Methods of tissue decellularization

Abstract

The present invention provides a method for producing a decellularized extracellular matrix (ECM) scaffold for at least a portion of a lobular organ lacking a common carotid artery, the method comprising: a) occluding afferent vessels to substantially seal the target lobular organ or portion thereof without the common carotid artery and / or major arteries in a non-human donor or a dead / brain-dead human donor; b) optionally: (i) flushing clots and / or blood from at least a portion of the occluded afferent vessels; and / or (ii) perfusing the organ or portion thereof to confirm occlusion of the afferent vessels; c) removing the sealed organ or portion thereof from the donor; and d) perfusing the sealed organ or portion thereof with a detergent and enzyme solution to obtain a decellularized ECM scaffold. Also provided are methods for producing artificial organs, and artificial organs produced by the methods. [Selection diagram] Figure 5

Description

[Technical Field]

[0001] The present invention Common artery and / or major Na The present invention relates to a method for producing a decellularized extracellular matrix (ECM) skeleton of a lobular organ lacking arteries, a skeleton obtained or obtainable by such a method, a method for regrowing said skeleton, and an artificial organ containing said skeleton. [Background technology]

[0002] Organ transplantation is an essential life-saving procedure for many patients. However, despite an increase in the number of organ donors, meeting the demand for organs remains impossible. Figures from the National Health Agency show that between March 2016 and March 2017, 411 patients died in the UK while on the active transplant waiting list. To reduce the likelihood of transplant rejection, donors and recipients must be tissue-matched, and even then, the vast majority of recipients require immunosuppression for the rest of their lives, which can lead to complications such as an increased risk of infection or damage to other organs.

[0003] Organ bioengineering, which involves seeding cells onto a supporting skeleton, is a developing area in the field of organ transplantation. Currently, the successful construction and transplantation of simple organs such as blood vessels, bladder, airway, and urethra have been possible. Typically, the construction of these organs involves seeding autologous cells onto a simple biomaterial skeleton. Due to their structural simplicity (including a hollow core and thin walls), once transplanted into a patient, these prosthetic organs can be supported by the diffusion of oxygen and nutrients from adjacent tissues until angiogenesis occurs. However, more complex organs cannot be supported by diffusion and require connection to the recipient's blood supply, posing a challenge in developing prosthetic organs that include importing and efferent vessels.

[0004] One possible solution to this problem, suggested in relation to kidney transplantation, is the use of an intact skeleton produced from the extracellular matrix (ECM) of a human or animal kidney. Donor kidneys were decellularized using lavage perfusion via the renal artery to create an ECM skeleton that retains an intact vascular network as well as various structural proteins and three-dimensional structures (Non-Patent Literature 1). While the skeleton shows promise for the development of artificial kidneys, the authors concluded that seeding and growth of cells on the skeleton is not yet sufficiently feasible to produce an artificial kidney for transplantation. Pancreatic perfusion-decellularization is performed by catheter insertion into the anterior hepatic portal vein, formed by the confluence of the splenic vein and superior mesenteric vein (Non-Patent Literature 2). The authors state that attempts to perfuse the organ via other circulatory systems, such as the pancreatic duct, have not been successful in producing a viable decellularized ECM skeleton.

[0005] Decellularized ECM scaffolds show promise in the development of artificial organs, particularly those with lobular structures. Common artery and / or major Na Significant technical challenges remain for organs with complex structures, such as those lacking arteries. One such organ is the thymus, which is formed from two lobes, each receiving its blood supply from branches derived from the subthyroid artery and the internal thoracic mammary artery. Common or main Na Because there are no arteries, veins, or ducts, it is not possible to use perfusion decellularization approaches that are used in relation to organs such as the kidney or pancreas. To date, it has only been possible to obtain the thymic skeleton by mechanically shaking or rotating the organ in a lavage solution, which has resulted in poor characterization of the entire skeleton and failure to maintain the fine reticular and vascular structures (Non-Patent Literature 3). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Katari R., Peloso A., Zambon JP., Sokeer S., Stratta RJ., Atala A., Orlando G. Renal Bioengineering with Scaffolds Generated from Human Kidneys, Nephron Exp. Nephrol. 2014;126:119-124

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problem to be Solved by the Invention

[0007] CommonArterial lobular organs typically develop as separate lobes, which then migrate to their final positions as independent, neovascularized structures, after which the lobes may adhere to one another via layers of connective tissue. Common to the entire organ, or actually to its lobes at times. no Due to the lack of vascular supply, it was not possible to apply a perfusion-decellularization approach to obtain the ECM skeleton from these organs. However, the inventors have developed a technique that enables whole-organ perfusion of such lobular organs. The method of the present invention creates an artificial perfusion system by closing the afferent vessel, thereby enabling the application of a perfusion-decellularization approach to the organ. [Means for solving the problem]

[0008] Therefore, in the first embodiment, the present invention is Common The present invention provides a method for producing a decellularized extracellular matrix (ECM) skeleton from at least a portion of a lobular organ lacking arteries; this method a) closes the afferent vessels within a non-human donor or a dead / brain-dead human donor jointly The and / or major Na Accompanied by arteries do not have The steps include: a) substantially sealing a target lobular organ or a portion thereof; b) optionally: (i) washing away coagulation and / or blood from at least a portion of the closed afferent vessel; and / or (ii) perfusing the organ or a portion thereof to confirm closure of the afferent vessel; c) removing the sealed organ or a portion thereof from the donor; and d) perfusing the sealed organ or a portion thereof with a washing agent and an enzyme solution to obtain a decellularized ECM skeleton. The novel artificial perfusion system approach provided by the method of the present invention preserves the fine reticular structure of the ECM, including the vascular network, Common This makes it possible to obtain a decellularized ECM skeleton from lobular organs without arteries. In embodiments of the present invention, the ECM skeleton is obtained from a portion of a lobular organ such as a single lobe or gland, the lobe or gland being Common It lacks arterial supply.

