JP2014501574A - Irreversible electroporation using tissue vasculature to treat abnormal cell populations or generate tissue scaffolds - Google Patents

Abstract

  The present invention relates to the field of biotechnology as well as medical treatment of diseases and disorders. Embodiments of the present invention are for treating a tumor deeply embedded in a tissue or organ, or for generating a scaffold from existing animal tissue without deorganizing the organ and damaging the existing vasculature It relates to the transport of irreversible electroporation (IRE) through the vasculature of organs. In particular, an in vivo non-thermal irreversible electroporation (IRE) administration method for treating tumors located in vascular tissues and organs is provided. Furthermore, embodiments of the present invention can be derived from natural sources produced using in vitro IRE to remove cell debris, maximize recellularization potential, and minimize foreign immune responses. Provide scaffolding and organization. Such engineered tissue can be used in a method of treating a subject in need of tissue replacement or augmentation.

Description

  This application is a U.S. filed on June 24, 2008. S. Patent provisional application No. 61 / 075,216; U.S. filed on April 29, 2008; S. Patent Provisional Application No. 61 / 125,840; U.S. filed on April 22, 2009; S. Patent Provisional Application No. 61 / 171,564; and U.S. filed on Apr. 9, 2009. S. U.S. filed on June 24, 2009, claiming depending on the advantage of the filing date of provisional patent application No. 61 / 167,997. S. No. 12 / 491,151, a continuation-in-part application (CIP), which is a U.S. patent application filed on April 29, 2008. S. U.S. filed on April 29, 2009, claiming depending on the advantage of filing date of provisional patent application No. 61 / 125,840. S. No. 12 / 432,295, a CIP application; this application is a U.S. patent filed on November 23, 2010; S. Claims depend on the advantage of the filing date of provisional application No. 61 / 416,534. The entire description of all such applications has been incorporated herein by reference.

  The present invention relates to the field of biotechnology as well as the medical field of diseases and disorders. Embodiments of the present invention can be used to treat a tumor that is deeply embedded in a tissue or organ or to scaffold from an existing animal tissue, such as human tissue, without deorganizing the organ and damaging the existing vasculature. For the transport of irreversible electroporation (IRE) through the vasculature of the organ to produce irreversible electroporation (IRE). Furthermore, embodiments of the present invention can be used to treat malignant and benign diseases through enhanced administration of therapeutic agents or gene constructs by facilitating reversible electroporation. Vascular electrical conduits can also be used to perform other therapies that rely on the transport of electrical energy to a target area of body or organ tissue without damaging the existing vasculature.

  Unnecessary soft tissue removal may include surgical removal, application of excessive amounts of ionizing radiation or other forms of energy (excessive heating and cooling), exposure to cytotoxic chemicals, or such This can be done by a number of means including combinations of these means. Such means are commonly used to destroy neoplasms. Known treatments in the art include surgical procedures for physical removal of abnormal cell populations, radiation to kill cells of abnormal cell populations, exposure to toxic chemicals (ie, chemotherapy), or Including a combination of such techniques. Each treatment modality is quite effective for a variety of cell proliferative diseases, but none of the techniques is very effective in treating all types of cell proliferative diseases and disorders.

  Surgical procedures are physically approachable and are effective in the removal of solid tumors on tissues and organs that allow sustained physical damage or can be regenerated, It is difficult to perform on tumors that are not easy to approach or that cannot be regenerated. In such cases, surgical procedures often require long recovery times and subsequent treatments and can be associated with considerable physical damage to the patient. At other times, the removal of the neoplasia that has grown on a large scale is prevented because the removal may also cause the patient to die. Similarly, radiation therapy can cause collateral damage to the tissue surrounding the tumor and can cause long lasting side effects that reduce the quality of life of the patient. Chemotherapy treatment can cause systemic damage to the patient, can cause substantial side effects that require a long recovery period, and can cause permanent damage to tissues and organs.

  Recent work by the inventors has focused on the removal of unwanted soft tissue (malignant tumors) by applying excessive amounts of electrical energy, using a technique termed irreversible electroporation (IRE). Successful regulation and / or removal of soft tissue sarcomas and malignant gliomas has been achieved. Irreversible electroporation (IRE) involves positioning an electrode in or near a target region to transport a series of low energy microsecond electrical pulses. Such pulses can permanently destabilize the cell membrane of the target tissue (eg, tumor) and kill the cells. When applied correctly, IRE does not cause significant damage to surrounding tissue, does not damage major blood vessels, and kills neoplastic cells non-thermally in an adjustable manner without requiring the use of drugs. .

  Other methods for treating a disease include altering a diseased tissue or organ. Over the last 20 years, organ transplantation has become the standard care for patients diagnosed with end-stage diseases such as cirrhosis, renal failure and the like. The surprising success of liver transplantation, showing survival rates of 90% and 75% after 1 year and 5 years, respectively, continued to increase the number of patients waiting for transplantation. Chan, S.M. C. et al, A decade of right river advert-to-adult living donor liver translation-The recipient mid-term outcomes. Anals of Surgery 248, 41 1-418, doi: 10.1097 / SLA. 0b013e318181854e6 (2008) ("Chan 2008").

  According to the United Network of Organ Sharing (UNOS), 108,000 people are currently awaiting organ transplantation in the United States alone, including kidney, liver, heart, lung, etc. There are more candidates. Of those candidates, more than 16,000 candidates who need immediate liver transplant (1 year) and at least 100,000 more with advanced liver disease that can benefit from organ transplant There are patients. In 2009, there were less than 7,000 liver transplants from live or dead donors. United Network of Organ Sharing, <http: // www. unos. org> (2010).

  Standard liver transplantation in the United States is usually based on organs removed from a donor determined to be brain death (brain stem death). Kootstra, G.M. Daemen, J .; H. C. & Oomen, A .; P. A. Categories of Non-Heart-Beating Donors, Transplant. Proc. 27, 2893-2894 (1995); Rigotti, P .; et al. Non-Heart-beating Donors-An Alternative Organ Source in Kidney Transplantation, Transplant. Proc. 23, 2579-2580 (1991); and Balupuri, S .; et al. The trouble with kids derived from the non-heart-beating donor: A single center 10-year experience. See Transplantation 69, 842-846 (2000).

  Despite improvements in transplant surgery and general medicine, the number of patients waiting for organ transplants continues to increase, but organ supply is not. The increase in the difference between organ supply and clinical demand is due to an increase in population age, an increase in diseases that require liver transplantation (infection with liver cancer and hepatitis virus), and a rapid decrease in organs after provision ( Several hours), due to various factors including mismatches caused by histocompatibility and other immune phenomena, size mismatches between organs and potential recipients (including pediatric patients), Transplantation is performed in end-stage liver disease (Murray, K.F. & Cariters, RL AASLD practice guidelines: Evaluation of the patient for liver transplantation. Hepatology 41, 1407-143. )); Non-alcoholic fatty liver disease (Amaraparkar, DN et al. How common is non-alcoholic fatty live disease in the Asia-Pacific region and area theol. En. 78. J. 78. J. 1111 / j.l440-1746.2007.05042.x (2007)); and the suitability of transplantation for patients with metabolic or congenital diseases (Miro, JM, Laguno, M., Moreno) , A., Rimola, A. & Hosp Clin, OLTHVIVG Management of end stag live disease (ESLD): What is the current role of orthotopic live translation (OLT)? J. Hepatol.44, S140-S145, doi: 10.016 / j.jhep.2005. This is a treatment that can only be performed on patients.

  Organ supply is delayed due to delays in acquisition. For example, the requirement for removal of organs consistent with brain death (brain stem death) requires the use of a hospital with means to maintain it with an artificial life support device. As a result, problems with organ donation can occur when intensive care is overused. Fabre. Report of the British Transplantation Society Working Party on Organ Donation. (1995). The use of life support devices for the protection of potential organ donations is ethically controversial (Feest, TG et al., Protocol for Incrementing Organic Donation after Cerebrovascular Diets in a Distr. General Hosp. 1133-1135 (1990); and Riad, H. & Nichols, A., An Ethical Debate Electrical Ventilation of Potential Organ Donors, Br. Med. J. 310, 714-715 (1995)), social, cultural. And refusal to provide in areas where religious pressure limits organ procurement is common.

  The increasing difference between organ donation and patient supply has increased interest in alternative organ sources. Perera, M .; , Mirza, D .; F. & Elias, E .; Liver transplantation: Issues for the next 20 years. J. et al. Gastroenterol. Hepatol. 24, S124-S131, doi: 10.1 1 1 1 / j. 1440-1746.2009.06081. x (2009). The development of artificial materials to replicate organ structure and function has had limited success. Large volumes of poorly organized cells and tissues cannot be transplanted due to initially limited diffusion of oxygen, nutrients and waste products. Folkman, J .; Self-Regulation of Growth In 3 Dimensions. Journal of Experimental Medicine 338 (1973); and Kaufman, D. et al. S. Hanson, E .; T.A. Lewis, R .; L. Auerbach, R .; & Thomson, J.M. A. Hematopoietic colony-forming cells, derived from human emblemic stem cells. Proc. Natl. Acad. Sci. U. S. A. 98, 10716-10721 (2001).

  Nevertheless, researchers have made some progress towards partial organ regeneration. For example, mouse kidney cells grown in a decellularized collagen matrix and transplanted into an athymic mouse developed into a nephron-like structure after 8 weeks. Atala, A .; Engineering organs. Curr. Opin. Biotechnol. 20, 575-592, doi: 10.016 / j. copbio. 2009.10.003 (2009) ("Atala 2009"). In addition, a 5 mm thick porous polyvinyl-alcohol (PVA) structure in which hepatocytes were injected after implantation into mice developed into a liver-like form over the course of one year. Kaufmann, P.A. M.M. et al. Long-term hepatocyte transplantation using three-dimensional matrixes. Transplant. Proc. 31, 1928-1929 (1999) ("Kaufmann 1999"). Cell survival and proliferation in each such structure is limited to a few millimeters from the nutrient source.

  For the development and differentiation of complete organs suitable for human transplantation, structures that provide microvascular structures are required to transport nutrients everywhere in the tissue, with a physical semi-rigid matrix for cellular organization and Fixing tools must be developed. Atala 2009; Kaufmann 1999; and Atala, A .; Experimental and clinical experience with tissue engineering techniques for mutual restructuring. See Urological Clinics of North America 29,485- + (2002). Traditional down-facing manufacturing techniques currently range from several centimeters in size (eg, vena cava) to as small as a few μm (most capillaries) in human organs. It is not possible to produce hierarchical vascular structures in the range. Although microfabrication technology can replicate some features of the complex structure of mammalian microvasculature, current processes have failed to scale to microscale. Structures characterized over a range of lengths are currently produced only through biological mechanisms, and relatively new fields of biological manufacturing are intended to use and regulate such processes. Has been developed as.

  Bioengineered tissue has been manufactured through many schemes, including direct printing and biospraying, in which cells and support matrix are simultaneously deposited to form a composite network. Mironov, V.M. et al. Organ printing: Tissue spheroids as building blocks. Biomaterials 30, 2164-2174, doi: 10.016 / j. biomaterials. 2008. 12.084 (2009); Nakamura, M .; et al. Biocompatible ink jet printing technique for designed seeding of individual living cells. Tissue Engineering 11, 1658-1666 (2005); and Campbell, P. et al. G. & Weiss, L. E. Tissue engineering with the aid of inkjet printers. Expert Opin. Biol. Ther. 7, 1123-1127, doi: 10.15517 / 147125988.7.1123 (2007). Centrifugal force was used to produce a crosslinked hydrogel with a cellular network of densely packed tubular structures. Kasyanov, V.M. A. et al. Rapid biofabrication of tubular tissue constructs by centrifugal casting in a decoupled natural scaffold with laser-machined micropores. J. et al. Mater. Sci. -Mater. Med. 20, 329-337, doi: 10.1007 / sl0856-008-3590-3 (2009). Dielectrophoretic (Albrecht, D.R., Sah, RL & Bhatia, SN Geometric and material detergents of patterning efficiency by dielectrophoretics. 1529 / biophysj.104.039511 (2004)) and magnetic field (Mironov, V., Kasyanov, V. & Markwald, R. R. Nanotechnology in vascular tissue biology: ation.Trends Biotechnol.26,338-344, doi: 10.016 / j.tibtech.2008.3.0001 (2008)) is used to guide the placement of cells within the synthetic matrix and within the bio-polymer Built-in cells were electrospun into the tissue structure (Stankus, JJ, Guan, JJ, Fujimoto, K. & Wagner, WR Microintegrating smooth cells into a biodegradable biomolecule. fiber matrix.Biomaterials 27,735-744, doi: 10.016 / j.biomaterials.2005.6.020 (2006)). Such techniques attempt to distribute the cells within a suitable synthetic-manufactured network, deviating from the reconstitution of the embedded cells into an optimal structure.

  The decellularization of existing tissue extends the concept of biological manufacturing that takes advantage of the body's natural programming and produces a complete organization that includes a functional vascular network. Rat hepatocyte extracellular matrix structures were generated using chemical decellularization and revaccination. Baptista, P.M. M.M. et al. Generation of a Three-Dimensional Liver Bioscaffold with an Intact Vascular Network for Whole Organ Engineering (Wake Forest Institue for Regnerative Medicine Harvard-MIT Division of Health, Science, and Technology Rice University, 2009). Decellularized rat hearts re-inoculated with multiple cell types are contractible and capable of generating pumping pressure. Ott, H.C. C. et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nature Medicine 14, 213-221, doi: 10.1038 / nml684 (2008). Challenges to chemical decellularization techniques are the potential for detergents that damage ECM components, the potential to generate and deposit toxicity, and the scale-up of such techniques from small rat organs to large organs. Including inherent difficulties. Such challenges must be overcome before decellularized organs can be successfully transplanted in a clinical setting.

