JP2008099737A - Bioartificial glomerulus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bioartificial glomerulus suitable for a continuous hemofiltration. <P>SOLUTION: The continuous hemofiltration has high antithrombogenicity and promotes the filtration by covering a hollow fiber inner face in the inner face of a hemofiltration apparatus using a patient's own vascular endothelial cell, and expanding the aperture of a fenestra (a window) of the cell membrane similarly to that of the glomerular endothelial cell. The aperture of the fenestra of the endothelial cell is continuously expanded by the application of actin filament interruption agent. The application dose is sufficient for sustaining the expansion of the aperture continuously at least for 7 days. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an artificial glomerulus, specifically, a bioartificial glomerulus using endothelial cells that enlarge the diameter of fenestra.

  The current dialysis therapy as an artificial kidney merely substitutes the glomerular filtration function intermittently and incompletely. Only 12 hours (about 7%) out of 168 hours per week are used for dialysis treatment, and its ability to excrete water and metabolites is significantly lower than that of living kidneys. Therefore, maintenance hemodialysis patients are severely restricted in water and food intake, and suffer from various complications resulting from such imperfections. In addition, the cost of treatment for such complications such as hospitalization and surgery is increasing year by year, which contributes to an increase in medical costs in Japan.

  In order to make current hemodialysis treatment more efficient, it is unavoidable to remove the strong limiting factor of 12 hours per week. However, on the other hand, patients are deprived of important time during the day every other day for hospital visits and treatment, and it is virtually impossible to spend more treatment time with the current dialysis technology. In order to resolve the contradiction between the necessity of a long treatment time to ensure the treatment efficiency close to the renal function and the freedom of the patient, a technological leap in the treatment system is necessary.

Already, the present inventor performed 10 liters (L) of continuous blood filtration per day from β ₂ which is a causative protein of dialysis amyloidosis from low molecular weight substances such as urea, creatinine and uric acid as compared with current hemodialysis. -It was clarified that even microglobulin can be maintained at a remarkably low value (see Non-Patent Document 1). Continuous blood filtration of 10L / day is the continuous removal of about 7 ml / min of filtrate from the blood, and generally does not require a hollow fiber module with a membrane area of about 1.8 m ² , and a membrane area of 0.2 Since a filtration module that can be stored in a breast pocket of ˜0.3 m ² suffices, troublesomeness associated with wearing can be minimized (Non-patent Document 2). Eating and drinking water and the resulting metabolites do not accumulate in the body beyond the day, but can be filtered and removed immediately like the kidneys, so there is less burden on the body and less complications occur. However, in order to allow such continuous wearing filtration, it is necessary for one filter to function for at least one week or more even if systemic anticoagulation therapy is minimized. Current continuous hemofilters using artificial materials are only required to function for up to 24 hours after a systemic anticoagulant therapy.
Saito A, Takagi T, Sugiura S, Ono M, Minakuchi K, Teraoka S, Ota K .: Maintaining low concentration of plasma β2-microglobulin through continuous slow haemodialysis. Nephrol Dial Transplant 10 (Suppl. 3): 52-56, 1995 Saito A: Research in the development of a wearable bioartificial kidney with a continuous hemofilter and a bioartificial tubule device using tubular epithelial cells. Artif Organs 2004; 28: 58-63

  As a result of studying various problems in view of the above-mentioned present situation, the present inventor has found that the inner surface of the continuous blood filter is capable of quickly removing excess water and accumulated metabolites, particularly in patients with congestive heart failure and chronic renal failure. The idea is to cover the inner surface of the hollow fiber with the patient's own vascular endothelial cells and enlarge the pore size of the cell membrane fenestra, similar to that of the glomerular endothelial cells, to facilitate filtration. As a result, research for developing a continuous blood filter (bioartificial glomerulus) having high antithrombotic properties was conducted, and the present invention was completed.

  The bioartificial glomerulus of the present invention is a bioartificial glomerulus comprising a hollow fiber blood filtration membrane coated with an inner surface using a patient's own endothelial cells and a filtration container containing the same as a blood filter, Sustained for 7 days, increase the number of cell membrane fenestra in the cell, expand its pore size to 0.5-2 times the glomerular endothelial cell phenestral pore size, or promote blood filtration by the effect of both It is characterized by that.

The endothelial cells are preferably vascular endothelial cells, or the endothelial cells are derived from endothelial progenitor cells collected from a patient.
The number of phenestra increases or the pore diameter of the endothelial cells increases by application of an actin filament blocking agent.

