Minimally invasive cell transplant procedure to induce development of in vivo organogenesis

Abstract

To provide a minimally invasive cell transplant procedure to induce the development of in vivo organogenesis.SOLUTION: Provided herein are methods and systems of transplanting cells and growing an ectopic tissue in a lymph node of a subject. In certain embodiments, the methods and systems provided herein enable minimally invasive cell transplantation to treat patients in need of cell transplantation. In certain embodiments, the methods and systems provided herein include the use of ultrasound endoscopy. In a specific embodiment, the disclosure provides a minimally invasive method of transplanting one or more cells and growing an ectopic tissue in a subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority claim This application claims priority to U.S. Provisional Application No. 62 / 832,492, filed April 11, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Introduction The present disclosure relates to minimally invasive methods for transplanting cells into the lymph nodes of a subject to generate functional ectopic tissues and organs. [Background technology]

[0003] background The demand for organ transplantation and / or organ regeneration is great. However, the shortage of available organs for transplantation to end-stage patients represents a major medical, social, and economic challenge worldwide. In addition, whole organ transplantation can have stringent requirements regarding the recipient's own health status. For example, there are currently approximately 30,000 end-stage liver disease (ESLD) patients annually in the United States who are ineligible for standard liver transplantation.

[0004] Alternative approaches to whole organ transplantation may include cell transplantation to regenerate failing organs. For example, hepatocyte transplantation (HT) can prolong and improve the quality of life of ESLD patients who have no further treatment options and may be considered unsuitable for standard liver transplantation. However, orthotopic cell therapy targeted to the diseased organ may be infeasible for many reasons, including the possible loss of an appropriate environment in the cirrhotic and fibrotic liver of end-stage disease and the loss of the thymus in complete DiGeorge syndrome.

[0005] For patients suffering from ESLD, there may be a significant challenge: most cell therapies aim to promote cell engraftment in the diseased native liver. The transplanted liver cells are generally injected into the spleen (splenic artery in patients or splenic parenchyma in rodents) or intrahepatically via the portal vein. The liver cells transplanted into the splenic artery can rapidly migrate actively or passively to the diseased liver after the initial splenic injection, and liver regeneration by the transplanted liver cells is expected to occur. However, due to the anatomical site of transplantation, this approach has significant limitations. Most of these ESLD patients (Takahashi et al., 2014) have splenomegaly and hypersplenism, in which the spleen may undergo aggressive cell capture followed by phagocytosis and cell destruction against any cells (e.g., red blood cells, white blood cells, and platelets) that enter the splenic parenchyma through the splenic arterial circulation. Moreover, the transplanted liver cells may be further directed to the liver through the splenic vein as a major component of the portal venous supply to the liver parenchyma. These cells can circulate from the portal triad to the hepatic sinusoids, and partial obstruction of small portal branches and hepatic sinusoids by hepatocytes results in transient portal hypertension and early ischemia, leading to the death of many transplanted cells (da Fonseca et al., 2008). The ability of transplanted hepatocytes to overcome the sinusoidal endothelial cell barrier and to engraft within the liver parenchyma may be very limited. In addition, ESLD patients may have a significant degree of liver fibrosis and cirrhosis, which may be a major limiting factor for subsequent cell growth within the hepatic lobule, which is already limited by progressive disorganized cytoarchitecture.

[0006] Thus, there is a need for novel cell-based methods of organ regeneration that can generate functional organs with appropriate anatomical characteristics. There is also a need for novel treatments for ESLD, metabolic liver disease and acute liver failure. Summary of the Invention [Means for solving the problem]

[0007] overview In certain embodiments, the present disclosure provides a minimally invasive method for transplanting one or more cells in a subject and growing ectopic tissue.In certain embodiments, the method includes: advancing an endoscope into, for example, the gastrointestinal tract, airway or urinary tract of a subject by intraluminal approach; inserting a needle attached to the endoscope through visceral side wall into the lymph node of the subject by transluminal approach; and delivering one or more cells through the needle into the lymph node, thereby allowing the one or more cells to engraft in the lymph node and generate ectopic tissue in the lymph node.

[0008] In certain embodiments, the advancing of the endoscope, the inserting of the needle, or both, are performed using radiological imaging or ultrasound imaging. In certain embodiments, the radiological imaging includes dynamic radiological imaging, computed tomography (CT), magnetic resonance imaging (MRI), or both. In certain embodiments, the lymph node is present in the abdominal or thoracic cavity of the subject. In certain embodiments, the lymph node is present in the mediastinal or retroperitoneal region of the subject.

[0009] In certain embodiments, the minimally invasive method of transplanting one or more cells and growing ectopic tissue in a subject of the present disclosure includes inserting a needle into a lymph node in the abdominal or thoracic cavity of the subject using ultrasound or radiation imaging, and delivering one or more cells to the lymph node through the needle, thereby allowing the one or more cells to engraft, expand, and differentiate into ectopic tissue in the lymph node.In certain embodiments, the method further includes advancing an endoscope through the gastrointestinal tract, airway, or urinary tract of the subject, and inserting the needle through a visceral side wall using a transluminal approach to reach the lymph node of the subject, where the needle is attached to the endoscope.In certain embodiments, advancing the endoscope, inserting the needle, or both, are performed using ultrasound imaging of the lymph node.In certain embodiments, the radiation imaging includes dynamic radiation imaging, computed tomography (CT), magnetic resonance imaging (MRI), or both. In one particular embodiment, the endoscope includes an ultrasound probe configured to detect lymph nodes.

[0010] In certain embodiments, the one or more cells comprise hepatocytes, pancreatic cells or islets, kidney cells or fragments, thymus cells or fragments, or lung cells or fragments.In certain embodiments, the one or more cells are autologous, allogeneic, or xenogeneic to the subject.In certain embodiments, the one or more cells are syngeneic to the subject.

[0011] In certain embodiments, the method disclosed herein further comprises isolating one or more cells from living donor tissue.In certain embodiments, the method disclosed herein further comprises recovering one or more cells from cryopreservation before delivery.In certain embodiments, the method further comprises administering an immunosuppressant to the subject to reduce immune rejection of one or more cells.

[0012] In certain embodiments, the method includes delivering one or more cells to at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more lymph nodes in the peritoneal or thoracic cavity.

[0013] In certain embodiments, the one or more cells comprise cells with an average diameter of about 20 μm.In certain embodiments, the one or more cells comprise at least about 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million or 100 million cells per delivery to a single lymph node.In certain embodiments, the needle delivers one or more cells in a suspension with at least about 10 million, 20 million, 25 million, 30 million, 40 million, 45 million, 50 million, 55 million, 60 million, 70 million, 80 million, 90 million or 100 million cells per mL. In certain embodiments, the needle delivers the one or more cells in a suspension having at least about 10, 20, 30, 40, or 50 million viable cells per mL. In certain embodiments, the one or more cells are delivered to the lymph node as a cell population, the cell population having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, or about 100% viable cells. In certain embodiments, the one or more cells are delivered to a lymph node as a cell population, and when the one or more cells pass through the needle, the delivery leads to a decrease in the percentage of cell viability in the cell population of less than about 20%, 15%, 10%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% or 0.5%.

[0014] In certain embodiments, the inner diameter of the needle is at most about 700 μm, 600 μm, 500 μm, 450 μm, 400 μm, 300 μm, 260 μm, 250 μm or 200 μm. In certain embodiments, the inner diameter of the needle is at most about 260 μm. In certain embodiments, the outer diameter of the needle is at most about 1 mm, 900 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 520 μm, 510 μm, 500 μm, 480 μm, 450 μm or 400 μm. In certain embodiments, the outer diameter of the needle is at most about 510 μm. In certain embodiments, the size of the needle is up to about 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or 27 gauge. In certain embodiments, the size of the needle is up to about 25 gauge. In certain embodiments, the needle does not exceed about 25 gauge, and the needle delivers one or more cells in a suspension having at least about 50 million viable cells per mL, and the one or more cells include at least about 65% viable cells.

[0015] In certain embodiments, the one or more cells comprise kidney cells, and the ectopic tissue is ectopic renal tissue. In certain embodiments, the method treats a liver disease or condition in a subject. In certain embodiments, the liver disease or condition is end-stage liver disease or liver fibrosis related to alcohol consumption, hepatitis A, B, C, or D infection, non-alcoholic fatty liver disease, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, biliary atresia, cystic fibrosis, Alagille syndrome, syphilis, brucellosis, parasitic infection, chemical exposure, chronic biliary disease, Budd-Chiari syndrome, Osler's disease, or right heart failure. In certain embodiments, the liver disease or condition is associated with a metabolic disorder, including tyrosinemia, maple syrup urine disease, phenylketonuria, Crigler-Najjar syndrome, oxalosis, hyperoxaluria, hemochromatosis, alpha-1 antitrypsin deficiency, Wilson's disease, familial intrahepatic cholestasis syndrome, galactosemia, glycogen storage disease, or familial amyloidotic polyneuropathy. In certain embodiments, the subject has undergone a portocaval shunt surgical procedure or a transjugular intrahepatic portosystemic shunt (TIPS) procedure, which reduces blood supply to the liver of the subject and induces hepatocellular dysfunction in the subject, and the portocaval shunt surgical procedure includes an end-to-side portocaval shunt, a side-to-side portocaval shunt, a mesenteric vena cava shunt with interposition of an H-type or C-type graft, or a mid or distal splenorenal shunt. In certain embodiments, the methods treat end stage liver disease in a subject.

[0016] In certain embodiments, the one or more cells comprise kidney cells or kidney tissue fragments, and the ectopic tissue is ectopic kidney tissue.In certain embodiments, the method treats the renal disease or condition of a subject.In certain embodiments, the renal disease or condition is end-stage renal disease.

[0017] In certain embodiments, the one or more cells comprise pancreatic cells or pancreatic islets, and the ectopic tissue is ectopic pancreatic tissue.In certain embodiments, the method treats the endocrine pancreatic disease or condition that reduces or eliminates the insulin secretion of the subject.In certain embodiments, the pancreatic disease or condition is type I diabetes, type II diabetes or chronic pancreatitis that reduces the insulin secretion of the subject.

[0018] In certain embodiments, the one or more cells comprise lung cells or lung tissue fragments, and the ectopic tissue is ectopic lung tissue.In certain embodiments, the method treats a pulmonary disease or condition of a subject.In certain embodiments, the pulmonary disease or condition is chronic obstructive pulmonary disease (COPD).In certain embodiments, the COPD is caused by tobacco smoke, pollutants and fumes, alpha-1-antitrypsin, cystic fibrosis, chronic asthma, emphysema, chronic bronchitis or idiopathic pulmonary fibrosis.

[0019] In certain embodiments, the one or more cells comprise thymocytes or thymus fragments, and the ectopic tissue is ectopic thymus tissue. In certain embodiments, the thymocytes or fragments are obtained from a donor subject, and the ectopic thymus tissue induces donor-specific tolerance in the subject to transplantation of cells from the donor subject. In certain embodiments, the disease or condition is age-related immune system dysfunction, and the ectopic thymus tissue regulates the immune function of the subject.

[0020] In certain embodiments, the lymph nodes are in proximity to the gastrointestinal tract and the endoscope is advanced along the gastrointestinal tract. In certain embodiments, the lymph nodes include one or more of periduodenal lymph nodes, perigastric lymph nodes, peripancreatic lymph nodes, mesenteric lymph nodes, ileocolic lymph nodes, mesocolic lymph nodes, gastric lymph nodes, hepatosplenic lymph nodes, splenic hilar lymph nodes, paraesophageal lymph nodes, paracardia lymph nodes, para-aortic lymph nodes, retroaortic lymph nodes, lateral aortic lymph nodes, pre-aortic lymph nodes, lesser curvature lymph nodes, common hepatic lymph nodes, splenic artery lymph nodes, celiac trunk lymph nodes, iliac lymph nodes, or retroperitoneal lymph nodes. In certain embodiments, the lymph nodes include periduodenal lymph nodes. In certain embodiments, the lymph nodes are in proximity to the airway and the endoscope is advanced along the airway. In certain embodiments, the lymph node comprises one or more lymph nodes in the mediastinal region. In certain embodiments, the lymph node comprises one or more lymph nodes of parasternal lymph nodes, intercostal lymph nodes, supradiaphragmatic lymph nodes, superior tracheobronchial lymph nodes, inferior tracheobronchial lymph nodes, bronchopulmonary lymph nodes, paratracheal lymph nodes, or intrapulmonary lymph nodes. In certain embodiments, the lymph node is in close proximity to the urinary tract, and the endoscope is advanced along the urinary tract. In certain embodiments, the lymph node comprises one or more lymph nodes in the retroperitoneal region. In certain embodiments, the lymph nodes include one or more of the external iliac lymph nodes, the internal iliac lymph nodes, the lumbar vena cava lymph nodes, the lumbar aortic lymph nodes, the superficial inguinal lymph nodes, the deep inguinal lymph nodes, the perivesical intercaval lymph nodes, the perivesical obturator lymph nodes, or the perivesical presacral lymph nodes.

[0021] In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal.

[0022] In certain embodiments, ectopic tissue forms within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 days after delivery of one of more cells into the lymph node.

[0023] The present disclosure further provides a method for treating liver disease in a subject who needs to treat liver disease.In a certain embodiment, the method includes: advancing an endoscope into the gastrointestinal tract, airway or urinary tract of the subject by intraluminal approach; using transluminal approach and utilizing ultrasound imaging, inserting the needle attached to the endoscope through the visceral side wall into the lymph node of the subject; and delivering one or more cells through the needle into the lymph node, so that one or more cells can be engrafted in the lymph node and generate ectopic liver tissue in the lymph node. In non-limiting embodiments, ectopic tissue forms within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 days following delivery of one or more cells into the lymph node.

[0024] The present disclosure provides a system for transplanting hepatocytes and growing an ectopic liver in a subject, the system comprising an endoscope and a needled syringe containing a suspension including a cell population, the suspension having about 25 million to about 100 million viable hepatocytes per mL, and the needle being at most about 25 gauge. In certain embodiments, the endoscope and needle are configured to advance together along the gastrointestinal tract, airway, or urinary tract of the subject. In certain embodiments, the syringe is configured to deliver one or more cells through the needle. In certain embodiments, the endoscope comprises an ultrasound probe. In certain embodiments, the ultrasound probe is configured to detect lymph nodes in the abdominal cavity of the subject. In certain embodiments, the suspension has at least about 30, 40, 45, 50, 55, 60, 70, 80, 90, or 100 million cells per mL. In certain embodiments, the cell population in the syringe has at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or about 100% viable cells. In certain embodiments, the cell population in the syringe has at least about 65% viable cells. In certain embodiments, the cell population in the syringe comprises at least about 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million or 100 million cells. In certain embodiments, the cell population in the syringe comprises at least about 50 million viable cells per mL, and the cell population comprises at least about 65% viable cells.

[0025] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0026] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0027] [Figure 1A] FIG. 1A is a photograph of an endoscopist performing endoscopic ultrasound on a laboratory animal for EUS-guided cell delivery to lymph nodes.

[0028] [Figure 1B] FIG. 1B is an ultrasonography photograph showing an ultrasound image of the fine needle aspiration needle reaching a nearby lymph node (LN).