[0009] In embodiments of the present invention, the target organ or a portion thereof can be removed from a living human donor prior to steps a to d described above. The decellularized ECM skeleton obtained in this embodiment of the present invention can be utilized as outlined below.

[0010] Substantially sealing a target organ or part thereof means that the vital blood vessels supplying the organ or lobe are closed, thereby creating an artificial perfusion system. Typically, one or two vessels will be left open for subsequent cannula insertion to allow perfusion of the organ. Preferably, only one vessel is left open for subsequent cannula insertion. Appropriate techniques for closing the vessels are well known to those skilled in the art. For example, the afferent vessel can be clamped, sutured, or plugged. In a preferred embodiment of the present invention, the afferent vessel is ligated.

[0011] The donor may be a non-human donor, preferably a non-human mammal such as a pig. Alternatively, the donor may be a deceased / brain-dead human donor, such as a brain-dead, beating, and respirable organ donor. The human donor may be a pediatric donor, an adult donor, or an elderly donor. Since the skeletal source may be homogeneous or heterogeneous, maximal decellularization is desirable. However, since the ECM has been shown to affect cell migration, proliferation, and differentiation, this must be balanced with the need to preserve the integrity and bioactivity of the ECM. The three-dimensional structure, surface topology, and composition of the ECM all contribute to these effects. While the term decellularization is not fully defined by quantitative metrics, several minimum criteria have been proposed, including less than 50 ng of dsDNA per 1 mg of dry weight of ECM, DNA fragment length less than 200 bp, and / or the absence of visible nuclear material in histological staining of the tissue. Decellularized ECM produced by the method of the present invention may satisfy any one or more of these criteria.

[0012] As described above, the method of the present invention optionally comprises the step of washing clots and / or blood from at least a portion of the closed inlet blood vessels. This can be done by washing the blood vessels with an inert buffer such as phosphate-buffered saline (PBS). Removing clots and / or blood from the closed inlet blood vessels helps to ensure that the created artificial perfusion system can be freely perfused subsequently with a detergent without any occlusion.

[0013] This method may additionally or alternatively comprise the step of perfusing the sealed organ or a portion thereof to confirm the closure of the inlet blood vessels. Typically, this perfusion is performed by cannulating the inlet blood vessels that are not closed during the step of sealing the organ or a portion thereof. This perfusion step can be performed using an inert buffer such as PBS, and can be used to identify any additional inlet blood vessels that still need to be closed, for example due to anatomical variations between individual donors. The perfusion step may further be beneficial for identifying any incomplete closure of the blood vessels, which is detrimental to the artificial perfusion created by the closure of the blood vessels, thereby allowing this to be corrected prior to perfusion-decellularization of the target organ or a portion thereof.

[0014] Many suitable detergents and enzyme solutions can be used for perfusion-decellularization of the target organ or a portion thereof. In particular, the detergent may be selected from one or more of sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS) and Triton X-100. In a preferred embodiment of the present invention, the detergent is SDC, which has been shown to generate a decellularized ECM scaffold that retains the fine natural network structure and vasculature of the ECM. Furthermore, this detergent is non-toxic and can simply be rinsed away with water after the perfusion-decellularization cycle(s) are completed. In contrast, SDS may require removal by an additional detergent.

[0015] The concentration of the washing agent can be optimized based on the volume of the organ or part of the organ being perfused, the perfusion cycle or the duration of the cycle, and the perfusion rate. For example, the organ or part of the organ may be perfused with about 1% to about 10% (w / v) of SDC, preferably about 1% to about 7% (w / v), and more preferably about 2% to about 6% (w / v) of SDC. In a preferred embodiment of the present invention, the organ or part of the organ may be perfused with about 4% (w / v) of SDC. Alternatively, the organ or part of the organ may be perfused with about 0.05% to about 1% (w / v) of SDS, preferably about 0.05% to about 0.5% (w / v), and more preferably the organ or part of the organ may be perfused with about 0.1% (w / v) of SDS. The organ or a portion thereof may be perfused with about 0.05% to about 5% (w / v) of Triton X-100, preferably about 0.1% to about 2% (w / v), and more preferably with about 1% (w / v) of Triton X-100. In embodiments of the present invention in which the washing agent used for perfusion-decellularization of the organ or a portion thereof is SDS, the organ or a portion thereof may be washed with another detergent such as Triton X-100 after the perfusion-decellularization step is complete.

[0016] The detergent and enzyme solutions used in the present invention preferably contain one or more enzymes, such as an endonuclease (e.g., deoxyribonuclease I) or a protease (e.g., trypsin). The detergent and enzyme may be provided in separate solutions or combined in a single solution.

