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    • 专利摘要:
    Procedure for the decellularization of biological organs and tissues. The present invention relates to the use of methyl salicylate for the decellularization of organs and/or tissues, preferably of nervous tissue. In addition, it also refers to the tissue and/or organ obtained by said use and its use for the preparation of an implantable prosthesis for the regeneration of tissues and/or organs. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2610573A1
    申请号:ES201531544
    申请日:2015-10-28
    公开日:2017-04-28
    发明作者:Manuel José GAYOSO RODRÍGUEZ
    申请人:Universidad de Valladolid；
    IPC主号:
    专利说明:

    The present invention relates to the use of methyl salicylate for decellularization.of organs and / or tissues, preferably of nervous tissue. Tissues and organsobtained through such use are useful for the regeneration of tissues and / or organs.Therefore the present invention can be framed in the field of medicine. 10 STATE OF THE TECHNIQUE
    Decellularization involves the removal of cells from an organ or tissue, trying to maintain the highest possible integrity of the remaining extracellular matrix, to generally use this matrix as a support or scaffold for repair 15 or, if possible, replacement of injured tissues or organs. Decellularization is carried out by destroying the cells and suppressing cell debris. The elimination of cells and their remains leads to the disappearance of antigens that produce both inflammatory and immune reactions, while the components of the extracellular matrix are proteins whose structure is very similar in different individuals of the same species and even between individuals of different species are proteins that are phylogenetically highly conserved that do not usually produce inflammatory or immune reactions, which will replace the damaged tissue or organ (Constantinou and Jiménez. 1991. Matrix 11: 1–9). The extracellular matrix once decellularized can serve as scaffolding or support 25 for subsequent recelularization with the cells of the recipient or other immunocompatibles that will not produce immune rejection. Decellularization and recelularization are being carried out in numerous organs and tissues, such as: heart valves, tendon, ligaments, blood vessels, urinary bladder, liver, kidney, lung, skeletal muscle, skin and nerves. (Gilbert et al. 2006. Biomaterials
    30 27; 3675–3683; Isaac Perea-Gil et al. 2015. Am J Transl Res; 558-573; Ott et al. 2008. Nat Med; 14: 213-221).
    Decellularization generally begins with the physical destruction of cells.After destroying the cells, cell debris is usually removed with
    35 enzymatic or detergent treatments that disorganize membranes


    cell phones and facilitate the removal of the remains while trying to preserve, as far as possible, the structure and functions of the extracellular matrix.
    The peripheral nerve has the ability to regenerate its nerve fibers after a
    5 section in favorable conditions. That is, if a nerve is sectioned for trauma, surgical resection of a tumor or other cause if the conditions are favorable, it can regenerate its nerve fibers and restore its functions. The most favorable circumstances are a clean section of the nerve without separation of the proximal and distal ends of the section. This ideal situation is not usually frequent and the
    10 proximal and distal ends are usually more or less separated. In these cases, if you try to pull the ends of the nerve to suture them, the tension prevents the regeneration of the nerve fibers, so it is necessary to interpose a material as a bridge between the ends of the sectioned nerve. The most effective so far is the autologous nerve implant, that is, a nerve is sacrificed
    15 usually sensory of the individual to recover a motor nerve. This method has as main drawbacks the morbidity caused and the shortage of the material available for the implant. For these reasons, different alternatives have been studied, such as the allogeneic implant, but it needs immunosuppression, as well as the construction of biocompatible implants, both biological and
    Collagen or biomaterials such as polymers of lactic acid, caprolactone or glycolic acid.
    For the beginning of decellularization, the destruction of cells, in the nerve and in other organs and tissues, methods such as: osmotic shock 25 with hypotonic solutions or with distilled water, sudden freezing and thawing and sonication have been employed (Gilbert et al. 2006. Biomaterials 27; 3675-3683). Among the first studies of nerve decellularization, the one carried out by Johnson et al. 1982. (Muscle & Nerve 5; 335-344.). The main objective was the ultrastructural and biochemical study of the extracellular matrix of the nerve. To get such
    30 cell-free matrix, the authors used strong detergents, based on the more or less complete destruction, produced by detergents on the junctions between lipids and proteins.
    Later Sondell et al. 1998 (Brain Research, 795; 44-54) obtained more specific results through a treatment that combines osmotic shock, with distilled water, and the use of non-ionic (Triton ™ X-100) and anionic detergents


    (deoxycholate). The extracellular matrix obtained in this way seems to correspond to the basal lamina that surrounds the nerve fibers so, according to these authors, these basal lamina tubes serve as support or scaffolding for regeneration, through its now empty interior, of Nerve prolongations interrupted. In this way, the basal lamina surrounds the regeneration units. Hudson et al. 2004 (Tissue Engineering, 10; 1346-1358) looked for what they called the optimization of the method by studying the specific action of each detergent on the cells and the extracellular matrix. His method begins with an osmotic shock with distilled water and uses amphoteric (sulfobetaine 10 and sulfobetaine 16) and anionic detergents (Triton ™
    10 X-200) to remove cell debris. With prostheses of this type, experimental results similar to those obtained with autologous or isogenic implants have been obtained and much more effective than those obtained with the method of Sondell et al. 1998
    15 The only implant obtained from human allogeneic nerve and commercially available is AVANCE®. This prosthesis comes from human donors and after freezing and thawing the nerve segment is decellularized with detergents and with CSPG (chondroitin sulfate proteoglycan) chondroitinase (Muir. 2010. Experimental Neurology 23; 102-111) .The CSPG consists of a nucleus of protein and a number
    20 variable glycosaminoglycans such as chondroitin sulfate.
    Until now, the decellularization of organs and tissues is based mainly on the action of different types of detergents on lipids and cell proteins.
    25 For the regeneration of tissues and organs, due to the importance of scaffolding having the correct three-dimensional shape, there is a need to produce a decellularized tissue or organ with the same three-dimensional interstitial structure, shape and size as that of the native. The reconstruction of an artificial organ using
    A decellularized organ will thus produce an artificial organ that functions as well as the native organ because it retains the same interstitial shape, size and structure that allows the cells to assume a morphology and structure comparable to those of the native organ. In addition, in the case of motor nerve regeneration, there is a need to achieve a decellularized motor nerve
    35 allogeneic to avoid the need to sacrifice sensory nerves of the individual.