  The actin filament interfering drug is selected from the group consisting of Cytochalasin B, Latruncyulin A, Swinholide A, Halichondramide / Dihydorohalichondramide, Jasplakinolide / Misakinolide.

It may be a blood filter that applies an anticoagulant drug to the hollow fiber to minimize systemic anticoagulation therapy.
The hollow fiber is preferably formed of a highly permeable blood filter membrane made of polysulfone, cellulose acetate, polyimide, or the like.

The application of the drug is performed by allowing the drug to be supported on a hollow fiber in advance or by flowing a fluid containing the drug prior to use.
In the method for producing a bioartificial glomerulus according to the present invention, the inner surface of a hollow fiber, which is a blood filtration membrane, is coated with the patient's own endothelial cells, then accommodated in a filtration container and used for blood filtration. Apply interfering drugs to increase the number of cell membrane fenestra in the cell, expand its pore size to 0.5-2 times the glomerular endothelial cell phenestral pore size, or promote blood filtration by the effects of both It is characterized by that.

  As an applied amount of the actin filament interfering agent, the agent expands the pore diameter of the plasma membrane fenestra of the endothelial cells by 0.5 to 2 times the phenestral pore diameter of the glomerular endothelial cells, and the expansion of the pore diameter continues at least. Enough to last for 7 days.

  The bioartificial glomerulus of the present invention is an artificial glomerulus that can be filtered efficiently for 24 hours continuously for at least 7 days by attaching cells that enable faithful reproduction of kidney function. That is, self-endothelial cells are engrafted on the inner surface of the blood filter, and the diameter of the fenestra is continuously expanded by the action of an actin filament-blocking agent, thereby providing a continuous blood filter with high antithrombotic properties.

When the bioartificial glomerulus of the present invention requires continuous hemofiltration to improve the pathology of excessive water retention and accumulation of metabolites such as chronic / acute heart failure and chronic / acute renal failure / multi-organ failure In addition, it is possible to prevent the adverse effects of continuously using an anticoagulant such as heparin and to provide a safe and simple continuous treatment system that functions for a long time.
Detailed Description of the Invention
The “artificial glomerulus” is a blood filter that mimics the filtration function of a glomerulus that scrapes waste products and moisture from blood as a kidney filtration device, and artificially performs filtration with the glomerulus. “Bioartificial glomeruli” are cells in which cells, tissues, biological materials, etc. are incorporated into such artificial glomeruli. “Bioartificial kidney” means bioartificial glomer.
ulus) and bioartificial tubules, which can be dialyzed efficiently and continuously for 24 hours by attaching cells that allow reproduction of kidney function. Useful tubules A substance reabsorption function can also be added. FIG. 1 shows a conceptual diagram of a bioartificial kidney aimed by the present inventor. The artificial kidney used for the current dialysis treatment performs only rapid dialysis within a limited time, and its effect is limited.
Bioartificial glomerulus The bioartificial glomerulus of the present invention is
The blood filter is a bioartificial glomerulus comprising a hollow fiber blood filtration membrane coated with an inner surface of the patient's own endothelial cells and a filtration container containing the same, and lasts for at least 7 days. It is characterized by increasing the number of cell membrane fenestra, expanding its pore size to 0.5 to 2 times the phenestral pore size of glomerular endothelial cells, or promoting blood filtration by the effects of both.

  The bioartificial glomerulus of the present invention is a blood filter using a hollow fiber engrafted with self-endothelial cells and a container containing the hollow fiber. In other words, heparin, etc. when continuous hemofiltration is required to improve the pathology of excessive water retention and accumulation of metabolites such as chronic / acute heart failure and chronic / acute renal failure / multi-organ failure It is a medical device that realizes a safe and simple continuous blood filtration system by preventing the harmful effects of continuous use of the anticoagulant.

  The capillaries constituting the glomerulus are fenestrated capillaries, and countless small holes exist in the endothelial cell membrane. This small pore is called fenestra (fenestra, window), and is open to the cell membranes of fenestrated cells, fenestrated capillary endothelial cells and glomerular endothelial cells. Or “fenestra diameter”) is approximately 30 to 80 nm in diameter. This facilitates material permeation inside and outside the blood vessel.