[0017] Perfusion-decellularization of an organ or part thereof is preferably performed using a decongestant-enzyme treatment (DET), which would be well-known to the skilled practitioner. As described above, They do not have a common artery. Target lobular organ or part thereof toThe primary afferent vessels are closed to substantially seal the organ or part thereof, while one or two vessels are left open for subsequent cannula insertion for perfusion of the organ or part thereof. Therefore, the lavage agent and enzymes are perfused into the organ or part thereof through the open vessel(s). Typically, the organ or part thereof is perfused for about 1 to about 4 hours, preferably about 1 to about 2 hours. In embodiments of the present invention, the organ or part thereof may require only about 1.5 hours of lavage agent perfusion.

[0018] The sealed organ or a portion thereof may be perfused with a washing agent at a rate of about 0.2 ml / min to about 1 ml / min, preferably at about 0.5 ml / min to about 0.9 ml / min. In a preferred embodiment of the present invention, the organ or a portion thereof may be perfused at a rate of about 0.6 to about 0.8 ml / min.

[0019] In embodiments of the present invention, a sealed organ or part thereof may be perfused for a single cycle, which has been shown to avoid the formation of deposits in the microvessels. For example, the organ or part thereof may be perfused with a lavage agent for a single cycle of about 1.5 hours. Alternatively, the organ or part thereof may be perfused for two cycles of about 1.5 hours each, using a second cycle of lavage agent perfusion.

[0020] A sealed organ or part thereof can float freely in the solution during perfusion, which has been shown to reduce tension and avoid the risk of catheters or cannulas coming into contact with the blood vessel wall. However, in alternative embodiments of the present invention, the organ or part thereof may simply be immersed in a suitable container and may not float freely. The organ or part thereof may be perfused within a bioreactor or a closed system.

[0021] As mentioned above, Common and / or major NaLobular organs lacking arteries are organs that develop as separate lobes that migrate to their final locations as independent angiogenic structures. Such organs include the thymus, thyroid gland, parathyroid gland, and salivary glands. Because there is no common arterial supply to the entire organ, or to its specific parts such as lobes or glands, applying perfusion-decellularization approaches to obtain the ECM skeleton from these organs or parts thereof has been impossible until now.

[0022] The thymus consists of two lobes that merge in the middle and are surrounded by a capsule that extends inward along with blood vessels. Each lobe has a dense outer cortex and a less dense inner medulla. In the two lung lobes, hematopoietic progenitor cells called thymocytes, derived from the bone marrow, mature into T cells. Once mature, T cells migrate from the thymus and form peripheral T cells, which play a role in directing many parts of the adaptive immune system. Loss of the thymus at a young age due to chromosomal abnormalities (such as DiGeorge syndrome) results in severe immunodeficiency and subsequent increased susceptibility to infection. The thymus supplies blood to the entire organ system. Common The thymus lacks arteries and instead receives blood supply from the internal mammary artery (also known as the internal mammary artery) and branches of the inferior thyroid artery, sometimes with branches from the superior thyroid artery. The vessels closed to seal the thymus may include the right or left common carotid artery, the right subclavian artery, the right internal mammary artery, the right costocervical trunk, the right aortic arch, the left aortic arch, the left costocervical trunk, and the left internal mammary artery. As previously described, one or two vessels remain open for subsequent organ perfusion. In a preferred embodiment of the present invention, only one vessel remains open. For example, if the right common carotid artery is closed, the thymus may be perfused via the left common carotid artery, or if the left common carotid artery is closed, the thymus may be perfused via the right common carotid artery.

[0023] The thyroid gland is a butterfly-shaped organ located in the front of the neck in humans. It consists of two lobes, left and right, connected by a narrow isthmus. The main functions of the thyroid gland are the production of iodine-containing thyroid hormones, triiodothyronine (T3) and thyroxine (T4), as well as the peptide hormone calcitonin. Thyroid hormones have a wide range of effects on the human body, including their impact on metabolism, cardiovascular health, and development, as well as their role in maintaining normal sexual function, sleep, and thought patterns. The thyroid gland supplies blood to the entire body. Common The thyroid gland lacks arteries and instead receives blood supply from the inferior thyroid artery, a branch of the subclavian artery's thyrocarotid trunk, as well as from the superior thyroid artery, branches of the external carotid artery, and sometimes from the thyroid Ima artery, which can vary in size from very small to the size of the inferior thyroid artery. The vessels that occlude the thyroid gland may include the left and right common carotid arteries after joining with the superior thyroid artery. If the two lobes of the thyroid gland are connected by vessels passing through the isthmus, the method of the present invention may be used to decellularize the ECM skeleton of the entire organ. Alternatively, this method may be applied to a single lobe of the thyroid gland to create a decellularized ECM skeleton of that lobe. Each thyroid lobe may be perfused via the right or left common carotid artery.

[0024] Humans typically have four parathyroid glands, usually divided into two pairs of glands located behind the left and right lobes of the thyroid gland. The two upper parathyroid glands are called superior parathyroids, while the two lower ones are called inferior parathyroids. The main function of the parathyroid glands is to maintain a very narrow range of calcium and phosphate concentrations in the body, allowing the nervous and muscular systems to function properly. The blood supply to the parathyroid glands is provided by the thyroid gland that covers them, and each parathyroid gland... Common or It lacks major arteries. The decellularization method of the present invention can be applied to individual parathyroid glands.

[0025] Mammalian salivary glands are exocrine glands that produce saliva through a system of ducts. Humans have three pairs of major salivary glands (parotid, submandibular, and sublingual glands) as well as hundreds of minor salivary glands. Each of the major salivary glands has its own... Common artery or The aorta is absent. The decellularization method of the present invention can be applied to the parotid gland, submandibular gland, or sublingual gland.