    DESCRIPTION OF THE INVENTION
    The present invention demonstrates the use of methyl salicylate for the
    5 decellularization of organs and tissues. As an example of the procedure of our invention, the decellularization of the peripheral nerve is described but such use can be extended to other tissues or organs, such as for example peripheral nerve, central nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.
    10 The method described here does not use any type of detergent, but it is methyl salicylate the agent that performs the decellularization. This fact allows, with similar results in terms of regeneration, these prostheses have physical qualities more suitable for surgical use since they have more consistency and
    15 stiffness than other prostheses obtained with other methods.
    Methyl salicylate (also called methyl ester 2-hydroxybenzoic or methyl 2-hydroxybenzoate), of molecular formula C8H8O3 is known to the expert by CAS number 119-36-8. The structural formula of methyl salicylate is as follows:
    In the present invention, pure methyl salicylate (100%) has been used, however, it is possible to use it diluted, for example in absolute alcohol, at a lower concentration
    25 provided that it retains its decellularizing properties, so that 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 could be used for example , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
    Methyl salicylate may be of natural origin, for example of plant origin (for example, of plants of the genus Gaultheria, for example Gaultheria procumbens; or


    of the genus Betula) or obtained by chemical synthesis, for example obtained by reaction of salicylic acid with methanol.
    The present invention also relates to salts of methyl salicylate or its
    5 derivatives that can be generated by chemical methods known to the person skilled in the art, for example, by a reaction with an acid in water or in an organic solvent or in a mixture of both. As the organic solvent, ether, ethyl acetate, ethanol, isopropanol or acetonitrile can be used. Examples of acid addition salts include mineral acid addition salts such as, by
    For example, hydrochloride, hydrobromide, iodhydrate, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and ptoluenesulfonate. Salt preparation can be carried out by methods known in the state of the art.
    15 Methyl salicylate has been used in the state of the art as an active ingredient in analgesic, anti-rheumatic, sunscreen creams and ointments and as a transparent histological and anatomical sections (that is, for the improvement of the visualization of cells, nuclei and cytoplasms cell phones).
    The present invention relates to a method of decellularization for organs and tissues that eliminates cells and cell debris while respecting the extracellular matrix. The procedure consists in the rupture of the cells by osmotic shock, preferably by immersion in distilled or purified water. Being the salicylate
    It is necessary to dehydrate the samples before subjecting them to the action of methyl salicylate, therefore, a dehydration is continued in alcohols of increasing concentration and from the absolute alcohol it is passed to methyl salicylate to extract cell debris. Finally, it is washed in absolute alcohol and hydrated in decreasing alcohols to water, for example, deionized, distilled or
    30 purified, preferably deionized. DNase can be used to remove, if any, excessive remains of nuclear material. The process can be repeated until returning to the water from where the pieces can be passed to a physiological liquid and used as a support for recelularization or put them in a suitable medium and keep them until they are used. For example, if the prosthesis obtained
    35 is not going to be used at that time it can, after immersion in a buffer of


    freezing and a slow decrease in temperature (for example -1ºC / min), keep frozen.
    The prosthesis (tissues and / or decellularized organs) can be recelularized “in vitro”
    5 with the desired cell type. For example, with Schwann cells, stem cellsmesenchymal bone marrow, mesenchymal stem cells of adipose tissue,etc.
    A person skilled in the art could introduce changes or modifications in the examples or particular embodiments described without departing from this invention.
    In a first aspect the present invention relates to the use of methyl salicylate for decellularization of tissues and / or organs.
    In a preferred embodiment of the first aspect of the invention, the tissue is selected from the list consisting of: nervous tissue, nerves and nervous tracts, dermis, choroids, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. Preferably it is nerve tissue, where the nerve tissue is a nerve, more preferably it is a peripheral nerve, more preferably a mixed peripheral nerve.
    In another preferred embodiment of the first aspect of the invention the use is an in vitro or ex vivo use.
    In the present invention "regeneration" is used as a synonym for reconstruction, that is, for total or partial repair of the structure and / or functionality of a tissue and / or organ.
    The term "in vitro" refers to the use being made outside the subject's body. That is, it is performed on a biological sample of a subject.
    The term "biological sample" in the present invention refers to any sample comprising tissues and / or organs of an individual, obtained by any method known to a person skilled in the art that serves this purpose. The biological sample in the present invention can be fresh or frozen.


    The term "ex vivo" refers to the method of the invention being performed outside the subject's body under conditions similar to those found in tissue and / or organ within the subject's body.
    In another preferred embodiment of the first aspect of the invention the use is a use inalive.
    The tissues and / or organs described in the present invention may be whole tissues and / or organs or fragments thereof of any size. In one embodiment
    10 preferred tissue and / or organ is isolated. In a preferred embodiment they are mammalian. Suitable mammals include humans, primates, dogs, cats, rodents (eg mice, rats), cows, horses, pigs, rabbits, goats and sheep. More preferably of human origin, where the human is of any age or sex. In another preferred embodiment they are of autologous, allogeneic origin,
    Xenogeneic (heterologous). In a preferred embodiment the tissues or organs are obtained from post mortem individuals, preferably from organ donors.
    "Decellularization" means the removal of cells as well as their fragments (for example, cell membranes, cytoplasmic components and / or
    20 nuclear), antigenic and / or inflammatory components. The decellularization of organs comprises eliminating the nuclear and cellular components of an isolated organ or part of an organ, leaving an interstitial structure that has the same size and shape of the native organ.
    The term "decellularized organ" as used herein refers to an organ or part thereof from which the entire cellular and tissue content has been removed, leaving an interstitial structure (the extracellular matrix). The organs are composed of various specialized tissues. The specialized tissue of an organ is the parenchyma and provides the function
    30 specific associated with the organ. Most organs also have a framework, the stroma, composed of non-specialized connective tissue that supports the parenchyma. The decellularization procedure eliminates the parenchyma, leaving the three-dimensional interstitial structure of connective tissue, composed mainly of collagen. The interstitial structure has the same shape and size as the
    35 native organ, providing the support framework that allows cells to join and grow on it. The decellularized organs can be rigid or