  Xenotransplantation or animal organ transplantation is one potential solution to future organ shortages. Keeffe, E .; B. Liver transplantation: Current status and novel approaches to live replacement. Gastroenterology 120, 749-762, doi: 10.1053 / gast. 2001.22583 (2001). Porcine xenografts show considerable potential but have not been widely approved and used. Pancreatic islet transplantation has recently been shown to reverse reverse diabetes mellitus (Cardona, K. et al. Long-term urine symbiotic populations in the United States of America). , 304-306, doi: 10.1038 / nml375 (2006); and Hering, B. J. et al. Prolonged diabetics reverse after intratransplantation of wild-type porci e islets in immunosuppressed nonhuman primates. Nature Medicine 12, 301-303, doi: 10.1038 / nml369 (2006)), the use of a T-cell resistance protocol is a potential for long-term kidney transplantation in non-human primates Proved. Yamada, K .; et al. Marked promotion of porcine renal xenograph survival in baboons through the use of alpha 1, 3-galactosyltransferase pair gene-knockout. Nature Medicine 11, 32-34, doi: 10.1038 / nml72 (2005).

  Furthermore, it has been demonstrated that pig liver is capable of restoring clotting while under short-term perfusion of ammonia-depleted human plasma. Chari, R.A. S. et al. Brief Report-Treatment of Hepatic Failure with ex-vivo Pig River Perfusion Followed by Liver Transplantation, N.M. Engl. J. et al. Med. 331, 234-237 (1994); and Makowka, L .; et al, The Use of a Pig Live Zenograf for Temporary Support of a Patient with Fulminant Hepatic Failure, Transplantation 59, 1654-1659 (95). Unfortunately, the mechanism of graft loss and rejection in such transplants is still not well known, and immune rejection remains the greatest obstacle to successful transplants. Yang, Y. et al. G. & Sykes, M .; Xenotransplantation: current status and a perspective on the future. Nat. Rev. Immunol. 7,519-531, doi: 10.1038 / nri2099 (2007).

  Artificial tissue is the most promising for treating some of today's most devastating diseases. Since artificial tissue and organ replacements can be developed in the laboratory, they can potentially be transported on a large scale at multiple disease stages, and this therapy significantly reduces patient waiting time. Artificial tissue concept using selective cell transplantation is the success of artificial bladder tissue for bladder reconstitution, injectable artificial chondrocytes and vascular grafts for the treatment of vesicoureteral reflex and urinary incontinence It has been experimentally and clinically applied to a variety of disorders, including general use. For human clinical use, the process includes in vitro inoculation and deposition of human cells on the scaffold. Once inoculated, the cells proliferate and migrate into the scaffold and are differentiated with the appropriate cell type for the particular tissue involved, but secreting extracellular matrix components forms the tissue You need to do. The three-dimensional structure of the scaffold, and in particular the pore size and density of the scaffold, is important in the successful growth and migration of cells seeded to produce important tissue. Therefore, the choice of scaffold is determined so that the cells can be performed in the manner required to produce the desired morphology and size of tissues and organs.

  To date, artificial tissue scaffolding has generally consisted of natural and synthetic polymers. Methods known in the art for forming artificial tissue scaffolds from polymers include solvent-casting, particle-leaching, gas forming of polymers, etc., phase separation, and solution casting. Electrospinning is another popular method for generating scaffolds for artificial tissues and organs, but widely used techniques have so far created fundamental manufacturing constraints that prevent clinical transplantation. have. Such constraints are caused by a certain lack of processes that can produce nano-, micro-, and millimeter-sized electrospun structures that sufficiently promote cell growth and function.

  Of fundamental importance to most artificial tissue scaffolds is the exchange of gases and nutrients. Essentially, this is accomplished by a microcirculation that provides oxygen and nutrients to the tissue and removes waste products at the capillary level. However, gas exchange in most artificial scaffolds is generally done passively by dispersion (generally over distances of less than 1 mm) or actively by elution of oxygen from certain types of material fibers. Microcirculation is difficult to manipulate, especially because the capillary cross-sectional dimensions are only 5-10 micrometers (μm) in diameter. Still, a manufacturing process for manipulating the tissue scaffold has not been developed and a network of blood vessels cannot be generated. Currently, artificial tissue scaffolds with circulation designed in a structure for gas exchange are not known. As a result, the scaffold for tissues and organs is limited in size and form.

  In addition to gas exchange, the scaffold of artificial tissue must exhibit mechanical properties similar to the original tissue to be replaced. This is true because the cells present in the native tissue sense a physiological temperament that can act to regulate tissue growth and function. Most natural hard and soft tissues are elastic or viscoelastic and can reversibly recover the temperament they depend on under normal working conditions. Therefore, it is particularly suitable for supporting structures such as bone, cartilage or blood vessels at the time of transplanting an artificial tissue structure having similar mechanical properties as a mature extracellular matrix of the original tissue into a host. .

  There are a number of physical, chemical, and enzymatic methods known in the art for producing scaffolds from natural tissue. The most common physical methods for producing the scaffold are snap freezing, mechanical force (eg, direct compression), and mechanical agitation (eg, sonication). The most common chemical methods for producing scaffolds are alkali or base treatment, use of nonionic, ionic, or zwitterionic detergents, hypotonic or hypertonic solutions And the use of chelating agents. Common enzymatic methods for producing scaffolds include the use of trypsin, endonuclease, or exonuclease. Currently from two or more of the above mentioned methods and especially other general classifications (ie physical, chemical, enzymatic) in order to fully decellularize the tissue to produce the scaffold It has been recognized in the art that more than one scheme must be used. Unfortunately, the method used is relatively powerful on tissue and may completely remove cytoplasmic components. Such powerful processing irreversibly degrades the resulting scaffold to destroy vasculature and nerve structures.

  Animal Preliminary-Most successful scaffolds used in both clinical experiments and human clinical applications are biological (natural) and made by decellularizing large animal (eg, pig) organs . In general, removal of cells from a tissue or organ for the production of the scaffold must leave a complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM). The tissue from which the ECM is obtained, the species of origin, the decellularization method and the terminal sterilization method for such biological scaffolds are very diverse. However, as mentioned in this application, the decellularization method is relatively powerful and causes substantial destruction or degradation of the extracellular scaffold. Once the scaffold is manufactured, it can be seeded with human cells to grow, migrate, and differentiate into specific tissues. The goal of most decellularization processes is to minimize the disruption that occurs in the scaffold and maintain the original mechanical and biological properties of the tissue. However, until now, such a goal has not been achieved. Snap cooling has often been used to decellularize tendon tissue, ligament tissue and nerve tissue. Due to the rapid cooling of the tissue, the ice crystals inside the cells destroyed the cell membrane and caused cell lysis. In order to prevent ice formation from ECM destruction, the rate of temperature change must be carefully adjusted. Although cooling is an effective method of cell lysis, subsequent steps to remove cytoplasmic components from the tissue must be followed.

  Although cells can be eluted by applying pressure directly to the tissue, this method is only effective for tissues or organs characterized by closely organized ECM (eg, liver, lung). Mechanical forces were also used to delaminate tissue layers from organs characterized by natural planes of detections such as the small intestine and bladder. Such a method is effective and results in minimal collapse for the three-dimensional structure of the ECM within such tissue. In addition, mechanical agitation and sonication can be used simultaneously with chemical treatment to assist in cell elution and removal of cell debris. Mechanical agitation can be applied using a magnetic agitation plate, an orbital shaker, or a low profile roller. Although there were no studies to determine the optimal magnitude or frequency of ultrasound for cell disruption, a standard ultrasound cleaner appeared to be effective. As mentioned above, the physical treatments currently used are generally insufficient to achieve complete decellularization and must be accompanied by secondary treatments, usually chemical treatments. Enzymatic treatments such as trypsin and chemical treatments such as ionic solutions and detergents disrupt the bonds that allow intracellular and extracellular linkages. They are therefore often used as a secondary step in decellularization after general disruption by mechanical means.

  Recently, improvements have been made in the IRE field and the concept of tumor treatment in IRE has been established, but the inventors have developed a book for improved apparatus and methods for removing diseased tissue using IRE. Recognized that field demand still exists. More specifically, we select cells from soft tissue using a tissue vascular bed as a physiological electrode ("Physiological Vascular Electrode" or "PVE"). Removed. The in vitro and in vivo application of this technique for the removal of unwanted cells is not only active and useful for the treatment of permeable and localized neoplasms, but also in tissue engineering, tissue regeneration, and biological The present invention can also be widely applied to organ transplantation using a tissue structure derived from a science.

  The present invention provides a novel application of irreversible and reversible electroporation through the use of existing vasculature in tissue as a conduit for pulse transport. Such a methodology reduces the energy required to induce non-thermal ablation of cells and tissues and dramatically improves the preservation of extracellular matrix for the development of vascularized tissue structures, only a few microns Cause separation of the "electrodes". It also has clinical implications in the treatment of solid tumors in vivo and other in vivo applications, including utilizing the reversible area of electroporation, where the electric field does not kill cells The permeability of the cells is temporarily increased to allow intracellular transport of exogenous substances.

  The use of IRE to decellularize tissue provides a controlled and accurate method for destroying cells of tissues or organs, including angiogenesis of the original tissue and other overall morphological characteristics It was assumed that there was no damage to the underlying extracellular matrix (ECM). Such a decellularized scaffold is suitable for inoculating cells of a suitable organism.

  When the step is performed extracellularly, the inoculated tissue is suitable for being transplanted into a living body as a replacement tissue. In addition to methods for producing scaffolds, the invention also provides methods for producing not only decellularized scaffolds themselves, but also artificial tissues and organs made from such scaffolds. Furthermore, the present invention provides the artificial scaffold applications and the artificial tissues and organs made from such scaffolds.

  More specifically, IRE leaves the fine vasculature intact, and optionally active perfusion of the organ, optionally in combination with IRE administration using the organ vasculature as a path for the electric field. Can be used to centrally remove tissue. Such an innovative methodology that uses the vasculature as a route for IRE pulse transport is suitable for treating tumors deeply embedded in tissue (eg, treatment from inside out) or complete It is a promising solution for the generation of a complex organ scaffold.

  Plate and needle electrodes can be used to transport IRE to treat tissues and organs. However, in some cases, plate electrodes may not be a preferred solution for removing tumors or treating deep organs within an organ. For example, plate electrodes are typically located on the external surface of the organ and can cause unwanted damage to healthy tissue located between the tumor and the external surface of the organ. A needle electrode is a viable method because a needle is inserted into the organ to treat a further internal portion of the organ. The needle electrode that appears to be most effective in transport therapy must puncture the organ. Furthermore, the low heat dose requirement limits the maximum processing area for each setting of treatment and should generally require a large number of needle holes to treat the entire organ. Moreover, such holes provide an alternative path for fluid and may not allow perfusion of downstream organ sections. Plate electrodes can be used to alleviate some of these problems, but can introduce their own constraints. Large plate electrodes can be used to treat the corresponding part of the liver in one treatment. However, the voltage relative to the distance ratio of the treatment to be applied varies considerably over the area to be treated because of the variation in tissue depth. Protocols associated with such transport systems are generally at risk of causing inadequate treatment of the organ.

  As an alternative to plates and needle electrodes, the inventors have used electrosurgery through the vasculature of organs that are mechanically perfused to induce non-thermal cell death in bulk tissue (eg, liver tissue). Apparatus, systems, and methods for delivering pulses are provided.

  A representative device for administering IRE directly into the vasculature of an organ is shown in FIG. 1, which is a schematic diagram of an embodiment of an IRE-mechanical perfusion coupling device according to the present invention. The connection to the vasculature and the perfusion system is made through the use of Luer lock connection tubes. A one-way valve inside the device can be used to prevent backflow and to electrically isolate the organ between mechanically imitating heart beats. Thus, such electroporation systems are internally connected to blood vessels and conduction structures (eg, hepatic veins, hepatic arteries, hepatic portal vein, and / or hepatic bile ducts) and do not delay sustained mechanical perfusion. Can be. In such a preferred center removal system, the protocol is capable of inducing 95% cell death while preserving the extracellular basement membrane of lobule units.

  Application of electrical energy through the vascular bed of in vitro and in vivo tissue can be used to remove unwanted cells as a means for treatment of disease and generation of useful biological tissue structures. In addition, such techniques can be used to transport electrical or thermal energy throughout the organ (or a specific area) and allow other phenomena. Other treatments that can also be applied through the vasculature include electro-chemotherapy, and electro-gene-therapy, radio frequency ablation, RF-induced thermal and pulsed nanosecond electric fields. Reversible electroporation. Indeed, all therapies that require a conductive pathway to transport energy can be tailored by applying the inventive principles described in this document.

  Embodiments of the present invention apply different amounts of energy (both reversible and irreversible electroporation) (eg, using organ vasculature as a conductive pathway) and are selective for general and neoplastic cells. Methods are provided that can be adjusted to have an effect on the removal of a wide range of desired tissue, including removal.

  Applying various amounts of energy using the vasculature of the organ as a conducting pathway is another treatment modality (eg, electrochemotherapy, ECT, electrogenetherapy, EGT). , Nanosecond pulsed electric fields (nsPEF), high-frequency irreversible electroporation (H-FIRE), high-frequency ablation (radio-frequency therapy) Can be used to achieve the desired therapeutic effect.

  According to the present invention, the effect of energy application (eg using the vasculature of the organ as a conductive pathway) diversifies / diversifies energy pulse form, voltage, amperage, duration, repetition rate and frequency. Alternatively, it can be controlled and adjusted by the design and positioning of the energy system.

  Another aspect of the present invention is to regulate and control the effects of energy application (eg, using the vasculature of an organ as a conductive pathway) through the design and use of an elaborate and precisely regulated vascular perfusion system. including.

  A method for adjusting the effect of energy application through, for example, a pulsed electric field according to the present invention can include diversifying the physiologically mimicking characteristics of a sophisticated and precisely regulated vascular perfusion system.

  Moreover, for example, the effect of energy application through a pulsed electric field can be adjusted by diversifying perfusion conditions and preservation conditions of in vitro organs and tissues.

  For example, energy application using IRE through a pulsed electric field and the resulting effects can be adjusted by diversifying the perfusion and storage conditions of in vivo organs and tissues according to embodiments of the present invention.

  The present invention also modifies the composition of the intravascular fluid that functions in part or in whole as an extension of the energy application system and protocol, for example, energy application using the vasculature of the organ as a conductive pathway. Including a method of adjusting the effect of.