[0026] The present invention also provides a decellularized ECM skeleton that can be obtained or obtained by the method of the present invention. The method of the present invention results in a high degree of preservation of the structure of the ECM and vascular structure, while Common It has been demonstrated that this method can successfully decellularize lobular organs that lack arteries. This method can be applied to the entire organ or to parts of it, such as lobes or glands.

[0027] In a further embodiment, the present invention provides a method for producing an artificial organ, the method comprising regrowing a decellularized ECM skeleton obtained or obtainable by the method of the present invention with stromal cells such as a combination of epithelial and mesenchymal cells, culturing the regrowthed skeleton in vitro for about 4 to about 7 days, and then optionally seeding the regrowthed skeleton with donor cells.

[0028] The decellularized ECM skeleton can be regrowed with epithelial and mesenchymal cells in a ratio of approximately 2:1 to approximately 5:1. Preferably, the skeleton is regrowed with epithelial and mesenchymal cells in a ratio of approximately 5:1. The stromal cells are typically obtained by ex vivo expansion of cells from an organ tissue donor (i.e., allogeneic or xenogeneic cells) and can be delivered to the skeleton by direct needle injection, which provides a successful distribution of cells on and / or inside the skeleton. However, in embodiments of the present invention, the stromal cells can be autologous cells. The cells can be delivered separately, simultaneously, or sequentially. Sequential delivery may involve delivering cell populations within at least 1, 2, 5, 10, 20, 30, 40, 50, or 60 minutes from each other, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 36, or 48 hours from each other. However, in a preferred embodiment of the present invention, epithelial cells and mesenchymal cells are delivered simultaneously, either from separate sources or pre-mixed together in appropriate ratios as described above. Typically, the cells are injected in the form of a cell suspension, which contains a mixture of epithelial cells and mesenchymal cells in a ratio of about 2:1 to about 5:1 and may have a volume of about 10 μl to about 100 μl, preferably about 10 μl to about 60 μl, more preferably about 30 μl to about 40 μl. For example, a single injection may contain about 2 × 10⁶ of cells in a volume of about 30 μl to about 40 μl. 6 Epithelial cells and approximately 0.4 × 10 6 It may contain a suspension of mesenchymal cells.

[0029] The repopulation of decellularized ECM scaffolds with endothelial cells, optionally in combination with mesenchymal cells, is a critical step in the revascularization of the scaffold. For example, endothelial cells (preferably including pericyte-like cells) and mesenchymal cells may be injected at multiple sites of the scaffold. Alternatively, endothelial cells (preferably including pericyte-like cells) and mesenchymal cells can be injected into one or more afferent blood vessels to revascularize the scaffold. The cells are injected as a composition, and the volume of the injection(s) is optimized to fill the volume of the scaffold. Similarly, the concentration of cells in the composition can be optimized based on the volume of the composition to ensure that a sufficient number of cells are injected to completely revascularize the scaffold.

[0030] The repopulated scaffold is typically maintained in vitro for about 4 to about 7 days to allow cell migration and initial differentiation (e.g., cell polarization, adhesion molecule up / down-regulation) on the reticular ECM of the scaffold. Next, donor cells from different origins (typically allogeneic or xenogeneic cells, but which can optionally be autologous) can be delivered, for example, by direct needle injection into or onto the previously injected scaffold region. For example, the donor cells can be postnatal thymus-derived Lin - / CD45 + / CD3 - / CD4 - / CD8a - thymocytes, fetal liver-derived CD34 + cells, umbilical cord blood (CBC)-derived CD34 + cells, adult bone marrow-derived CD34 + cells or mobilized peripheral blood (adult)-derived CD34 +The donor cells may be hematopoietic stem cells (HSCs) containing cells. The donor cells may be iPSC-derived stromal cells and / or endothelial cells, and may be patient-specific (i.e., autologous) in combination with HSCs or T cell progenitor cells. Alternatively, the donor cells may originate from any lineage (e.g., endoderm, mesoderm, or neural crest) and may include thymocytes, thymic epithelial cells, mesenchymal cells, dendritic cells, endothelial cells, pericytes, macrophages, or B cells. Typically, the organ is maintained in vitro for an additional 24 hours or more after delivery of the donor stem cells and before transplantation to the recipient. It will be recognized that the skeleton may not need to be fully aggregated with seeded cells for it to be useful for transplantation to the subject. For example, the skeleton may have had cells seeded over at least 70, 80, 90, 95, or 99% of its surface.

[0031] Optionally, endothelial cells grown in vitro can be further delivered to the organ before transplantation into the recipient. Without being constrained by theory, such cells are thought to contribute to accelerating angiogenesis in the transplanted organ. In fact, such endothelial cells can be delivered to the organ via an artificial perfusion system created during skeletal construction. Delivery of such endothelial cells can help re-endothelialize the organ's vascular tree. Optionally, donor stem cells can be delivered via the same pathway.

[0032] In another embodiment of the present invention, a decellularized ECM skeleton can be regrown with stromal cells to form a regrowing skeleton that can be transplanted into a recipient. Once transplanted into the recipient, the regrowing skeleton can be angiogenically regenerated and subsequently regrow directly in vivo with autologous stem cells such as HSCs from the blood circulation. Mesenchymal cells and / or immune cells resident at the transplantation site can also invade the skeleton and help support remodeling and / or the survival and / or differentiation and functionality of the seeded cells. The regrowing skeleton may have been seeded with cells covering at least 70, 80, 90, 95, or 99% of its surface prior to transplantation.