    semi-rigid, presenting the ability to alter its shape. Examples of decellularized organs include, but are not limited to, peripheral nerve, nervous tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.
    In the present invention, "decellularized tissue" is understood as that tissue to which the entire cellular content has been removed leaving the interstitial structure, ie the extracellular matrix. When we refer to tissues in the present invention, we also refer to parts thereof.
    The term "isolated organ or tissue" as used herein refers to an organ that has been removed from an individual, preferably a mammal. Suitable mammals include humans, primates, dogs, cats, rodents (for example, mice, rats), cows, horses, pigs, rabbits,
    15 goats and sheep. The term "isolated organ" also includes an organ removed from a subject that needs an artificially reconstructed organ. Suitable organs may be any organ or part of the organ required for implantation in a subject. Examples include, but are not limited to, peripheral nerve, nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver,
    20 pancreas, spleen, ureter and urethra.
    An organ or part of an organ can be isolated from the subject that requires an artificial reconstructed organ. For example, a diseased organ of a subject can be removed and decellularized, provided that the disease affects the parenchyma of the
    25 organ, but that does not harm the connective tissue, for example by tissue necrosis. The diseased organ can be removed from the subject and decellularized. The decellularized organ, or part of the organ, can be used as a three-dimensional scaffolding to reconstruct an artificial organ. An autologous artificial organ can be reconstructed using the subject's own decellularized organ as scaffolding and
    30 recelularizing it with a population of cells derived from the subject's own tissues. For example, cell populations derived from the skin, bone marrow, adipose tissue, liver, pancreas, arteries, veins, umbilical cord and placental tissues of the subject. If the cells are not autologous they cause their immunological rejection, unless an immunosuppressive treatment is established.


    An allogeneic artificial organ can be reconstructed using as scaffolding, the decellularized organ of another individual of the same species and recelularizing it with populations of cells derived from the subject itself. In this case, the scaffolding would be allogeneic and autologous cells. For example, different populations of cells can be derived from mammals such as primates, dogs, cats, rodents (eg mice, rats), cows, horses, pigs, rabbits, goats and sheep. The recelularization would be performed with populations of cells derived from the subject himself, in this case the xenogeneic (heterologous) prosthesis and autologous cells would be considered. Standard procedures for the isolation of an organ
    10 targets are well known to those skilled in the art and can be used to isolate the organ.
    An organ, or part of an organ, can also be derived from a human corpse or from mammalian species other than the subject, such as organs from
    15 primates, dogs, cats, rodents (for example mice, rats), cows, horses, rabbits, pigs, goats and sheep. Standard procedures for the isolation of a target organ are well known to those skilled in the art and can be used to isolate the organ.
    In a second aspect the present invention relates to a method for decellularizing a tissue or an in vitro organ comprising the following steps:
    to. rupture of the cells of an organ or tissue;
    b. dehydration of the organ or tissue of step (a), with gradation alcohols
    growing; 25 c. Immersion of the organ or tissue of step (b) in methyl salicylate;
    d. Immersion of the organ or tissue of step (c) in absolute alcohol;
    and. hydration of the decellularized organ or tissue of step (d) with decreasing gradation alcohols;
    Hereinafter we will refer to it as the "first method of the invention" or the "first method of the present invention".
    In a third aspect the present invention relates to a method for decellularizing a tissue or an organ comprising the following steps:
    to. rupture of the cells of an organ or tissue; 35 b. dehydration of an organ or tissue with increasing gradation alcohols;


    C. decellularization of the organ or tissue of step (b) by contact with methyl salicylate;
    d. removal of methyl salicylate from step (c) with absolute alcohol;
    and. hydration of the decellularized organ or tissue of step (d) with alcohols
    5 of decreasing gradation;where at least one of the stages is performed in the body of the lifeless subject.
    Hereinafter we will refer to it as the "second method of the invention" or the "second method of the present invention". In a preferred embodiment of the method
    10 second of the invention the breaking of step (a) is performed in the lifeless body of a subject. In a preferred embodiment at least one of the steps is performed by perfusion.
    In the present invention, the tissues and / or organs obtained from a "lifeless subject" are obtained from corpses from medical-legal autopsies and from deceased organ donors, all obtained according to current legislation.
    In the present invention, tissues and / or organs from a "lifeless subject" can be obtained at the time of death or after hours or days after the same.
    The tissues and / or organ fragments in the present invention can also come from biopsies of living organisms.
    In a preferred embodiment of the second and third aspects of the invention the steps
    (a) to (e); that is, (a), (b), (c), (d) and (e); they are performed more than once, preferably twice.
    Preferably in a preferred embodiment of the second and third aspects of the invention step (d) is performed at least three times, for at least 12 hours one of the washes. In this step, methyl salicylate is extracted.
    In a preferred embodiment of the second and third aspects of the invention, the tissue is selected from the list consisting of: peripheral nerve, central nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, Bladder, ureter and urethra. Preferably the peripheral nerve is a mixed peripheral nerve.