[0029] [Diagram 2] FIG. 2 shows a diagram of a surgeon injecting hepatocytes directly into the periduodenal lymph nodes of an experimental animal.

[0030] [Diagram 3] FIG. 3 is a bar graph summarizing the percentage of cell viability of different batches of hepatocytes isolated from donor animals and passed through needles of different gauges.

[0031] [Figure 4] FIG. 4 shows photographs of the open abdominal cavity of the experimental animals before and after the portacaval shunt procedure, as well as a diagram of the portacaval shunt procedure (far right).

[0032] [Diagram 5] FIG. 5 is a flow chart showing the experimental design for the preclinical study according to the present disclosure.

[0033] [Figure 6A] FIG. 6A shows positive staining for CK-18, a marker for hepatocytes, in normal liver tissue.

[0034] [Figure 6B]FIG. 6B shows the presence of CK-18 immunostaining signals in lymph nodes 6 days after endoscopic ultrasound (EUS) injection of hepatocytes in a preclinical study.

[0035] [Figure 6C] FIG. 6C shows the presence of CK-18 immunostaining signals in lymph nodes 6 days after direct injection of hepatocytes in a preclinical study.

[0036] [Figure 7A] FIG. 7A shows the presence of liver tissue in lymph nodes approximately 90 days after transplantation of autologous liver cells into the lymph nodes by endoscopic ultrasound (EUS) injection.

[0037] [Figure 7B] FIG. 7B shows the presence of liver tissue in the lymph node approximately 90 days after allogeneic liver cells were transplanted into the lymph node by endoscopic ultrasound (EUS) injection.

[0038] [Figure 7C] FIG. 7C shows the presence of liver tissue and fumarylacetoacetate hydrolase (FAH)-positive liver cells in the lymph node approximately 60 days after transplantation of autologous liver cells into the lymph node by endoscopic ultrasound (EUS) injection.

[0039] [Figure 7D] FIG. 7D shows the presence of liver tissue and FAH-positive liver cells in the lymph node approximately 150 days after allogeneic liver cells were transplanted into the lymph node by endoscopic ultrasound (EUS) injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description For purposes of clarity, and not of limitation, the detailed description of the subject matter of this disclosure is divided into the following subsections: 1. Overview; 2. Definition; 3. Cell delivery techniques; 4. Generation of ectopic tissue from transplanted cells; 5. Diseases and symptoms; 6. Subjects; 7. Systems; and 8. Kit.

[0041] 1. Overview In summary, the present disclosure relates to a method and system for minimally invasive procedures that induce the occurrence of in vivo organogenesis.Provided herein is a method and system for transplanting cells by delivering cells to the lymph nodes of a subject and growing ectopic tissue in the subject.The process of growing ectopic tissue with appropriate anatomical and functional characteristics in lymph nodes from delivered cells or tissue fragments is called in vivo organogenesis.The minimally invasive procedures enabled by the present disclosure can generate ectopic tissue that can complement or enhance the normal function of one or more organs for a subject.

[0042] As discussed above, one of the problems associated with current transplantation therapies, particularly orthotopic organ transplantation, may be that many patients with end-stage disease, such as end-stage liver disease or end-stage renal disease, are no longer suitable for major surgery that may be necessary for orthotopic organ transplantation or other types of cell transplantation. There may be significant risks associated with major surgery, and given the deteriorating health of these patients, prognosis may be poor. In one embodiment, the present disclosure solves this problem by providing a minimally invasive procedure (e.g., endoscopic ultrasound (EUS)). The present method and system can generate anatomically intact and fully or at least partially functional organs at heterotopic sites using lymph nodes as natural bioreactors. The methods and systems provided herein can minimize the morbidity and mortality of patients with end-stage organ disease, which may often be associated with additional surgical procedures. The present disclosure may also allow at least some of the surgical procedures described herein to be performed on an outpatient basis, thereby lowering the cost of the initial procedure.

[0043] As used herein, the term "ectopic tissue" may refer to tissue that is present in an ectopic (non-native) location in the body and has one or more morphological and / or functional properties similar or the same as a healthy organ or tissue normally found in its natural location in the body. The term "functional ectopic tissue" may refer to ectopic tissue that has one or more functions of a healthy organ or tissue normally found in its natural location in the body. The term "ectopic location" may be used to describe a location relative to a natural location in a subject, for example, an ectopic location may refer to a location that is different from the natural location in a subject's body. For example, liver tissue is not normally found in lymph nodes, so a liver in a lymph node is ectopic in a healthy, normal mammalian body, i.e., an ectopic liver. According to some embodiments of the present disclosure, ectopic tissue can be grown in a lymph node that includes a cell population that morphologically resembles liver cells and collectively can perform one or more functions that a healthy, natural liver can perform.

[0044] In certain embodiments, the method includes advancing an endoscope into a body lumen or closed body cavity of the subject. In certain embodiments, the method includes advancing an endoscope into the gastrointestinal (GI) tract, airway, or urinary tract of the subject.

[0045] In certain embodiments, the endoscope is advanced into the body lumen of the subject by intraluminal approach.As used herein, the term "endoluminal approach" refers to any approach, such as method and device, known in the art for inserting an endoscope into the body lumen of the subject.For example, but not by way of limitation, the method includes advancing an endoscope into the gastrointestinal (GI) tract, airway or urinary tract of the subject by intraluminal approach.

[0046] In certain embodiments, the method may further include inserting a needle into a lymph node of a subject, the needle being attached to an endoscope. In certain embodiments, the needle attached to the endoscope is inserted into the lymph node through a visceral wall. Non-limiting examples of visceral walls include anatomical structures surrounding hollow organs and / or ductal organs, such as the stomach, duodenum, trachea, bronchus, or bladder. In certain embodiments, the method further includes inserting a needle through a transluminal approach. As used herein, the term "transluminal approach" refers to any approach, e.g., method and device, known in the art for inserting a working instrument (e.g., a needle) into a lumen by using an endoscope.

[0047] In certain embodiments, the method further comprises delivering one or more cells to the lymph node via a needle. In certain embodiments, the method comprises delivering a single cell to the lymph node. In certain embodiments, the method comprises delivering a cell population to the lymph node. In certain embodiments, the cell population comprises one cell type. In certain embodiments, the cell population comprises at least two cell types. In certain embodiments, the one or more cells delivered to the lymph node can engraft in the lymph node and generate ectopic tissue.

[0048] In certain embodiments, the steps of advancing the endoscope, inserting the needle, or both are performed by minimally invasive or non-invasive methods. For example, ultrasound imaging or other detection methods can be applied in the methods provided herein to locate the target lymph node. In the above process, ultrasound imaging or other methods, such as minimally invasive or non-invasive detection approaches, can be applied to either advance the endoscope, locate the suitable target lymph node, or monitor the insertion of the needle through the visceral side wall or into the target lymph node.

[0049] In another aspect, the present disclosure provides a method comprising inserting a needle into lymph nodes in the abdominal, pelvic or thoracic cavity of a subject using ultrasound. The method may further comprise delivering one or more cells to those lymph nodes via the needle. Localization of lymph nodes under ultrasound guidance may be performed by any available technique. For example, but not by way of limitation, ultrasound imaging may be used to identify and locate lymph nodes suitable for cell injection. In certain embodiments, ultrasound spectroscopy may be used to detect lymph nodes. In certain embodiments, ultrasound imaging or ultrasound spectroscopy may be used in conjunction with other detection methods for lymph node localization.

[0050] In certain embodiments, the method includes delivering a cell population to a lymph node of a subject, and the population of hepatocytes engrafts in the lymph node to generate an ectopic liver. In certain embodiments, the method further includes reducing the blood supply to the liver of the subject. It has been discovered by the present disclosure that reducing the blood supply to the liver of the subject benefits the growth of an ectopic liver in the lymph node.

[0051] 2.Definition In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that as used in the specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] In this application, unless otherwise stated, the use of "or" means "and / or". As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may indicate that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B and / or C" or "A, B, C or any combination thereof" may mean "individually A; individually B; individually C; A and B; B and C; A and C; and A, B and C". The term "or" may be used conjunctively or disjunctively, unless the context clearly dictates disjunctive use.

[0053] Furthermore, use of the term "including" as well as other forms (e.g., "include," "includes," and "including") is non-limiting.

[0054] References herein to "some embodiments," "certain embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0055] As used in the specification and claims, the words "comprising" (and any form of comprising (e.g., "comprise" and "comprises")), "having" (and any form of having (e.g., "have" and "has")), "including" (and any form of including (e.g., "includes" and "include")), or "containing" (and any form of containing (e.g., "contains" and "contain")) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0056] As used herein, the term "about" with respect to a numerical value and its grammatical equivalents may include the numerical value itself and a range of values ​​plus or minus 10% from the numerical value.

[0057] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method by which the particular value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within one standard deviation or within more than one standard deviation, as is customary in the art. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes 10 and any amount between 9 and 11. In yet another example, the term "about" with respect to a numerical reference may also include a range of values ​​of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term "about" may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where a particular value is described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for that particular value.

[0058] 3. Cell Delivery Technique In certain embodiments, the disclosed methods and systems utilize an endoscope to transluminally deliver cells to a lymph node of a subject. In certain embodiments, the lymph node is located adjacent to or within a body lumen or closed cavity accessible by use of an endoscope. In certain embodiments, the lymph node is located in the abdominal, pelvic, or thoracic cavity of a subject.

[0059] As used herein, the term "endoscope" may refer to any device that can be inserted into the body of a subject, e.g., a human subject or a non-human mammalian subject, e.g., a dog, pig, horse, donkey, rabbit, cow, mouse, or rat, to view the interior of the subject. Sometimes, the field of view provides visual inspection, e.g., when the endoscope is equipped with illumination optics or is otherwise assisted by illumination. In certain embodiments, the endoscope of the present disclosure includes, is attached to, or is associated with one or more sensing probes to provide various detection modes for examining the interior of a subject. For example, the endoscope may have illumination optics for visual inspection, an ultrasound probe for ultrasound-mediated detection (e.g., ultrasound imaging), or detectors for radiation illumination, infrared signals, radiofrequency signals, or fluorescent signals.

[0060] The endoscopes described herein may include one or more of the following components: a rigid or flexible tube that travels along the luminal passages (e.g., gastrointestinal, respiratory or urinary tracts) of a subject; a light delivery system for illuminating the organ or object under examination; a lens system that transmits the image from the objective lens to an observer, an eyepiece, or a video system that displays the image captured by the endoscope inside the body; and one or more passages for entering medical instruments or remote control devices. The light source may be outside the body, or may be small and mounted inside the endoscope. When the light source is outside the body, the light may be transmitted through a fiber optic system. There may be an optical system, e.g., an optical fiber, for transmitting the captured optical image inside the body. Additionally or alternatively, the endoscopes provided herein may be equipped with (e.g., comprised as part of or attached to) other detection devices for various purposes.

[0061] The method provided herein may include advancing an endoscope in a body lumen or closed body lumen of a subject. Non-limiting examples of body lumens include the gastrointestinal (GI) tract (e.g., esophagus, stomach, duodenum, small intestine, large intestine, colon, bile duct, rectum, anus), airways (e.g., nose, lower airways), ear, urinary tract, cervix, uterus, and fallopian tube. Non-limiting examples of closed body cavities include the abdominal cavity and pelvic cavity. In certain embodiments, the method disclosed herein includes advancing an endoscope in the gastrointestinal tract, airways, or urinary tract of a subject.

[0062] In certain embodiments, the method disclosed herein comprises advancing an endoscope along a body lumen or in a closed body cavity to deliver cells to at least one lymph node located adjacent to or located in a body lumen or closed body cavity of a subject.In certain embodiments, the location of the lymph node is selected based on the purpose of cell transplantation.In certain embodiments, the lumen and / or closed body cavity is selected based on lymph nodes suitable for cell transplantation.

[0063] In certain embodiments, the method disclosed herein comprises advancing an endoscope along the gastrointestinal tract to deliver cells to one or more lymph nodes adjacent to the gastrointestinal tract.An endoscope (e.g., esophagoscope or gastroscope) can be used to deliver cells to lymph nodes adjacent to the upper or lower part of the gastrointestinal tract.For example, an esophagoscope can be used to deliver cells to lymph nodes close to the esophagus, and a gastroscope can be used to deliver cells to perigastric and / or periduodenal lymph nodes. Non-limiting examples of lymph nodes in proximity to the gastrointestinal tract to which cells can be delivered using the methods provided herein include periduodenal lymph nodes, perigastric lymph nodes, peripancreatic lymph nodes, mesenteric lymph nodes, ileocolic lymph nodes, mesocolic lymph nodes, gastric lymph nodes, hepatosplenic lymph nodes, splenic hilar lymph nodes, paraesophageal lymph nodes, paracardia lymph nodes, para-aortic lymph nodes, retroaortic lymph nodes, extra-aortic lymph nodes, pre-aortic lymph nodes, lesser curvature lymph nodes, common hepatic lymph nodes, splenic artery lymph nodes, coeliac axis lymph nodes, iliac lymph nodes, and retroperitoneal lymph nodes. The endoscope can be inserted through the subject's mouth or, in certain embodiments, through the subject's nose. Alternatively, an endoscope may be inserted through the subject's anus, for example, a proctoscope, sigmoidoscope, or colonoscope may be used to deliver cells to lymph nodes near the rectum or colon. In certain embodiments, the endoscope is flexible so that it can be moved along the gastrointestinal tract, and laboratory tests are performed to locate lymph nodes suitable for cell transplantation purposes.

[0064] In certain embodiments, the methods disclosed herein include advancing an endoscope along the subject's airway to deliver cells to one or more lymph nodes in close proximity to the airway.For example, but not by way of limitation, a bronchoscope or laryngoscope is used to target lymph nodes close to the trachea or bronchi or larynx of the lungs of a subject.Non-limiting examples of lymph nodes in close proximity to the airway that can be delivered to cells using the methods provided herein include parasternal lymph nodes, intercostal lymph nodes, supradiaphragmatic lymph nodes, superior tracheobronchial lymph nodes, inferior tracheobronchial lymph nodes, bronchopulmonary lymph nodes, paratracheal lymph nodes, and intrapulmonary lymph nodes.A bronchoscope or laryngoscope can be inserted through the subject's mouth, or in certain embodiments, through the subject's nose.

[0065] In certain embodiments, the methods disclosed herein include advancing an endoscope along the urinary tract to deliver cells to one or more lymph modes in proximity to the urinary tract of a subject. For example, but not by way of limitation, a cystoscope is used to target lymph nodes close to the urethra or bladder. The cystoscope can be inserted through the urethra of a subject. Non-limiting examples of lymph nodes in proximity to the urinary tract to which cells can be delivered using the methods provided herein include one or more of the following lymph nodes: external iliac lymph nodes, internal iliac lymph nodes, caval lumbar lymph nodes, aortic lumbar lymph nodes, superficial inguinal lymph nodes, deep inguinal lymph nodes, perivesical intercaval lymph nodes, perivesical obturator lymph nodes, or perivesical presacral lymph nodes.