Normally, arterial pressure (40-50mmHg) is applied to the glomerulus, but because glomerular endothelial cells have windows, plasma components (water, electrolytes, waste products, etc.) are easily filtered from the capillary wall. . The bioartificial glomerulus of the present invention increases the number of cell membrane fenestra of the endothelial cells covering the inner surface of the hollow fiber membrane by applying a drug, or the pore diameter is 0.5 to 2 times the fenestral pore diameter of the glomerular endothelial cells. A blood filter that promotes blood filtration by the effects of both, or both.
-Filtration membrane A blood filtration membrane consists of the filtration substance which ultrafilters the blood, and the container which accommodates this. The filtration material is required to be a porous material, but filtration membranes based on hollow fiber membranes have been used for hemodialysis so far, and the filtration membrane of the bioartificial glomerulus of the present invention also has such a filtration membrane. Hollow fiber is adopted due to many advantages.

<Hollow fiber>
In the artificial glomerulus of the present invention, a hollow fiber membrane having a relatively uniform distribution of fine pore diameters and an extremely large substance carrying capacity is preferably used. The hollow fiber membrane has a structure that is generally used for filtration and separation of substances, and a large number of micropores are formed on the side surface of the membrane between the inner surface and the outer surface. The micropores have a diameter of 0.001 μm to several μm, preferably 0.03 to 1 μm. In the membrane body using the hollow fiber, the carrying amount or accommodating capacity of the substance per unit surface area is extremely large as compared with other porous substances due to its structural characteristics. Therefore, it can be used as a carrier having a very large carrying capacity even in the case of a minute medical device.

As the hollow fiber material, polysulfone, polyethersulfone, polyacrylonitrile, polyvinyl alcohol, cellulose acetate and the like are used. The base material may be any hollow fiber membrane for hemodialysis, but a synthetic polymer hollow fiber membrane for an artificial kidney is preferably employed. Therefore, it is desirable that the hollow fiber is formed of a film made of cellulose acetate, polysulfone, polyimide, or ethylene / vinyl alcohol copolymer.

  Japanese Patent No. 2916446 discloses a method for producing an asymmetric microporous hollow fiber as an ultrafiltration hollow fiber suitable for blood filtration. This hollow fiber has a structure having an inner surface having a continuous porous sponge structure in which the pore diameter increases sequentially toward the outside and a microporous barrier layer. For this reason, the hollow fiber is used for artificial kidneys, filters for artificial dialysis, and the like. Since such hollow fiber is excellent in fluid permeability, mechanical strength and processability, it is suitable as a material for the hollow fiber membrane of the present invention.

<Endothelial cells>
In the bioartificial glomerulus of the present invention, endothelial cells from patients, preferably vascular endothelial cells, more preferably endothelial precursor cells derived from peripheral blood of patients (for example, vascular endothelial cells) are used. The reason why the endothelial cells are affixed to the inner surface of the filtration membrane of the hollow fiber is to secure a continuous and sufficient amount of filtration and acquire blood compatibility and antithrombotic properties as in the case of the glomerulus. This basically eliminates the need for anticoagulant measures, which are necessary when using artificial materials for continuous blood filters. In addition, unnecessary rejection in blood filtration can be avoided by using the patient's own endothelial cells.

  In general, engraftment and maintenance of autologous endothelial cells in a confluent monolayer on the surface of the filtration membrane will achieve hemocompatibility and antithrombotic properties, but it will significantly increase the ability to obtain a sustained and sufficient amount of filtration. It is customary to result in suppression. Glomerular endothelial cells, called fenestrated cells, allow filtration through a thin cytoplasm and a large fenestra in the cell membrane. For this reason, only glomerular endothelial cells can obtain a sufficient filtrate while forming a confluent monolayer on the filtration membrane with endothelial cells. However, the use of such glomerular endothelial cells for the bioartificial glomerulus of the present invention is clearly not realistic from the viewpoint of availability and ethics.

  Desirably, the endothelial cells other than the glomeruli are engrafted and maintained in a monolayer confluent on the surface of the filtration membrane. To develop a bioartificial glomerulus that has high blood compatibility and antithrombogenicity and can use high filtration performance, a confluent cell monolayer is formed on the inner surface of the filtration membrane of the hemofilter using the patient's own vascular endothelial cells. It is conceivable to form a large fenestra.