    In the present invention the term "subject" refers to a mammal, preferably primate, more preferably to a human, where the human is of any age or sex. The individual can also be a dog, cat, rodent (for example, mouse, rat),
    5 rabbit, cow, horse, pig, goat and sheep.
    In a preferred embodiment of the second and third aspect of the invention in step
    (a) cell rupture is performed by osmotic shock, sonication or by freezing and thawing. Preferably the osmotic shock is performed
    10 by immersion (or perfusion) in a hypotonic solution, distilled water, double distilled water or purified water, preferably in distilled water.
    Sonication procedures include, but are not limited to, acoustic horns, piezoelectric crystals or any other method for producing stable sonic waves, for example with sonication probes. Sonication should be carried out at a frequency that selectively removes cell membranes and / or cellular material without destroying the interstitial structure. The sonication frequencies that are appropriate will depend on the size and type of isolated organ that is decellularized. Typical sonication frequencies are between 40 kHz and 50 kHz. 20 However, it is foreseeable that a wide range of frequencies, from the subauditives to the ultrasonic ones (between approximately 7 Hz and 40 MHz, preferably between 7 Hz and 20 MHz), will provide a sonically increased dissociation of the tissues. Variations in the type of sonication are also contemplated in the invention and include pulsating versus continuous sonication. The
    25 power levels of the sonication source are between 10-4 and approximately 10 watts / cm2.
    If necessary, because the deoxyribonucleic acid (DNA) residues are more than 50 ng / mg dry weight, it can be treated with nuclease (phosphodiesterase) (for example, use S1 Nuclease (Thermo Scientific) which has DNase and RNase activity ) or with deoxyribonuclease (DNase), for example at a concentration of 0.1mg / ml and subsequent washing with distilled water. Therefore, in another preferred embodiment of the second and third aspects of the invention, the method further comprises after step (e) the use of nuclease or (DNase) on the hydrated organ or tissue (is
    35 say, in one step (f)).


    In a more preferred embodiment of the second and third aspects of the invention, the method further comprises a step (g) where the decellularized organ or tissue is introduced into a liquid selected from the group consisting of distilled water, double distilled water, purified water, alcohol (of any gradation), physiological buffer
    5 and a half culture. Preferably it is in distilled water. Subsequently it can be introduced into another liquid from those selected above.
    In an even more preferred embodiment of the second and third aspects of the invention at least one of the above mentioned steps is carried out under stirring (to improve the diffusion of the liquids). Preferably by mechanical agitation. In a particular embodiment, the mechanical stirring process involves the use of a mechanical stirring plate (for example an orbital motion plate). The isolated organ or tissue, or part thereof, is introduced into a container (sealed or not) with an adequate volume of fluid and stirred on the plate at a suitable speed. 15 The proper speed to agitate the isolated organ will depend on its size. For example, a rotation speed between about 50 revolutions per minute (r.p.m.) and about 150 r.p.m. A large organ will require faster speed compared to a smaller organ, as is known to the person skilled in the art. The volume of fluid in
    20 that the insulated organ is introduced and the time it remains in it will also depend on the size of the isolated organ, as is known by the person skilled in the art.
    In the method of the invention any of the steps (a), (b), (c), (d) (e) or (f) can be performed at least twice. Preferably, steps (a) to (e) are carried out at least twice.
    The times and volumes of solutions in which the tissue and / or organ are submerged or perfused will vary depending on the size thereof.
    In a preferred embodiment of the second aspect of the invention in step (a) to break the cells, the osmotic shock will be used, by immersing the piece in distilled or purified water for at least 24 hours. Water will be changed at least three times, preferably four times. The volume of liquids of
    35 immersion will be at least 20 times the volume of the piece and the times will depend


    of the size of the pieces that are processed, as is known by the person skilled in the art.
    In another preferred embodiment of the second or third aspect of the invention in step
    5 (b) dehydration is carried out in alcohols of increasing concentration,preferably alcohols of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, more preferably70, 96 and 100% (absolute alcohol). More preferably the pieces thetissue or organ to decellularize at each concentration of alcohol, at least 30 min
    10 and at least three changes are made.
    In another preferred embodiment of the second or third aspect of the invention in step
    (c) immersion in methyl salicylate or contact with methyl salicylate is
    make with at least three changes in said liquid, preferably in the last of the 15 which will remain for about 6-24 hours, preferably 18-22 hours.
    In another preferred embodiment of the second or third aspect of the invention in step
    (e) hydration is carried out in alcohols of decreasing concentration preferably 100, 99, 98, 97 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78,
    20 77, 76, 75, 74, 73, 72, 71 or 70%, more preferably 100, 96 and 70%. Preferably, at each concentration of alcohol, 30 min will be maintained and at least three changes will be made.
    In another preferred embodiment of the second or third aspect of the invention in step
    25 (e) the last step is to distilled water (ie after the last concentration of alcohol used), preferably with at least three changes between 15-45 minutes, more preferably 30 min.
    In another preferred embodiment of the second aspect of the invention all steps 30 must be performed under continuous agitation.
    The tissue and / or organ can be maintained before step (a) and / or after step (e) in a suitable cell culture medium, for example, depending on the tissue and / or organ the most suitable one will be used as the person skilled in the art knows, for example,
    35 but not limited to them, RPMI 1640, half of Fisher, half of Iscove, half of McCoy, half of Dulbecco and the like.