[0066] In certain embodiments, the method disclosed herein comprises advancing an endoscope in a closed body cavity (e.g., abdominal cavity, pelvic cavity) to deliver cells to at least one lymph node in or near the closed body cavity. In certain embodiments, the endoscope is inserted into the closed body cavity through a small incision, for example, a small incision on the surface of the abdomen. In certain embodiments, the closed body cavity is the abdominal cavity or the pelvic cavity. Non-limiting examples of lymph nodes in or near the abdominal or pelvic cavity include splenic lymph nodes, hepatic lymph nodes, gallbladder lymph nodes, omentum foramen lymph nodes, right / left gastric lymph nodes, pyloric lymph nodes, super pyloric lymph nodes, infrapyloric lymph nodes, postpyloric lymph nodes, superior pancreatic lymph nodes, inferior pancreatic lymph nodes, superior / inferior pancreaticoduodenal lymph nodes, inferior mesenteric lymph nodes, sigmoid lymph nodes, superior rectal lymph nodes, mesocolonic lymph nodes, left colonic lymph nodes, right colonic lymph nodes, midcolic lymph nodes, appendix lymph nodes, ileocolic lymph nodes, retrocaecal lymph nodes, pretectal lymph nodes, superior mesenteric lymph nodes, left lumbar lymph nodes, extra-aortic lymph nodes, and pre-aortic lymph nodes.

[0067] In certain embodiments, the methods and systems disclosed herein use ultrasound to locate lymph nodes for cell implantation and / or to guide the advancement of an endoscope, the insertion of a needle, or any combination thereof through a body lumen or a closed body cavity. Ultrasound is a sound wave with a frequency higher than the upper limit of human hearing, which may be from about 20 kHz to several gigahertz. Ultrasound imaging (or ultrasound examination) may be performed in various formats depending on the intended purpose of applying the methods and systems provided herein. Non-limiting examples of ultrasound examination may include ultrasound Doppler, contrast ultrasound examination, molecular ultrasound examination, elastography, interventional ultrasound examination, and compression ultrasound examination. In certain embodiments, the ultrasound imaging technology used herein has high spatial and / or temporal resolution for the purpose of locating lymph nodes suitable for injection using techniques such as those described in International Patent Publication Nos. WO2018222724A1 and WO2018134729A1, each of which is incorporated herein by reference.

[0068] In certain embodiments of the present disclosure, the methods disclosed herein use endoscopy in combination with ultrasound imaging, where the endoscope is attached to an ultrasound probe (e.g., as part of the endoscope or as a separate part). The terms "endoscopic ultrasound", "endoscopic ultrasound" or "EUS" used interchangeably herein may refer to a medical procedure that combines ultrasound and endoscopy to detect (e.g., obtain images of) and / or manipulate internal organs in the chest, abdomen and pelvis or any other internal body structure. The EUS devices and EUS techniques used in the methods and systems provided herein may be those currently commercially available and / or as described in U.S. Patent Publication Nos. US20060106306A1 and US20070237373A1 and International Patent Publication No. WO1998009247A1, each of which is incorporated herein by reference in its entirety.

[0069] In certain embodiments, ultrasound imaging of target lymph nodes (e.g., lymph nodes to which one or more cells are delivered) is performed using other imaging techniques, such as radiation imaging. Radiation imaging can include dynamic radiation imaging (fluoroscopy), computed tomography (CT) or magnetic resonance imaging (MRI). For example, but not by way of limitation, computed tomography (CT) is performed to obtain anatomical information of the subject's whole body or some local organs or tissues. In certain embodiments, magnetic resonance or any other available medical technique is used together with or instead of ultrasound imaging to locate the target lymph nodes in the subject's abdominal, pelvic or thoracic cavities.

[0070] The methods and systems provided herein may include using a needle to deliver one or more cells to a lymph node. The needle may be part of a syringe that may be configured to contain the cells to be delivered and push the cells through the delivery needle. The needles provided herein may be configured to have a certain sharpness and stiffness. For example, the needle is inserted into a lymph node. In certain embodiments, the needle is also configured to penetrate the wall of the gastrointestinal tract (e.g., the wall of the esophagus, stomach, intestine or colon), the wall of the airway, the wall of the urinary tract (e.g., the wall of the urethra or bladder), so that the needle can reach lymph nodes outside the tract. Any suitable needle known in the art may be used with the methods disclosed herein.

[0071] In certain embodiments, the needle is attached to an endoscope, for example, as part of the endoscope or as a separate device. The needle may be a needle used in fine needle aspiration (FNA) procedures. The FNA needle may be commercially available or may be specially designed for the application of the teachings of the present disclosure. FNA needles are typically used for biopsy purposes, for example, to make an incision in tissue and aspirate tissue fragments for diagnostic purposes. Instead, in the methods and systems provided herein, the FNA needle may be used for cell delivery purposes. In certain embodiments, the FNA needle is manipulated through a linear array echoendoscope EUS / FNA device. An exemplary EUS / FNA device may be configured to control and measure the advancement of the FNA needle (e.g., a hollow needle with a removable rigid stylet) within a semi-rigid protective sheath. The EUS / FNA device may also have a handle with a port for inserting or withdrawing the stylet and for attaching a syringe into which viable cells can be inserted. In certain embodiments, a needle is inserted into a lymph node acoustically identified using an EUS probe of an ultrasound endoscope device, while still in a protective sheath. The needle can then be advanced out of the sheath and transluminally inserted into the target lymph node under direct ultrasound guidance. A stopcock attached to the tip of a syringe can help create and hold a vacuum in the needle body. Once the tip of the needle reaches the target lymph node and the stylet is removed, a syringe containing viable cells in culture medium can be connected to the needle handle. When the stopcock is opened, the cells are immediately injected from the needle, which is placed in the lymph node parenchyma, under direct vision. The needle can have an adjustable spacer / slider at the distal part of the handle that can change the length of the sheath exiting the endoscope, which can ensure additional safety and precision when injecting near the lymph node.

[0072] In certain embodiments, the needle is configured, for example, the size of the needle is configured to facilitate injection of cells into lymph nodes. In certain embodiments, the size (e.g., gauge) of the needle is selected based on the location of the target lymph node, the type of cells to be delivered, and the amount of cells to be delivered. In certain embodiments, the thinner the needle, the easier it is to reach and insert into the target lymph node. In certain embodiments, it is easier to insert a thinner needle into a small target lymph node, which may otherwise tent around a thicker needle. In certain embodiments, the size of the needle, for example, the inner diameter of the needle, is configured to allow cells to be pushed out without clogging the needle. As described herein, the size of the needle refers to the size of the tip of the needle, not the center of the needle, which is connected to the other part of the syringe. As disclosed herein, the outer diameter of the needle refers to the first complete diameter from the tip to the outside of the needle wall, and the inner diameter of the needle refers to the first complete diameter from the tip to the inside of the needle wall.

[0073] In certain embodiments, the needle has an inner diameter of at most about 700 μm, 600 μm, 500 μm, 450 μm, 400 μm, 300 μm, 260 μm, 250 μm or 200 μm. In certain embodiments, the needle has an inner diameter of at most about 260 μm. In certain embodiments, the inner diameter of the needle can be at most about 700 μm, 600 μm, 500 μm, 450 μm, 400 μm, 300 μm, 260 μm, 250 μm or 200 μm. In certain embodiments, the inner diameter of the needle is about 260 Jim. The needle may have an outer diameter of at most about 1 mm, 900 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 520 μm, 510 μm, 500 μm, 480 μm, 450 μm, or 400 μm. In certain embodiments, the outer diameter of the needle is at most about 510 μm. In certain embodiments, the outer diameter of the needle is at most about 1 mm, 900 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 520 μm, 510 μm, 500 μm, 480 μm, 450 μm, or 400 μm. In certain embodiments, the outer diameter of the needle is about 510 μm. In certain embodiments, the needle is a certain gauge as defined according to ISO7864:2016. For example, but not by way of limitation, the needle is about 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, or 27 gauge (ga). In certain embodiments, the needle is at most about 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, or 27 ga. The needle may be at most about 25 ga. As described herein, when comparing needle sizes based on a particular gauge size, the comparison is made between the outer diameter of the needle and the outer diameter of a standard hypodermic needle of the particular gauge as defined by ISO7864:2016. In certain other embodiments, the needle has a relatively small outer diameter and a relatively large inner diameter. In certain embodiments, the needle has a non-standard size, for example, having a thin wall while maintaining a large inner diameter and a small outer diameter.

[0074] The total number of cells and the concentration of cells in the needle syringe are selected based on several factors, including the size of the cells to be injected, the type of ectopic tissue to be generated, the proliferative potential of the cells, the target mass of ectopic tissue, the size of the injection needle, and the size of the target lymph node.

[0075] In certain embodiments, a syringe as described herein contains at least about 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 3 billion, 5 billion, 8 billion, 10 billion, 20 billion, 50 billion, or 100 billion cells for injection. In certain embodiments, a syringe as described herein has about 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 3 billion, 5 billion, 8 billion, 10 billion, 20 billion, 50 billion or 100 billion cells for injection. In certain embodiments, a syringe as described herein has at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 3, 5, 8, 10, 20, 50, or 100 billion cells for injection. In certain embodiments, the syringe contains about 50 million to about 200 million cells for injection.

[0076] In certain embodiments, the syringe as described herein contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 million hepatocytes for generating an ectopic liver. In certain embodiments, the syringe as described herein has about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 million hepatocytes for generating an ectopic liver. In certain embodiments, the syringe as described herein has at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 million hepatocytes for generating an ectopic liver. In certain embodiments, the syringe contains about 50 million to about 200 million cells for generating an ectopic liver.

[0077] In certain embodiments, a syringe as described herein holds a cell suspension having at least about 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 3, 5, 8, or 10 billion cells per mL. In certain embodiments, a syringe as described herein holds a cell suspension having about 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 3, 5, 8 or 10 billion cells per mL. In certain embodiments, a syringe as described herein holds a cell suspension having at most about 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 3, 5, 8, or 10 billion cells per mL.

[0078] In certain embodiments, the syringe as described herein contains a hepatocyte suspension having at least about 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 million cells per mL for generating an ectopic liver. In certain embodiments, the syringe as described herein contains a hepatocyte suspension having at least about 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 million cells per mL for generating an ectopic liver. In certain embodiments, a syringe as described herein contains a hepatocyte suspension having at most about 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 million cells per mL for generating an ectopic liver.

[0079] In certain embodiments, the needle for injecting the hepatocytes is selected to be no greater than about 25 gauge, and the needle delivers cells including hepatocytes in a suspension having at least about 50 million viable cells per mL. In certain embodiments, the cell population in the syringe has at least about 65% viable cells.

[0080] Without wishing to be bound by any particular theory, the viability of cells delivered to lymph nodes may be important for the formation and function of ectopic tissue. In certain embodiments, the viability percentage in the cell population needs to be controlled. The syringe, e.g., needle, can be configured to maintain the viability of cells during the delivery process. For example, the size of the needle and the cell concentration of the suspension can be set so that the viability percentage is not significantly reduced when the cells pass through the needle during the cell delivery process. In certain embodiments, the cell delivery leads to a decrease in the cell viability percentage in the cell population of about 20%, 15%, 10%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% or less than 0.5% when the cells pass through the needle. In certain embodiments, the percentage of cell viability decreases by less than about 10% when the cells pass through the needle during cell delivery (eg, compared to cell viability prior to injection).

[0081] In certain embodiments, the method provided herein comprises reducing blood supply to the liver of a subject and delivering one or more hepatocytes to a lymph node of the subject. The reduction of blood supply to the liver of a subject can induce hepatocyte dysfunction, for example, the dysfunction or death of at least some liver cells, and as a result, the reduction or loss of liver function. Without wishing to be bound by a particular theory, the reduction or loss of liver function in a subject after reducing blood supply to the liver can have a compensatory effect on the growth of ectopic liver in lymph nodes after transplanting hepatocytes into the lymph nodes. In other words, the reduction of blood supply to the liver, in certain embodiments, promotes the formation of ectopic liver in lymph nodes and / or, in certain embodiments, improves the functional performance of ectopic liver in lymph nodes. In certain embodiments of the above method, cell delivery, when performed together with the reduction of blood supply to the liver, is performed by direct injection of hepatocytes (e.g., via major surgery or percutaneous injection) or EUS-guided injection of hepatocytes.

[0082] To reduce blood supply to the liver, surgical methods can be used, which can include redirecting blood inflow from the portal vein. For example, a transjugular intrahepatic portosystemic shunt (TIPS) procedure is performed to establish a direct connection between the inflow portal vein and the outflow hepatic vein, thus reducing blood flow through the liver. TIPS refers to an artificial flow path in the liver that establishes a connection between the portal vein and the hepatic vein. TIPS has been used as a stand-alone treatment to alleviate the adverse visceral and systemic hemodynamic effects of progressive portal hypertension resulting from end-stage liver disease. In certain embodiments, the method disclosed herein includes using TIPS in combination with hepatocyte transplantation in lymph nodes to generate heterotopic liver in lymph nodes.

[0083] A transjugular intrahepatic portosystemic shunt may be surgically placed in the liver through major surgery or minimally invasive procedures that require opening of the abdominal cavity. In certain embodiments, a transjugular intrahepatic portosystemic shunt is placed under fluoroscopic guidance. Access to the liver may be obtained through the internal jugular vein in the neck. Once access to the jugular vein is confirmed, a guidewire and introducer sheath may be placed to facilitate placement of the shunt. This process may allow access to the patient's hepatic veins by proceeding from the superior vena cava through the inferior vena cava and finally into the hepatic vein. Once the catheter is placed in the hepatic vein, the wedge pressure may be obtained and the pressure gradient in the liver may be calculated. Carbon dioxide may then be injected to locate the portal vein. A special needle known as a Colapinto may then be advanced through the liver parenchyma to connect the hepatic vein to the large portal vein near the center of the liver. A flow path for the shunt may then be created by inflating an angioplasty balloon in the liver along the path created by the needle. The shunt may be completed by placing a special mesh tube known as a stent or endograft to maintain a passage between the higher pressure portal vein and the lower pressure hepatic veins. After this procedure, fluoroscopic images can be obtained to demonstrate placement.

[0084] In certain other embodiments, in order to reduce blood supply to the liver, a portocaval shunt procedure is performed to establish communication between the portal vein and the inferior vena cava.Portocaval shunt can be placed by surgical methods, such as but not limited to the procedure described in Example 2.Other surgical procedures that can be used can include end-to-side portocaval shunt or side-to-side portocaval shunt (SSPCS), mesenteric vein-inferior vena cava shunt (MCS) by interposition of H-type or C-type graft, and spleno-renal shunt (SRS) (middle or distal).

[0085] In certain embodiments, hepatocytes are delivered to lymph nodes after reducing blood supply to the liver.In certain embodiments, hepatocyte transplantation is performed immediately after reducing blood supply to the liver.In certain embodiments, hepatocyte transplantation is performed, for example, about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks or 4 weeks after reducing blood supply to the liver.In certain embodiments, such intervals cannot be longer than a certain time to avoid complete liver failure, which may be life-threatening.For example, but not by way of limitation, the intervals are shorter than about 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks or 4 weeks.Alternatively, hepatocyte transplantation can be performed immediately before reducing blood supply to the liver. In certain embodiments, the interval can be less than about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 2 weeks or 3 weeks.The interval between two steps can depend on the degree of reduction in blood supply to the liver and the health of the subject undergoing the procedure.The determination of the interval between two steps as described herein may require a medical evaluation by a physician on an individual basis.