  More specifically, aspects of embodiments of the present invention include the following aspects or combinations thereof. In a first aspect, comprising a method of removing cells comprising: positioning one or more electrodes in, on or near an organ or tissue; mechanically perfusing said tissue or organ with a perfusate; and An electric field is administered into the tissue or organ with sufficient power for a time sufficient to cause electroporation of the target cell. In a second aspect, such a method can include: inserting the electrode into the vasculature of the organ or tissue; mechanically perfusing the tissue or organ with a perfusate through the vasculature And an electric field is administered into the vasculature with sufficient power for a time sufficient to cause electroporation of the target cells.

  In the third aspect, such a method includes inserting a first electrode into an artery of the organ, inserting a second electrode into a vein of the organ, and tissue between the electrodes through the vasculature of the organ. Alternatively, electroporation of the cells can be included. Additionally or alternatively, other physiological pathways such as ureters or common bile ducts can be used for energy applications. Such methods can include non-thermal irreversible electroporation of target cells.

  The fifth aspect includes any one of the first to fourth aspects, wherein cells from the tissue or organ by mechanical perfusion, physiological perfusion, or physical, chemical, or enzymatic techniques, or some combination thereof It further includes removing debris.

  In a sixth aspect, a tissue scaffold formed by removing the target cells from a natural tissue source by administering irreversible electroporation to the target cells during perfusion of the tissue according to an embodiment of the present invention. Provided. In the seventh aspect, such a scaffold of the sixth aspect can include electroporation administered through the vasculature of the tissue. In the eighth aspect, the scaffold of the sixth or seventh aspect can include the extracellular matrix and vascular structures of natural tissue sources. In a ninth aspect, the scaffold of any of the sixth to eighth aspects can provide the tissue scaffold having a thickness of at least one size of about 1-8 cm, or 5-10 cm. .

  In a tenth aspect, under conditions that allow irreversible electroporation to be administered to target cells during tissue perfusion to obtain a tissue scaffold and allow living cells to grow on or in the scaffold, It includes an artificial tissue formed by removing the target cells from a natural tissue source by re-inoculating the scaffold with the living cells.

  In an eleventh aspect, the artificial tissue of the tenth aspect can include electroporation administered within the vasculature of the natural tissue. In the twelfth aspect, the scaffold of artificial tissue of the tenth aspect or the eleventh aspect can include a tissue scaffold from an animal (any animal including a human) and a living cell derived from a human. A thirteenth aspect includes the artificial scaffold of any of the tenth to twelfth aspects, wherein the tissue scaffold can include an extracellular matrix and a vascular structure of a natural tissue source. A fourteenth side includes the artificial tissue scaffold of any of the tenth to thirteenth side, wherein the artificial tissue scaffold has a thickness of at least one size of about 1-10 cm. Have. The fifteenth aspect can include the scaffold of artificial tissue of any of the tenth to fourteenth aspects, which includes the heart, lung, liver, kidney, pancreas, spleen, gastrointestinal tract, bladder, prostate, It is the ovary, brain, ears, eyes or skin or parts thereof, or all other organs or parts thereof.

  In a sixteenth aspect of the present invention, comprising a device for removing target cells of a tissue or organ comprising: a perfusion system suitable for transporting perfusate from an artery of said tissue or organ vasculature to a vein; A first electrode and a second electrode suitable for insertion into the artery and vein of tissue or organ vasculature, respectively; and sufficient to perform electroporation of a target cell located between the first and second electrodes A voltage source that activates communication with the electrode to apply a voltage between the first and second electrodes during perfusion with sufficient voltage for a long period of time. In the seventeenth aspect of the present invention, the apparatus of the sixteenth aspect can include electroporation that is non-thermal irreversible electroporation. In an eighteenth aspect of the present invention, the device according to the sixteenth aspect or the seventeenth aspect is electrically coupled with an adjuvant macromolecule in a perfusate (either in vivo in a patient's blood or extracellular in a synthetic perfusate). Including perforations, secondary effects of the electroporated cells can be promoted.

  In the nineteenth aspect, the electrode can be inserted into living tissue as described in the third aspect, wherein the electrical energy parameter applied through the target region is a mild of the tissue. It is set to intentionally induce an increase in temperature that induces transport, such as thermal or thermal destruction (radio frequency ablation).

  In a twentieth aspect of the invention, an aspect of the invention is administered to induce an electric field through the vasculature with sufficient power for a time sufficient for an electrical pulse to cause electroporation in a target cell; It can be adapted to be performed on live animals or organs under mechanical perfusion to allow the introduction of therapeutic gene constructs to treat diseases that occur within the target area. Such therapy would be helpful, for example, by administering gene therapy for islets of pancreas in type I diabetes to spur insulin production (disease tissue, non-cancerous) ).

  In a twenty-first aspect of the invention, an aspect of the invention provides for electroporation of a target cell to allow introduction of a genetic construct to replace the target cell to treat systemic disease. Can be tuned to be performed on a live animal or organ under mechanical perfusion so that for a sufficient amount of time, an electrical pulse is administered with sufficient power to induce an electric field through the vasculature . Such therapy can be applied in the context of immune regulation in which genes are introduced into tissues such as muscles to produce desired antibodies (healthy tissues used as blood vessels in therapy).

  In a twenty-second aspect of the invention, an aspect of the invention provides for the introduction of a macromolecule to allow the introduction of a macromolecule to treat a disease either within the target region or with a systemic disease. Performed in a live animal or organ under mechanical perfusion so that an electrical pulse is administered to induce an electric field through the vasculature with sufficient power for a time sufficient to cause electroporation Can be adjusted as follows. Such therapies are useful in increasing drug influx to treat a range of diseases, not only killing cancer cells alone, but all others that can treat non-cancerous diseases. Other drugs are also possible. Such an aspect is similar to the twentieth aspect and the twenty-first aspect, but uses a drug instead of a gene.

  In a twenty-third aspect of the invention, an aspect of the invention provides for an animal or a living animal under mechanical perfusion such that administration of a cross electric field through the vasculature results in thermal ablation of the target area or the entire organ. Execution can be coordinated with organs.

  In the twenty-fourth aspect, any one of the methods of the first aspect to the twenty-third aspect includes electrochemotherapy (ECT), electrogene therapy (EGT), nanosecond pulsed electric fields (nsPEF), A therapy selected from high-frequency irreversible electroporation (H-FIRE), radio-frequency ablation, or acute hyperthermia.

  A twenty-fifth aspect comprises the method of the other aspects referred to herein, comprising or further comprising administering energy into the vasculature for a sufficient amount of time with sufficient power to cause target cell death. Including.

  In a twenty-sixth aspect, a first electrode is inserted into a blood vessel of the organ, a second electrode is inserted into another blood vessel of the organ, and electroporation is administered between the electrodes through the vasculature of the organ. Other aspects of the method referred to in this application.

  Similarly, the twenty-seventh aspect is that a first electrode is inserted into a blood vessel of the organ, a second electrode is inserted into a fluid conduit of the organ, and electroporation is administered between the electrodes through the vasculature of the organ. And other aspects of the method referred to in this application.

  According to a twenty-eighth aspect of the present invention, the first electrode is inserted into a blood vessel of the organ, the second electrode is inserted into another blood vessel of the organ, and the treatment region of the organ is the first and second blood vessels. The method according to other aspects mentioned herein, wherein the electroporation is located between and between the electrodes through the vasculature of the organ.

  In the twenty-ninth aspect, a first electrode is inserted into a blood vessel of the organ, a second external ground pad, a plate electrode, or a conductive gel outside the organ is used as the second electrode, and electroporation is performed on the vessel of the organ. Including the methods of other aspects mentioned herein, administered between the electrodes through a structure.

  Aspects of the invention can include electroporation to induce non-thermal irreversible electroporation of target cells. Certain aspects of the invention can be tailored to include removing cellular debris from the tissue or organ by mechanical perfusion, physiological perfusion, or physical, chemical, or enzymatic techniques, or some combination thereof. . Also, certain aspects of the invention can be tailored to further include observing through electrical measurements to sense or observe electroporation.

  The accompanying drawings illustrate some obvious aspects of embodiments of the invention and should not be used to limit or define the invention. Together with the written description, the drawings are provided to illustrate the clear principles of the invention.

  FIG. 1 is a schematic view of an embodiment of an IRE-mechanical perfusion coupling device according to the present invention.

  FIG. 2 is a graph showing potential and electric field contours represented by numerical expressions of a conventional experiment.

  3A and 3B are graphs showing the numerical results of two different simulations with leaflet level detail (model 2): (FIG. 3A) of external electrode set at 1500V (top) or ground (bottom) One was charged (FIG. 3B), the central venule of each leaflet was charged at 1500 V, and the external electrode was grounded.

  FIG. 4A shows a simulated electric field distribution with a single leaflet when a 50V 100 microsecond pulse is administered to the bile duct and the central venule and portal arteriole are grounded.

  4B is a schematic representation of a hepatic lobule showing a complex vascular network of functional units of blood vessels (see Slieman, TA The Digestive System, http://tonyslieman.com/phyty_230_lecture_digestive.html). .

  FIGS. 5A-5B show (FIG. 5A) the position of the electrode on actively perfused liver tissue and (FIG. 5B) after treatment with 99, 100 μs, 1500 V / cm pulse and 4 hours of perfusion, resulting in It is the photograph which showed the produced | generated lesion, The area | region of the approximate value of the said electrode is a black outline.

  6A-6C are schematic diagrams of the numerical model used to determine the therapeutic electric field threshold, with numerical results depicting an electric field of 379 ± 142 V / cm within the therapeutic margin.

  FIGS. 7A-7B are graphs showing that lesions that occurred after the pulse was applied at 1 Hz were statistically larger (α = 0.1) than lesions that occurred at 0.25 and 4 Hz rates. It is.

  8A-8B are photomicrographs showing a histological comparison of untreated liver tissue against a region that received light IRE treatment showing preservation of connective tissue and blood vessels.

  9A-9C are photomicrographs (FIG. 9A) showing the portion of the liver that has not been treated after 24 hours of perfusion. After 24 hours of perfusion, the same liver segment (FIG. 9B) treated with 90 1500 V / cm, 100 μs pulses at 4 Hz using a needle electrode (FIG. 9B) shows 10 × magnification and (FIG. 9C) shows 20 × magnification. .

  FIGS. 10A-10B are photomicrographs (FIG. 10A) after 24 hours of perfusion, untreated liver portion and (FIG. 10B) after perfusion for 24 hours at 20 × magnification, using a needle electrode at 1 Hz. The same liver treated with 100 1500 V / cm, 100 μs pulses is shown.

  FIG. 11 is a photograph of a perfusion electrode according to the present invention.

  FIG. 12 is a photograph showing a kidney with a prototype electrode and a perfusion system with renal veins and arteries attached simultaneously.

  FIG. 13 is a graph showing impedance measurements from the kidney before pulsing, showing substantial electrical resistance and capacitance.

  FIG. 14 is a photograph of a monitor showing the resulting waveform from a 50 microsecond pulse having an amplitude of 1000V.

  FIG. 15 is a graph showing the extinction time and maximum transported current for pulses transported with amplitudes between 100 and 2000V.

  FIG. 16 is a photograph showing the kidney portion 30 minutes after pulsing, in which the red portion shows the living tissue and the white portion shows the area removed by the IRE pulse.

  Reference will now be made to detail various exemplary embodiments of the invention. It will be understood that the following discussion of exemplary embodiments is not intended to limit the invention. Rather, the following discussion is provided so that the reader will understand the specific aspects and features of the present invention in more detail.

  According to embodiments of the present invention, a viable decellularized tissue scaffold undergoes non-thermal irreversible electroporation (N-TIRE) on an organ under continuous perfusion. It can be achieved by using it. Electroporation (reversible and / or irreversible) is transported to tissues and organs using known techniques including plate electrodes or needle electrodes, or even by transporting electrical charges through the organ or tissue vasculature. Can be done.

  More specifically, electroporation can be used as a therapy to treat many medical conditions. Irreversible electroporation, including non-thermal IRE, is a method of killing unwanted cells using an electric field in tissue while preserving ECM blood vessels and nerves. When applied across cells, certain electric fields have the ability to decellularize cell membranes through a process termed “electroporation”. The process was known to be “irreversible electroporation” when the electric field allowed the cells to survive and temporarily penetrate the cell membrane. Reversible electroporation has become an important tool in biotechnology and medicine. Other electric fields can cause the cell to die after the cell membrane is permeated. Such a necrosis process was known as “irreversible electroporation”. Non-thermal irreversible electroporation is a new and minimally invasive surgical technique that removes unwanted tissue, such as tumor tissue. The technique is easy to apply, can be observed and adjusted, is not affected by local blood flow, and does not require adjuvant drugs. A minimally invasive process involves positioning needle electrodes in or around the target area to transport a series of short and intensive pulses that cause structural changes in the cell membrane that promote cell death . The voltage is applied to electroporate tissue without inducing substantial Joule heating that significantly impairs the main blood vessels and ECM. In setting the specific tissue type and pulse conditions, the primary parameter for determining the irreversibly electroporated volume is the electric field distribution within the tissue. Recently, IRE animal experiments have moved away from specific modes of non-thermal cell removal such as preserving key structures such as extracellular matrix, major blood vessels, and myelin sheath, non-scarring, as well as promoting beneficial immune responses. A number of beneficial effects have been demonstrated.

  Electroporation is a non-linear biophysical process where a pulsed electric field is applied that leads to an increase in cell permeability, possibly through the formation of a lipid bilayer nanoscale pore. Weaver, J. et al. C, Electroporation of biological membranes from multicellular to nanoscales, IEEE Trns. Dieletr. Electr. Insul. 10, 754-768 (2003). If the pulse length and intensity do not exceed certain criteria, such permeability is reversible and cell health and function are maintained. When the critical electric field strength threshold is exceeded (approximately 500-700 V / cm with normal settings of pulse parameters), the cell membrane cannot be recovered and cell death will occur. Garcia, P .; et al. Intracranial non-irreversible electroporation: in vivo analysis. J Membr Biol 236, 127-136 (2010); et al. Sequential finite element model of tissue electropermeabilization. Iee Transactions on Biomedical Engineering 52, 816-827, doi: 10.1109 / tbme. 2005.8455212 (2005); and Lee, E .; W. et al. Advanced Hepatic Ablation Technology for Creating Complete Cell Death: Irreversible Electroporation. Radiology 255, 426-433, doi: 10.1148 / radiol. See 10090337 (2010) ("Lee 2010").