It has been shown in hepatic sinusoid endothelial cells that phenestra of endothelial cells is formed and maintained by intracellular actin filaments and inhibition of the actin filaments can increase the number of phenestra (Steffan AM, Gendrault JL , Kirn A. Increase in the number of fenestra in mouse endothelial liver cells by altering the cytoskeleton with cytochalasin B. Hepatology 1987; 7: 1230-1238: Braet F, Spector I, Shochet N, Crews P, Higa T, Menu E, De zanger R, Wisse E. The new anti-actin agent dihydrohalichondramide reveals fenestra-forming centers in hepatic endothelial cells. BioMed Central Cell Biology, HYPERLINK "http://www.biomedcentral.com/1471-2121/3/7" http : //www.biomedcentral.com/1471-2121/3/7 ). However, the diameter of hepatic sinusoid endothelial cell phenestra did not increase, and only a few minutes later was restored. For this reason, such endothelial cells are not practical for the present invention.

In contrast, in the present inventors' study, rat glomerular endothelial cells (rat glomerular cells) were used by changing the concentration and action time of actin filament blockers such as Cytochalasin B.
endothelial cells), changes in fenestra diameter of human umbilical vein endothelial cells (HUVEC) and filtration under hydrostatic pressure were measured. In addition, changes in diameter and filtration amount after several days of Cytochalasin B treatment were searched. There have been no studies in the past that have taken into account changes in the amount of filtrate. From such studies by the present inventors, in rat glomerular endothelial cells and HUVEC, even after 7 days of Cytochalasin B treatment, there is no cell damage and the phenestra diameter is expanded, and the increase in filtrate volume under hydrostatic pressure is maintained. It became clear. From this, it became possible to continuously increase the diameter of fenestra of vascular endothelial cells, thereby increasing the amount of filtrate, and the bioartificial glomerulus of the present invention was completed.

  During blood filtration, blood that flows to the filter passes through a lumen in a hollow fiber that is a filtration membrane. It is the inner tube inner wall of the hollow fiber that attaches the endothelial cells to the hollow fiber membrane. The wall surface of the inner tube has a large number of small holes leading to the outside of the hollow fiber, and water, electrolytes, low-molecular waste products, and the like go out from the small holes into the external dialysate. Such small pores are covered with a single layer of endothelial cells and thus become clogged, but this time they are filtered through a number of fenestras vacated in the cell membrane of the endothelial cells.

  The bioartificial glomerulus of the present invention mimics the glomerulus of the kidney. As described above, it is originally desirable to use glomerular endothelial cells. However, in the present invention, the endothelial cells that are easy to obtain, handle and proliferate, In particular, vascular endothelial cells are preferably used. If possible, it is also desirable to use vascular endothelial cells or vascular endothelial precursor cells collected from peripheral blood as the endothelial cells. The collected cells or cells derived therefrom are used for the endothelial cells used in the present invention. Vascular endothelial cells are isolated from peripheral blood or umbilical vein blood. Prostaglandins released from peripheral blood vascular endothelial cells or human umbilical cord blood vein endothelial cells (HUVEC) on the inner surface of the membrane of the hollow fiber, protein factors represented by t-PA, The effect of suppressing thrombus formation and acute occlusion is expected by cell regulatory factors such as nitric oxide.

  Furthermore, such vascular endothelial cells can be derived from vascular endothelial progenitor cells under certain inducing conditions. Vascular endothelial progenitor cells are present in peripheral blood, bone marrow, and the like, and have high proliferative ability, vascular endothelium-like function, and differentiation ability into vascular endothelial cells including vascular endothelial cells. Vascular endothelial progenitor cells are a cell that forms a colony by culturing a mononuclear cell fraction separated from bone marrow, peripheral blood or umbilical cord blood by Ficoll density gradient centrifugation under culture conditions suitable for culturing vascular endothelial progenitor cells. To be separated. By applying remarkable cell engineering techniques, it is possible to isolate and proliferate vascular endothelial progenitor cells and to induce differentiation into more differentiated vascular endothelial cells. The stem cell-derived differentiated vascular endothelial progenitor cells obtained from bone marrow can be differentiated into vascular endothelial cells of any organ. Similarly, if somatic stem cells are found in the peripheral blood, vascular endothelial progenitor cells may also be induced therefrom.