[0033] The decellularized or regrowthed ECM skeleton described above may be further treated to enhance cell adhesion to the skeleton and / or cell growth on the skeleton. Such treatments may include the application of proteins such as growth factors or extracellular matrix proteins, including, for example, one or more of collagen, elastin, fibronectin, laminin, or proteoglycans. In embodiments of the present invention, cells seeded on the skeleton may be modified to produce growth factors such as VEGF, which enable faster angiogenesis. Cell modification typically refers to genetic modification of cells, and standard techniques known in the art may be utilized.

[0034] The present invention also provides artificial organs comprising a decellularized ECM skeleton obtained or potentially obtained by the present invention. These artificial organs can be manufactured by regrowing the decellularized ECM skeleton of the present invention with stromal cells as described above. The artificial organs can be used, for example, in therapies to provide transplantable organs for treating congenital or acquired conditions.

[0035] As described above, the cells used to regrow the decellularized ECM skeleton of the present invention can be obtained from donor tissue that can be cultured to provide the desired cell type and grown in vitro before being seeded onto the skeleton. In this way, a single tissue donor can provide multiple prosthetic organs. Since the cells grown on the skeleton are key to the potential immunogenicity of the prosthetic organ (the skeleton itself is not immunogenic since the cells have been removed from it and therefore does not need to be obtained from a tissue-compatible donor), this provides a significant advantage, especially when suitable tissue donors are scarce.

[0036] An artificial organ comprising a decellularized ECM skeleton obtained by the method of the present invention may be a thymus and may be used to treat conditions such as DiGeorge syndrome, a congenital chromosomal disorder that causes many symptoms, including a deficiency or hypoplasia of the thymus, leading to severe immunodeficiency. The artificial organ of the present invention may be used as an alternative source of mature cells for adoptive cell transfer, i.e., for the production of therapeutic cells such as T cells.

[0037] In a further embodiment, the present invention provides an in vitro method for testing a compound for its ability to induce a pharmacological, immunological, or toxicological response, the method comprising contacting the compound with an artificial organ comprising the decellularized ECM skeleton of the present invention.

[0038] Artificial organs can be cultured in vitro for 1, 2, 3, 4, 5, or 6 days. In embodiments of the present invention, artificial organs can be cultured in vitro for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. Preferably, artificial organs are cultured in vitro for up to 8 weeks. Artificial organs may be cultured under standard culture conditions. However, in preferred embodiments of the present invention, artificial organs are perfused with cell culture medium during the culture period, for example, by culturing the organs in a bioreactor.

[0039] Furthermore, the present invention provides a method of organ transplantation, which comprises surgically transplanting an artificial organ containing the decellularized ECM skeleton of the present invention into a patient. Also provided is a method of treating a disease, which comprises surgically transplanting an artificial organ containing the decellularized ECM skeleton of the present invention into a patient in need. In particular, the artificial organ containing the decellularized ECM skeleton of the present invention can be used to induce immune tolerance in conventional organ transplantation.

[0040] As used herein, the terms “patient” or “recipient” may include any mammal, including humans. In particular, a patient may be a pediatric patient, such as a neonatal or infant. A patient may have an acquired or congenital disorder requiring organ replacement. For example, an organ may be missing, diseased, damaged, or otherwise impaired in function. A patient may have received a transplant of another organ prior to or concurrently with receiving an artificial organ comprising the decellularized ECM skeleton of the present invention. Without being constrained by theory, it is conceivable that cells that regrow the decellularized ECM skeleton (e.g., AIRE+ cells) may provide Treg cells and induce donor-organ resistance.

[0041] The present invention is described in detail hereby only by examples with reference to the figures. [Brief explanation of the drawing]