  Application of pulses that permanently destabilize cell membranes, inducing necrosis in an accurate and adjustable manner with submillimeter resolution (Edd, JF, Horowitz, L., Davalos, RV. , Mir, L.M. & Rubinsky, B. In vivo results of a new focal tissue abduction technique: Irreversible electronation. )) Is a non-thermal irreversible electroporation. n, N-TIRE) (Davalos, RV, Mir, LM & Rubinsky, B. Tissue ablation with reversible electroporation. 10.1007 / s 10439-005-8981-8 (2005) ("Davalos 2005")). Said technique does not rely on a thermal mechanism (Davalos 2005) and appears to preserve the structure of the underlying extracellular matrix. Studies show that nerve conduits and bile ducts are not damaged within the removal area. Maor, E .; , Ivorra, A .; Leor, J .; & Rubinsky, B.M. The effect of irreversible electroporation on blood vessels. Technology in Cancer Research & Treatment 6,307-312 (2007). In addition, in vivo hepatotomy areas showed minimal damage and minimal vasculitis in small blood vessels (Lee 2010).

Cryogenic oxygen perfusion (HOPE) is a whole organ preservation method that mechanically transports a nutrient-rich blood substitute that is oxygenated to the whole organ below physiological temperatures. Dutkowski, P.A. , De Rougemont, O. & Clavien, P.A. A. Machine perfusion for 'Marginal' river graphs. Am. J. et al. Transplant. 8, 917-924, doi: 10.1111 / j. 1600-6143.2008.02165. x (2008); and Monbaliu, D .; & Brassil, J.A. Machine p
erfusion of the liver: past, present and future. Curr. Opin. Organ Transpl. 15, 160-166, doi: 10.1097 / MOT. Please refer to 0b013e328383342b (2010). This method is based on a study conducted to reach a storage time of 72 hours (Yamamoto, N. et al. 72-Hour Preservation of Porcine Liver Continuous Conjugation of the United States of America and the United States). 51,288-292 (1991) and has been successfully demonstrated to improve renal preservation quality and transplant success rates with warm ischemia {Olschewski, P. et al. The influenza. of storage temperature burning machine perfu sion on preservation quality of marginal donors. Cryobiology 60, 337-343, doi: 10.016 / j. cryobiol. & Minor, T. Use of a New Modified HTK Solution for Machine Preservation of Marginal Live Grafts.Journal of Surgical Research 160, 150.1.01.200. } Schoon et al. And Blockmann e. al. demonstrated the ability of the perfusion fluid to extend organ quality using normal temperature perfusion maintained at or near physiological temperatures.Schoon, MR et al. Liver translation after organ preservation with normotherm. extracorporal perfusion.Annals of Surgery 233,114-123 (2001); and Blockmann, J. et al. Please refer to 0b013e3181a63cl0 (2009).

  Such organ preservation methods can be used to separate the N-TIRE tissue removal effect from the immune response observed in vivo and spontaneous degradation of post mortem tissue. Recently, taking advantage of N-TIRE's ability to destroy cells without destroying the extracellular matrix (Davalos, R. V. Irreversible Electro- mation to Create Tissue Scaffolds. United States patent (2009); and Phillips, M .; , Maor, E. & Rubinsky, B. Non-Thermal Irreversible Electrophoresis for Tissue Decellularization. Making the use of deformation laboratory models to be a viable means for Flip was determined.

  The results of N-TIRE treatment are clinically meaningful and can be expected through many modeling tissue removal / decellularizations that can be widely used in tissue engineering. Davalos, R.A. V. & Rubinsky, B.M. Temperature condensations burning irreversible electroporation. International Journal of Heat and Mass Transfer 51, 5617-5622, doi: 10.016 / j. ijheatmasstranfer. 2008.4.0046 (2008); F. & Davalos, R.A. V. Mathematical Modeling of Irreversible Electroporation for Treatment Planning. Technology in Cancer Research & Treatment 6, 275-286 (2007); and Neal, R. et al. E. , II & Davalos, R .; V. The Feasibility of Irreversible Electrophoresis for the Treatment of Breast Cancer and Other Heterogeneous Systems. See Anals of Biomedical Engineering 37, doi :: 10.1007 / sl 0439-009-9796-9 (2009).

  The artificial scaffold is replated with the recipient's cells, resulting in a functional organ structure. Such an organ regeneration method would be used to reduce the supply shortage of transplanted organs.

  Embodiments of the present invention relate to a method, system and apparatus for irreversible electroporation (IRE) transport through the vasculature of an organ. IREs administered in this way in vitro or in vivo can be used to treat tumors that are deeply embedded in vascularized organs. The application of embodiments of the present invention also allows for decellularization of organs and through the vasculature of tissues and organs to generate scaffolds from existing tissue from which cellular debris has been removed without damaging the extracellular matrix. Including using IRE.

  In general, the removal process involves delivering a series of low energy (intensive but short) electrical pulses to the target tissue. Such a pulse renders the target tissue cell membrane unrecoverable enough to kill cells affected by the electric field. The treatment is non-thermal and essentially only affects the cell membrane of the target tissue and does not affect nerves or blood vessels within the treated tissue.

  The present invention provides a decellularized scaffold that can be formed using non-thermal-irreversible electroporation (IRE). The organ or tissue can be perfused during and / or after the process, which is a common technique in the medical field.

  After manufacturing the scaffold, tissue engineering can include in vitro inoculation and attachment of human cells to the scaffold. To date, the most successful scaffolds for tissue engineering have been natural and have been created by chemically and / or mechanically decellularizing large animal (eg, pig) organs.

  Embodiments of the present invention provide a method for producing a decellularized tissue scaffold. In general, a tissue containing cells and the underlying scaffold can be treated in vivo or in vitro with an electric field of power for a period sufficient to kill the cells of the tissue. The electric field is administered directly into the vasculature of the organ. The electric field is induced through the vasculature of the organ by the walls of the vascular system. Preferably, the electrical pulse is administered in a manner that avoids disruption to the underlying scaffold and the vasculature of the organ or tissue.

  The method is suitable for manufacturing a tissue scaffold for use with artificial tissue. Although the source of tissue is not limited, in an exemplary embodiment, the tissue is a anatomy similar to a relatively large animal or human (in important tissues), such as a pig, cow, horse, monkey, or ape. It is derived from an animal recognized as having a scientific structure. In an embodiment, the tissue source is a human, which is an application for reducing the likelihood of rejection of the scaffold-based artificial tissue.

  In a preferred embodiment, the method does not damage vascular structures such as capillaries. In an embodiment, the method does not damage a neural tube present in the tissue prior to treatment with the electric field. As used herein, the term “untouched” means a condition that allows an element to perform its original function on a substantial scale. Thus, for example, an intact capillary is a capillary that can carry blood, and an intact neural tube is a neural tube that can accommodate neurons. In embodiments, cells of other glandular structures remain intact, including not only the vasculature and nervous system, but also examples such as bile ducts or milk ducts. In such embodiments, the cells may remain as part of the scaffold and prosthetic tissue, or suitable treatment techniques or cells seeded on the scaffold or of the body containing the prosthetic tissue Can be removed by cells.

  According to an embodiment of the invention, the tissue is exposed to an electric field that is sufficient in time and power to cause cell necrosis of the tissue, but insufficient to significantly destroy the scaffold in which the cells are present. To do. Moreover, in a preferred embodiment, the electric field does not cause irreversible tissue damage as a result of heating of the tissue. Various schemes for providing such an electric field are possible. In an exemplary embodiment, one or more electrical pulses are applied to cause cell membrane disruption as a result of electricity and little as a result of heat. When more than one pulse was used, the pulses, among others, were separated by a period of time that allowed the tissue to cool, preventing thermal damage from occurring on a significant scale. For example, one or more electrical pulses can be applied to the associated tissue for a range of about 5 microseconds (μs) to about 200 seconds, with the pulses being at a lower repetition rate over a period of 1 to 24 hours. Can be applied at. For convenience, short-term processing is desired. Thus, in a preferred embodiment, electrical pulses are applied for a period of about 1 to 10,000 μs. Also, although there is no limit on the number of pulses delivered to the tissue, in a preferred embodiment, about 1 to about 100 pulses are applied to the tissue. For example, in an exemplary embodiment, about 10 to 1000 pulses of 100 μs each in a period are applied to the tissue for cellular removal.

  Thereafter, the organ can be processed to form a decellularized scaffold by adjusting perfusion parameters to remove cell debris. Mechanical decontamination of cell debris may be performed for a period of time that is essential to achieve the desired result of removal of the debris, for example, a period of 4, 12, 24, 48, or 72 hours. Perfusion parameters including cardiac contraction pressure, diastolic pressure, and temperature can be adjusted to improve cell debris removal efficiency. A mild detergent may also be added if it is essential to increase or facilitate the removal of cell debris. Preferred embodiments provide a perfusion protocol that results in the removal of 99% cell debris and DNA structure.

  Embodiments of the present invention apply varying amounts of energy (both reversible and irreversible electroporation) to produce a wide range of favorable tissue removal effects, including selective removal of general and neoplastic cells Provide a method that can be adjusted to do.

  According to the present invention, the effects of energy application can be adjusted and adjusted by changing a number of parameters including, for example: energy voltage, amperage, duration and frequency; energy supply system design and location Adjustment; Design and use of sophisticated and precisely regulated vascular perfusion systems; Physiological mimicking characteristics of sophisticated and precisely regulated vascular perfusion systems; Perfusion and storage conditions for ex vivo organs and tissues; In vivo organs and Tissue perfusion and storage conditions; and / or composition of intravascular fluid that acts in part or in whole as an extension of the energy application system and protocol.

  Such efforts to ensure the production of tissue scaffolds, or treatment of in vivo or in vitro tumors, or the biotransport of suitable macromolecules in diseased or otherwise targeted tissues. There are various parameters that can be observed or adjusted with the use of a non-thermal IRE. For example, embodiments of the present invention may be used in the context of islet gene therapy with type I diabetes that stimulates insulin production, ie, non-cancerous diseased tissue. The present invention can also be applied to immunomodulation by introducing genes into tissue such as muscle to produce the desired antibody, ie, using healthy tissue as a conduit for therapy. Parameters that can be changed for a particular application include voltage gradient. In an embodiment, the pulse generates a voltage gradient in the range of about 10 vol / cm to about 10,000 vol / cm. The voltage gradient (magnetic field) is a function of the distance between the electrode and electrode geometry, which is modified depending on the tissue sample, tissue characteristics and other factors. In some embodiments, two electrodes were used and the electrodes were positioned at a spacing of about 5 mm to 10 cm. Typical electrode diameters are in the range of 0.25 to 1.5 mm, and usually 2 or 4 electrodes are used. In an embodiment, one bipolar electrode is used. The “electrode” can also include an insulated portion (including the use of a non-conductive coating) and a conductive portion (eg, an end) to ensure proper application of electrical current and the tissue The generation of excessive heat in a part of can be minimized.

  Suitable electric fields and durations of exposure are those reported in the literature as suitable for medical treatment for tumor removal. Exemplary exposure parameters include: 90 90 microsecond (μs) pulses at 1.5 kV / cm at a frequency of 1 Hz or 4 Hz; 80 at 2.5 kV / cm at a frequency of 1 Hz or 4 Hz 100 μs pulses; one 200 ms pulse at 400 V / cm; 10 100 μs pulses at 3800 V / cm at a frequency of 10 pulses per second; 1000-1667 V / cm at a frequency of about 1 Hz or 4 Hz 90 100 μs pulses in the range; and 80 100 μs pulses in the range 1000-3000 V / cm at 1 Hz or 4 Hz. In general, the frequency of pulsing may be as low as half of the pulse width or may be spaced far away. Any suitable frequency that allows electroporation without significant thermal damage to the tissue is also acceptable. The current can be applied to either DC or AC. Indeed, it is within the skill of the art to adjust such parameters applicable to achieve a particular desired result. A certain number of pulses can be administered, for example 1-1000 pulses. The pulse can have a period of time, such as 1 microsecond to 1 second. The voltage can be an amount to achieve reversible or irreversible electroporation depending on the desired effect, and can include, for example, 500 V / cm to 5000 V / cm, such as 1500 V / cm. In fact, to achieve IRE with a capillary bed, much less voltage may be required, for example, in the order of about 1 V / cm to about 1000 V / cm.

The shape and size of the electrodes are not critical in the practice of the present invention. One skilled in the art can select any form and size suitable for transmitting the desired electric field within the tissue. For example, the electrode may be a plate type, a wire type, or a needle type, and may be a circular shape, an elliptical shape, a rectangular shape, a rectangular shape, a diamond shape, a hexagonal shape, an octagonal shape, or the like. In a wire-type electrode, a diameter smaller than the vascular structure into which the wire is inserted is preferable so that the electrode does not block the vascular structure. In some designs and indications, the use of electrodes that are part of an occlusive device that provides separation of part of the circulation of an organ or tissue is preferred. Similarly, the surface area of the electrode is not critical in the practice of the invention. Thus, for example, the surface region may be about 0.5 cm ² , about 1 cm ² or more. In an electrode that is inserted directly into the vasculature of an organ or tissue, preferably the electrode is a long wire electrode that is long enough to provide a suitable distance electrode within the vasculature. The diameter of the wire-type electrode is also preferably adjusted for insertion into various types of organ vasculature so that contact between the electrode and the inner wall of the vasculature can be avoided or minimized Can be done. In some embodiments, there is at least a partial separation between the electrode and the walls of the vasculature. In wire-type electrodes, the target region blood vessels “upstream” and “downstream” to reduce the potential of the combined energy loss and current moving across the target region bounds between the electrodes. Can be temporarily blocked. Such an embodiment performed in vivo would deliver the pulse within a time period that does not induce unacceptable ischemic effects in the target area and certain “downstream” areas. More specifically, such a technique clamps the blood vessels above and below the target organ or tissue so that the rest of the blood vessels do not conduct current (insulated blood vessel walls). Having clamping will "open" the circuit). This is more appropriate for gene or drug delivery applications that are not aimed at killing cells, and there must be a suitable window in which this can be done without damaging the tissue. Such a method of the invention can be used, for example, for plaque removal, for example, occlusion of the carotid artery for about 10 minutes to remove plaque from the carotid artery.

