune dysfunction in in vitro simulated microgravity environment: role of cellularnucleotide nutrition," In Vitro Cell Dev. Biol. Anim, 38(4):213-217 (Apr. 2002).
  • KABASHIMA-NIIBE A et al "Mesenchymal stem cells regulate epithelial- mesenchymal transition and tumor progression of pancreatic cancer cells," Cancer Sci., 104(2): 157-164 (Feb. 2013).
  • KRAMPERA M et al, "Role for INF- ⁇ in the immunomodulatory activity of human bone marrow mesenchymal stem cells," Stem Cells, 24(2):386-398 (Feb. 2006).
  • NEEDLEMAN, SB and WUNSCH, CD "A general method applicable to the search for similarities in the amino acid sequence of two proteins," /. Mol. Biol , 48(3):443-453 (1970).
  • NICHOLS JE et al, "Design and development of tissue engineered lung," Organogenesis, 5(2):57-61 (Apr-Jun. 2009).
  • NICHOLS Neurotrophic and neuro-protective potential of a novel subpopulation of peripheral blood-derived CD 133+ ABCG2+CXCR4+ mesenchymal stem cells: development of autologous cell-based therapeutics for traumatic brain injury," Stem Cell Res. Ther., 4(1):3 (Jan. 2013).
  • NICHOLS JE et al, "Production and assessment of decellularized pig and human lung scaffolds," Tissue Eng. Part A., 19(17-18):2045-2062 (Sept. 2013).
  • NICHOLS JE et al, "Production and utilization of acellular lung scaffolds in tissue engineering," /. Cell. Biochem., 113(7):2185-2192 (Jul. 2012).
  • PLETT PA et al., "Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells," Exp. Hematol., 32(8):773-781 (Aug. 2004).
  • SHEELA "Artificial vital organs and medical bionics market is expected to reach USD 43.3 billion globally in 2018: transparency market research," Wall Street J., Jun. 27, 2013.
  • TASCIOTTI E et al. , "Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications," Nature Nanotechnol, 3(3):151— 157 (Mar. 2008).
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the invention have been described herein in terms of illustrative embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods, and/or the steps or the sequence of steps of the methods without departing from the spirit, scope, and concept of the invention. More specifically, it will be apparent that certain compounds, which are chemically- and/or physiologically-related, may be substituted for one or more of the compounds described herein, while still achieving the same or similar results. All such substitutions and/or modifications, as apparent to one or more of ordinary skill in the relevant arts, are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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Abstract

L'invention concerne des méthodes de décellularisation d'un organe isolé, ou d'une partie de celui-ci, par agitation mécanique de l'organe isolé avec un fluide contenant un détergent qui élimine la membrane cellulaire entourant l'organe isolé, en présence d'une source régulable d'air afin de réguler l'élimination du détergent de l'organe décellularisé, tout en préservant la matrice extracellulaire et l'échafaudage. L'invention concerne également des procédés de production de tissus acellulaires imprégnés de mésoparticules pour la production d'organes et de tissus mammifères appropriés pour une greffe humaine,entre autres.
PCT/US2016/057977 2015-10-20 2016-10-20 Appareil et procédés de production de tissus acellulaires pour la régénération d'organes WO2017070392A1 (fr)

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CN108548735A (zh) * 2018-05-23 2018-09-18 东北大学 一种霍普金森压杆子弹电磁回收装置
WO2019100454A1 (fr) * 2017-11-27 2019-05-31 大连理工大学 Charpente poreuse décellularisée pour modèle tumoral tridimensionnel, et son procédé de construction et ses applications
US20210155904A1 (en) * 2018-04-18 2021-05-27 The Board Of Regents Of The University Of Texas System Production of a bioengineered lung
CN114796615A (zh) * 2022-04-20 2022-07-29 诺一迈尔（苏州）医学科技有限公司 一种软骨脱细胞基质及其制备方法

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Publication number Priority date Publication date Assignee Title
WO2019100454A1 (fr) * 2017-11-27 2019-05-31 大连理工大学 Charpente poreuse décellularisée pour modèle tumoral tridimensionnel, et son procédé de construction et ses applications
US20210155904A1 (en) * 2018-04-18 2021-05-27 The Board Of Regents Of The University Of Texas System Production of a bioengineered lung
CN108548735A (zh) * 2018-05-23 2018-09-18 东北大学 一种霍普金森压杆子弹电磁回收装置
CN108548735B (zh) * 2018-05-23 2024-04-16 东北大学 一种霍普金森压杆子弹电磁回收装置
CN114796615A (zh) * 2022-04-20 2022-07-29 诺一迈尔（苏州）医学科技有限公司 一种软骨脱细胞基质及其制备方法
CN114796615B (zh) * 2022-04-20 2023-08-25 诺一迈尔（苏州）医学科技有限公司 一种软骨脱细胞基质及其制备方法

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