    The methods of the invention may further comprise drying or freezing of the decellularized organ or tissue. The dried or frozen decellularized organ or tissue can be stored at a suitable temperature or equilibrated in a
    5 balancing fluid before use.
    The balancing of the decellularized organ takes place in a balancing fluid. Examples of equilibration fluid include, but are not limited to, distilled water, alcohol (of any gradation), physiological buffer and culture medium
    10 (for example, but not limited to, RPMI 1640, half of Fisher, half of Iscove, half of McCoy, half of Dulbecco and the like.)
    Drying procedures of the decellularized organ or tissue include freeze drying or lyophilization of the organ to remove residual fluid. He
    The lyophilized decellularized organ or tissue can be stored at a suitable temperature until its use is required.
    A fourth aspect of the invention relates to decellularized tissue or organ (or fragments thereof) according to the method described in the second or third
    Aspect of the present invention. Hereinafter we will refer to it as the "tissue and / or organ of the invention" or "the tissue and / or decellularized organ of the invention".
    In the present invention the tissue or organ of the invention is also called "scaffolding", "three-dimensional scaffolding", "three-dimensional structure", "prosthesis" or
    25 "graft".
    The tissue and / or organ grafts (or parts thereof) of the present invention provide a natural tissue and / or substitute organ and retain the biological properties for the promotion of cell regeneration through the graft. The
    The native structure of the extracellular matrix is conserved, in particular, in the case of nerve grafts, the basal and endoneural lamina retain their natural and original structure, thus the scaffolding of the invention promotes the regeneration of Schwann axons and cells. leading them to the distal nerve.
    The decellularized tissues and organs of the invention are immunologically tolerated.


    The decellularized tissues and organs of the invention can be part of a suture, a tube, a sheet or a scaffold for proper positioning in the subject's body.
    5 Tissue and / or organ can be frozen before decellularization and then stored until later use.
    In a preferred embodiment of the fourth aspect of the invention the tissue is selected
    10 from the list consisting of: peripheral nerve, nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. Preferably the peripheral nerve is a mixed peripheral nerve.
    The tissue and / or decellularized organ of the fourth aspect of the invention can also
    15 understand a growth factor. Preferably the growth factor is selected from the list consisting of: nerve, epidermal, endothelial, fibroblastic, insulin-like growth factors, brain-derived neurotrophic factor, neuregulins and neurotrophins.
    20 Neurotrophins (or neurotrophic factors) promote the survival and functionality of the nerve or glia cells. They include a factor that participates in neuronal differentiation, induces neural proliferation, influences synaptic function and / or promotes neuronal survival. Examples of neurotrophins are: ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), FGF, brain-derived
    25 neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), glia derived neurotrophic factor (GDNF), NT-4, NT-5, NT-6, NT-7, Purpurin and the like. Other factors that have neurotrophic activities although not classified as neurotrophins would be contemplated in the present invention. For example, these "neurotrophin-like factors" would include epidermal growth factor (EGF), heparin-binding
    30 neurite-promoting factor (HBNF), insulin-like growth factor 2 (IGF-2), fibroblastic growth factor (a-FGF and b-FGF), platelet derived growth factor (PDGF), NSE, and activin
    TO.
    The tissue and / or decellularized organ of the fourth aspect of the invention can also
    35 understand cytokines and / or hormones. For example, but not limited to, growth hormone, erythropoietin, thrombopoietin, interleukin 3, interleukin 6, interleukin


    7, macrophage colony stimulating factor, c-kit ligand stem cell factor, osteoprotegerin ligand, insulin, insulin-like growth factors, fibroblast growth factor, nerve growth factor, ciliary neurotrophic factor, growth factor derived from platelets and bone morphogenetic protein.
    The tissue and / or decellularized organ of the fourth aspect of the invention can also comprise materials or substances that favor its implantation. For example, it may comprise phorbol ester phorbol myristate acetate (PMA) for the reduction of the immune response mediated by CD4 + T cells. The tissue and / or organ
    Decellularized from the fourth aspect of the invention can also comprise an antibiotic and / or in immunosuppressant.
    The tissue and / or decellularized organ of the fourth aspect of the invention can also comprise at least one preservative.
    15 A preservative is understood to mean that substance that maintains the properties of the drug by inhibiting germ contamination can be an ionic preservative
    or non-ionic. The preservative used will not be toxic, will be chemically stable, and compatible with the tissue and / or organ. As preservatives can be used
    Those known in the state of the art, for example, the preservative may refer to ethyl alcohol, benzoic acid, sodium benzoate, ascorbic acid, potassium sorbate, methylparaben, ethylparaben or butylparaben. "Germs" means any cell that can grow and multiply in the composition of the invention, for example bacteria, fungi or yeasts.
    Another preferred embodiment relates to the use where the composition further comprises an antioxidant.
    The term "antioxidant" refers to that substance that is capable of retarding or
    30 prevent oxidation. As antioxidant agents, those known in the state of the art can be used, for example tocopherol, ascorbic acid, sodium ascorbate, tartaric acid, butylhydroxyanisole, citric acid, vitamin A or vitamin E.
    The tissue and / or decellularized organ produced by the methods of the invention can be used as three-dimensional scaffolding to reconstruct an artificial organ.


    For the reconstruction (or regeneration) of the artificial organ, preferably autologous cell populations can be used to avoid immunosuppressive treatment, but both allogeneic and xenogenic cell populations can also be used. The procedures for the isolation and culture of cells used to reconstruct an organ are known to the person skilled in the art. Cells can be isolated using techniques known to those skilled in the art. For example, the tissue or organ can be broken down mechanically and / or treated with digestive enzymes and / or with chelating agents that weaken the connections between neighboring cells, making it possible to disperse the tissue in a suspension of individual cells without appreciable levels of cell breakage. Enzymatic dissociation can be achieved by crushing the tissue and treating the crushed tissue with any of several digestive enzymes, either alone or in combination. These include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase and / or hyaluronidase, DNase, pronase and dispase. Mechanical rupture can also be achieved by various procedures, including, but not limited to, scraping the surface of the organ, the use of crushers, mixers, sieves, homogenizers, pressure cells or sonicators, among others. Preferred cell types include, but are not limited to, mesenchymal stem cells from bone marrow or adipose tissue, nerve cells, choroid, renal, urothelial cells, adipose tissue cells, skeletal or smooth muscle, myocytes (muscle stem cells), fibroblasts, chondrocytes, adipocytes, myofibroblasts and ectodermal cells, including ductile and skin cells, hepatocytes, islet cells, cells present in the intestine and other parenchymatous cells, osteoblasts and other bone-forming cells or cartilage Isolated cells can be cultured in vitro in order to increase the number of cells available for infusion in three-dimensional scaffolding. If an immune response occurs in the subject after implantation of the reconstructed artificial organ where allogeneic or xenogeneic cell populations have been used, the subject can be treated with immunosuppressive agents.
    such as cyclosporine or FK506, in order to reduce the likelihood of rejection.
    The invention also relates to the use of a tissue and / or a decellularized organ
    as three-dimensional scaffolding to rebuild an organ. Through the use of a decellularized organ, the interstitial structure of connective tissue is preserved.
    This allows the cell populations of the patient to whom the