In the bioartificial glomerulus of the present invention, vascular endothelial cells derived from other organs and tissue sites are used instead of glomerular endothelial cells. Since the properties of vascular endothelial cells (for example, vascular endothelial growth factor dependency, transporter expression) differ from organ to organ, they cannot be used as replacement cells for glomerular endothelial cells as they are. Even with the same fenestrated cell, the phenestral diameter of the cell membrane must be adjusted to the same or close to that of glomerular endothelial cells. Therefore, a fenestra diameter suitable for blood filtration is ensured by using a drug that seems to have an effect of enlarging the fenestra diameter or increasing the number of fenestras.
-Drugs The endothelial cells are enlarged in their fenestra pore size by applying an actin filament blocking drug. The effect of anti-actin filament drugs on phenestra of endothelial cells is considered to be a phenomenon that is conventionally observed only in the sinusoid endothelial cells of the liver, and has been studied in sinusoid endothelial cells. However, the study by the present inventor has revealed that the diameter of fenestra is increased in glomerular endothelial cells and HUVEC, and that the effect on fenestra, which is considered transient in liver sinusoid endothelial cells, has been revealed for a long time. It was.

In addition to Cytochalasin B shown in the experiment of the present inventor (Act 2), an actin filament blocking agent that seems to have an effect of expanding the diameter of phenestra of endothelial cells or increasing the number of phenestra is as follows. Although it does not limit, for example, latrunculin A, swinholide A, jasplakinolide / misakinolide, halichon-dramide / dihydrohalichondramide etc. are mentioned. Furthermore, VEGF (Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestra- tion induced by vascular endothelial growth factor. J Cell Sci 2995; 108: 2369-2379: Chen J, Braet F, Brodsky S, Weinstein T, Romanov V , Noiri E, Gollgorsky MS. VEGF-induced mobilization of caveolae
Am J Physiol Cell Physiol 2002; 282: C1053-C1063) ⁾ and Angiotensin II (Otani a, Takagi H, Suzuma K, Honda Y. Angitensin II potentiates vascular endothelial growth facto
Circ Res 1998; 82: 619-628), Atrial Natriuretic Peptide (ANP) also promotes endothelial cell permeability, but these are transient after administration. It is a sexual effect, and continuous administration is difficult to apply to its treatment due to its influence on other functions of the patient.

  Therefore, from the viewpoint of avoiding damage to cells and ensuring the sustainability of the expansion effect, the preferred actin filament interfering drug is preferably at least 1 from the group consisting of Cytochalasin B, Latruncyulin A, Swinholide A, Halichondramide / Dihydorohalichondramide, Jasplakinolide / Misakinolide. It is desirable to select the species. These agents may be used alone or in combination of two or more.

  Furthermore, other drugs such as anticoagulant drugs may be used in combination. For example, an anti-coagulant drug may be further applied to the hollow fiber to provide a blood filter with enhanced antithrombogenicity. When the hollow fiber is used as a blood filter agent, it is desirable that the drug to be carried is an anticoagulant. In particular, argatroban (antithrombin drug) and sarpogrelate hydrochloride (antiplatelet drug), which are extremely effective in suppressing the formation of initial thrombus, are preferred. Therefore, the bioartificial glomerulus of the present invention is desirably a blood filter that applies an anticoagulant drug to the hollow fiber and minimizes systemic anticoagulation therapy. In particular, wearing continuous blood filtration is required to be easily performed by a small and highly functional filtering means, but the bioartificial glomerulus of the present invention establishes a so-called antithrombotic circuit to meet such a demand. .

The application of the drug is performed by allowing the hollow fiber to carry the drug in advance or by flowing a fluid containing the drug prior to use. The method of such application is appropriately selected based on the drug used. For example, the above-mentioned anticoagulant drug may be preloaded on a hollow fiber in a necessary amount, and the actin filament blocking drug flows into the hollow fiber by flowing a fluid containing the drug prior to use. It is preferable to enlarge the diameter of the phenestra of the attached endothelial cells. Alternatively, an actin filament interfering agent may be carried on the blood inflow side of the hollow fiber, and the diameter of the phenestra of the endothelial cells is enlarged during use.
・ Blood filter (filtration container)
The current blood filtration apparatus is also used as a cartridge type in which a hollow fiber filtration membrane is housed in a filter, but the bioartificial glomerulus of the present invention is also preferably such a cartridge type device. Therefore, the filtration container that houses the hollow fiber membrane may be made of the same material and material as the conventional cartridge. In the case of a wearable type or an implantable type for continuous blood filtration, it is desirable to take a form suitable for that purpose and to make it more compact and manufactured from a biocompatible material.