  In some situations, heat is generated when tissue is exposed to an electric field. In order to avoid tissue damage due to heat, the amount of energy transferred to the tissue was set below a threshold level per unit time. Time and temperature parameters are known in the art for IRE, and any suitable combination can be used. For example, the temperature of the tissue can be observed during the process for removal, and the electrical pulse is adjusted to maintain the temperature at 100 ° C. or lower, such as 60 ° C. or lower, or body temperature. In an embodiment, the temperature may be maintained at a normal physiological temperature for a particular organ or subject. In some embodiments, the temperature is maintained at 50 ° C. or lower.

  In some embodiments, the method adjusts the voltage, pulse length, and / or number of pulses applied to obtain an average irreversible electroporation across the biological cells of the tissue to produce a non-target Achieving irreversible electroporation of the biological cells within the tissue at a level that minimizes damage to the tissue. The protocol can include parameters that are the same or changed through the administration of electrical charge. Similarly, in some embodiments, the duration of the applied voltage is adjusted by the ratio of current to voltage to achieve irreversible electroporation of the identified target cell, thereby allowing the cell membrane to Collapsed in a way that causes death. Additional exemplary parameters are listed below.

  Example 1-Multiphysics modeling

  Preliminary multiphysics modeling was performed to establish the feasibility of using the vasculature as a route for the transport of electrical pulses. The electric field distribution, which is the main factor for the development of porosity (reversible or irreversible) in the cell membrane, was modeled using the finite element method. Three liver tissue models have been developed to show liver structure at different levels of detail. Comsol Multiphysics was used to calculate the electrical potential (Φ) according to Laplace's equation:

  Here, σ is electrical conductivity.

Model 1 : First, a 2 cm thick homogeneous structure having a 1 cm diameter plate electrode on one of the side surfaces of the tissue was formed using the liver as a model. Such a model was used to relate experimental lesions to electric field strength. A single 10 microsecond 1500 V / cm pulse was delivered to the upper electrode and the lower electrode was grounded. As shown in FIG. 2, the results of this model require that, for the electrical parameters used, a simulated electric field strength of about 400 V / cm produces lesions that are as wide as the experimental results. Showed that.

Model 2 : The liver is a complex organ with many sets of vasculature, lymph and bile ducts. The hepatic portal vein and the arterial branches of the liver transport blood to the lobule, the basic functional unit that functions structurally to filter blood, remove bacteria, and produce bile. To do. The blood transported through the leaflets eventually reaches the large central venule that flows into the hepatic vein and returns it to the circulatory system. Under perfusion, perfusate replaces blood as the fluid transported through the vasculature. However, it was mentioned that bile production was found to last up to 24 hours. To understand how the complex vasculature affects the electric field distribution when two different modes of voltage application were used, a second multi-scale model was fabricated. The first aspect used a typical electrode located outside the tissue, the second aspect used a central venule as a voltage source and an external electrode as ground. The leaflets were formed into a hexagonal model with an equivalent radius of 1 mm surrounded by a 5 micron connective tissue layer and a 200 micron diameter central venule, consistent with porcine histology. Stimulation was again with a single 10 microsecond 1500 V / cm pulse.

  The electric field distribution within the tissue did not change significantly to the additional heterogeneity generated by the lobular epithelial layer when an external electrode was used. Most significant changes appeared in close proximity to the higher conductivity central venules. The central venule of each leaflet was charged at 1500 V, and a very different electric field distribution was formed. The tissue region that reached an electric field sufficient to be irreversibly electroporated spread only approximately 0.5 cm into the tissue. Although this aspect of electric field application is not sufficient for overall decellularization, it can be an effective means of treating tumors close to the surface of highly vascularized organs. The model shown in FIG. 3A shows an external electrode charged at either 1500 V (upper part) or ground setting (lower part), and the electric field distribution using such an aspect is added to the inhomogeneity. It was proved that there was no significant change with sex. The model shown in FIG. 3B shows the central venule of each leaflet charged at 1500 V, with the external electrode grounded. The model shown in FIGS. 3A-B may not be completely physiologically accurate because fluid flows easily from the portal arterioles and portal veins through the leaflets to the central venule. Was mentioned.

Model 3 : Model 3 includes portal arterioles, portal venules, and bile ducts at the apex of a single leaflet. The central vein is surrounded by bulk liver tissue. The magnitude of the applied 10 microsecond pulse was varied until the electric field distribution developed in the range seen in bulk liver tissue.

  The results from such a simulation are shown in FIG. 4A. Results from such simulations indicate that IRE development that induces an electric field within a single leaflet is possible using the vasculature of the organ. Furthermore, it has been found that 50V pulses are very sufficient to increase the electric field relative to the IRE induction level. This is significantly less than the 1500V pulse that is essential to induce IRE when an electric field is applied across the entire tissue structure. It can be seen from FIG. 4B and the histological sample that there is a complex microvasculature network that contacts the portal arterioles and venules from the central vein. . Between the vasculature are hepatocytes that secrete bile into the bile ducts. Models 1 and 2 assume that such a microvasculature network exhibits considerable resistance to current flow and that the internal structure of the leaflets can be modeled as a heterogeneous tissue. This is a reasonable estimate of where vascular occlusion can occur if red blood cells (in vivo) or epithelial cells are damaged by an electric field.

  An approximation of the resistance (R) of the vasculature and microvasculature is easily obtained when the fluid conductivity (ρ) is known by solving the relational equation:

  Here, L and A are the length of the blood vessel and the cross-sectional area of the blood vessel, respectively. The results presented in Table 2 show that when a blood vessel is filled with a phosphate buffer solution, a vessel with a diameter of 10 μm and a length of 100 μm can have a resistance as high as 90 kΩ. Although very striking, this resistance is not high enough to prevent current flow through the microvasculature. Furthermore, the large number of microvessels connected in parallel everywhere in the organ will drastically reduce the overall electrical resistance.

  One embodiment of the invention is to adjust the scaffold by administering IRE during active perfusion of the organ or tissue. Electrodes can be located in or near the tissue with application of electrical pulses that generate irreversible (or reversible) electroporation of the cells through the target area. One way to administer the electrical pulse is through the vasculature of the organ itself. The position of the electrode defines the area to be treated; therefore, the area to be treated may be just a part of the whole tissue or organ used as the starting material. The electrical pulses pass irreversibly or reversibly (if irreversible) through the membrane of the cell to be treated and cause cell death. The length of time of the electrical pulse, the applied voltage, and the resulting membrane permeability are all adjusted within a specified range. Application of electrical pulses that cause cell death can still protect some or all of the organ, preferably the vasculature of the organ associated with microcirculation and macrocirculation. In some embodiments, the microcirculation structure can be partially or totally compromised, but a larger structure was maintained.

  In the in vitro and ex vivo embodiments of the present invention, secondary techniques for removing intracellular material can be used. For example, all known physical, chemical or enzymatic techniques can be used to remove cell debris from the irreversibly permeabilized cells. Similarly, the artificial tissue can be attached to an artificial perfusion system that can pump a liquid composition (eg, a detergent-containing liquid composition) through the artificial tissue, and can remove cell debris from the scaffold. It is important that such secondary treatments can be applied under relatively mild conditions that allow removal of cell debris but also allow the scaffolding structure (including blood vessels and neural structures) to remain. is there. The use of a non-thermal IRE allows such a mild process and improves the final manufactured scaffold compared to a process that does not rely on a non-thermal IRE.

  In the method of the in vitro embodiment, the debris remaining from the irreversibly permeabilized cells may remain in situ and through natural processes such as the body's own circulation and immune system. Can be removed.

  The concept of irreversible electroporation to decellularize tissue is different from other forms of decellularization used in the field. Irreversible electroporation differs from cell lysis using chemical and physical methods or osmotic imbalances because it uses electricity to kill cells. Irreversible electroporation is a more gentle method because it only destroys the cell membrane of cells in the targeted tissue and does not damage the underlying extracellular matrix (ECM). Conversely, chemical and physical methods can damage essential structures such as ECM, blood vessels, and nerves. The type IRE described in this application uses electrical pulses to provide a specific means, ie, an effective means to induce cell death by lethal disruption of the cell membrane.

  Irreversible electroporation can be used to decellularize tissue in a minimally invasive process that does not substantially affect or affect the ECM. Non-selective modes of decellularization are acceptable in the tissue engineering field, and some methods of inducing osmotic imbalance, cooling, or chemical decellularization result in sonication. provide.

  One exemplary embodiment of the present invention is that the cells of a tissue are irreversible by applying a pulse and voltage of a very precisely determined length during active perfusion of the organ or tissue. A method of electroporation. This can be done by measuring and / or observing changes in electrical impedance in real time to obtain irreversible cell damage without thermal damage, noting the decrease at the start of electroporation and adjusting the current in real time . Therefore, the method can include a computer device and a sensor for observing the effect of electrical processing. In embodiments where voltage is applied, the observation of the impedance provides the user with information about the presence and absence of porosity. Such measurements show the progress of pore formation and indicate whether irreversible pore formation has occurred leading to cell death.

  In other embodiments, the onset and magnitude of electroporation of cells in tissue may be correlated to changes in the electrical impedance of the tissue (used herein to mean voltage versus current). Provide a method. At a given point, the electroporation becomes irreversible. A decrease in the resistance of a group of biological cells occurs when the membrane of the cell becomes permeable due to pore formation. By observing the impedance of the biological cells in the tissue, it is possible to detect not only the average time at which porous formation of cells occurs, but also the relative degree of cell membrane permeability resulting from the porous formation. By testing cells in a given tissue with a step-wise increase in voltage, the point at which irreversible electroporation occurs can be determined. Thereafter, such information can be used to establish that the cells of the tissue have undergone irreversible electroporation. Such information can also be used to adjust the electroporation process by determining the selection of the voltage magnitude. Other image techniques can be used to observe how much the region has been processed (eg, ultrasound, MRI, CT, etc.).

  The present invention provides irreversible electroporation that occurs simultaneously on multiple cells providing direct induction of the actual occurrence of electroporation and an average degree of electroporation induction on multiple cells. Such a discovery is equally useful in irreversible electroporation of biological tissue (a mass of biological cells with adjacent membranes) for similar reasons. The advantages of such a process include a high level of control over the starting point of electroporation.

  One feature of embodiments of the present invention is that during electroporation of the tissue, the current and pulse length are adjusted to a predetermined range as the magnitude of the current depends on the degree of electroporation, so that the cells and Obtaining irreversible electroporation of target cells of the tissue that minimizes cell damage around the tissue. Other characteristics of embodiments of the present invention are that the pulse length and current are higher than the intracellular electrical regulation that causes cell death and lower than can cause thermal damage to the surrounding tissue. It is to be adjusted precisely within the range. Another characteristic of embodiments of the present invention is that measuring current (real time) through a circuit gives an average overall measure of electroporation achieved by the cells in the tissue. .

  Another feature of the embodiment is that the exact electrical resistance of the tissue can be calculated from a voltage measurement of the crossing time to the probe electrode and a measurement of the crossing current using a circuit attached to the electrode of the electroporation. Including: calculating an accurate electrical resistance of the tissue from a voltage measurement of the crossing time to the probe electrode and a measurement of the crossing current using a circuit attached to the electrode of the electroporation; Can be used to map the electroporation distribution of the tissue. In irreversible electroporation it is possible to carry out current or EIT measurements in substantial time (several minutes or more) after said electroporation to prove that it is actually irreversible, As a result, it is preferable.

  In embodiments of the method, it is preferred to remove cell debris from the decellularized scaffold after primary cell disruption to a non-thermal IRE. In such embodiments, all known techniques for doing so can be used, including all known physical, chemical, and / or enzymatic methods. In one exemplary embodiment, removal of cellular material is performed using a suitable agent (e.g., water, pH adjusted) using general diffusion, transport or a mixture of both through the remaining free vasculature. Or at least partially through perfusion of tissue scaffolding with aqueous solutions of one or more chelating agents).

  In an in vitro method, it is preferable to sterilize the scaffold, particularly when the scaffold is used to produce tissues and organs that have been treated for transplantation into a host. In order to reduce the patient's risk of rejection (eg due to DNA fragments), sterilization and / or debris removal after decellularization is commonly done for scaffolds used as implants. When the scaffold requires several types of sterilization, methods published in the literature can be used for sterilization of the scaffold.

In an in vitro method, the method of producing a decellularized tissue scaffold results in a decellularized tissue scaffold isolated from its natural environment. In an in vivo method, the method of producing a decellularized tissue scaffold results in a tissue scaffold that is free of the usual cytoplasmic components. Therefore, in the aspect of the present invention, a scaffold for the artificial tissue is provided. The treated tissue scaffold comprises a natural scaffold that has been removed from its natural environment / cell trait components have been removed. The artificial tissue scaffold of the present invention preferably comprises at least a portion of the vasculature (ie, arteries, veins, capillaries) and conductive structures (ie, conduits) present in the tissue in its native state, preferably Most, and more preferably substantially all or all. In embodiments, the tissue scaffold comprises at least a portion, preferably most, and more preferably substantially all or all of the neural structures present in the tissue in its native state. In embodiments, the scaffold further comprises such cells that make up such vascular structures and / or such neural structures. Preferably, the artificial tissue scaffold comprises a reduced number of the cells naturally present in the scaffold. Most of the cells of origin, more preferably substantially all of the cells of origin, and most preferably all of the cells of origin are not found in the artificial scaffold. In embodiments, the remaining cells are cells that contain blood vessels or neural structures.
In a preferred embodiment, some, most or all cell debris from the cell is not found in the scaffold of the artificial tissue. Similarly, in an embodiment, the tissue scaffold includes some or all of the neurons originally present in the tissue. However, in an embodiment, the neuron is destroyed, but the neural tube in which the neuron is present remains intact.