[0086] Graft rejection may be a problem associated with any transplantation procedure involving non-self organ or cell transplants.Immunosuppression can be applied to prevent or ameliorate the graft rejection induced by the procedure according to the method of the present disclosure.For example, immunosuppressant drugs can be administered to subjects immediately before cell transplantation or immediately after cell transplantation is completed, or about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 2 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, 4 years or more after the procedure.

[0087] Various immunosuppressive approaches can be applied herein. For example, but not limited to, the subject is administered an immunosuppressant for induction immunosuppression, which can include any immunosuppressive drug administered immediately after transplantation in an intensive dose to prevent acute rejection. Non-limiting examples of related drugs can include methylprednisolone, Atgam, thymoglobulin, OKT3, basiliximab, solumedrol and daclizumab. The subject can also be administered an immunosuppressant for maintenance immunosuppression, which can include any immunosuppressive drug administered before, during or after transplantation with the intention of long-term maintenance. For example, but not limited to, prednisone, cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, prograf or rapamycin can be used for maintenance immunosuppression as described herein. In certain embodiments, the subject may also be administered an immunosuppressant for anti-rejection immunosuppression, which may include any immunosuppressive drug administered for the purpose of treating acute rejection episodes in the early or specific follow-up period after transplantation, for example, up to 30 days after the diagnosis of acute rejection.Non-limiting examples of related drugs may include methylprednisolone, Atgam, OKT3, thymoglobulin, basiliximab or daclizumab.Other examples of immunosuppressants that may be used in the method may also include other steroids (e.g., corticosteroids, dexamethasone and prednisone), Cox-1 and Cox-2 inhibitors, macrolide antibiotics (e.g., rapamycin and tacrolimus) and other substances that limit, reduce or suppress the activity of B cells, T cells and / or other natural immunity.

[0088] 4. Generation of Ectopic Tissue from Transplanted Cells The methods and systems provided herein can be applied to transplant various cell types, including but not limited to liver cells, kidney cells or kidney tissue fragments, pancreatic cells or pancreatic islets, thymus cells or thymus fragments, or lung cells or lung tissue fragments. Cells transplanted into lymph nodes can engraft and form ectopic tissue that can complement or enhance the function of one or more normal organs of the subject.

[0089] The cells transplanted into lymph node can be homogenous or heterogeneous cell populations depending on the purpose of cell transplantation.For example, in certain embodiments, a homogenous population of liver cells is delivered to lymph node to generate ectopic liver.In certain embodiments, when growing ectopic liver, a heterogeneous population of cells, such as liver cells and other liver parenchymal cells, is delivered to lymph node.In certain embodiments, a heterogeneous population of kidney cells or kidney fragments is delivered to lymph node to generate ectopic kidney in lymph node. In certain embodiments, fetal kidney from a donor subject, or renal organoids generated by in vitro differentiation methods (e.g., methods described in International Patent Publication Nos. WO2014182885A2, WO2019006127A1 and WO2018227101A1, each of which is incorporated herein by reference) are obtained, processed into small fragments (e.g., chopped or ground), and resuspended in liquid to obtain a solution that is delivered to lymph nodes. The solution contains the various cell types that constitute the fetal kidney or renal organoid. In certain embodiments, a heterogeneous population of pancreatic cells is delivered to lymph nodes to generate ectopic pancreas.

[0090] The methods and systems provided herein may include delivering a therapeutically effective amount of cells to a lymph node of a subject. The term "therapeutically effective amount" as used herein, when referring to a cell population to be transplanted or delivered, may refer to an amount of suitable cells in a cell population, e.g., an amount of cells to be transplanted, or an amount of a composition containing cells to be transplanted, effective to produce a desired therapeutic effect in a subject receiving cell transplantation to a lymph node, with a reasonable benefit / risk ratio applicable to any medical treatment. For example, and not by way of limitation, the amount of cell population to be transplanted into a subject is an amount sufficient to produce a statistically significant and measurable change in one or more symptoms of the disease or condition (e.g., end-stage liver disease or end-stage renal disease) for which the transplantation is intended to treat. The determination of a therapeutically effective amount depends on the intended medical purpose for which the method or system is applied. The therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, and the severity and type of the subject's condition, and the administration of other pharmacologic active agents.

[0091] Depending on the intended purpose of applying the methods and systems provided herein, various amounts of cells can be delivered to lymph nodes to grow ectopic tissue. At least about 1, 2, 3, 4, 5, 7, 9, 10, 15, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 300, 500, 750, 800, 900, 1, 5, 10, 20, 30, 500, or more cells can be delivered per lymph node. In certain embodiments, about 10, 15, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, or 100 million cells can be delivered to a single lymph node. In certain embodiments, about 50 to about 200 million cells can be delivered to a single lymph node.

[0092] The delivered cell population may have at least about 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or about 100% viable cells. To maintain the cell viability level in the delivered cell population, available measures to enhance cell viability may be applied. In certain embodiments, the viability status of the cell population (e.g., viability percentage or other cell viability parameters) may be measured prior to delivery of the cells to ensure that a viable cell population is delivered and, as a result, functional ectopic tissue can be generated in the lymph node.

[0093] In non-limiting embodiments, ectopic tissue can be formed at least about 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 110 days, 120 days, 130 days, 140 days, 150 days, 160 days, 170 days, 180 days, 190 days or about 200 days after transplantation of various cell types disclosed into lymph nodes. For example, but not by way of limitation, hepatocytes can be delivered to lymph nodes by EUS, and the engrafted hepatocytes can form liver tissue about 60, about 90 or about 150 days after hepatocyte transplantation. In non-limiting embodiments, after transplantation of the various disclosed cell types into lymph nodes, ectopic tissue can be formed within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 days. For example, but not by way of limitation, hepatocytes can be delivered to lymph nodes by EUS, and the engrafted hepatocytes can begin to form liver tissue within about 60, about 90 or about 150 days after transplantation of the hepatocytes.

[0094] The cells to be delivered can be obtained from different sources by several different methods. The cells can be allogeneic, xenogeneic or autologous to the subject. The cells can be obtained directly from living donor tissue. For example, hepatocytes are isolated and prepared for transplantation according to the methods described in U.S. Patent Nos. 9,125,891 B2 and 6,610,288 B1, U.S. Patent Publication Nos. 20120045764 A1 and 20040110289 A1, each of which is incorporated herein by reference. In certain embodiments, the cells to be delivered are obtained from in vitro sources. Stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, trophoblast stem cells or any other type of pluripotent or multipotent stem cells) can be cultured in vitro and differentiated into a certain cell type or cell population suitable for the purpose of applying the methods and systems provided herein. For example, as described above, kidney organoids or pancreatic islets are obtained using in vitro differentiation methods and prepared for transplantation procedures described herein.Delivered cells can be stored, for example, cryopreserved, before transplantation.Standard cryopreservation and recovery protocols can be used as long as cell viability level is suitable for subsequent cell delivery and ectopic tissue formation in lymph nodes.In certain embodiments, delivered cells are genetically modified so that one or more genes of delivered cells are modified, or the cells are modified to express one or more exogenous genes.For genetic modification of delivered cells, any available gene editing method (for example, homologous recombination, transposase / transposon, zinc finger nuclease, TALEN and CRIPSR, for example, CRISPR-Cas9 technology) can be applied.

[0095] The cells can be prepared for implantation and suspended in a solution. In certain embodiments, the suspension containing the cell population can further comprise a pharma- ceutically acceptable excipient, diluent or carrier. As used herein, the term "pharma-ceutically acceptable" can refer to a compound, material, composition and / or dosage form that is suitable for use in contact with the tissue of a subject (e.g., a human subject or a non-human animal) without excessive toxicity, irritation, allergic reaction or other problem or complication, within the scope of sound medical judgment, and with a reasonable benefit / risk ratio.

[0096] As used herein, the term "solution" may include pharmaceutically acceptable carriers or diluents in which the cells of the present invention can be viable. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer, solvent and / or dispersion medium. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid enough to be easily syringable. The solution may be stable under the conditions of manufacture and storage, and may be protected against the contaminating action of microorganisms such as bacteria and fungi, for example, by using paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. The solution of the present disclosure may be prepared by incorporating the viable functional cells as described herein and other ingredients listed above, as necessary, into a pharmaceutically acceptable carrier or diluent, followed by filtration sterilization.

[0097] Non-limiting examples of substances or materials that may serve as pharma- ceutically acceptable excipients for purposes of this disclosure include sugars (e.g., lactose, glucose, and sucrose); starches (e.g., corn starch and potato starch); cellulose and its derivatives (e.g., sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc); cocoa butter and suppository wax; oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil); glycols (e.g., , propylene glycol); polyols (e.g., glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); esters (e.g., ethyl oleate and ethyl laurate); agar; buffers (e.g., magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; bulking agents (e.g., polypeptides and amino acids); serum components (e.g., serum albumin, HDL, and LDL); C2-C12 alcohols (e.g., ethanol); and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," "pharmaceutical acceptable carrier," and the like are used interchangeably herein. As used herein, the term "pharmaceutically acceptable excipient" may refer to a pharmaceutically acceptable material, composition or vehicle (e.g., liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc magnesium, calcium or zinc stearate or stearic acid) or solvent encapsulating material involved in holding or delivering the compound, material or cell to an organ or part of the body.Each excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, eg, the cells to be implanted, and not injurious to the subject.

[0098] After being delivered to lymph nodes, the cells of the present disclosure can engraft, proliferate, and generate ectopic tissue in lymph nodes. As used herein, the term "engraft" or its grammatical equivalents can refer to the process in which one or more cells are transplanted into a target lymph node, survive in the lymph node, and become biologically active (e.g., perform cellular functions). As used herein, the term "proliferate" or its grammatical equivalents can refer to the process in which cells undergo one or more mitosis and produce several progeny cells, and this proliferation process can exponentially increase the number of cells. In certain embodiments, the cells proliferate at a high level, and the mass of ectopic tissue that is ultimately generated is much larger than the original cell mass delivered to the lymph node. In certain embodiments, the cell proliferation is at a moderate level. In certain embodiments, the increase in mass is, for example, at least about 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 7.5-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 17.5-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 1000-fold compared to the mass of the originally transplanted cells. In certain embodiments, the number of cells is increased by at least about 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 7.5-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 17.5-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 1000-fold, as compared to the mass of cells originally transplanted, for example.

[0099] In certain embodiments, vascularization may occur in the lymph node receiving the cell transplant, for example, there may be blood vessels in the lymph node, in certain embodiments, in the ectopic tissue, that infiltrate and form a vascular network. In certain embodiments, the infiltrating vascular network may lay the foundation for blood supply to the ectopic tissue. In certain embodiments, the infiltrating vascular network may help transport metabolic products or materials to and / or from the ectopic tissue. For example, but not by way of limitation, an ectopic liver may produce glycogen, glucose, and / or bile, which may be transported to the main bloodstream by the vascular network formed in the transplant site (lymph node). In certain embodiments, an ectopic pancreas may produce and secrete insulin, glucagon, and / or somatostatin, which may also be introduced into the main bloodstream of the subject's body by the vascular network formed in the lymph node. In certain embodiments, the lymphatic circulation system also serves as a transport route for substances produced by the ectopic tissue. Thus, in certain embodiments, the lymphatic system may supply such substances to the blood circulatory systems of other parts of the body through their crosstalk.

[0100] 5. Diseases and symptoms The above method and system can be applied to treat disease or condition of a subject.In certain embodiments, the above method and system are used in growing one or more ectopic tissues in lymph nodes, which ectopic tissues can complement or enhance the function of the organ of the subject, and as a result, can ameliorate or cure one or more symptoms of the disease or condition that the subject suffers from.

[0101] A. Liver Disease and Symptoms In certain embodiments, the methods and systems can be used to deliver hepatocytes to lymph nodes of a subject, allowing the hepatocytes to populate the lymph nodes and generate an ectopic liver. An ectopic liver can have one or more functions that a normal, healthy liver organ can perform, such as, for example, producing bile, which can help remove waste products and break down fats in the small intestine during digestion; producing plasma proteins, such as albumin; producing cholesterol, phospholipids, and lipoproteins, which help transport fats around the body; controlling blood sugar by converting excess glucose into glycogen for storage (glycogen synthesis) and depolymerizing glycogen when glucose is needed (glyconolysis); and regulating excess carbohydrates and proteins. These include, but are not limited to, the conversion of lipids into fatty acids and triglycerides; deamination and transamination of amino acids; the conversion of non-nitrogen-containing portions of amino acids into glucose or lipids; the oxidation of triglycerides to produce energy; the processing of hemoglobin for use in its iron content (the liver stores iron); the conversion of toxic ammonia to urea; hemodialysis to remove certain drugs and other toxic substances; the synthesis of clotting factors necessary for blood clotting; fighting infection by producing immune factors and removing bacteria from the bloodstream; and the clearance of bilirubin from red blood cells.

[0102] In certain embodiments, the methods and systems can be used to treat several different liver diseases or conditions. Such liver diseases or conditions can include liver failure or a decline in one or more liver functions. Non-limiting examples of liver diseases and / or disorders that can be treated by the methods of the present disclosure include acute liver failure, cirrhosis, liver cancer, hepatitis, fatty liver disease, and non-alcoholic fatty liver disease. The liver condition can be associated with metabolic disorders (e.g., pediatric metabolic disorders), including, but not limited to, tyrosinemia, maple syrup urine disease, phenylketonuria, Crigler-Najjar syndrome, oxalosis, hyperoxaluria, hemochromatosis, alpha-1 antitrypsin deficiency, Wilson's disease, familial intrahepatic cholestasis syndrome, and familial amyloid polyneuropathy. The methods and systems may be of particular use in treating end-stage liver disease and / or liver fibrosis, such as that caused by hepatitis B infection, hepatitis C infection, alcohol consumption, cirrhosis, non-alcoholic steatohepatitis or hemochromatosis.

[0103] In certain embodiments, the methods and systems provided herein, when applied to a subject in need of amelioration of one or more symptoms associated with liver disease or liver condition, ameliorate such symptoms, e.g., restore one or more liver functions, and / or in certain embodiments, prolong the survival time of a subject who experienced a life-threatening liver disease or liver condition prior to cell transplantation. For example, a subject who has undergone a hepatocyte transplant according to the present disclosure may have improved results of liver function tests (e.g., alanine transaminase (ALT) test, aspartate aminotransferase (AST) test, alkaline phosphatase (ALP) test, albumin test, bilirubin test). Such improvement may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 100% recovery when compared to the test results of other healthy subjects or the same subjects prior to having liver disease or liver condition. In certain embodiments, the lifespan of a subject receiving a hepatocyte transplant is extended by at least about 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 18 years, 20 years, 25 years, 30 years, 40 years, 50 years, 60 years or more.