Production method The production method of the bioartificial glomerulus of the present invention comprises:
The inner surface of the hollow fiber, which is a blood filtration membrane, is coated with the patient's own endothelial cells, then housed in a filtration container, and when used for hemofiltration, an actin filament interfering agent is applied to the cell membrane fenestra. It is characterized by increasing the number, expanding the pore diameter to 0.5-2 times the phenestral pore diameter of glomerular endothelial cells, or promoting blood filtration by the effects of both.

  The applied amount of the actin filament interfering drug is 0.5 to 2 times the pore diameter of the cell membrane fenestra of the endothelial cells (based on the average value of the fenestra pore diameter), preferably 0. It is desirable that the amount be 8 to 1.2 times, more preferably substantially 1 time, and the amount is sufficient to continue the expansion of the pore size for at least 7 days. Alternatively, the effect of increasing the number of cell membrane fenestra may be obtained by applying an actin filament interfering agent, and thus the agent is applied to increase the number of cell membrane fenestra in the cell, or the pore size is expanded, or Both effects promote blood filtration.

  It is substantially insufficient in less than 7 days to achieve continuous hemofiltration in patients with renal failure that the enlargement of the plasma membrane fenestra diameter of the endothelial cells coated on the hollow fiber inner surface continues for at least 7 days. Because. Therefore, it is desirable that one hemofilter function for at least one week or more during continuous filtration. Such enlargement of the fenestra pore size and the duration of the expansion are required for effective continuous filtration. In this sense, the bioartificial glomerulus of the present invention is suitable not only for intermittent hemodialysis, but also for an artificial kidney aiming at a substitute for complete renal function.

A patient's vascular endothelial progenitor cells are collected as follows. From the patients with end-stage renal disease who fully explained the significance, specific methods, and problems of this treatment method and obtained consent for treatment, centrifuge blood cell fractionator such as Cube system for Leukapheresis ¹⁾ Process liters and collect the mononuclear cell fraction. The collected mononuclear cells are separated by a concentration gradient method using Lymphoprep medium or separated using a cell filtration device ²⁾ . CD133 positive mononuclear cells are collected by magnetic cell sorting and cultured using EBM-2 medium. As proof of endothelialization,
(1) mRNA expression of ecNOS, Flk-1 / KDR (VEGFR-2) and CD31 is evaluated by RT-PCR, and (2) NO secretion reaction by stimulation of VEGF or Acetylcholine. The collected endothelial cells are seeded in a hollow fiber module at a density of 10 ⁷ to 10 ⁸ cells / ml by rotating every 90 ° C. four times. After sowing, the hollow fiber module is cultured in a CO ₂ incubator. When a confluent monolayer is formed, Cytochalasin B is added to the medium at a concentration of 10 μg / ml and cultured for 2 hours. Cytochalasin B-added medium is thoroughly washed with Cytochalasin B-free medium and used for continuous filtration treatment of patients.

Reference 1) Hernandez DA, et al. Human endothelial cell cultures from progenitor cells obtained by leukapheresis. Am Surgeon2000; 66: 355-359
2) Aoki M, et al. Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device.Stem Cells 2004; 22: 994-1002 www.StemCells.com
In order to engraft the endothelial cells obtained from the patient in the hollow fiber, for example, in the case of a membrane area of 0.4 m ² polysulfone membrane hollow fiber device (1600 hollow fibers, inner diameter 300 nm), the density of vascular endothelial cells is 10 ⁶ / mL Thus, the present inventors have confirmed that a confluent monolayer is formed within 24 hours when the hollow fiber is seeded four times every hour. For small continuous filters, a membrane area of 0.2 m ² polysulfone membrane hollow fiber should be used. In order to promote the establishment of endothelial cells on the inner surface of the hollow fiber, the surface of the hollow fiber may be coated with a cell adhesion protein represented by collagen, fibronectin, laminin, an adhesive polymer or the like before cell seeding.

Blood is filtered through the glomeruli of the kidneys, and substances other than blood cells and proteins are transferred to the tubules as raw urine, what is needed in the tubules is reabsorbed, and waste products are secreted to form urine. It is excreted outside the body. The living kidney also exhibits metabolism, blood pressure / electrolyte regulation, and endocrine function, but is considered one of the most difficult organs to regenerate because of the many cell types involved in these functions.