[0042] [Figure 1] Figure 1 shows a schematic diagram of the surgical procedure for ligating specific arterial branches to enable the formation of an artificial perfusion system around the thymus. Nodes 1-9 are formed on the afferent vessels (node ​​6 on the left aortic arch should not be tightened at this stage), and then a catheter is inserted into the left common carotid artery. The left internal jugular vein is incised, followed by the left aortic arch being incised below node 6, and then the right internal jugular vein is severed. The aortic arch is irrigated and blood clots removed by slowly flushing a buffer such as PBS through the catheter, and then node 6 on the left aortic arch is tightly closed. Next, the cannula-inserted / catheter-inserted thymus is removed by severing the obturator arteries near nodes 1-9, and then the associated veins are severed, starting with the subclavian vein. [Figure 2] Figure 2 shows the rat thymus before (A, B) and after (C, D) decellularization by perfusion with a washing agent and enzyme solution (DET), both by microscopic examination (B, D) and histological examination (hematoxylin and eosin, H&E; A, C). [Figure 3]Figure 3 shows the histology of the decellularized thymic skeleton of the present invention: staining with hematoxylin and eosin (H&E) (A), picrosilius red (collagen, B), and Masson's trichrome (ECM, C) demonstrates the absence of cell nuclei as well as the preservation of the thymic capsule and reticular ECM. [Figure 4] Figure 4 shows macroscopic microscopic examination of rat thymic skeletons before (Panel A) and after (Panel B) decellularization by the method of the present invention. H&E staining of the thymus at low magnification (4X, Panel C) and high magnification (Panel D, scale bar = 100 μm) shows preservation of parenchymal lobules and vascular structures. [Figure 5] Figure 5 shows the appearance of the thymic skeleton obtained by the method of the present invention before (A), immediately after (B), and after 4 days of culture (C) injection of stromal cells. The excess thymic tissue (darker areas in the image) allows for connection to a cannula. [Figure 6] Figure 6 shows the histological features (H&E) of the regrowing stromal skeleton cultured for 4 days (Panel A). Immunohistochemical (IHC) images demonstrate healthy stromal cells that are mostly caspase-3 negative and positive for the progenitor / stem cell marker p63 (Panel B). Only a few cells are caspase-3 positive and show signs of apoptosis (yellow*) (Panel C). The regrowing stromal cells express cytokeratin 5 (CK5), CK8, and α6 integrin (CD49f) (Panel E). Furthermore, since most epithelial cells (EpCAM positive) are proliferating (Ki67 positive) (Panel D), it is indicated that the stromal cells regrow the stromal skeleton and maintain the progenitor cell phenotype within the 3D structure (scale bar = 50 μm). [Figure 7] Figure 7 shows macroscopic (panel A, scale bar = 2 mm) and histological (H&E) images of regrowing skeletons engrafted in NSG mice and matured in vivo at 8 weeks (B), 11 weeks (C), and 21 weeks (D), respectively. H&E staining demonstrates the presence of small round cells of hematopoietic origin interacting with organized stromal cells. Progressive stromal cell maturation is present, particularly as indicated by the presence of Hassall bodies within the skeleton at 11 weeks. Scale bar = 50 μm. [Figure 8] Figure 8 shows the immunohistochemistry of regrowthed skeletons transplanted into mature NSG mice in vivo, demonstrating the maturation of human stroma (EpCAM and ECadherin-positive cells, red) expressing functional markers such as HLA-DR (green) (A). Anti-human vimentin (green) recognizes organized human mesenchymal cells (B), while anti-human CD3 (green) demonstrates the presence of mature thymocytes in contact with human thymic stromal cells (C). Detection of AIRE-1-positive cells in the transplanted musculoskeletal system (3,3'-diaminobenzidine, DAB staining) demonstrates the functional maturation of thymocytes, which play a crucial role in eliminating autoreactive T cells (D). [Figure 9] Figure 9 shows that cells within the regrowthed, decellularized ECM skeleton differentiate into the correct lymphoid lineage. A. FACS analysis of mature, dissociated, genetically engineered human thymic skeletons in vivo shows that the majority (80%) of CD45+ cells developed from CD34+ progenitor cells are CD3+, lymphoid lineages; B. Single-positive CD4 and CD8 cells express maturation markers such as CD3+ and TCRab+. [Figure 10] Figure 10 shows that single and double CD4+ and CD8+ thymocytes are produced at rates comparable to those in normal tissue. A. FACS analysis of dissociated fresh human thymus showing single and double positivity for CD4+ and CD8+ thymocytes. B. FACS analysis of thymocytes generated in vitro from TN progenitor cells within a genetically engineered thymic skeleton (rat) at 6 days; this demonstrates that the genetically engineered human thymus supports T cell development ex vivo. [Figure 11] Figure 11 shows macroscopic microscopic examination of pig thymus before (left panel) and after (right panel) perfusion decellularization (two cycles of 4 hours of perfusion with 0.6–0.8 ml / min of 4% SDC). [Figure 12] Figure 12 shows histological analysis of porcine thymus after perfusion-decellularization demonstrating the preservation of ECM and vascular structure: H&E left panel; Masson's trichrome (MT); picrosilius red (PS) demonstrates the preservation of ECM proteins and vascular structure. [Figure 13] Figure 13 shows macroscopic microscopic examination of human thymus before (left panel) and after (right panel) perfusion decellularization (two cycles of 4 hours of perfusion with 0.6–0.8 ml / min of 4% SDC). [Figure 14] Figure 14 shows H&E staining of the human thymic skeleton after perfusion decellularization, demonstrating the removal of cellular components and the preservation of large and small vascular structures. [Examples]

[0043] Example 1 The rat thymus was sealed by ligating the right common carotid artery, right subclavian artery, right internal mammary artery, right costocervical trunk, right aortic arch, left aortic arch, left costocervical trunk, left internal mammary artery, and left subclavian artery (see Figure 1) and then excised from the subjects. The sealed thymus was decellularized in parallel using the following washing agent-enzyme perfusion (DET) treatment, in which it was perfused by inserting a cannula into the left common carotid artery while it was freely suspended in solution: a. Perfuse with MilliQH2O at 0.2 mL / min for 96 hours at 4°C; b. Perfuse with 4% SDC at a rate of 0.2 mL / min for 1.5 hours at RT; c. Perfuse with MilliQH2O at 0.2 mL / min for 24 hours at RT; d. Perfuse with 0.1 mg / ml DNA-degrading enzyme (warm 37°C) at a rate of 0.2 mL / min for 30 minutes; and Perfuse with PBS at 0.2 mL / min for 1 hour under RT.

[0044] The skeleton was then treated for sterilization (gamma irradiation, 1782 Gy).

[0045] The parallel decellularization of the thymus exemplifies the efficiency and reproducibility of the method of the present invention. As shown in Figure 2, decellularization results in the formation of clear thymic tissue while preserving the capsule. Histological analysis of the decellularized thymus by H&E staining, as shown in Figures 2C and 3A, demonstrates the absence of any cell nuclei, DNA, or cellular debris. Figures 3B and 3C show the preservation of the capsule and reticular ECM of the thymic structure. The thymic ECM is detected as a homogeneous reticular tissue, and the staining intensity reflects a higher abundance of ECM in the organ capsule (red in Figure 3B and blue in Figure 3C).