[0104] B. Renal Disease and Conditions In certain embodiments, the above method and system can be used to deliver kidney cells or kidney tissue fragments to a subject's lymph node, allowing the kidney cells to populate the lymph node and generate an ectopic kidney. The ectopic kidney can have one or more functions that a normal healthy kidney organ can perform. For example, the ectopic kidney can perform to some extent or fully the main functions of the kidney: urine production, which may include filtering low molecular weight substances (e.g., metabolic waste products) from blood to produce ultrafiltrate that ultimately becomes urine, while leaving cells and large proteins in blood; and reabsorption of certain substances (e.g., ions, glucose) from the ultrafiltrate into peritubular capillaries. In certain embodiments, the ectopic kidney can also participate in maintaining systemic homeostasis (e.g., but not limited to, acid-base balance, electrolyte concentration, extracellular fluid volume, and blood pressure) as a normal kidney can perform.

[0105] In certain embodiments, the method and system are used in treating several different renal diseases or conditions. Such renal diseases or conditions may include renal failure or one or more renal function declines. Non-limiting examples of renal diseases and / or disorders that can be treated by the method of the present disclosure include acute renal failure, chronic renal disease, glomerulonephritis, lupus, polycystic kidney disease, nephropathy, nephrosis, renal malformation and renal cancer. The method and system can be particularly used in treating end-stage renal disease (renal failure), such as those caused by diabetes, autoimmune diseases (e.g., lupus nephropathy and IgA nephropathy), genetic diseases (e.g., polycystic kidney disease), nephrotic syndrome and urinary tract problems.

[0106] In certain embodiments, the methods and systems provided herein, when applied to a subject in need of amelioration of one or more symptoms associated with a renal disease or condition, ameliorate such symptoms, e.g., restore one or more renal functions, and / or in certain embodiments, prolong the survival time of a subject who experienced a life-threatening renal disease or condition prior to cell transplantation. For example, and not by way of limitation, a subject who has received transplantation of kidney cells or kidney fragments according to the present disclosure has improved results in renal function tests (e.g., blood tests for serum creatinine, glomerular filtration rate (GFR), blood urea nitrogen (BUN)). Such improvement can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100% recovery when compared to the test results of other healthy subjects or the same subjects prior to having a renal disease or condition. In certain embodiments, the lifespan of a subject receiving a kidney cell or kidney fragment transplant may be extended by at least about 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 18 years, 20 years, 25 years, 30 years, 40 years, 50 years, 60 years or more.

[0107] C. Pancreatic Diseases and Conditions In certain embodiments, the methods and systems are used to deliver pancreatic cells (e.g., pancreatic β, α, δ, ζ or γ cells) or islets to a lymph node of a subject, thereby allowing the pancreatic cells or islets to engraft in the lymph node and generate an ectopic pancreas. The ectopic pancreas may have one or more functions that a normal, healthy pancreatic organ may perform. For example, but not by way of limitation, the ectopic pancreas secretes one or more of insulin, glucagon, somatostatin or pancreatic polypeptide. In certain embodiments, the ectopic pancreas secretes one or more endocrine hormones in appropriate response to physiological stimuli. In certain embodiments, the ectopic pancreas secretes insulin in response to an increase in blood glucose concentration and / or secretes glucagon in response to a decrease in blood glucose concentration. This may help maintain blood glucose homeostasis. Such secretory responses may be proportional to changes in physiological stimuli. For example, the greater the increase in blood glucose, the more insulin the ectopic pancreas will secrete, at a ratio that may be at least partially equivalent or similar to that of a normal, healthy pancreas in another healthy subject or the pancreas of the same subject prior to the onset of pancreatic disease.

[0108] In certain embodiments, the method and system are used in treating several different pancreatic diseases or conditions. Such pancreatic diseases or conditions may include pancreatic insufficiency or reduced one or more pancreatic functions. The pancreatic diseases or conditions treated by the methods and systems provided herein may be endocrine pancreatic diseases or conditions. Non-limiting examples of pancreatic diseases and / or disorders that may be treated by the methods of the present disclosure include acute pancreatitis, chronic pancreatitis, hereditary pancreatitis and pancreatic cancer. The method and system may be particularly used in treating pancreatic insufficiency or other conditions that require pancreatic transplantation.

[0109] In certain embodiments, the methods and systems provided herein, when applied to a subject in need of amelioration of one or more symptoms associated with a pancreatic disease or condition, ameliorate such symptoms, e.g., restore one or more pancreatic functions, and / or in certain embodiments, extend the survival time of a subject who experienced a life-threatening pancreatic disease or condition prior to cell transplantation. For example, and not by way of limitation, a subject who has received a transplant of pancreatic cells or islets according to the present disclosure may have improved glycemic control. Such improvement may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 100% recovery when compared to the test results of other healthy subjects or the same subject prior to having a pancreatic disease or condition. In certain embodiments, the lifespan of a subject receiving a pancreatic cell or islet transplant may be extended by at least about 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 18 years, 20 years, 25 years, 30 years, 40 years, 50 years, 60 years or more.

[0110] D. Immune System Dysfunction / Immunodeficiency Disorders and Immunodeficiency Conditions In certain embodiments, the above method and system are used to deliver thymocytes or thymus fragments to lymph nodes of a subject, allowing the thymocytes or thymus fragments to engraft in the lymph nodes and generate ectopic thymus.In certain embodiments, the ectopic thymus complements or enhances one or more functions that normal healthy thymus organs can perform.For example, but not by way of limitation, the ectopic thymus can participate in the immune regulation of the body by participating in the growth, development, maturation and selection of T cells. The generation of an ectopic thymus according to the present disclosure can be used in enhancing or regulating immune system function in subjects with immune system dysfunction, for example, elderly or aged subjects (e.g., subjects over the age of 50, 55, 60, 65, 70, 75, 80, or 85 years) or subjects with an immunodeficiency disorder or condition (e.g., X-linked agammaglobulinemia (XLA), common variable immunodeficiency (CVID), severe combined immunodeficiency (SCID), severe burns, chemotherapy, radiation, diabetes, malnutrition, acquired immune deficiency syndrome (AIDS), leukemia, severe viral infections, and multiple myeloma).

[0111] In certain embodiments, thymocytes or thymus fragments from a donor subject are introduced into the lymph nodes of a recipient subject prior to organ or other cell transplantation from the same donor subject. The generation of ectopic thymus in lymph nodes can induce tolerance to the donor subject in the recipient subject, which can be beneficial for subsequent organ or cell transplantation. In these embodiments, thymocyte transplantation can be performed at least about 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, or 1 year prior to organ or other cell transplantation, so that appropriate immune tolerance to the donor subject can be established in the recipient. Thymocyte transplantation can be applied with any type of organ transplantation or any other type of cell transplantation as described herein, and can reduce graft rejection responses seen in recipients who otherwise receive the same organ or cell transplantation without thymocyte transplantation according to the present disclosure.

[0112] The life span of a subject receiving a cell transplant according to the present disclosure may be extended by at least about 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 12 years, 15 years, 18 years, 20 years, 25 years, 30 years, 40 years, 50 years, 60 years or more.

[0113] E. Pulmonary Disease and Symptoms In certain embodiments, the methods and systems are used to deliver lung cells or lung tissue fragments to a lymph node of a subject, allowing the lung cells or lung fragments to engraft in the lymph node and generate an ectopic lung. The ectopic lung may have one or more functions that a normal healthy pancreas and lung may perform. For example, the ectopic lung may be a patient with chronic obstructive pulmonary disease (COPD). The ectopic lung may increase the lung function mass that may be significantly reduced by progressive fibrosis in COPD patients. In certain embodiments, patients who have undergone lung reduction procedures that would not be candidates for standard lung transplants may be suitable for transplantation of lung cells into peribronchial lymph nodes.

[0114] In certain embodiments, the method and system are used in treating several different pulmonary diseases or pulmonary conditions. Such pulmonary diseases or pulmonary conditions may include pulmonary insufficiency or one or more reduced pulmonary functions. Non-limiting examples of pulmonary diseases and / or pulmonary disorders that can be treated by the method of the present disclosure include chronic obstructive pulmonary disease (COPD). COPD can be caused by tobacco smoke and also smoking, tobacco pollutants (chemicals, dust or toxic substances) and fumes, genetic disorders (e.g., alpha-1-antiytrypsin, cystic fibrosis), chronic asthma, emphysema, chronic bronchitis or idiopathic pulmonary fibrosis.

[0115] In certain embodiments, the methods and systems provided herein may ameliorate one or more symptoms associated with a pulmonary disease or condition, e.g., restore one or more lung functions, and / or in certain embodiments, extend survival time of a subject who experienced a life-threatening pulmonary disease or condition prior to cell transplantation.

[0116] 6. Subjects The subject that can receive the cell transplantation according to the present disclosure can be any human patient (e.g., an ESLD patient, a renal failure patient, a type I diabetes patient, or a patient waiting for an organ transplant). In certain embodiments, the subject is in a certain medical treatment stage.

[0117] The subject receiving the cell transplant according to the present disclosure may be of any age, and may be an adult, a newborn, an infant, or a child. In certain embodiments, the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or 99 years of age, or within a range therein (e.g., 2-20 years, 20-40 years, or 40-90 years). Further, the subject may be male or female.

[0118] The subject's eligibility to receive cell transplantation according to the present disclosure can be determined by a certified physician. In certain embodiments, a comprehensive evaluation of the subject's health status may be required before cell transplantation. In certain embodiments, the requirements for the subject's health status may be significantly lower than other organ or cell transplantation procedures. Without wishing to be bound by a particular theory, the minimally invasive procedure according to the present disclosure may significantly reduce the risk of surgical procedures compared to the major surgery usually performed for organ transplantation. Furthermore, considering that cells are transplanted into one or more lymph nodes rather than into a diseased organ, the requirements may be even significantly lower since there may be no prerequisite for a diseased organ.

[0119] Any of the methods and systems disclosed herein may also be used on non-human subjects, such as laboratory animals or farm animals, for research or veterinary purposes. Non-limiting examples of non-human subjects include dogs, goats, guinea pigs, hamsters, mice, pigs, non-human primates (e.g., gorillas, apes, orangutans, lemurs, or baboons), rats, sheep, or cows.

[0120] 7. System The present disclosure further provides a system for transplanting cells into lymph nodes and growing functional ectopic tissue. The system may include an endoscope and a syringe with a needle containing a suspension including one or more cells. The endoscope and needle may be configured to be advanced together along a body lumen (e.g., the gastrointestinal tract, the airway, or the urinary tract) or a closed body cavity (e.g., the abdominal, pelvic, or thoracic cavity) of a subject, and the syringe may be configured to deliver the one or more cells through the needle.

[0121] As discussed above, in certain embodiments, the suspension may have at least about 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 3, 5, 8, or 10 billion cells per mL. In certain embodiments, a syringe as described herein holds a cell suspension having about 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 3, 5, 8 or 10 billion cells per mL. In certain embodiments, a syringe as described herein holds a cell suspension having at most about 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 3, 5, 8, or 10 billion cells per mL.

[0122] In certain embodiments, the syringe as described herein has a hepatocyte suspension having at least about 30, 40, 45, 50, 55, 60, 70, 80, 90, or 100 million cells per mL for generating an ectopic liver. In certain embodiments, the syringe as described herein has a hepatocyte suspension having at least about 30, 40, 45, 50, 55, 60, 70, 80, 90, or 100 million cells per mL for generating an ectopic liver. In certain embodiments, a syringe as described herein contains a hepatocyte suspension having at most about 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 million cells per mL for generating an ectopic liver.

[0123] As discussed above, in certain embodiments, the needle may have an inner diameter of at most about 700 μm, 600 μm, 500 μm, 450 μm, 400 μm, 300 μm, 260 μm, 250 μm or 200 μm. In certain embodiments, the needle has an inner diameter of at most about 260 μm. In certain embodiments, the inner diameter of the needle is about 700 μm, 600 μm, 500 μm, 450 μm, 400 μm, 300 μm, 260 μm, 250 μm or 200 μm. In certain embodiments, the inner diameter of the needle is about 260 μm. The needle may have an outer diameter of at most about 1 mm, 900 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 520 μm, 510 μm, 500 μm, 480 μm, 450 μm, or 400 μm. In certain embodiments, the outer diameter of the needle is at most about 510 μm. In certain embodiments, the outer diameter of the needle is at most about 1 mm, 900 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 520 μm, 510 μm, 500 μm, 480 μm, 450 μm, or 400 μm. In certain embodiments, the outer diameter of the needle is about 510 μm. In certain embodiments, the needle is a certain gauge as defined according to ISO7864:2016. For example, but not by way of limitation, the needle is about 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or 27 gauge (ga). In certain embodiments, the needle is at most about 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or 27 ga. For example, but not by way of limitation, the needle of the system can be at most about 25 ga. In certain embodiments, the needle has a non-standardized size, e.g., having a thin wall while maintaining a large inner diameter and a small outer diameter.

[0124] The system and method provided herein may be particularly useful for cells with an average diameter of about 20 μm, such as hepatocytes.Without wishing to be bound by a particular theory, the size of cells to be delivered may determine the concentration of cells used in suspension, the inner diameter of the needle used for cell delivery, and the cell viability level in the syringe before cell delivery and after cell delivery.For cells with an average diameter of less than 20 μm, the needle size may be smaller than 25 ga, and the cell concentration and total cell number of the suspension may be higher than that of cells with an average diameter of about 20 μm, such as parameters discussed above.On the other hand, for cells with an average diameter of more than 20 μm, the needle size may be larger than 25 ga, and the cell concentration and total cell number of the suspension may be lower than that of cells with an average diameter of about 20 μm, such as parameters discussed above.

[0125] 8. Kit The present disclosure further provides a kit comprising materials useful for carrying out any of the methods disclosed herein. In certain embodiments, the kit comprises a container comprising one or more cells disclosed herein. In certain embodiments, the cells may be provided in a frozen container. In certain embodiments, the cells may be provided in a solution, e.g., in a medium. In certain embodiments, the container may comprise about 10 million to about 500 million cells. In certain embodiments, the container may comprise a homogenous cell population. Alternatively, the container may comprise a heterogenous cell population. Non-limiting examples of cells that may be provided in the container include liver cells, kidney cells or kidney tissue fragments, pancreatic cells or pancreatic islets, thymus cells or thymus tissue fragments, or lung cells or lung tissue fragments.

[0126] In certain embodiments, the kit may further comprise a second container comprising a solution for introducing cells into a lymph node as disclosed herein.

[0127] In certain embodiments, the kit may further include a device (or system) for delivering cells to lymph nodes as disclosed herein. In certain embodiments, the kit may further include an endoscope as disclosed herein. In certain embodiments, the endoscope is coupled to a needle. For example, but not by way of limitation, the kit may include a needle and / or an endoscope for delivering cells to lymph nodes as disclosed herein. Non-limiting examples of such delivery devices are disclosed in the system section above. EXAMPLES

[0128] The following examples are provided to further illustrate certain embodiments of the present invention, but are not intended to limit the scope of the invention, and it will be understood that by their exemplary nature, other procedures, methodologies or techniques known to those skilled in the art may be substituted.

[0129] Example 1. Isolation and viability testing of hepatocytes for transplantation This example demonstrates that when a cell suspension is delivered through a needle of a certain size according to the present disclosure, the viability of isolated hepatocytes is not significantly affected.This example demonstrates that hepatocytes isolated and delivered according to an exemplary method of the present disclosure can have a viability suitable for transplantation.