  Although the current artificial kidney and the treatment using it are technically established, the function of the current artificial kidney is far from the function of the living kidney, and it is necessary to replace the body waste and part of the electrolyte adjustment. Only. In other words, patients suffer from various complications because they are poor in selectivity and remove useful substances such as amino acids and hormones, and cannot replace the metabolic and endocrine functions of living kidneys. For these reasons, it is essential to develop a continuous and highly efficient system for next-generation artificial kidney treatment. Development of new artificial kidney treatment systems such as portable artificial kidney treatment and dialysate regeneration type peritoneal dialysis is underway. Development of a bioartificial kidney system using patient-derived glomerular / tubule cells or cells with similar functions enhanced by gene transfer is aimed at.

In order to develop a bioartificial glomerulus that has high blood compatibility and antithrombotic properties, yet can be used for high filtration performance, the inner surface of the filtration membrane of the hemofilter is confluent by using the patient's own vascular endothelial cells. It is necessary to form a layer and form a large fenestra. The present invention is a bioartificial glomerulus capable of expanding the number and diameter of fenestra holes (windows) in the cell membrane of glomerular endothelial cells and extending the duration thereof by adding a drug. It is. The application of such a drug and the obtained effect are important in maintaining the dialysis efficiency of the bioartificial glomerulus for a long period of time. The increase in the number of fenestra by adding a drug has been reported for liver cells, but there was no mention of enlargement of the fenestra diameter, and the duration was also short.

  The present inventor uses an actin filament interfering agent such as Cytochalasin B, and changes the concentration and action time of rat glomerular endothelial cells, human umbilical vein endothelial cells (HUVEC) of fenestra diameter. The change and the amount of filtration under hydrostatic pressure were measured. We also searched for changes in diameter and filtration volume after several days of Cytochalasin B treatment. There have been no studies in the past that have taken into account changes in the amount of filtrate. From the study of the present inventors, in rat glomerular endothelial cells and HUVEC, even after 7 days of Cytochalasin B treatment, no cell damage is observed, the phenestral diameter is in an expanded state, and the increase in filtrate volume under hydrostatic pressure is maintained. It became clear.

Even when HUVEC was used, the same results as those obtained with rat glomerular endothelial cells were obtained. As a result, sustained enlargement of the fenestra diameter of fenestrated endothelial cells was observed by the action of Cytochalasin B or Latrunculin A. From this, it was found that the same effect can be obtained with vascular endothelial cells.

The bioartificial glomerulus of the present invention is a bioartificial tubule (Saito A, Artif produced by the present inventor).
In conjunction with Organs 28, 2004), it can be a pioneer in developing a complete artificial kidney (Figure 1).

The materials used herein are only preferred examples within the scope of this invention. In addition, the device names used in the following examples, the concentration of the materials used, the amount used, the processing time, the processing conditions, the numerical conditions such as the processing temperature, the processing method, and the like are merely preferred examples within the scope of the present invention. The following description will be made with reference to some drawings, which are merely schematically shown so that the present invention can be understood.
[Example]

In order to enlarge the phenestral diameter of rat glomerular endothelial cells, Cytochalasin B 10 μg / ml, which is one of actin filament blockers, is added to the medium in which the endothelial cells are seeded for 2 hours. The diameter of fenestra was markedly increased, and the enlargement of fenestra was maintained for at least 5 days without cell damage (FIGS. 2-A and B).

Next, actin filament staining with Fluorescence-phalloidin, Cytochalasin B
In the group to which Actin was added, interruption of actin filaments was confirmed, and spotted actin filaments were scattered (FIGS. 3A and 3B).

After treatment with 10 μg / ml Cytochalasin B for 2 hours, seeding rat glomerular endothelial cells on day 4 in transwell, and measuring water transfer to the contralateral side under the same hydrostatic pressure at the stage of confluence did. In an experiment using Transwell, Cytochalasin B (CB) was added to the medium for 2 hours, and after 4 days, a Horse radish Peroxidase filtration experiment was conducted. The group treated with Cytochalasin B showed significant water transfer compared to the non-treated group. It was seen. From this, it was found that about 6 times the filterability was maintained as compared with the group to which Cytochalasin B was not added (FIG. 4).

  As an experiment using endothelial cells other than rat glomerular endothelial cells, Cytochalasin B or Latrunculin A (LA) was added to HUVEC (human umbilical cord blood vein endothelial cells), and a filtration experiment was performed in the same manner (FIGS. 5-A to 5). 5-C).