[0046] Example 2 The rat thymus was extracted and decellularized according to the protocol outlined in Example 1. The rat weighed 173 g.

[0047] Macroscopic images of the decellularized thymus are shown in Figure 4, before (4A) and after (4B) decellularization using H&E staining. The low-magnification (Figure 4C) and high-magnification (Figure 4D) areas show that vascular structures are preserved throughout the organ tissue, as well as in the parenchymal lobules. No DNA or cell debris was detected. This indicates whole-organ (both lobules) decellularization via a single cannula at the histological level.

[0048] Example 3 The thymic skeleton was obtained according to the protocol outline of Example 1. Next, following ex vivo expansion from a tissue thymic donor, the thymus was regrowthed by simultaneously delivering a 5:1 combination of thymic epithelial cells (TECs) and thymic mesenchymal cells (TMCs), respectively, by direct needle injection. In the thymic lobes shown as clear tissue in Figures 5A-C, cells were injected using a needle syringe (one injection per lobe, 40 μl each, 2 M TEC: 0.4 M TMC per injection). Extrathymic tissue (darker areas in Figures 5A-C) was made available for connection to a cannula maintained during culture.

[0049] The regenerated skeleton was maintained under in vitro conditions to allow cell migration and early differentiation (e.g., cell polarization, up / down regulation of adhesion molecules) on the reticular ECM of the skeleton over a period of 4 days. For in vitro culture, the skeleton was connected to a cannula and treated with 10% fetal bovine serum (FBS), insulin (5 μg / mL), and 3,3,5-triiodo-L-thyronine (T3) (2 × 10⁻¹⁶). -9 M), hydrocortisone (0.4 μg / mL), cholera toxin (1 × 10⁻⁶) -10 The samples were placed in petri dishes immersed in cFAD (epithelial) medium consisting of DMEM and Ham's F12 medium (v / v 3:1) supplemented with M), and 1% penicillin / streptomycin.

[0050] Figure 5 shows the appearance of the skeletal structure of TEC and TMC cells before injection (Figure 5A), immediately after injection (Figure 5B), and 4 days after culture (Figure 5C). Histological analysis of the skeletal structure by H&E staining 4 days after culture is shown in Figure 6A, which reveals that the cells adhere to and follow the skeletal ECM. Only a small area of ​​the skeletal structure has not yet regrowed, demonstrating the presence of intact matrix within the skeletal structure (black*). Immunohistochemical (IHC) images demonstrate that healthy stromal cells were negative for caspase-3 and mostly positive for the progenitor / stem cell marker p63 (Figure 6B). Only a very small number of cells (yellow*) were caspase-3 positive and showed apoptosis (Figure 6C). Regrowing stromal cells expressed cytokeratin 5 (CK5), CK8, and α6 integrin (CD49f) (Figure 6E). Furthermore, the proliferation of more epithelial cells (EpCAM-positive) (Ki67-positive) (Figure 6D) indicated that stromal cells regrow their skeletal structure, maintaining the phenotype of the progenitor cells within the 3D structure.

[0051] Example 4 Repeated rat thymic skeletons were prepared according to the protocol outlined in Example 3 above. After 4 days of in vitro culture with stromal cells, the regenerated skeletons were subcutaneously transplanted into NSG mice for 8 and 11 weeks.

[0052] Histological analysis (H&E) of two thymic skeletons matured in vivo at 8 weeks (Figure 7B), 11 weeks (Figure 7C), and 21 weeks (Figure 7D) demonstrated the presence of hematopoietic-derived small round cells interacting with organized thymic stromal cells. In particular, progressive stromal cell maturation was observed, as indicated by the presence of Hassall bodies within the skeleton at 11 weeks (Figure 7C) and an increase in thymocyte density (Figure 7D).

[0053] Example 5 Repeated rat thymic skeletons were prepared according to the protocol outlined in Example 3 above and regrowthed in human stromal cells identified by EpCAM and EC adherin-positive cells. After 4 days of in vitro culture with stromal cells, the regrowthed skeletons were subcutaneously transplanted into NSG mice for 11 weeks.

[0054] Immunohistochemical analysis demonstrated the presence of regrowthable human stroma, identified by EpCAM and EC adherin-positive cells. Functional maturation of stromal cells was indicated by the upregulation of MHC class II surface receptors (HLA-DR-positive cells) (Figure 8A). Anti-human vimentin immunostaining detected the presence of mesenchymal cells (Figure 8B), while anti-human CD3 immunostaining indicated the presence of mature thymocytes within the same region (Figure 8C). Detection of autoimmune response element 1 (AIRE-1)-positive cells in the transplanted thymic skeleton demonstrates the functional maturation of thymocytes, which play a crucial role in eliminating autoreactive T cells and thereby inducing tolerance (Figure 8D). This demonstrates the ability of human cultured stromal cells to survive for extended periods in vivo and organize into a functional structure similar to that of the native human thymus (i.e., Hassall body and HLA-DR expression).

[0055] Example 6 When a portion of the thymic skeleton, which had been regenerated and transplanted into NSG mice in vivo, was dissected and analyzed by FACS, a high survival rate of human cells was observed (63.9%, center panel, Figure 9A); the majority of viable cells were CD45+ (92.9%, right panel, Figure 9A). Importantly, 80% of the CD45+ cells were also CD3+ (Figure 9B, left panel), demonstrating that the thymic skeleton is an inducible microenvironment for the lymphoid system. The presence of single positive CD4+ and CD8+ cells expressing TCRab demonstrates that mature, functional T cells are produced within the skeleton in vivo (Figure 9B, center and right panels).