[0130] Liver cells were isolated according to the following protocol. First, the left liver lobe was surgically obtained from donor dogs, placed in a double-bagged container, and transported for further processing under ice-based refrigeration (static storage with Belzer's solution at 4°C). Liver cells were isolated using the two-step collagenase perfusion method reported by Seglen (Seglen PO, Preparation of isolated rat liver cells. Methods Cell Biol, 1976;13:29-63). Briefly, a portal vein cannula or any major blood vessel was cannulated. In a culture dish, the liver was perfused with EDTA solution (0.02%) at 37°C for 10 minutes at a flow rate of 50 ml / min. Collagenase solution (37°C) was then recirculated through the liver sample at the same flow rate. After 10 minutes, the liver capsule was broken, and the digested liver parenchyma was suspended in ice-cold Hank's Balanced Salt Solution or Plasma-Lyte A solution containing calcium gluconate and human serum albumin. The resulting liver cell suspension was filtered and washed three times.

[0131] Trypan blue exclusion testing was used to ascertain the viability of isolated hepatocytes in suspension at various steps throughout the experiments described below. In these experiments, different batches of suspensions with different cell concentrations were tested for cell viability before and after passing through EUS FNA needles of various gauges. Figure 3 is a plot summarizing the cell viability results obtained from the experiment with two batches of cells. As shown in this figure, most, but not all, of the solutions tested had similar cell viability levels before and after passing through any of the EUS needles tested.

[0132] Needles used in testing: 19 gauge (19ga) needle (Covidien: Ref# DSN-19-01): Approximate dead volume is 1.2 mL. 22 gauge (22ga) needle (Covidien: Ref#N22-05): Approximate dead volume is 0.5mL. 25 gauge (25ga) needle (Covidien: Ref# DSN-25-01): Approximate dead volume is 1.1 mL.

[0133] Batch #1: Donor ID#:6303. Initial viability after liver digestion and isolation of hepatocytes into single cells: 59.5%. Total of 4.24 billion viable hepatocytes (5.3M / mL in a total of 800 mL solution).

[0134] Cell viability after 3 cycles of washing and ready for transplant: 72.04%. Total of 429 million viable hepatocytes (2.86M / mL in a total of 150mL solution used out of 800mL).

[0135] The 429 million viable hepatocytes were resuspended in approximately 20 ml and tested for viability using 19, 22 and 25 ga EUS needles. Table 1 summarizes the viability scores obtained in the various groups tested in this experiment. Controls are solutions that had not been passed through any EUS needles prior to post-test viability assessment and were otherwise handled identically.

[0136] [Table 1]

[0137] Batch #2: Donor ID#:6302. Initial viability after digesting the liver and isolating the hepatocytes into single cells: 67.5%. For a total of approximately 5 billion isolated live hepatocytes (5.5 M / mL in a total of 900 mL solution), 600 ml of solution (approximately 3.3 billion cells) was used in this experiment.

[0138] Cell viability after 3 wash cycles and ready for transplantation: A total of 624 million viable hepatocytes (4.16M / mL in 150mL solution) with a viability of 71.7%. Store in UW overnight for transplantation the next day. A total of 567 million viable hepatocytes (8.1M / ml in 70ml solution) with a viability of 76.3%. Fourteen tubes were cryopreserved at 107 hepatocytes / ml, 2 ml per tube. A total of 1.3 billion cells (9.1M / ml in 150 ml solution) with a viability of 69.7%.

[0139] Hepatocytes were resuspended at 25M / ml and 50M / ml and tested for viability using 19, 22 and 25ga EUS needles. Tables 2 and 3 summarize the viability scores obtained in the various groups tested in this experiment. Controls were solutions that had not been passed through any EUS needles prior to post-test viability assessment and were otherwise handled identically.

[0140] [Table 2]

[0141] [Table 3]

[0142] Example 2. Portocaval shunt in dogs as a model of liver failure This example describes an exemplary surgical procedure to create an animal model for studying regenerative medicine to treat liver failure.

[0143] As shown in Figure 4, a complete portocaval shunt to block the main blood flow to the liver can be performed, which can induce liver injury. With the use of an appropriate surgical stapler, this procedure can be performed with little blood loss. Following mobilization of the main portal vein (PV) in the hepatic porta, the common bile duct (CBD) and the main hepatic artery (HA) are isolated. A proximal incision of the main PV towards the confluence between the superior mesenteric vein (SMV) and the splenic vein (SV) is also performed. The infrahepatic inferior vena cava (IVC) towards the renal vein is further mobilized with a distal incision. After a complete ligation and transection of the main PV just before the bifurcation (right and left branches) with an articulated endovascular staple, the PV is mobilized caudally towards the IVC. The PV is fully mobilized caudally towards the IVC and an end-to-side anastomosis (continuous suture of 6-0 Prolene) is performed between the PV and the IVC. This step includes partial clamping of the IVC and complete clamping of the proximal PV. Once complete hemostasis was achieved, the abdominal cavity was closed. After the animals had fully recovered from general anesthesia, they were extubated in the OR.

[0144] Example 3. Preclinical study of EUS-guided delivery of hepatocytes in lymph nodes for organogenesis This example illustrates the treatment of liver failure in a large animal (canine) model by EUS-guided delivery of hepatocytes to the periduodenal lymph nodes (LNs).

[0145] The preclinical studies in a large animal model (dog) described herein have been approved by IACUC protocols under USDA guidelines and are performed in a fee-for-service lab at Allegheny Health Network, Highmark, Pittsburgh, Pa. These studies involve surgical construction of a complete portocaval shunt (PCS) followed by hepatocyte transplantation in a dog model of induced liver failure.

[0146] Briefly, two groups of animals are tested to confirm that hepatocyte transplantation into the periduodenal lymph nodes (PDLN) induces the development of new ectopic liver buds (organogenesis) with normal liver cytoarchitecture and full function. The control group undergoes a complete PCS procedure followed by a sham injection of normal saline into the LN. The study group undergoes the same initial PCS procedure followed by autologous or allogeneic hepatocyte transplantation into the PDLN by EUS approach. Both groups receive a nonspecific immunosuppressive regimen consisting of tacrolimus and prednisone. Both groups are followed for 6 months after the initial procedure. Animals in the control group experience progressive liver failure and have a 30% mortality rate over the duration of the study. Animals in the study group do not show long-term signs of liver failure and do not die. These animals show signs of enlargement of the PDLN site where the hepatocytes were initially transplanted. At the end of the study necropsy, the animals demonstrate the development of ectopic liver tissue in their PDLNs with normal anatomical and histological features that demonstrate clinical and laboratory evidence of sustainable liver function over the duration of the study.

[0147] As shown in the study flow diagram in Figure 5, group 1 serves as the donor group for groups 4-7 who will receive allogeneic transplantation of hepatocytes (HT). Group 2 will undergo a PCS procedure along with a placebo injection (saline) into the LNs. Group 3 is the autologous control group. Groups 4 and 5 will undergo direct injection of HT into the LNs (as shown in Figure 2) at low dose levels (75 M HT; 25 M hepatocytes / mL, 1 mL of solution injected into each of three LNs) or medium dose levels (150 M HT; 50 M hepatocytes / mL, 1 mL of solution injected into each of three LNs). Groups 6 and 7 will undergo HT transplantation via a minimally invasive endoscopic ultrasound (EUS) approach (as shown in Figures 1A and 1B) at both low and medium doses as outlined above.

[0148] The primary endpoint is at 90 days (N=2 sacrifices from each of Groups 2-7), with further long-term safety follow-up from Days 91 to 180, and sacrifice of study animals at Day 120 (N=1 from each of Groups 2-7), Day 150 (N=1 from each of Groups 2-7), and at the end of study at Day 180 (N=1 from each of Groups 2-7).

[0149] Lymph nodes are collected from sacrificed animals at each time point, fixed in paraformaldehyde, and embedded in optimal cutting temperature compound or paraffin. Sections are prepared and stained with hematoxylin and eosin (H&E) or hematoxylin along with anti-fumarylacetoacetate hydrolase (FAH) immunostaining. FAH is highly expressed in hepatocytes and can be used to identify hepatocytes. Sections are imaged and assessed histologically for the presence of engrafted hepatocytes and the formation of ectopic liver tissue.

[0150] statistical analysis The primary endpoints are the size and weight (g) of lymph nodes with newly engrafted hepatocytes at 90 days. A full-sized liver in a medium-large dog (23.7 kg) weighs approximately 767±48 g, which also serves as an estimate for the weight of a fully engrafted ectopic liver and the standard deviation of liver weight within treatment groups. Four of the six treatment groups receive allogeneic hepatocytes at a dose of either 75 M (low dose) or 150 M (medium dose) by direct injection or endoscopic infusion into the lymph nodes. Two-way analysis of covariance (ANCOVA) of the four allogeneic groups, adjusting for total animal weight (kg), is used to examine differences in main effect endpoints and interactions between dose level and infusion type. Five dogs per treatment group provided 92% power to detect an endpoint difference of 77 g between the two levels of each main effect (effect size of 0.8) using an F-test with α=0.05 (2-tailed) and 75% power to detect an interaction between dose level and injection type when the effect size was 0.6. Based on previously reported findings (Kam et al., 1987, Sohlenius-Sternbeck, 2006), without wishing to be bound by any particular theory, there may be an underlying linear relationship as well as a significant correlation between liver weight and total weight. This is a conservative estimate of the overall power since it does not take into account covariate adjustments.

[0151] One-way ANOVA with the same endpoints and covariates will be used to perform an overall F-test for treatment group differences, as well as valid pairwise comparisons between the six groups. The six groups include the four allogeneic groups described above, plus a group that received 75 M autologous hepatocytes by endoscopic injection, and a control group that received endoscopic injection of saline. Five dogs per group would provide 100% power to detect at least one difference of 154 g (20% engraftment) against the null hypothesis of equal means when using an overall F-test with α = 0.05 (two-sided). This sample size would achieve 90% power to detect a difference of at least 230 g (30% engraftment) against any step-down test, using the Tukey-Kramer (pairwise) multiple comparison procedure to hold the significance level at 0.05. These power calculations are derived by PASS12 (Hintze, 2013).

[0152] Surgical and experimental methods (I) Line Installation and Animal Support A PE catheter is inserted into the right jugular vein to initiate 0.9% NaCl IV support fluid infusion. Paralytic agents are administered and maintained throughout the duration of the procedure via syringe pump or bolus therapy. A peripheral artery is inserted with a PE catheter. Additionally, a double-lumen long-term central venous access (superior vena cava) is placed via a right neck incision and direct access to the right external jugular vein. After the abdominal procedure is completed, a permanent central venous access is inserted and exposed in the posterior neck. An electrocardiac register is monitored with electrodes placed on the animal's body surface. Rectal temperature is monitored continuously. Animal support follows SOP ANI-017 Guidelines for Performing Survival Surgery in USDA Regulated Species and ANI-032 Thermal Regulation of the Anesthetized Patient.

[0153] (II) Partial resection of the left liver lobe for subsequent hepatocyte isolation The animal is stabilized under general anesthesia and then prepped and draped in a sterile fashion. The abdominal cavity is entered through a midline incision. First, the hepatic hilum is incised and the left lateral segment (LLS) is isolated. The left portal vein (PV) and left hepatic artery (HA) are isolated and circumscribed with vascular tape. The LLS is resected by controlled parenchymal transection in conjunction with articulated endovascular staples. A partial resection of the left lateral segment removes approximately 20% of the total liver volume. Once the left lateral segment of the liver is removed from the surgical field, the specimen is processed in a back-table (BT) procedure under sterile conditions. This BT involves flushing the left PV and left HA with cold lactated Ringer's solution (LR) to remove all blood (flushing step), followed by infusion of Belzer's solution for later liver preservation. The liver segments are packed in double plastic bags and placed in an ice cooler before being transported to the laboratory where cell isolation will be performed (to obtain hepatocytes).

[0154] (III) Portocaval shunt procedure After completing the left lateral segment resection, an extended hepatic hilar resection is performed and a complete portacaval shunt is performed according to the procedure as described in Example 2.

[0155] (IV) Hepatocyte isolation for transplantation The liver cells are isolated according to the procedure described in Example 1. The resulting liver cell suspension is filtered and washed. Trypan blue exclusion test is used to confirm the viability of the isolated liver cells. In the autologous transplant group, in which animals receive their own liver cells, the animals remain under general anesthesia for an additional 5 hours while the cells are isolated and prepared for subsequent injection.

[0156] (V) Hepatocyte transplantation by direct surgical injection After obtaining samples for quality control (cell viability, cell number, culture and sensitivity), the surgeon will perform the hepatocyte transplantation. Determined for each assigned experimental group, cells will be injected intraparenchymally (25 × 10 cells in a small volume (1–3 ml) of injection medium) directly into the mesenteric and periduodenal lymph nodes by direct surgical approach or via endoscopic injection (described below). 6 ~50×10 6Cell yield in viable cells / ml). Adequate hemostasis is achieved completely after heterotopic hepatocyte injection into the lymph nodes. After meticulous hemostasis is achieved, the peritoneal cavity is lavage-perfused with a solution of antibiotic and antimycotic agents (Neomycin 500 mg / L, Polymyxin 15,000 units / kg / L, Bacitracin 1000 units / kg / L and Amphotericin B 4 mg / kg / L). The abdominal wall is closed in a three-layer fashion without the use of drains. Sealing sutures are placed at the skin closure. The animal is allowed to recover adequately from the general anesthesia procedure and is further extubated in the operating room. The animal is then transferred to a suitably equipped animal facility for post-operative care.

[0157] (VI) Hepatocyte transplantation via intraluminal endoscopic approach After completion of PCS, endoscopic injections are performed in animals under general anesthesia. A certified pancreaticobiliary endoscopist with extensive experience in EUS performs these procedures. A linear ultrasound endoscope (Olympus GF-UCT 180) is used for these experiments. This endoscope is inserted through the animal's mouth and advanced into the stomach and duodenum. An endoscopic ultrasound (EUS) probe assists and guides the location of the periduodenal lymph nodes (LNs). The LNs are accessed directly via a transgastric and / or transduodenal approach. Once the LNs are properly entered by a fine needle aspiration (FNA) needle (Boston scientific, needles ranging from 19 to 25G), the surgeon assists the endoscopist in performing the hepatocyte transplantation after successful needle insertion into the LNs via this EUS-guided procedure. Pre-isolated hepatocytes are injected into the LNs at a concentration of 25 × 10 6 cells / mL or 50 x 10 6 The hepatocytes are maintained in a solution containing 1000 cells / mL. The hepatocytes in this solution are transplanted directly into the periduodenal LN using a fine needle aspiration (FNA) needle (Boston scientific, needles ranging from 19-25 G). Once this surgical procedure is completed, the animal is allowed to recover properly from the general anesthesia procedure and is further extubated in the operating room. The animal is subsequently transferred to a suitably equipped animal facility for post-operative care.

[0158] (VII) Recovery and Post-Operative Care Allow the animal to recover from anesthesia with appropriate monitoring while resuming natural ventilation. Animals will be monitored 24 h per day by trained preclinical facility personnel for the first 2-3 days after surgery (longer if necessary), then at least daily for the duration of the study. Postoperative care of the animals will be performed under the guidance of the study director in consultation with a veterinarian.