  In some embodiments, the artificial scaffold includes cell debris from cells that are originally (ie, naturally) in the scaffold. As discussed above, in such embodiments, the cell debris can be removed using known means. Alternatively, some or all of the cell debris may remain in and on the scaffold. In embodiments where cell debris remains on the scaffold, it is removed later by the action of new cells seeded on the scaffold and / or during the new cell inoculation, infiltration, and growth steps. obtain. For example, when new cells are seeded on a scaffold containing cell debris, only the new cell penetration and growth activity, or a combination with the perfusion means for the application and support of the new cells Can result in removal of the cell debris.

  The present invention provides an artificial tissue scaffold comprising a vascular structure that functions to provide nutrients and gas for cells to grow on and within the scaffold. The use of a non-thermal IRE to generate the artificial scaffold allows maintenance of such critical structures and provides an improved scaffold for use in medical, veterinary and research activities. . As such, the present invention provides an artificial scaffold that can have relatively large dimensions. That is, the artificial scaffold is already known in the art because re-inoculated cells growing in the original scaffold do not need to be close (ie within 1 mm) to the external surface to obtain nutrients and gases. It may be thicker than other scaffolds. The artificial scaffold can have any desired range of thicknesses, with the only limitation being the ability to generate sufficient electric fields to effect decellularization. However, such a limitation is not a significant physical limitation, because the position of the electrode that affects the IRE is easily adjusted and adjusted according to the requirements of the practitioner of the present invention.

  The artificial scaffolds of the present invention may have a thickness that is close to or similar to the thickness of the tissues and organs from which they are derived. Exemplary thicknesses are relatively thin (ie, 1 mm or less) to intermediate thickness (ie, about 5 mm to 1 cm) and relatively thick (ie, 5 cm or more, eg, 10-20 cm). Range).

  Provided below is an example of providing an artificial scaffold from a tissue IRE during active perfusion by the method of the present invention. Ideally an IRE performed ex vivo must be performed because the tissue is perfused in the bioreactor. The perfusion of tissue within a bioreactor has been published in the literature, and the parameters described in the literature can generally be applied within this context. IRE is able to kill the cells in the target area without damaging major blood vessels, connective tissue, nerves and surrounding tissues, and therefore for cell removal that is perfectly suitable for manufacturing scaffolds. This is a specific mode. Generally, mild enzymes or chemicals (non-ionic detergents, amphoteric detergents, chelating agents, enzymatic methods) can be used to allow removal of DNA fragments after decellularization (in vivo IRE The removal of cells can be accomplished by the body's natural system).

  One approach to performing an ex vivo IRE in a bioreactor perfusion system includes: (a) Attaching a freshly cut organ to the bioreactor perfusion system to maintain a physiological environment or to achieve specific results Maintained under other temperature conditions; (b) inserting an electrode into a target area, such as within the vasculature of the organ; (c) the organ with saline or other suitable perfusate And (d) administering an electrical pulse through one or more electrodes and administering reversible or irreversible electroporation through the vasculature. Once the unwanted cells are removed, the cellular debris is removed using chemical (eg, non-ionic detergent) or physical techniques to remove cellular components / debris (especially when extracellular). Thereafter, the scaffold can inoculate inoculated cells within the targeted / treated area. Further perfusion of the organ can be administered nutrients and / or growth media to the inoculated cells (Perfusion during IRE, Edd et al, 2006) and the bioreactor perfusion system Can be maintained at optimum conditions (37 ° C.). One or more of the steps of such a method can be performed and can also be performed to obtain a preferred volume of artificial tissue. For example, to process multiple areas of the organ or to process the entire organ, electroporation is repeated on multiple smaller areas of the organ, allowing a substantial intermittent pause that allows cell debris to be washed away from the organ. A period of perfusion can be performed after the IRE is performed without a period or IRE. Similarly, if a plate or needle electrode is used, perfusion can be performed prior to positioning or insertion of the electrode, with a wire-type electrode positioned within the vasculature, and properly positioning the electrode. After positioning, it is preferable to initiate active perfusion. Indeed, one or more such steps may be omitted, changed, rearranged with other steps, or appropriately replaced with other steps.

Example II-IRE during active perfusion

  A cross-bred piglet was killed by overdose of barbiturate. The liver was removed and placed on ice within 15 minutes after death. A vascular anastomosis device with a perfusion system was formed by inserting a connecting tube of a Luer-lock type syringe into the portal vein, hepatic artery and main hepatic vein, and fixed with zip ties. Prior to placing on the perfusion system, the liver was rinsed with lactated Ringer's solution (LRS) to remove blood / coagulum.

  The liver was perfused for 4 and 24 hours using a VasoWave® Perfusion System (Smart Perfusion, Denver, NC). This system generated cardiac imitation pulse waves to generate physiological cardiac contractions and diastole pressures and flow rates within the organ. This system can adjust the oxygen content of the perfusate above and below physiological standards. The perfusate composed of the modified LRS was transported to the portal vein and hepatic artery and returned to the system through the hepatic vein for reuse. All livers were actively mechanically perfused within 1 hour after death.

  Under perfusion, a low energy pulse was delivered to the liver tissue using an ECM 830 Square Wave BTX Electrophoresis System (Harvar Apparatus, Cambridge, Mass.). Two metal plate electrodes of 2 cm diameter were attached to a pair of ratcheting vice grips (38 mm, Irwin Quick-Grips) using Velcro. A high voltage wire was used to connect the electrode to the BTX unit. The electrode was gently clamped to the liver and the center-to-center distance of the electrode was measured. The voltage output at the BTX unit was adjusted so that the applied electric field of the approximate value was 1000 V / cm.

  99 individual 100 μs square pulses were administered at repetition rates of 0.25, 0.5, 1.0 and 4.0 Hz. The repetition rate test was performed arbitrarily and repeated a minimum of 3 times. A false control was performed in which no pulse was delivered and the electrode was positioned on the tissue. Two additional tests were performed with needle electrodes positioned at 0.5 cm intervals using a voltage to distance ratio of 1500 V / cm at a rate of 1 Hz and 4 Hz. The settings for such a protocol can be seen from FIG. 5A. All N-TIRE treatments were completed within 2 hours after death. Surface lesions occurring at each treatment site were measured at the end of the 24 hour perfusion period.

  After N-TIRE treatment and mechanical perfusion, the liver was separated from the VasoWave® system, sectioned to preserve the lesions, and the tissue was fixed by impregnation in 10% natural buffered formalin solution. After fixation, the tissue was cared for, treated with normal paraffin embedding, compartmentalized with 4 micrometers, and stained with hematoxylin-eosin (H & E) and trichrome stains. . A veterinary pathologist who was unaware of the N-TIRE processing parameters evaluated the tissue compartment.

  Numerical modeling can be used to predict the electric field distribution and provide insight into the N-TIRE processing region in the tissue. Ed, J. et al. F. & Davalos, R.A. V. Mathematical modeling of irreversible electroporation for treatment planning. Technology in Cancer Research and Treatment 6, 275-286 (2007) ("Edd 2007"). This was chosen as a method related to lesion volume with an effective electric field threshold for lesions occurring in the liver. The method for predicting the N-TIRE region is similar to that described by Ed and Davalos (Edd 2007). In order to understand the effective electric field threshold for inducing N-TIRE in the liver, finite element simulations were performed using Comsol Multiphysics 3.5a (Comsol, Stockholm, Sweden). . The numerical model was constructed using 2 cm diameter plates, each 1 mm thick, located above and below the tissue. The model is described in Garcia et al. (Garcia, P. et al. Intracranial non-irreversible electroporation: in vivo analysis. J Membr Biol 236, 127-136 (2010)). The electric field distribution was obtained by solving Laplace's equation.

  Here, σ is the electrical conductivity of the tissue, and ψ is a potential.

The electrical boundary condition by the tissue in contact with the energized electrode is ψ = V _o . The electrical boundary condition at the contact point of the other electrode is ψ = 0. The boundary where the analyzed region is not in contact with the electrode has been treated as electrical insulation. The electrical boundary condition by the tissue in contact with the energized electrode is ψ = V _o . The electrical boundary condition at the contact point of the other electrode is ψ = 0. The boundary where the analyzed region is not in contact with the electrode has been treated to be electrically isolated.

  The conductivity change due to electroporation and temperature was modeled by calculating the dynamic conductivity according to the following formula:

(Boone, K., Barber, D. & Brown, B. Review-Imaging with electricity: report of the European Concerned Action on Impedance Tom. E.n.E. D., Lebar, A.M. & Miklavic, D. Feasibility of Employing Model-based optimized of Efficient and Efficient Efficient Efficient 3-781 (2007); Sel, D. et al. Sequential fine element element of tissue electropermeabilization.IEEE Trans Biomed Eng 52, 816-827, doi: 10.1 109 / TB52.200. , N. et al., The Course of Tissue Permeabilization studyed on a mathematical model of a subtumorous tumor in small animal.

  In order to obtain a simulation of the electric field with which the tissue was exposed, such a numerical model was calculated for the pulse parameters used in the liver. Thereafter, the N-TIRE electric field threshold was found by measuring the area of the lesion and determining the electric field value in such a region in the model.

  Surface lesions appeared during perfusion within 30 minutes of starting treatment. The area of the liver surface lesion generated by the plate electrode was the same type, although larger than the area from the needle electrode. In FIG. 5B, a 3.3 cm surface lesion obtained after 4 hours of treatment and generated from an applied voltage of 1500 V is seen. The numerically modeled size of such lesions is that generated within the region of tissue to which an electric field of 379 ± 142 V / cm or higher is applied. The results of the numerical model for such a test can be seen from FIGS.

  In the frequency test, the average applied voltage with respect to the distance between the plates was 962 V / cm. Lesions from such trials occurred over 22 hours after death and had an average diameter of 2.5 cm (125% electrode diameter); this occurred at 0.25 Hz and 936 V / cm It has a minimum lesion of 2 cm and a maximum lesion of 3.2 cm that occurred at 1.0 Hz and 950 V / cm. Although not significantly significant, the results suggest that the lesion size is the largest average at 1 Hz and decreases as the frequency increases or decreases. Lesions that occur after treatment applied at 0.25 and 4 Hz are statistically smaller (α = 0.1) than lesions that occur for treatment applied at 1 Hz (FIGS. 7A-7B).

The analysis of the processed tissue shows a uniform processing region that extends cylindrically through the tissue. It was calculated the calculated processing volume of between 1.97Cm ³ and 6.37Cm ³ to the tissue thickness which corresponding 0.628cm and 0.792cm.

  In histological experiments from 24 hours after treatment, the treated area shows cell death (FIG. 8B) compared to the control (FIG. 8A). More specifically, FIGS. 8A-8B show a histological comparison in the area of untreated liver tissue versus the maintenance of connective tissue and blood vessels in the gently IRE treated area. Untreated sample stained with H & E (FIG. 8A) and 99 100 μs, 1000 V / cm pulses administered with H & E at 10 × magnification using plate electrodes for 24 hours cardiac imitation perfusion (FIG. 8B).

  The hepatic lobule in pigs is further widened by connective tissue, including blood vessels and bile structures, and has an important cord architecture that terminates in hepatic venules. In the region adjacent to energy transport, the hepatocyte code was well conserved with some vacuolar hepatocytes (discovery expected in a 24 hour ex vivo mechanical perfusion cycle). The sinusoidal structure in the untreated area is opened, reflecting the flow of perfusate between the hepatic artery / portal vein and hepatic vein. N-TIRE treatment disrupts hepatic cords and induces cell degradation (FIG. 8B). There is also preservation of the main liver lobule, including broader connective tissue and blood vessels. In the region of N-TIRE treatment, the cell code was not clear, and the sinusoidal intima was fractionated with varying degrees.

  Like humans, pigs have liver lobes substantially separated by a thin band of fibrous connective tissue that connects between portal triads. Such microstructure affects the distribution of lesions induced by electroporation. Lesions are confined within structural lobules that are lesions at the edges of the electroporation field or are normal or almost normal. Thus, when considering the process of developing a central liver lesion with electroporation or the subsequent development of an intact connective tissue / conduit / vascular matrix for tissue processing, the band of connective tissue is due to electropulsing. Acting as an insulator is an important discovery.

  9A-9C are photomicrographs (FIG. 9A) showing the portion of the liver that was not treated after 24 hours of perfusion. After 24 hours of perfusion, the same liver segment (FIG. 9B) treated with 90 1500 V / cm, 100 μs pulses at 4 Hz using a needle electrode (FIG. 9B) shows 10 × magnification and (FIG. 9C) shows 20 × magnification. .

FIG. 9A shows a portion of an untreated porcine liver with a general sinusoidal cell code aligned from portal tracts to the central vein. Cell morphology is well preserved. Some vascular hyperemia with red blood cells is seen, and there is also a minor centrilobular biliary stasis. Slightly damaged porcine leaflets were observed in the area where plate electroporation was performed (FIG. 9B). In the center of the leaflet, the destruction of the code structure and the alteration and community of some cells appear.
A higher resolution photograph of this region can be seen from FIG. 9C, and cellular changes are more easily seen. Such treated areas show minor lesions composed of cells separated from the hepatocyte code distribution and code basement plate. Minor cholestasis is seen (dark pigment).

  Administration of N-TIRE treatment with either a needle electrode or a plate electrode produced several distinct liver lobe lesions. The severity of lesions in each leaflet ranges from a minor level to a moderately serious level. Minor lesions consist of a small population of hepatocytes isolated from the basement membrane. Such cells exhibit a loss of tissue and cytoplasm / organ organ communities in fine intracellular structures (FIGS. 9A-9C).

  A moderately serious lesion is easily grasped (FIGS. 10A to 10B). Cells affected by N-TIRE treatment show varying degrees of cell swelling and nucleolysis (FIG. 10B). Within individual leaflets, most cells are affected. In some lobules, there is a flank nuclear pycnosis with a small population of hyperchromic cells isolated from the basement membrane and cellular alterations. In some lobules, centrilobular cholestasis with bile pigment aggregation is seen in the peripheral sinus space. In all cases, as mentioned, connective tissue bridging bands were observed in intact bile ducts and vasculature, and lobes with substantial N-TIRE-induced tissue damage appeared even more spread.

This study reports the effect of non-thermal irreversible electroporation in actively perfused organs for this use in making a decellularized tissue scaffold for organ transplantation. The results reported in this application localized tissue volume to 6.37 cm ³ in a single N-TIRE process. This can be easily expanded to a larger volume by performing multiple treatments with the goal of generating decellularized structures for partial and whole liver transplants. After 24 hours of perfusion, a large amount of cell debris generally remains in the tissue structure. Removal of such debris is essential to minimize the immune response of the recipient and remove cellular components through longer and more effective perfusion protocols and / or using perfusion by the body's own mechanisms. Can be achieved by maximizing.