[0056] Example 7 Thymic skeletons were regrowthed in vitro with stromal cells and maintained in culture for 7 days, after which immature triple-negative thymocytes (TNs) were injected. Analysis by FACS 7 days after injection into the thymic skeleton revealed maturation to both single and double-positive CD4+ and CD8+ cells (Figure 10B). Importantly, the single-positive CD4 / CD8 ratio in the skeleton was similar to that of the native thymus (Figures 10A, B).

[0057] The project leading to this application is funded by the EU's Horizon 2020 Research and Innovation Programme under grant agreement number 639429.

[0058] References Fan Y., Tajima A., Goh SK., Geng X., Gualtierottii G., Grupillo M., Coppola A., Bertera S., Rudert WA., Banerjee I., Bottino R., Trucco M.Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor-Specific Immune Tolerance to Allografts,Molecular Therapy 2015;23:1262-1277 Goh SK.,Bertera S.,Olsen P.,Candiello J.,Halfter W.,Uechi G.,Balasubramani M.,Johnson S.,Sicari B.,Kollar E.,Badylak SF.,Banerjee I.Perfusion-decellularised pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.Biomaterials 2013;34(28):6760-6772 Katari R.,Peloso A.,Zambon JP.,Sokeer S.,Stratta RJ.,Atala A.,Orlando G.Renal Bioengineering with Scaffolds Generated from Human Kidneys,Nephron Exp.Nephrol.2014;126:119-124

Claims

1. a) Closing the afferent vessel to seal the target lobular organ or part thereof in a non-human donor or a dead / brain-dead human donor that does not have a common and / or major artery; c) the step of removing the sealed organ or a portion thereof from the donor; and d) Perfusing the sealed organ or a portion thereof with a washing agent and enzyme solution to obtain a decellularized ECM skeleton. Includes, b) (i) washing away coagulation and / or blood from at least a portion of the closed afferent vessel; and / or (ii) perfusing the organ or a portion thereof to confirm the closure of the afferent vessel. After step a) and before step c), including or not including step b), A method for producing an artificial organ comprising the steps of: regrowing a decellularized extracellular matrix (ECM) skeleton obtained by a method for producing a decellularized extracellular matrix (ECM) skeleton of at least a portion of a lobular organ without a common artery, together with stromal cells; culturing the regrowthed skeleton in vitro for 4 to 7 days; and subsequently seeding the regrowthed skeleton together with donor cells, or not seeding the regrowthed skeleton together with donor cells.

2. The method according to claim 1, wherein the detergent is selected from one or more of sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS), and Triton X-100.

3. The method according to claim 2, wherein the SDC concentration is 1% to 10% (w / v), or the SDS concentration is 0.05% to 1% (w / v), or the Triton X-100 concentration is 0.05% to 5% (w / v).

4. The method according to any one of claims 1 to 3, wherein the sealed organ or a part thereof is perfused with a washing agent for 1 to 4 hours.

5. The method according to any one of claims 1 to 4, wherein the sealed organ or a part thereof is perfused with a washing agent at a rate of 0.2 ml / min to 1 ml / min.

6. The method according to any one of claims 1 to 5, wherein the sealed organ or a portion thereof is perfused for a single cycle.

7. The method according to any one of claims 1 to 6, wherein the sealed organ or a part thereof is freely suspended in the solution during perfusion.

8. The method according to any one of claims 1 to 7, wherein the lobular organ or part thereof that does not have a common artery is the thymus or its lobe, the thyroid gland or its lobe, the parathyroid gland or a salivary gland.

9. The method according to claim 8, wherein the afferent vessels closed to seal the thymus include the right or left common carotid artery, the right subclavian artery, the right internal mammary artery, the right costocervical artery, the right aortic arch, the left aortic arch, the left costocervical artery, the left internal mammary artery, and the left subclavian artery, the left internal mammary artery, the left costocervical artery, and the left aortic arch.

10. The method according to claim 9, wherein the right common carotid artery is closed and the thymus is perfused through the left common carotid artery, or the left common carotid artery is closed and the thymus is perfused through the right common carotid artery.

11. The method according to claim 8, wherein the afferent vessel closed to seal the thyroid gland includes the right or left common carotid artery after joining with the superior thyroid artery.

12. The method according to claim 11, wherein each thyroid lobe is perfused via the right or left common carotid artery.

13. The method according to claim 1, wherein the decellularized ECM skeleton is regrowthed in epithelial cells and mesenchymal cells in a ratio of 2:1 to 5:

1.

14. An artificial organ obtained by the method described in any one of claims 1 to 13.

15. An artificial organ according to claim 14 for use in therapeutic purposes.

16. The artificial organ according to claim 14, wherein the artificial organ is a thymus and is intended for use in the treatment of DiGeorge syndrome.

17. in testing the ability of a compound to induce a pharmacological, immunological, or toxicological response. An in vitro method comprising contacting the artificial organ described in claim 14 with the compound.

18. A transplantable organ, including the artificial organ described in claim 14, for surgical implantation into a patient.

19. A transplantable organ comprising the artificial organ according to claim 14, for surgical implantation in a patient who needs it to treat a disease.

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