[0159] Animals are monitored frequently and closely. Each animal is assessed based on the following species-specific criteria for pain assessment and relief: vocalization, depression, and >50% increase in respiration (based on a dog's average respiratory rate of 20 / min). In addition, heart rate is monitored. Temperature is also monitored.

[0160] Pain Management and Monitoring Animals are initially monitored hourly for the following indicators of post-operative pain and distress using a pain scoring system including, but not limited to, overall activity level, surgical wound, appetite and attitude toward food.

[0161] Postoperative pain is treated with the indicated analgesic dosing based on the following indicators: an increase of approximately 10-15% in heart rate; and an increase of approximately 40% in respiratory rate. Pain is managed by scheduled administration of buprenorphine or butorphanol and ketoprofen, or other adjunctive analgesic methods as determined in consultation with a veterinarian.

[0162] All animals receive analgesia with buprenorphine (0.01 mg / kg IV q6–8 h) postoperatively. This can be supplemented with ketoprofen (1–2 mg / kg IV) and butorphanol (0.1 mg / kg IV) if additional or alternative analgesia is required. IV q6h) is used for complementation.

[0163] Further supportive and preventive measures Animals will receive daily IV antibiotics for the first week with additional medications in the post-operative period. Post-operative animals will receive maintenance IV fluids that are adjusted according to clinical and laboratory parameters.

[0164] In addition, gastric motility (e.g., ileus) is monitored throughout the postoperative period.

[0165] At the completion of the surgical procedure, a central line will be placed and this IV access will be kept clean and maintained for the duration of the study unless there are signs of infection, distress to the animal, or trauma to the dog.

[0166] Immunosuppressive (IS) therapy: Administer solumedrol (1 g IV) to the animals in the operating room prior to hepatocyte transplantation. The post-operative IS regimen is as follows: Prograf (approximately 0.3 mg / kg) Po q 12 hours for the duration of the study (monitor and adjust accordingly to prevent toxicity) Prednisone 20mg po qd x 1 week Prednisone 10mg po qd x 1 week Prednisone 5 mg po qd throughout the study

[0167] Example 4. Preclinical study of EUS-guided delivery of hepatocytes in lymph nodes for organ formation This example illustrates the treatment of liver failure in a large animal (canine) model by EUS-guided delivery of hepatocytes to the periduodenal lymph nodes (LNs).

[0168] The preclinical studies in a large animal model (dog) described herein were approved by IACUC protocols under USDA guidelines and were performed in a fee-for-service laboratory at Allegheny Health Network, Highmark, Pittsburgh, Pa. In these studies, a complete portocaval shunt (PCS) was surgically constructed, followed by hepatocyte transplantation in a dog model of induced liver failure.

[0169] Briefly, animal groups were tested to confirm that hepatocyte transplantation into the periduodenal lymph node (PDLN) could induce the development of new ectopic liver buds (organogenesis) with normal liver cytoarchitecture and full function. Study groups underwent a complete surgical PCS procedure followed by autologous or allogeneic hepatocyte transplantation into the PDLN via EUS approach or direct injection. All groups received a nonspecific immunosuppressive regimen consisting of tacrolimus and prednisone. The groups were followed up until 150 days after the initial procedure.

[0170] Animals received HT either by direct injection (as shown in Figure 2) or by a minimally invasive endoscopic ultrasound (EUS) approach (as shown in Figures 1A and 1B) into the LNs. Animals received 75 million HT (25M hepatocytes / mL, 1 mL of solution was injected into each of three LNs) or 150 million HT (50M hepatocytes / mL, 1 mL of solution was injected into each of three LNs).

[0171] Animals were sacrificed on days 6, 60, 90 or 150.

[0172] Figure 6A shows positive staining for CK-18, a marker for hepatocytes, in normal liver tissue, and Figures 6B and 6C show the presence of CK-18 immunostained hepatocytes in lymph nodes 6 days after transplantation by EUS and direct injection, respectively. These initial results demonstrate the presence of hepatocytes in lymph nodes in both the direct injection and EUS injection groups. Primary hepatocytes delivered to lymph nodes by EUS successfully formed hepatic tissue.

[0173] Figures 7A-7D show the formation of liver tissue after transplantation of hepatocytes by EUS. Lymph nodes were collected from sacrificed animals at each time point, fixed in paraformaldehyde, and embedded in paraffin. Sections were prepared and stained with hematoxylin and eosin (H&E) or hematoxylin along with anti-fumarylacetoacetate hydrolase (FAH) immunostaining. FAH is highly expressed in hepatocytes and can be used to identify hepatocytes. Sections were imaged and assessed histologically for the presence of engrafted hepatocytes and the formation of ectopic liver tissue.

[0174] Figure 7A shows the histological image of lymph nodes after transplantation of autologous hepatocytes by EUS. Approximately 25 × 10 per lymph node 6 Autologous hepatocytes were transplanted by EUS, and lymph nodes were harvested approximately 90 days after transplantation. Sections of lymph nodes were stained with hematoxylin and eosin (H&E). High magnification of H&E staining (right) shows that liver tissue (arrow) was formed from the engrafted hepatocytes.

[0175] Figure 7B shows lymph node histology after transplantation of allogeneic hepatocytes by EUS. Approximately 50 × 10 per lymph node. 6 Allogeneic hepatocytes were transplanted by EUS, and lymph nodes were harvested approximately 90 days after transplantation. Sections of lymph nodes were stained with H&E. Liver tissue (arrows) was formed from the engrafted hepatocytes in various parts of the lymph nodes.

[0176] Figure 7C shows the histological image of lymph nodes after transplantation of autologous hepatocytes by EUS. Approximately 50 × 10 per lymph node 6 Autologous hepatocytes were transplanted by EUS, and lymph nodes were harvested approximately 60 days after transplantation. Sections of lymph nodes were stained with H&E (right panel) or hematoxylin and anti-fumarylacetoacetate hydrolase (FAH) immunostaining (left panel). Liver tissue (arrows) was formed in various parts of the lymph nodes, and the liver tissue contained FAH-positive hepatocytes.

[0177] Figure 7D shows lymph node histology after transplantation of allogeneic hepatocytes by EUS. Approximately 25 × 10 per lymph node. 6 Allogeneic hepatocytes were transplanted by EUS, and lymph nodes were harvested approximately 150 days after transplantation. Sections of lymph nodes were stained with H&E (right panel) or hematoxylin and anti-fumarylacetoacetate hydrolase (FAH) immunostaining (left panel). Liver tissue (arrows) was formed in various parts of the lymph nodes, and the liver tissue contained FAH-positive liver cells.

[0178] Animals in the study group did not show signs of long-term liver failure or death. The animals showed signs of enlargement of the PDLN site where the hepatocytes were initially transplanted. At the end of the study necropsy, the animals showed the development of ectopic liver tissue in the PDLN with normal anatomical and histological features. The ectopic liver in the PDLN showed evidence of sustainable liver tissue for the duration of the study.

[0179] Surgical and experimental methods (I) Line Installation and Animal Support A PE catheter was inserted into the right jugular vein to initiate 0.9% NaCl IV support fluid infusion. Paralytic agents were administered and maintained by syringe pump or bolus therapy throughout the duration of the procedure. PE catheters were inserted into peripheral arteries. Additionally, a double-lumen long-term central venous access (superior vena cava) was placed through a right cervical incision and direct access to the right external jugular vein. After the abdominal procedure was completed, a permanent central venous access was inserted and exposed in the posterior neck. Electrocardiograms were monitored with electrodes placed on the animal's body surface. Rectal temperature was monitored continuously. Animal support followed SOP ANI-017 Guidelines for Performing Survival Surgery in USDA Regulated Species and ANI-032 Thermal Regulation of the Anesthetized Patient.

[0180] (II) Partial resection of the left liver lobe for subsequent hepatocyte isolation After the animals were stabilized under general anesthesia, they were prepped and draped in a sterile fashion. The abdominal cavity was entered through a midline incision. First, the hepatic hilum was incised and the left lateral segment (LLS) was isolated. The left portal vein (PV) and left hepatic artery (HA) were isolated and circumscribed with vascular tape. The LLS was resected by controlled parenchymal transection in conjunction with articulated endovascular staples. Approximately 20% of the total liver volume was removed by partial resection of the left lateral segment. Once the left lateral segment of the liver was removed from the surgical field, the specimen was processed in a back-table (BT) procedure under sterile conditions. This BT included flushing the left PV and left HA with cold lactated Ringer's solution (LR) to remove all blood (flushing step), followed by infusion of Belzer's solution for later liver preservation. The liver segments were packed in double plastic bags and placed in an ice cooler before being transported to the laboratory where cell isolation was performed (to obtain hepatocytes).

[0181] (III) Portocaval shunt procedure After completing the left lateral segment resection, an extended hepatic hilar incision was performed. A complete portacaval shunt was performed according to the procedure as described in Example 2.

[0182] (IV) Hepatocyte isolation for transplantation Hepatocytes were isolated according to the procedure described in Example 1. The obtained hepatocyte suspension was filtered and washed. Trypan blue exclusion test was used to confirm the viability of isolated hepatocytes. In the autologous transplant group in which animals received their own hepatocytes, the animals were kept under general anesthesia for another 5 hours while cells were isolated and prepared for subsequent injection.

[0183] (V) Hepatocyte transplantation by direct surgical injection After obtaining samples for quality control (cell viability, cell number, culture, and sensitivity), the surgeon performed the hepatocyte transplantation. Cells were injected intraparenchymally (25 × 10 cells in a small volume (1–3 ml) of injection medium) directly into the mesenteric and periduodenal lymph nodes by direct surgical approach or via endoscopic injection (described below), as determined for each assigned experimental group. 6 ~50×106 Cell yield in viable cells / ml). Adequate hemostasis was achieved completely after heterotopic hepatocyte injection into lymph nodes. After achieving meticulous hemostasis, the peritoneal cavity was perfused copiously with a solution of antibiotic and antimycotic agents (Neomycin 500 mg / L, Polymyxin 15,000 units / kg / L, Bacitracin 1000 units / kg / L and Amphotericin B 4 mg / kg / L). The abdominal wall was closed in a three-layer fashion and no drains were used. Sealing sutures were placed at the skin closure. The animals were allowed to recover adequately from the general anesthesia procedure and were further extubated in the operating room. The animals were then transferred to a properly equipped animal facility for post-operative care.

[0184] (VI) Hepatocyte transplantation via intraluminal endoscopic approach (EUS) After completion of PCS, endoscopic injections were performed in animals under general anesthesia. A certified pancreaticobiliary endoscopist with extensive experience in EUS performed these procedures. A linear ultrasound endoscope (Olympus GF-UCT 180) was used for these experiments. The endoscope was inserted through the animal's mouth and advanced into the stomach and duodenum. An endoscopic ultrasound (EUS) probe assisted and guided the location of the periduodenal lymph nodes (LNs). The LNs were reached directly via a transgastric and / or transduodenal approach. Once the LNs were properly entered by a fine needle aspiration (FNA) needle (Boston scientific, needles ranging from 19 to 25 G), the surgeon assisted the endoscopist in performing hepatocyte transplantation after successful needle insertion into the LNs via this EUS-guided procedure. Pre-isolated hepatocytes were injected into the LNs at a concentration of 25 × 10 6 or 50×10 6 The hepatocytes were maintained in a solution containing 1000 cells / mL. The hepatocytes in this solution were directly transplanted into the periduodenal LNs using fine needle aspiration (FNA) needles (Boston Scientific, needles ranging from 19 to 25 G). Once this surgical procedure was completed, the animals were allowed to recover properly from the general anesthesia procedure and further extubated in the operating room. The animals were then transferred to a properly equipped animal facility for post-operative care.

[0185] (VII) Recovery and Post-Operative Care Animals were allowed to recover from anesthesia with appropriate monitoring while natural ventilation was resumed. Animals were monitored 24 hours per day by trained preclinical facility personnel for the first 2-3 days after surgery (longer if necessary), and then at least daily for the duration of the study. Postoperative care of the animals was performed under the guidance of the study director in consultation with a veterinarian.

[0186] The animals were monitored frequently and closely. Each animal was assessed based on the following species-specific criteria for pain assessment and relief: vocalization, depression, and >50% increase in respiration (based on the average dog respiratory rate of 20 / min). In addition, heart rate was monitored. Body temperature was also monitored.

[0187] Pain Management and Monitoring Animals were initially monitored hourly for the following indicators of post-operative pain and distress using a pain scoring system including, but not limited to, overall activity level, surgical wound, appetite and attitude towards food.

[0188] Postoperative pain was treated with the indicated analgesic doses based on the following indicators: an increase of approximately 10-15% in heart rate; and an increase of approximately 40% in respiratory rate. Pain was managed by scheduled administration of buprenorphine or butorphanol and ketoprofen, or other adjunctive analgesic methods as determined in consultation with a veterinarian.

[0189] All animals received postoperative analgesia with buprenorphine (0.01 mg / kg IV q6–8 h), which was supplemented with ketoprofen (1–2 mg / kg IV) and butorphanol (0.1 mg / kg IV q6 h) if additional or alternative analgesia was required.

[0190] Further supportive and preventive measures Animals received daily IV antibiotics for the first week, with additional medications during the postoperative period. Postoperatively, animals received maintenance IV fluids adjusted according to clinical and laboratory parameters.

[0191] In addition, gastric motility (e.g., ileus) was monitored throughout the postoperative period.

[0192] At the completion of the surgical procedure, a central line was placed and this IV access was kept clean and maintained for the duration of the study unless there were signs of infection, distress to the animal, or trauma to the dog.