    scaffolding or cells grown in vitro in the scaffolding or perfused join the three-dimensional scaffolding. The conservation of a three-dimensional interstitial structure equal to the organ in vivo creates the optimal environment for cell-cell interactions and for the development and differentiation of cell populations.
    Therefore, a fifth aspect of the invention refers to the use of the decellularized tissue or organ of the fourth aspect of the invention as a prosthesis, that is, for the preparation of a prosthesis. Preferably an implantable prosthesis.
    In the present invention, prostheses are understood as tissue and / or organ that replaces a damaged tissue and / or organ and that fulfills its same function.
    A sixth aspect of the invention relates to the prosthesis comprising the decellularized tissue or organ of the fourth aspect of the invention. In one embodiment
    In particular, the prosthesis is selected from the list consisting of: peripheral nerve prosthesis, nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. Preferably the prosthesis is mixed peripheral nerve.
    The present invention also relates to the use of the prosthesis of the sixth aspect of the invention for the regeneration of tissues and / or organs. Preferably for the regeneration of peripheral nerve, nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter or urethra. More preferably the peripheral nerve is mixed peripheral nerve.
    A seventh aspect of the invention relates to a composition comprising the tissue and / or organ of the fourth aspect of the invention. Preferably the composition further comprises at least one growth factor. More preferably the growth factor is selected from the list consisting of: growth factors
    30 nervous, epidermal, endothelial, fibroblastic, insulin-like, brain-derived neurotrophic factor, neuregulins and neurotrophins (for example: ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), FGF, brain-derived neurotrophic factor (BDNF) , Neurotrophin-3 (NT-3), glia derived neurotrophic factor (GDNF), NT-4, NT-5, NT-6, NT-7, Purpurin and the like, Other factors that have neurotrophic activities
    Although they are not classified as neurotrophins, they would be contemplated in the present invention. For example, among these "neurotrophin-like factors" would include the


    epidermal growth factor (EGF), heparin-binding neurite-promoting factor (HBNF), insulin-like growth factor 2 (IGF-2), fibroblastic growth factor (a-FGF and b-FGF), platelet derived growth factor (PDGF ), NSE, and activin A.
    The composition of the seventh aspect of the invention can also comprise cytokines and / or hormones. For example, but not limited to, growth hormone, erythropoietin, thrombopoietin, interleukin 3, interleukin 6, interleukin 7, macrophage colony stimulating factor, c-kit ligand stem cell factor, osteoprotegerin ligand, insulin, growth factors similar to insulin growth factor
    10 of fibroblasts, nerve growth factor, ciliary neurotrophic factor, platelet-derived growth factor and bone morphogenetic protein.
    The composition of the seventh aspect of the invention may further comprise an antibiotic, a cytokine and / or in immunosuppressant.
    An eighth aspect of the present invention relates to the use of the composition of the seventh aspect of the invention for the manufacture of a medicament. Therefore the present invention also relates to the composition of the seventh aspect of the invention for use as a medicament.
    The term medicament and pharmaceutical composition and are used interchangeably in this invention. The term "pharmaceutical composition" herein refers to any substance used for relief, treatment or cure of diseases in humans or animals. The pharmaceutical composition of the
    The invention can be used both alone and in combination with other pharmaceutical compositions. In the context of the present invention, the pharmaceutical composition or medicament is characterized by comprising the composition of the seventh aspect of the invention in a therapeutically active amount.
    In the present invention the expression "therapeutically effective amount" refers to the amount of tissue and / or organ or fragments thereof calculated to produce the desired effect and, in general, will be determined, by the characteristics of the tissues, the route, form and frequency of administration thereof, and other factors, including the age, condition of the patient, as well as the severity of the
    35 alteration or disorder.


    The term "vehicle", like the excipient, refers to a substance that is used in the pharmaceutical composition or medicament to dilute any of the components of the present invention comprised therein to a certain volume or weight. The function of the vehicle is to facilitate the incorporation of others
    5 elements, allow a better dosage and administration or give consistency and formto the composition. When the presentation form is liquid, the vehiclePharmacologically acceptable is the diluent. The vehicles pharmaceuticallyacceptable that can be used in the pharmaceutical composition of the presentInvention are the vehicles known to those skilled in the art.
    In another particular embodiment, said pharmaceutical composition is prepared in solid form or in aqueous suspension, in a pharmaceutically acceptable diluent.
    The therapeutic composition provided by this invention can be administered.
    15 by any appropriate route of administration for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen. In a particular embodiment, the administration of the therapeutic composition provided by this invention is carried out for example parenterally, intra-intravenously, intravenously, orally, intraperitoneally or subcutaneously,
    20 preferably intravenously.
    The composition of the invention can be administered in one or several doses for the duration of the treatment in order to optimize the therapeutic effect.
    A ninth aspect of the invention relates to the use of the composition of the seventh aspect of the invention for the preparation of a medicament for the regeneration and / or repair of tissues and / or organs. Preferably the tissues and / or organs to be regenerated are selected from the list consisting of: peripheral nerve, central nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver,
    30 pancreas, spleen, bladder, ureter and urethra. More preferably the peripheral nerve is mixed peripheral nerve.
    Therefore the present invention also relates to the composition of the seventh aspect of the invention for use in therapy, preferably in therapy of
    35 repair and / or regeneration of tissues. Preferably the tissues and / or organs to be regenerated are selected from the list consisting of: peripheral nerve, tracts of the