  Besides the production of decellularized tissue scaffolds, such a method is an ideal scaffold for studying the effects of pulse parameters such as pulse length, repetition rate, and field intensity on the whole organ structure. I will provide a. Furthermore, since there is a direct regulation on the electrical properties of the perfusate, to test the effects of N-TIRE in diseased organs or cancerous organs with unique electrical or physical properties Can be provided as a model. The method of the invention can be applied to treat a diseased tissue or part of an organ, or to remove unnecessary or diseased tissue, after which the organ or tissue is regenerated. An inoculation can be made. For example, patients with liver disease can be treated by targeting only damaged areas of the liver through the vasculature, allowing the body to be decellularized and re-inoculated.

  The resulting scaffold from N-TIRE with perfusion maintains the vasculature essential for perfusion into structures that are much larger than the nutrient dispersion limitations present in non-vascular structures. Since the temperature of the perfusate used may be as low as 4 ° C., the thermal aspects associated with Joule heating are negligible. This provides an ideal scaffold for evaluating the effects on cells and tissues of the electric field separated from the effects of thermal damage. Furthermore, the lower temperature of the organ compared to in vivo application does not induce thermal damage, allowing higher voltages to be applied to obtain an appropriate electric field to decellularize thicker structures. To do. This is important because the thickness of the human liver can exceed 10 cm in some areas.

  As shown and described herein, when planning to decellularize tissues and organs that have undergone active perfusion, the treatment area of the decellularized tissue is a numerical value. Can be predicted through modeling. From the lesion size and numerical model used in this application, the protocol exposes the entire tissue to an electric field of 379 ± 142 V / cm when decellularizing the whole organ for an implantable scaffold It should be. This will ensure complete cell killing, allowing the desired cells to be globally re-inoculated into the scaffold to minimize the rejection effect of the recipient. The threshold found in this application is a little lower than the threshold in previous studies, as a result of the increased unique sensitivity of the cells to the unique pulse parameters used when under perfusion or the pulse. It was mentioned that could be.

  Sustained and active mechanical perfusion in the decellularization process is also useful for recellularization. Once the decellularization step is complete, the scaffold can be re-inoculated without risk of damage with removal of the newly generated scaffold from the perfusion system. Since the arterial and venous supply can be handled individually, multiple cell types can be transported simultaneously to other areas of the organ. Similarly, retrograde perfusion through the bile system can be an ideal route for transporting hepatocytes in the revaccination process.

  The microscopically visible lesions clearly demonstrate the mechanism and morphology for cellular stripping using electroporation. Furthermore, it is very interesting that at 24 hours there is only a modest loss of leaflet structure when using the electroporation parameters described herein. More severe conditions of energy transport are expected to change this, but induce damage to important connective tissue and vascular structures. By adding adjuvant cytotoxic agents, enzymes, and detergents into the perfusion fluid, the stringency and occasional nature of cell stripping can also be adjusted. Logically, it is better to create mild conditions that preserve critical structures for tissue engineering goals than to rapidly remove cells and matrix. It is clear that the ability to handle perfusion periods and perfusion conditions with a cardiac imitation system is useful for such a gradual and progressive approach to liver decellularization and ultimate recellularization.

Example III—IRE managed by vasculature with active perfusion

Normal kidneys were obtained from post-mortem pigs through standard and applied methods. When obtained, the renal arteries and veins of the kidney are separated and can be anastomosed to a cardiovascular emulation system (CVES) (VasoWave 2.3 ^™ , Smart Perfusion LLC, Denver, NC) It was connected so as to be suitable. The organ was connected to CVES and the kidney was washed with 1.5 liters of Krebs-Hensleit / glutathione physiological solution for 5 minutes.

  The kidney was then anastomosed to the arterial and vascular connector to the CVES to initiate perfusion (arterial systolic pressure with a physiological train of pulses delivered at 60 beats per minute): 110 mmHg, cardiac expansion pressure 60 mmHg). Perfusion was performed at 25 ° C. The renal veins were left unconnected, but the end of the vascular tissue was raised to produce a low back pressure (which is approximately 1 mmHg). Perfusion was stabilized at 15 minute intervals. The Krebs solution conductivity was measured at 0.72 S / m.

  As can be seen from FIG. 11, custom-made energy application electrodes can be manufactured for specific applications. In this embodiment, it has an end exposed through a hole through a small fraction of a foldable pipe near the center point (eg, a bare wire or a portion of an enamel coated wire with an exposed end). An electrode was generated by inserting and bonding an insulated wire and sealing the site. The enamel at the end of the wire was removed to create an electrical connection within the tubing and most of the wire was insulatively coated for safety externally. In addition, a shortened packaged pipe was applied depending on the length of the pipe for further electrical insulation. After that, a luer lock connecting tube was inserted into the end of the pipe to enable connection to the perfusion system. Such electrodes exhibit an electrical resistance of less than 1 ohm between the pulse generation system and the perfusion fluid. As can be seen from FIG. 11, the small diameter wire is located in the pipe and can be approached from the outside, allowing pulses to be transported simultaneously with perfusion.

  FIG. 12 is a graph showing a kidney attached simultaneously to a prototype electrode and perfusion system by a renal vein and artery. One electrode was positioned in line between the arterial supply from CVES and the renal vascular connector. One end of the venous electrode was connected to a renal venous vascular connector, and the other end of the electrode piping was supported by a syringe to form the back pressure. This also prevented the electrical current by finding a path of minimal resistance through the perfusion fluid backflow through the CVES, or by sending current partially through the system and the kidney.

  The resistance and capacitance of the vascular network were measured with an LCR meter. The resistance and capacitance values at low frequency (100 Hz) were 372.6 k Ohms and 0.248 nano-Farads, respectively. This calculates an RC time constant of 92.4 microseconds and a complete extinction time (5 * RC) of approximately 462 microseconds.

  The organ exhibited substantial electrical resistance and capacitance, as shown in FIG. 13, which provides impedance measurements from the kidney prior to pulsing. The trend observed between 100 and 10,000 Hz (FIG. 13) showed square wave pulses with a large DC component, and the RC time constant should have been even greater. Single 50 microsecond pulses were transported through the vasculature using BTX pulse regenerators (Harvar Apparatus) at voltages of 100, 500, 750, 1000, 1500, and 2000V. The transported current and the extinction time of the pulse were recorded. In all cases, the extinction time was less than expected.

  The resulting waveform is shown in FIG. More specifically, FIG. 14 provides the resulting waveform from a 50 microsecond pulse having an amplitude of 1000V. The disappearance of a pulse is a characteristic of tissue that has both a resistance and a capacitance component. A representative pulse voltage and current plot can be seen from FIG. The annihilation time decreased significantly between 100V and 500V and remained relatively constant at a voltage between 500 and 2000V. In all cases, the transported current was even greater (23-53%) than expected based on circulation.

  FIG. 15 is a graph showing the extinction time for a maximum current transported and a pulse transported with an amplitude between 100 and 2000V. Trends show that for 500 V and higher pulses, the resistance of the tissue decreases due to electroporation. Thereafter, 200 pulses at 1000V, with a repetition rate of one pulse every second, were applied to the organ.

  TTC colorant (2% of Krebs solution) was administered by perfusion for 15 minutes after pulsing was complete, and the organ was maintained on the perfusion system for an additional 30 minutes. The TTC colorant is used to indicate viable and non-viable tissue by forming a reaction product (red formazan) that develops color through the activity of tissue dehydrogenase. Viable tissue capable of oxygen breathing is stained in red; non-viable tissue or ischemic tissue is a pale color when compared. As can be seen from FIG. 16, at 30 minutes after pulsing, the kidney fraction is shown as a red area in the living tissue and a white area as removed by the IRE pulse. Fractionated organs were fixed in formalin for fixation and histological examination. It is clear from this figure that irreversible electroporation was applied to the large volume of the kidney. Thrombus in the vasculature can protect the entire organ from the application of electroporation. Such a problem can be solved by washing the vasculature of the organ immediately after surgical removal.

  The vasculature and microvasculature of the organ is used to transport short but intense electrical pulses to bulk tissue, maintaining the structure and function of the underlying extracellular matrix, and non-thermal cell death Can cause. Histological staining was performed on the overall health of the tissue (H & E), endothelial cell damage and removal (vWF), and the structure of the extracellular matrix (trichrome) and de-generation generated as a result of such preliminary experiments. It can be used to assess cellularization. The 4 hour total perfusion protocol can be used as a baseline for the decellularization protocol and extends beyond tissue construction, such as treating solid tumors deep inside vascularized organs .

  If an artificial scaffold is manufactured, the scaffold can be constructed so as to be regenerated further in vivo, or can be constructed in a tissue and an organ suitable for transplantation into the living body. A method for producing an artificial tissue can include: inoculating important cells into an artificial scaffold according to the present invention, and inoculating the inoculated scaffold with the inoculated cells penetrating the scaffold matrix. Exposed to conditions that allow it to grow. Inoculation of the scaffold can be done by any technique known in the art. Similarly, exposing the inoculated scaffold to conditions where the inoculated cells can penetrate and grow the scaffold using all techniques known in the art to achieve the results. Can be made. For example, incubating the inoculated scaffold in a commercial growth medium in a commercial incubator at about 37 ° C. under in vitro conditions, and if necessary, growth suitable for the growth medium Factors, antibiotics, etc. can be supplemented. Those skilled in the art can appropriately select appropriate inoculation and propagation techniques and parameters without a detailed description of the present application. In other words, with respect to cell inoculation and growth, the scaffold of the present invention generally behaves like other natural scaffolds known in the art. Although the scaffolds of the present invention have beneficial features that other scaffolds do not have, such features do not significantly affect cell seeding and growth.

  Artificial tissue has been developed as a replacement for tissue that is damaged, diseased, or otherwise defective. An important goal in the field of tissue engineering is to develop medical / therapeutic applications in human health. In view of the difficulty and ethical issues surrounding the use of human tissue as a source of scaffolds, tissue from large animals is usually used as a source for natural scaffolds. Thereafter, the xenotypic scaffold is inoculated with human cells for in vivo use in humans. Although the presently described artificial tissues are limited to human tissues based on animal scaffolds, it is envisaged to form the primary component of artificial tissues. Therefore, in an embodiment, the artificial tissue of the present invention is a tissue containing human cells on and in a scaffold derived from animal tissue other than human tissue.

  For certain in vivo applications, animal tissue was applied in vivo to a non-thermal IRE, and the treated tissue had cell debris removed by the host animal body. Thus, in certain in vivo embodiments, such removal is possible in the host animal's body itself (such a concept applies to the in vivo construction of the scaffold in humans as well). Therefore, the secondary cell debris removal step is unnecessary. Thereafter, the treated tissue is inoculated in vivo with, for example, human cells, so that the inoculated cells penetrate the scaffold and grow. Regenerated tissue is removed by appropriate infiltration and growth, and preferably contaminated host cells are washed and transplanted into a recipient animal, eg, a human. In such a situation, it is preferred that the host animal has a compromised immune system that cannot recognize or well recognize the inoculated cells as external cells. For example, genetically mutated animals with a compromised immune system can be used as host animals. Alternatively, for example, the host animal can be given an immunosuppressant to reduce or eliminate the animal's immune response to the inoculated cells.

  The recipient or host animal can be any animal, including humans, companion animals (ie, pets such as dogs or cats), livestock (eg, cattle, pigs, sheep), or sport animals (eg, horses). It's okay. Therefore, the present invention is applicable to both human and animal health care and research fields.

The practitioner still has the choice of cells to inoculate in vivo or in vitro or ex vivo. Many cell types can be obtained, and one skilled in the tissue engineering field can easily determine the type of cells used for tissue inoculation. For example, fibroblasts, chondrocytes, or hepatocytes can be used. In an embodiment, mesenchymal stem cells such as embryonic stem cells and living stem cells are used as cells for inoculating the scaffold. The source of the seeded cells is not particularly limited. Thus, the seeded cells may be autologous tissue, syngeneic or isogenic, allogenic, or xenogenic. A feature of the present invention is that it produces scaffolds and tissues with reduced immunogenicity (compared to scaffolds and tissues currently known in the art), so that the inoculated cells are autologous (see above). Preferably associated with the recipient of the artificial tissue).
In certain embodiments, it is preferred that the inoculated cells are stem cells or other cells that are capable of differentiating from an important tissue to an appropriate cell type.

  Alternatively or in addition, the in vivo method of generating a scaffold and the in vivo method of generating an artificial tissue can include processing tissue in the vicinity of a non-thermal IRE-treated cell with irreversible electroporation. . As part of the irreversible electroporation, one or more genetic elements, proteins or other substances (eg, drugs) can be inserted into the treated cells. The genetic element may be a coding region or other when it is expressed, reduces the interaction of the seeded cell with the cell, or otherwise produces an anti-inflammatory or other anti-inflammatory substance. Information can be included. Short term expression of such genetic elements can enhance the ability to grow artificial tissue in vivo without damage or rejection. Proteins and other substances can affect irreversibly electroporated cells or directly as products secreted from the cells after electroporation.

  Certain embodiments of the invention relate to the use of a human scaffold for use in the production of human artificial tissue. Together with other artificial tissues, such artificial tissues can be constructed in vitro, in vivo, or partially in vitro and partially in vivo. For example, a tissue donor has part or all of a tissue to which a non-thermal IRE has been applied and after constructing a tissue engineering scaffold for transplantation of recipient cells, these cells are grown be able to. By penetration and growth of the transplanted cells, the tissue can be removed and transplanted into a recipient as needed. Of course, as an ethical concern, the provider's organization must be an organization that is not fatal to the donor's life and health. For example, the tissue may be part of the liver. The artificial tissue can be regenerated to a fully functioning liver by removal from the host and transplantation by the recipient, and the remaining part of the host liver can regenerate the removed part.