[0193] Immunosuppressive (IS) therapy: Animals were administered solumedrol (1 g IV) in the operating room prior to hepatocyte transplantation. The postoperative IS regimen was as follows: Prograf (approximately 0.3 mg / kg) Po q 12 hours over the course of the study (monitored and adjusted accordingly to prevent toxicity). Prednisone 20mg po qd x 1 week Prednisone 10mg po qd x 1 week Prednisone 5 mg po qd throughout the study

[0194] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure can be used in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

[0195] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the inventions of which are incorporated herein by reference in their entireties for all purposes. In certain embodiments, for example, the following are provided: (Item 1) 1. A minimally invasive method of transplanting one or more cells and growing ectopic tissue in a subject, said method comprising: (a) advancing an endoscope via an endoluminal approach into the gastrointestinal tract, respiratory tract or urinary tract of the subject; (b) inserting a needle attached to the endoscope through a visceral wall into a lymph node of the subject using a transluminal approach; and (c) delivering the one or more cells through the needle into the lymph node, thereby allowing the one or more cells to engraft in the lymph node and generate the ectopic tissue in the lymph node. A method comprising: (Item 2) 2. The method of claim 1, wherein the step of advancing the endoscope, the step of inserting the needle, or both, is performed with the aid of radiological or ultrasonic imaging. (Item 3) 3. The method of claim 2, wherein the radiation imaging comprises dynamic radiation imaging, computed tomography (CT), magnetic resonance imaging (MRI), or both. (Item 4) 4. The method according to any one of items 1 to 3, wherein the lymph node is located in the abdominal or thoracic cavity of the subject. (Item 5) 5. The method of claim 4, wherein the lymph node is in the mediastinal or retroperitoneal region of the subject. (Item 6) 1. A minimally invasive method of transplanting one or more cells and growing ectopic tissue in a subject, said method comprising: (a) inserting a needle into a lymph node in the abdominal or thoracic cavity of the subject using ultrasound or radiological imaging; and (b) delivering said one or more cells through said needle to said lymph node, thereby allowing said one or more cells to engraft in said lymph node and generate said ectopic tissue in said lymph node. A method comprising: (Item 7) 7. The method of claim 6, further comprising advancing an endoscope through the gastrointestinal, respiratory or urinary tract of the subject and inserting the needle through a visceral side wall using a transluminal approach to reach the lymph node of the subject, wherein the needle is attached to the endoscope. (Item 8) 8. The method according to any one of items 2 to 7, wherein the step of advancing the endoscope, the step of inserting the needle, or both, are performed with the aid of ultrasound imaging of the lymph node. (Item 9) 9. The method of claim 8, wherein the radiation imaging comprises dynamic radiation imaging, computed tomography (CT), magnetic resonance imaging (MRI), or both. (Item 10) 10. The method of any one of items 1, 2, 7, 8 or 9, wherein the endoscope is equipped with an ultrasound probe configured to detect the lymph nodes. (Item 11) 11. The method of any one of items 1-10, wherein the one or more cells comprise liver cells, pancreatic cells or pancreatic islets, kidney cells or kidney tissue fragments, thymus cells or thymus tissue fragments, or lung cells or lung tissue fragments. (Item 12) 12. The method of any one of items 1-11, wherein the one or more cells are autologous, allogeneic or xenogeneic to the subject. (Item 13) 12. The method of any one of items 1-11, wherein the one or more cells are syngeneic to the subject. (Item 14) 12. The method of any one of items 1 to 11, further comprising isolating the one or more cells from viable donor tissue. (Item 15) 12. The method of any one of items 1 to 11, further comprising recovering the one or more cells from cryopreservation prior to said delivering. (Item 16) 16. The method of any one of items 1-15, wherein the method comprises delivering the one or more cells to at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more lymph nodes in the peritoneal or thoracic cavity. (Item 17) 17. The method of any one of items 1 to 16, further comprising administering an immunosuppressant to the subject to reduce immune rejection of the one or more cells. (Item 18) 18. The method of any one of items 1 to 17, wherein the one or more cells comprise cells with an average diameter of about 20 μm. (Item 19) 19. The method of any one of items 1 to 18, wherein the one or more cells comprise hepatocytes and the ectopic tissue is ectopic liver tissue. (Item 20) 20. The method of item 18 or 19, wherein the one or more cells comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 million cells per delivery to a single lymph node. (Item 21) 21. The method of any one of items 18-20, wherein the needle delivers the one or more cells in a suspension having at least about 10, 20, 25, 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 million cells per mL. (Item 22) 22. The method of any one of items 18-21, wherein the needle delivers the one or more cells in a suspension having at least about 10, 20, 30, 40, or 50 million viable cells per mL. (Item 23) 23. The method of any one of items 18-22, wherein the one or more cells are delivered to the lymph node as a cell population, wherein the cell population has at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or about 100% viable cells. (Item 24) 24. The method of any one of items 23, wherein the one or more cells are delivered to the lymph node as a cell population, and when the one or more cells pass through the needle, the delivery results in a decrease in cell viability in the cell population of less than about 20%, 15%, 10%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% or 0.5%. (Item 25) 24. The method according to any one of items 18 to 23, wherein the inner diameter of the needle does not exceed about 700 μm, 600 μm, 500 μm, 450 μm, 400 μm, 300 μm, 260 μm, 250 μm or 200 μm. (Item 26) 26. The method according to any one of items 18 to 25, wherein the inner diameter of the needle does not exceed about 260 μm. (Item 27) 27. The method according to any one of items 18 to 26, wherein the outer diameter of the needle does not exceed about 1 mm, 900 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 520 μm, 510 μm, 500 μm, 480 μm, 450 μm or 400 μm. (Item 28) 28. The method according to any one of items 18 to 27, wherein the outer diameter of the needle does not exceed about 510 μm. (Item 29) 29. The method of any one of items 18-28, wherein the needle size is no more than about 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or 27 gauge. (Item 30) 30. The method of any one of items 18 to 29, wherein the needle size does not exceed about 25 gauge. (Item 31) 31. The method of any one of items 18-30, wherein the needle is no greater than about 25 gauge, the needle delivers the one or more cells in a suspension having at least about 50 million viable cells per mL, and the one or more cells comprise at least about 65% viable cells. (Item 32) 32. The method of any one of items 19 to 31, wherein the method treats a liver disease or condition in the subject. (Item 33) 33. The method according to item 32, wherein the liver disease or condition is end-stage liver disease or liver fibrosis related to alcohol consumption, hepatitis A, B, C or D infection, non-alcoholic fatty liver disease, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, biliary atresia, cystic fibrosis, Alagille syndrome, syphilis, brucellosis, parasitic infection, chemical exposure, chronic biliary tract disease, Budd-Chiari syndrome, Osler's disease or right heart failure. (Item 34) 33. The method of item 32, wherein the liver disease or condition is associated with a metabolic disorder comprising tyrosinemia, maple syrup urine disease, phenylketonuria, Crigler-Najjar syndrome, oxalosis, hyperoxaluria, hemochromatosis, alpha-1 antitrypsin deficiency, Wilson's disease, familial intrahepatic cholestasis syndrome, galactosemia, glycogen storage disease or familial amyloidotic polyneuropathy. (Item 35) 33. The method according to any one of items 19 to 32, wherein the subject has undergone a portocaval shunt surgical procedure or a transjugular intrahepatic portocaval shunt (TIPS), which reduces blood supply to the liver of the subject and induces hepatocellular dysfunction in the subject, and the portocaval shunt surgical procedure includes an end-to-side portocaval shunt, a side-to-side portocaval shunt, a mesenteric vena cava shunt with interposition of an H-type or C-type graft, or a mid or distal splenorenal shunt. (Item 36) 36. The method of claim 35, wherein the method treats end stage liver disease in the subject. (Item 37) 17. The method of any one of items 1 to 16, wherein the one or more cells comprise kidney cells or kidney tissue fragments and the ectopic tissue is ectopic kidney tissue. (Item 38) 38. The method of claim 37, wherein the method treats a renal disease or condition in the subject. (Item 39) 39. The method of claim 38, wherein the renal disease or condition is end stage renal disease. (Item 40) 17. The method of any one of items 1 to 16, wherein the one or more cells comprise pancreatic cells or pancreatic islets and the ectopic tissue is ectopic pancreatic tissue. (Item 41) 41. The method of claim 40, wherein the method treats an endocrine pancreatic disease or condition that reduces or eliminates insulin secretion in the subject. (Item 42) 42. The method of claim 41, wherein the pancreatic disease or condition is type I diabetes, type II diabetes or chronic pancreatitis, which leads to reduced insulin secretion in the subject. (Item 43) 17. The method of any one of items 1 to 16, wherein the one or more cells comprise a lung cell or a lung tissue fragment and the ectopic tissue is ectopic lung tissue. (Item 44) 44. The method of claim 43, wherein the method treats a pulmonary disease or condition in the subject. (Item 45) 45. The method of claim 44, wherein the pulmonary disease or condition is chronic obstructive pulmonary disease (COPD). (Item 46) 46. ​​The method according to item 45, wherein said COPD is caused by tobacco smoke, pollutants and fumes, alpha-1-antitrypsin, cystic fibrosis, chronic asthma, emphysema, chronic bronchitis or idiopathic pulmonary fibrosis. (Item 47) 17. The method of any one of items 1 to 16, wherein the one or more cells comprise thymocytes or thymic tissue fragments and the ectopic tissue is ectopic thymic tissue. (Item 48) 48. The method of claim 47, wherein the thymocytes or thymic tissue fragments are obtained from a donor subject, and the ectopic thymic tissue induces donor-specific tolerance in the subject to transplantation of cells from the donor subject. (Item 49) 48. The method of claim 47, wherein the disease or condition is age-related immune system dysfunction and the ectopic thymus tissue regulates immune function in the subject. (Item 50) 50. The method of any one of items 1 to 49, wherein the lymph node is in proximity to the gastrointestinal tract and the endoscope is advanced along the gastrointestinal tract. (Item 51) 51. The method of item 50, wherein the lymph nodes comprise one or more of the following lymph nodes: periduodenal lymph nodes, perigastric lymph nodes, peripancreatic lymph nodes, mesenteric lymph nodes, ileocolic lymph nodes, mesocolic lymph nodes, gastric lymph nodes, hepatosplenic lymph nodes, splenic hilar lymph nodes, paraesophageal lymph nodes, paracardia lymph nodes, para-aortic lymph nodes, retroaortic lymph nodes, extra-aortic lymph nodes, pre-aortic lymph nodes, lesser curvature lymph nodes, common hepatic lymph nodes, splenic artery lymph nodes, celiac trunk lymph nodes, iliac lymph nodes, or retroperitoneal lymph nodes. (Item 52) 51. The method of claim 50, wherein the lymph nodes include periduodenal lymph nodes. (Item 53) 50. The method of any one of items 1 to 49, wherein the lymph node is in proximity to an airway and the endoscope is advanced along the airway. (Item 54) 54. The method of claim 53, wherein the lymph nodes include one or more lymph nodes in the mediastinal region. (Item 55) 55. The method of claim 53 or 54, wherein the lymph nodes comprise one or more of the parasternal lymph nodes, intercostal lymph nodes, supradiaphragmatic lymph nodes, superior tracheobronchial lymph nodes, inferior tracheobronchial lymph nodes, bronchopulmonary lymph nodes, paratracheal lymph nodes, or intrapulmonary lymph nodes. (Item 56) 50. The method of any one of items 1 to 49, wherein the lymph node is in proximity to the urinary tract and the endoscope is advanced along the urinary tract. (Item 57) 57. The method of claim 56, wherein the lymph nodes include one or more lymph nodes in the retroperitoneal region. (Item 58) 58. The method of item 56 or 57, wherein the lymph nodes include one or more of the external iliac lymph nodes, the internal iliac lymph nodes, the lumbar vena cava lymph nodes, the lumbar aortic lymph nodes, the superficial inguinal lymph nodes, the deep inguinal lymph nodes, the perivesical intercaval lymph nodes, the perivesical obturator lymph nodes, or the perivesical presacral lymph nodes. (Item 59) 58. The method according to any one of items 1 to 57, wherein the subject is a human. (Item 60) 58. The method according to any one of items 1 to 57, wherein the subject is a non-human animal. (Item 61) 61. The method of any one of items 1-60, wherein the ectopic tissue forms within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 days after delivery of the one or more cells into the lymph node. (Item 62) 1. A method of treating liver disease in a subject in need thereof, the method comprising: (a) advancing an endoscope via an endoluminal approach into the gastrointestinal tract, respiratory tract or urinary tract of the subject; (b) inserting a needle attached to the endoscope through a visceral side wall into a lymph node of the subject using a transluminal approach and with the aid of ultrasound imaging; and (c) delivering said one or more cells through said needle to said lymph node, thereby enabling said one or more cells to engraft in the lymph node and generate said ectopic liver tissue in said lymph node. A method comprising: (Item 63) 63. The method of item 62, wherein the ectopic tissue forms within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 days after delivery of the one or more cells into the lymph node. (Item 64) 1. A system for transplanting hepatocytes and growing an ectopic liver in a subject, the system comprising an endoscope and a syringe with a needle containing a suspension containing a cell population, the suspension having about 25 million to about 100 million viable hepatocytes per mL, and the needle being at most about 25 gauge. (Item 65) Item 65. The system of item 64, wherein the endoscope and the needle are configured to be advanced together along the gastrointestinal tract, respiratory tract, or urinary tract of the subject. (Item 66) 66. The system of claim 64 or 65, wherein the syringe is configured to deliver the one or more cells through the needle. (Item 67) 67. The system of any one of items 64 to 66, wherein the endoscope is equipped with an ultrasound probe. (Item 68) 68. The system of any one of items 64 to 67, wherein the ultrasound probe is configured to detect lymph nodes in the subject's abdominal cavity. (Item 69) 69. The system of any one of items 64-68, wherein the suspension has at least about 30, 40, 45, 50, 55, 60, 70, 80, 90 or 100 million cells per mL. (Item 70) 70. The system of any one of items 64-69, wherein the cell population in the syringe has at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or about 100% viable cells. (Item 71) 70. The system of any one of items 64 to 69, wherein the cell population in the syringe has at least about 65% viable cells. (Item 72) 72. The system of any one of items 64-71, wherein the population of cells in the syringe comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 million cells. (Item 73) 73. The system of any one of items 64-72, wherein the cell population in the syringe comprises at least about 50 million viable cells per mL, and the cell population comprises at least about 65% viable cells. (Item 74) A kit for carrying out the method according to any one of items 1 to 63.

Claims

1. A composition for use in a method of treatment, the method comprising transplanting the one or more cells into a subject to grow ectopic tissue, the method comprising: (a) advancing an endoscope into a body lumen of the subject via an endoluminal approach; and (b) delivering said composition via a needle into a lymph node, thereby allowing said one or more cells to engraft in said lymph node and generate said ectopic tissue in said lymph node. A composition comprising:

2. The composition described in claim 1, wherein the lymph nodes include at least three lymph nodes in the abdominal or thoracic cavity of the subject.

3. The composition described in claim 1 or claim 2, wherein the one or more cells comprise at least 10 million cells per delivery to a single lymph node.

4. A composition described in any one of claims 1 to 3, characterized in that 50 million to 200 million cells are delivered to a single lymph node.

5. The composition described in any one of claims 1 to 4, wherein the one or more cells include hepatocytes.

6. The composition of any one of claims 1 to 5, wherein the method treats end stage liver disease in the subject.

7. The composition described in any one of claims 1 to 6, wherein the lymph nodes include periduodenal lymph nodes.

8. A system for transplanting cells into a lymph node of a subject and growing ectopic tissue, comprising an endoscope and a syringe comprising a needle and one or more cells in suspension contained within the syringe, the endoscope and the syringe configured to advance together along a body lumen of the subject.

9. The system of claim 8, wherein the suspension contains at least 30 million and at most 500 million cells per mL.

10. The system of claim 9, wherein the suspension contains at most 80 million cells per mL.

11. The system of any one of claims 8 to 10, wherein the needle is 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 or 27 gauge.

12. A system described in any one of claims 8 to 10, wherein the needle has an inner diameter of up to 700 μm.

13. (a) the one or more cells comprise at least 65% viable cells. (b) the one or more cells comprise cells having an average diameter of 20 μm or more than 20 μm; and / or (c) the one or more cells comprise hepatocytes and the ectopic tissue is ectopic liver tissue. A system according to any one of claims 8 to 12.

14. A system described in any one of claims 8 to 13, wherein the size of the needle and the cell concentration of the suspension are configured so that when the cells pass through the needle, the cell viability percentage in the cells decreases by less than 20%.

15. The system of claim 8, wherein the suspension is an aqueous buffer solution containing calcium gluconate and human serum albumin.

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