    central nervous system, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. More preferably it is mixed peripheral nerve.
    The decellularized tissue or organ may be pretreated prior to use for the purpose.
    5 to enhance the binding of cells to the decellularized organ. For example, I couldtreat the decellularized organ with, for example, collagens, elastic fibers, fiberslattices, glycoproteins, glycosaminoglycans (eg heparan sulfate,Chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.).It can also be pretreated with mesenchymal, endothelial and / or cells
    10 parenchyma to allow better adhesion of the cells to the tissue and / or organ of the invention.
    The prosthesis, preferably implantable, may also comprise mesenchymal cells (for example mesenchymal cells of adipose tissue), endothelial or
    15 parenchyma for growth in decellularized tissue or organ. In the case of nerve cells you can also comprise Schwann cells.
    The present invention also relates to a method of tissue and / or organ regeneration comprising the use of methyl salicylate to decellularize an organ.
    20 and / or tissue and implantation of tissue and / or decellularized organ in a subject. The tissue or organ may be of the same subject to which it is to be implanted or of another individual of the same species or of another species.
    The present invention also relates to the in vitro use of the tissue and / or organ of the fourth aspect of the invention or of the composition of the seventh aspect of the invention.
    The present invention also relates to a kit comprising the tissue and / or decellularized organ of the fourth aspect of the invention, the prosthesis of the sixth aspect of the invention or the composition of the seventh aspect of the invention. The kit may also comprise one or more solutions useful for resuspending or rehydrating the tissue and / or organ of the fourth aspect of the invention, for example sterile saline or a pharmacologically acceptable buffer. In addition, the kit can also comprise cells (for example mesenchymal cells, particularly mesenchymal cells of adipose tissue; endothelial or parenchymal for growth in decellularized tissue or organ or also in the case of nerves, Schwann cells) or other components useful for regeneration, for example, cytokines and / or understand


    nerve, epidermal, endothelial, fibroblastic, insulin-like growth factors, brain-derived neurotrophic factor, neuregulins and neurotrophins.
    The present invention also relates to the use of the tissue regeneration kit
    5 and / or organs. Preferably for the regeneration of peripheral nerve, tract of thenervous system, optic nerve, dermis, choroid, heart, kidney, liver, pancreas,spleen, bladder, ureter or urethra. More preferably the peripheral nerve is nervemixed peripheral.
    In the present invention the terms "subject", "individual" and "patient" are used interchangeably.
    Here, including the appended claims, the singular forms of words such as "a", "a" and "the" include their corresponding plural references unless the context clearly indicates otherwise.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and characteristics of the
    The invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES
    25 FIG. 1 Cross section of a normal nerve (A), an acellular prosthesis (B) and a regenerating prosthesis (C). The arrows indicate the cell nuclei. EXAMPLES
    The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
    EXAMPLE 1 Use of methyl salicylate as a peripheral nerve 35 decellularizing agent


    The objective is the decellularization, that is, the removal of cells and cellular debris, from an organ or tissue, conserving the extracellular matrix. As an example we will take the decellularization of a nerve segment. The chemicals used were obtained, except those detailed by Sigma-Aldrich (Madrid).
    5The part object of the process was subjected to the following steps:
    1. A rat was anesthetized, with a lethal dose (0.6 ml / kg) of ketamine
    (Imalgene ™ 1000, MERIAL LABORATORIES). 10
    2. A 1 cm long segment of a sciatic nerve was taken.
    3. To break up the cells, the osmotic shock was used, by immersing the piece in distilled or purified water for at least 24 hours. He changed the
    15 water four times. The volume of the immersion liquids was at least 20 times the volume of the piece and the times depended on the size of the pieces that were processed.
    4. Dehydrated in alcohols of increasing concentration: 70, 96 and 100%. The
    20 pieces were kept at each concentration of alcohol, at least 30 min and at least three changes were made.
    5. Immersed in pure methyl salicylate, with three changes, in the last of the
    which remained overnight, about 18 hours. 25
    6. Salicylate was extracted by absolute alcohol in which at least three changes were made and remained in the last change until the next day (about 18 hours).
    30 7. Hydrated in alcohols of decreasing concentration: 100, 96 and 70%. In each alcohol concentration it was maintained for 30 min and three changes were made.
    8. The pieces were transferred to distilled water, with three changes of 30 min. Yes it is
    necessary, because the DNA residues are more than 50 ng / mg dry weight, it can be treated with 0.1 mg / ml DNase and washed with distilled water.


    All these steps from 1 to 8 were performed in continuous agitation on an orbital shaker with a frequency of 30 rpm.
    9. From this point the process was repeated, that is to say it goes back to step 3.5
    10. From the end of the last wash, in distilled water or in deionized water, the decellularized nerve segment can, after washing in a physiological solution, be implanted, for example, in a neurectomy performed in another rat. The prosthesis will be recelularized by the cells of the recipient animal thus repairing
    10 the lesion caused by the neurectomy.
    Figure 1 shows a section of the normal sciatic nerve (A), a section of decellularized prosthesis (B) and a section of the same prosthesis with regenerated nerve fibers (C). Note the absence of nuclei in the decellularized prosthesis.
    15 The foregoing description is that performed with a prosthesis implanted in the peripheral nerve. If it is desired to decellularize a solid organ such as the lung, the intestine, the liver, it is necessary to perfuse by the appropriate route with the mentioned liquids, for example: by air (lung), digestive tract (intestine), via
    20 blood (liver).
    Therefore, in the present invention the utility of methyl salicylate for peripheral nerve decellularization is demonstrated but the expert can extrapolate, without undue experimentation, its use for other organs of interest, such as
    25 for example, other nerves, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter or urethra.