  In this respect, the present invention has been described with respect to scaffolds for artificial tissues, artificial tissues, and methods for producing them. Similarly, it is important to note that the present invention includes artificial organs and methods of making them. This is generally taken to mean the part of a multicellular animal whose organs perform a distinct function or set of functions of said animal. It is also considered that organs are generally made of tissue and made of multiple types of tissue. Since the present invention can be applied to both tissues and organs in general and both of these categories are not critical to understanding the practice of the present invention, the terms “tissue” and “organ” Are used interchangeably in this application and are not intended to distinguish both.

  Many of the concepts included in the present invention may be referred to made with various exemplary concepts related to artificial tissue. For example, in the construction of an artificial organ, the initial organ can be completely depleted of cells using irreversible electroporation before re-inoculation (this relates in particular to organs having at least one size of less than 1 mm); The organ is irreversibly electroporated in fractions and re-inoculated so that human cells can penetrate a small fraction at a time; the organs are incremental slices that induce the cells in steps. Irreversibly electroporated in order to prevent viable human cells from coming into contact with the surviving animal cells to be replaced; the organ is electroreversibly electroporated in whole or in fractions and the human cells in the organ The whole organ can be irreversibly electroporated to kill the animal cells, and then human cells are transplanted on top of the organ and Permeates the Yaforudo, it can be replaced with other cells (by the animal cells die, the human cells fills the interior, to produce a new organ substituted).

  The provided isolated artificial tissues and organs can provide a method of using them. The present invention takes into account the use of the artificial tissue in both in vitro and in vivo settings. Thus, the present invention provides the use of said artificial tissue for research purposes and therapeutic or medical / veterinary purposes. There are a large number of practical technologies in research settings. An example of such an application is testing the efficacy of various removal techniques (including radiation treatment, chemotherapy treatment, or combinations) in the laboratory and optimizing treatment methods avoiding the use of sick patients The use of said artificial tissue in an ex vivo cancer model. For example, a recently removed liver (eg, porcine liver) can be attached to a bioreactor or scaffold and the tissue removed to treat the liver. Furthermore, due to the lack of a significant number of immune cells, tumors can be grown in the removed organs either by spontaneous means (carcinogens) or by introduction of tumorigenic cell lines into the tissue. . Such tumors can then be increased to gain knowledge and treated to facilitate optimization of treatment techniques in a physiologically relevant orthotopic setting. Another example of in vivo use is for artificial tissue.

  The artificial tissue of the present invention has an in vivo use. Among various uses, mention may be made of methods of in vivo treatment of a subject (in this application “patient” is used interchangeably and includes both humans and animals). For certain embodiments, in general, a method of treating a subject includes implanting a prosthetic tissue according to the present invention within or on the surface of the subject, wherein the implanting of the tissue comprises the subject. Cause a detectable change in the person. The detectable change may be any change that is detectable using natural sensations or artificial devices. Any type of treatment is envisioned by the present invention (disease or disorder, cosmetic treatment of skin wounds, etc.), and in many embodiments the treatment is a therapeutic treatment of the disease, disorder or other pain of the subject. Would be. Thus, the detectable change may be detection of a change, eg, an improvement in at least one clinical symptom of the disease or disorder that the subject is affected. Exemplary in vivo treatment methods include organ regeneration after tumor treatment, preparation of a surgical site for implantation of a medical device, skin transplantation, and a tissue or organ that has been damaged or destroyed by a disease or disorder ( For example, the replacement of a part or all of the liver) is included. Exemplary organs or tissues include: heart, lung, liver, kidney, bladder, spleen, pancreas, ovary, brain, ear, eye, or skin. In view of the fact that the subject is a human or an animal, the present invention may be either medical or veterinary.

  For example, the treatment method may be a method of regenerating a malfunctioning tissue related to a subject's disease. The method can expose a tissue to a non-thermal IRE to kill treated tissue cells and generate a tissue scaffold. The method can further include inoculating the tissue scaffold with cells from outside the subject such that the inoculated cells grow in and on the tissue scaffold. The proliferation of the cells produces a regenerated tissue that contains healthy and functional cells. Such a method does not require removal of the tissue scaffold from the subject. Rather, the scaffold is constructed from the original tissue and re-inoculated with healthy and functional cells. The entire process of scaffold generation, artificial tissue generation and subject treatment can be performed in vivo with the exception of expansion of the seeded cells as much as possible, and can be performed in vitro if necessary.

  In yet another exemplary embodiment, a tissue scaffold was constructed by removing tissue in the donor animal using a non-thermal IRE. The artificial tissue remains in the donor's body so that there are no cell debris from the tissue scaffold. After an appropriate time, the artificial tissue is removed from the donor's body and transplanted into the recipient's body. Transplanted scaffolds are not replated with external cells. Rather, the scaffold is naturally re-inoculated by the recipient's body to produce functional tissue.

  In yet another embodiment of the present invention, the wire electrode is used to allow a pulsed electric field or a continuous electric field to be used for irreversible electroporation or thermal damage to the target region, either in vivo or ex vivo. Can be induced. When irreversible electroporation is shown in such an example that it is possible in most of the organs to which active perfusion is applied, as an electrical conduit sufficient to transport an electrical pulse to the tissue It has been demonstrated to provide the natural vasculature of tissues / organs. If irreversible electroporation is a phenomenon that exhibits lower energy pulse parameters than irreversible electroporation, it is clear that irreversible electroporation is also possible through the vasculature. Such a technique is very useful if the practitioner does not remove the target region but wishes to introduce a therapeutic macromolecule such as a gene or drug into the target region. The macromolecule used and the desired therapeutic effect are left to the practitioner. In addition, radiofrequency ablation is a commonly used heat ablation that heats tissue using a continuous crossed magnetic field, and its use in medicine has increased, and the vascular electrode technique described in the present invention has been increased. Introducing it reduces the wettability.

  The present invention eliminates some of the most problems that currently arise from transplantation. The method described herein reduces the risk of rejection (as compared to traditional organ transplantation) because the only cells remaining from the donor organ are those associated with the formation of blood vessels and nerves. . At the same time, blood vessel, nerve and glandular structures are maintained. The present invention provides a relatively fast, effective and simple way to produce artificial tissue with substantially natural structure and function and low immunogenicity. Therefore, the artificial tissue of the present invention has a relatively low possibility of rejection and is very suitable for therapeutic and cosmetic applications. In embodiments where a human organ is used as the source of the scaffold (eg, from a living donor or cadaver), the risk of rejection is very low.

  In summary, the inventor has developed a new process using newly manufactured tools (electrodes and perfusion system) that can be used for advanced and selective removal of unwanted soft tissue including cells, tissues and organs. Established. Special embodiments focus on the application of pulsed electrical energy, regulation of perfusion fluid content and flow parameters, pulse energy transport schemes, and therapeutic effects within the fluid contained within the vascular tissue or vascular bed. Including the use of other adjustments to prescribe. It was further confirmed that cell removal from intact tissue is possible and useful for the generation of new tissue matrices for various applications. Such results presented in this application are based on experiments performed ex vivo. However, the fluid to be perfused is similar in composition and conductivity to blood. This indicates that such experimental results are indicative of the reaction seen in vivo.

  For example, the energy transport methods described herein that transport directly through the vasculature of the organ can also be used to induce other therapeutic responses. Near-lethal doses of electrical pulses (irreversible electroporation) can be used to treat local diseases by introducing genes or drugs into specific organs or areas of organs. Similarly, radiofrequency ablation techniques can use the vasculature to transport radio frequency energy and destroy diseased tissues or tumors through acute hyperthermia. Such a method can be improved by transporting a physiologically acceptable fluid (by co-perfusion) along with other electrical properties to increase such effects.

  Because this energy is applied through the vasculature, an adjustable large treatment volume can be achieved. Since the energy is not transported by the needle electrode and no hole is required in the organ, the entire structure of the organ is not destroyed. Furthermore, as theoretically expected and experimentally proven, the transported energy disappears directly across the vascular bed or capillary bed rather than across bulk tissue. Conventional therapy using a needle or plate electrode uses a similar voltage to carry a current in excess of 1 Amp and only affects a small volume. In contrast, energy transport using the renal vascular network causes a current of approximately 50 mA (less than 200 times the power) and can treat even larger volumes.

The invention has been described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Those skilled in the art will recognize that such features may be used alone or in some combination based on the given application or design requirements and specification. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. Where numerical ranges are provided herein, each numerical value between the upper and lower limits of this range is also specifically described. Such lower and upper limits of the range were similarly included or excluded from the range independently as well. As referred to herein, the singular forms “a” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a pulse” includes a plurality of such pulses, and reference to “the sample” refers to one or more samples and those skilled in the art. Including its known equivalents. It is intended that the specification and examples be considered as exemplary in nature, and modifications that do not depart from the essence of the invention are intended to be included within the scope of the art. In addition, all the references mentioned in the present description are incorporated herein as references.

Claims (28)

A method of removing cells from a tissue or organ, including:
Inserting one or more electrodes into the vasculature of an organ or tissue;
The tissue or organ is mechanically perfused with perfusate through the vasculature; and an electric field is applied within the vasculature with sufficient power for a time sufficient to cause electroporation of the target cells. Methods for removing cells from tissues or organs including:
Placing one or more electrodes in, on or near the organ or tissue;
The tissue or organ is mechanically perfused with a perfusate; and an electric field is applied in the tissue or organ with sufficient power for a time sufficient to cause electroporation of the target cell.   A therapy selected from electrochemotherapy (ECT), electrogene therapy (EGT), nanosecond pulsed electric field (nsPEF), radio frequency irreversible electroporation (H-FIRE), radio frequency ablation, or acute hyperthermia. The method according to 1 or 2.   3. The method of claim 1 or 2, further comprising energizing the vasculature with sufficient power for a time sufficient to cause necrosis of the target cell.   The first electrode is inserted into an artery of the organ, the second electrode is inserted into a vein of the organ, and electroporation is provided between the electrodes through the vasculature of the organ. The method according to any one of the above.   The first electrode is inserted into a blood vessel of the organ, the second electrode is inserted into another blood vessel of the organ, and electroporation is provided between the electrodes through the vasculature of the organ. 5. The method according to any one of 4.   A first electrode is inserted into a blood vessel of the organ, a second electrode is inserted into a fluid duct of the organ, and electroporation is provided between the electrodes through the vasculature of the organ. Item 5. The method according to any one of Items 1 to 4.   A first electrode is inserted into a blood vessel of the organ, a second electrode is inserted into another blood vessel of the organ, and a treatment region of the organ is located between the first blood vessel and the second blood vessel; The method according to claim 1, wherein is provided between the electrodes through the vasculature of the organ.   A first electrode is inserted into a blood vessel of the organ, a second external grounding pad, a plate electrode or a conductive gel outside the organ is used as the second electrode, and electroporation is performed through the vasculature of the organ. The method according to claim 1, wherein the method is applied between electrodes.   Performed in an organ or live animal under mechanical perfusion, and an electrical pulse is applied to induce an electric field through the vasculature with sufficient power for a time sufficient to cause electroporation of the target cell The method according to any one of claims 1 to 9, which enables the introduction of a therapeutic gene construct for treating a disease occurring in the target region.   Performed in an organ or live animal under mechanical perfusion, and an electrical pulse is applied to induce an electric field through the vasculature with sufficient power for a time sufficient to cause electroporation of the target cell 11. The method according to claim 10, which enables the introduction of a therapeutic gene construct for altering cells to treat systemic diseases.   Performed in an organ or live animal under mechanical perfusion, and an electrical pulse is applied to induce an electric field through the vasculature with sufficient power for a time sufficient to cause electroporation of the target cell 11. The method of claim 10, which allows for the introduction of a therapeutic macromolecule to treat a disease within the target region or systemic disease.   11. The method of claim 10, wherein the method is performed on an organ or living animal under mechanical perfusion and applying a cross electric field through the vasculature produces a target area or whole thermal ablation of the organ.   14. The method according to any one of claims 1 to 13, wherein the electroporation comprises non-thermal irreversible electroporation of target cells.   15. The method of any one of the preceding claims, further comprising removing cellular debris from the tissue or organ by mechanical perfusion, physiological perfusion, physical, chemical or enzymatic techniques, or any combination thereof. The method according to item.   16. The method according to any one of the preceding claims, further comprising sensing and observing electroporation by observing through electrical measurements.   A tissue scaffold formed by removing target cells from a natural tissue source by providing irreversible electroporation to the target cells during perfusion of the tissue.   The scaffold of claim 17, wherein the electroporation is provided through the vasculature of the tissue.   19. A scaffold according to claim 17 or 18, comprising the extracellular matrix and vascular structures of natural tissue sources.   20. A scaffold according to any one of claims 17 to 19, wherein the tissue scaffold has a thickness of at least one size of about 1-10 cm.   To obtain a tissue scaffold, the target cells are subjected to irreversible electroporation during perfusion of the tissue, and under conditions that allow growth of cells surviving in or on the scaffold. An artificial tissue constructed by removing the target cells from a natural tissue source by replating the cells into the scaffold.   23. The prosthetic tissue of claim 21, wherein the electroporation is provided within the vasculature of the natural tissue.   The artificial tissue scaffold according to claim 21 or 22, wherein the tissue scaffold is derived from an animal, and the living cells are derived from a human.   24. The artificial tissue scaffold of any one of claims 21 to 23, wherein the tissue scaffold comprises an extracellular matrix and vascular structures of natural tissue sources.   25. The artificial tissue scaffold of any one of claims 21 to 24, wherein the fabricated tissue scaffold has a thickness of at least one size of about 5-10 cm.   The artificial tissue according to any one of claims 21 to 25, which is a heart, lung, liver, kidney, pancreas, spleen, gastrointestinal tract, bladder, prostate, ovary, brain, ear, eye or skin or a part thereof. Scaffold. A device for removing target cells of a tissue or organ, including:
A perfusion system suitable for transporting perfusate from an artery to a vein of said tissue or organ vasculature;
A first electrode and a second electrode suitable for insertion into the artery and vein of the tissue or organ vasculature, respectively; and to perform electroporation of a target cell between the first electrode and the second electrode A voltage source that activates communication with the electrode to apply a voltage between the first electrode and the second electrode during perfusion with a sufficient voltage for a sufficient time.   28. The apparatus of claim 27, wherein the electroporation comprises non-thermal irreversible electroporation of target cells.