    权利要求:
    Claims (28)
    [1]
    1. Use of methyl salicylate for the decellularization of a tissue and / or an organ in
    vitro.5
    [2]
    2. Use according to claim 1 wherein the tissue is selected from the list consisting of: peripheral nerve, central nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra .
    Use according to claim 2 wherein the peripheral nerve is mixed peripheral nerve.
    [4]
    4. Method for decellularizing a tissue or an in vitro organ comprising the following steps:
    to. rupture of the cells of an organ and / or tissue;
    15 b. dehydration of the organ and / or tissue of step (a) with alcohols of increasing gradation;
    C. Immersion of the organ or tissue of step (b) in methyl salicylate;
    d. Immersion of the organ or tissue of step (c) in absolute alcohol;
    and. hydration of the decellularized organ or tissue of step (d) with alcohols 20 of decreasing gradation.
    [5]
    5. Method according to claim 4 wherein in step (a) the cell rupture is performed by osmotic shock, sonication or by freezing and thawing.
    [6]
    6. Method according to any of claims 4 or 5 which further comprises a step (f) wherein nuclease or DNase is used on the hydrated organ or tissue of step (e).
    Method according to any one of claims 4 to 6 which further comprises a step (g) wherein the decellularized organ or tissue is introduced into a liquid selected from the group consisting of: distilled water, double distilled water, purified water, buffer Physiological and culture medium.
    Method according to any one of claims 4 to 7 wherein at least one of the steps is carried out under stirring.

    [9]
    9. Method according to any of claims 4 to 8 wherein steps (a) to (e) are carried out at least twice.
    5 10. Method to decellularize a tissue or organ in a lifeless subject thatIt comprises the following stages:
    to. rupture of the cells of an organ and / or tissue in a lifeless subject;
    b. dehydration of the organ and / or tissue with increasing gradation alcohols;
    C. decellularization of the organ or tissue of step (b) by contact with methyl salicylate 10;
    d. removal of methyl salicylate from step (c) with absolute alcohol;
    and. hydration of the decellularized organ or tissue of step (d) with decreasing gradation alcohols;
    where at least one of the stages is performed by perfusion. fifteen
    [11]
    eleven.  Method according to claim 10 wherein in step (a) the cell rupture is performed by osmotic shock.
    [12]
    12.  Method according to any of claims 10 or 11, further comprising
    20 in one step (f) the use of nuclease or DNase on the hydrated passageway organ or tissue (e).
    [13]
    13. Method according to any of claims 10 to 12, further comprising a step (g) where tissue and / or decellularized organ is introduced into a liquid
    25 selected from the group consisting of: distilled water, double distilled water, purified water, physiological buffer and culture medium.
    [14]
    14. Method according to any of claims 10 to 13 wherein at least one of
    The stages are performed in agitation. 30
    [15]
    15. Method according to any of claims 10 to 14 wherein the steps (a) to
    (e) are performed at least twice.
    [16]
    16. Method according to any of claims 4 to 15 wherein the tissue is selected from the list consisting of: peripheral nerve, nervous system tract

    central, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.
    [17]
    17. Method according to claim 16 wherein the peripheral nerve is a peripheral nerve5 mixed.
    [18]
    18. Tissue and / or decellularized organ according to the method described in any of claims 4 to 17.
    19. Tissue and / or decellularized organ according to claim 18 which further comprises at least one growth factor.
    [20]
    20. Tissue and / or decellularized organ according to claim 19 wherein the growth factor is selected from the list consisting of: growth factor
    15 nervous, epidermal, endothelial, fibroblastic, insulin-like, brain-derived neurotrophic factor, neuregulins and neurotrophins.
    [21]
    21. Use of the tissue and / or decellularized organ according to any of the claims
    18 to 20 as prostheses. twenty
    [22]
    22. Use of the tissue and / or decellularized organ according to claim 21 wherein the prosthesis is selected from the list consisting of: peripheral nerve prosthesis, central nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.
    [23]
    23. Use of the tissue and / or decellularized organ according to claim 22 wherein the prosthesis is a mixed peripheral nerve prosthesis.
    [24]
    24. Prosthesis comprising the tissue and / or organ according to any of the 30 claims 18 to 20.
    [25]
    25. Prosthesis according to claim 24 wherein the prosthesis is selected from the list consisting of: peripheral nerve, central nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra .
    [26]
    26. Prosthesis according to claim 25 wherein the prosthesis is a mixed peripheral nerve.

    [27]
    27. Composition comprising the tissue and / or decellularized organ according to any of claims 18 to 20.
    Composition according to claim 27 further comprising at least one growth factor.
    [29]
    29. Composition according to claim 28 wherein the growth factor is selected from the list consisting of: epidermal nerve growth factor,
    10 endothelial, fibroblastic, insulin-like, brain-derived neurotrophic factor, neuregulins and neurotrophins.
    [30]
    30. Use of the composition according to any of claims 27 to 29 for the
    Preparation of a medicine. fifteen
    [31]
    31.  Use according to claim 30 for the preparation of a medicament for the regeneration of tissues and / or organs.
    [32]
    32  Use according to claim 31 wherein the tissues and / or organs to be regenerated are
    20 selected from the list consisting of: peripheral nerve, central nervous system tract, optic nerve, dermis, choroid, heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.
    [33]
    33. Use according to claim 32 wherein the peripheral nerve is mixed peripheral nerve. 25
    [34]
    34. In vitro use of the tissue and / or organ according to any of claims 18 to 20.

     DRAWINGS
    Fig. 1
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    WO2017072378A1|2017-05-04|
    ES2610573B1|2018-02-12|
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    CN109908403A|2019-01-08|2019-06-21|王伟|A kind of acellular nerve allografts and preparation method thereof|
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