Add Cytochalasin B 10 μg / dl into a confluent monolayer culture of HUVEC,
Or it culture | cultivated without adding and observed with the scanning electron microscope (Scanning electron microscopy; SEM) seven days later. From the SEM image (15000 times) in FIG. 5-B, a clear enlargement of fenestra was observed without cell damage.

On the other hand, Latrunculin A (LA) was added to the HUVEC culture solution and allowed to react for 2 hours. After changing the medium, the cells were cultured for 4 days and observed with SEM (× 15000). As a result, even in HUVEC, a clear enlargement of the fenestra diameter was recognized by the addition of LA (FIG. 5-C).

  Endothelial phenestra is formed and maintained by intracellular actin filaments, and it has been shown in liver sinusoid endothelial cells that inhibition of actin filaments can increase the number of phenestra, but the diameter of phenestra has increased. Moreover, the state of the increase was supposed to return to the original state after only a few minutes. The above results indicate that characteristics suitable for the function of the bioartificial glomerulus are imparted.

FIG. 1 shows a schematic diagram of a bioartificial kidney. A and V in the figure mean arteries and veins, respectively. FIG. 2-A is a SEM photograph of rat glomerular endothelial cells cultured in the absence of Cytochalasin B. (Scale bar = 1 μm) FIG. 2-B is a SEM photograph of rat glomerular endothelial cells cultured in the presence of 10 μg / ml Cytochalasin B (2 hours). This is the same magnification as in FIG. 2-A (Scale bar = 1 μm). FIG. 3-A shows the result of actin filament staining with Fluorescence-phalloidin in the absence of Cytochalasin B. FIG. 3B shows the result of actin filament staining with Fluorescence-phalloidin in the presence of Cytochalasin B. Fig. 4 shows treatment with 10 μg / ml Cytochalasin B for 2 hours, seeding rat glomerular endothelial cells on day 4 in transwell, and water transfer to the contralateral side under the same hydrostatic pressure when confluent Was measured. OD is the optical density. In the endothelial cell group (Cells with CB) treated with Cytochalasin B (CB), significant water migration was observed as compared with the non-treated endothelial cell group (Cells without CB). FIG. 5-A shows an SEM image (15000 times) after 7 days when Cytochalasin B is not added to a confluent monolayer culture of HUVEC. FIG. 5-B shows an SEM image (15000 times) after 7 days of adding Cytochalasin B 10 μg / dl to a confluent monolayer culture of HUVEC. A clear expansion of the fenestra is observed. FIG. 5-C is a photomicrograph obtained by adding Latrunculin A (LA) to the HUVEC culture solution and reacting for 2 hours, then changing the medium and culturing for 4 days and observing with SEM (× 15000). Also in HUVEC, the enlargement of the fenestra diameter was recognized with the addition of LA.

Claims (10)

  The blood filter is a bioartificial glomerulus comprising a hollow fiber blood filtration membrane coated with an inner surface of the patient's own endothelial cells and a filtration container containing the same, and lasts for at least 7 days. A bioartificial glomerulus that increases the number of cell membrane fenestra, enlarges its pore size to 0.5-2 times the phenestral pore size of glomerular endothelial cells, or promotes blood filtration by the effect of both .   The bioartificial glomerulus according to claim 1, wherein the endothelial cells are vascular endothelial cells.   The bioartificial glomerulus according to claim 1 or 2, wherein the endothelial cells are derived from endothelial progenitor cells collected from a patient.   The bioartificial glomerulus according to any one of claims 1 to 3, wherein the number of the phenestra is increased or the pore diameter of the endothelial cells is increased by application of an actin filament-blocking agent.   The bioartificial glomerulus according to claim 4, wherein the actin filament interfering drug is selected from the group consisting of Cytochalasin B, Latruncyulin A, Swinholide A, Halichondramide / Dihydorohalichondramide, Jasplakinolide / Misakinolide.   The bioartificial glomerulus according to claim 1, wherein the bioartificial glomerulus is a hemofilter that applies an anticoagulant drug to the hollow fiber to minimize systemic anticoagulation therapy.   The bioartificial glomerulus according to any one of claims 1 to 6, wherein the hollow fiber is formed of a membrane made of polysulfone, cellulose acetate, or polyimide.   The bioartificial glomerulus according to claim 4 or 6, wherein the drug is applied to the hollow fiber in advance, or the fluid containing the drug is allowed to flow prior to use. The inner surface of the hollow fiber, which is a blood filtration membrane, is coated with the patient's own endothelial cells, then housed in a filtration container, and when used for hemofiltration, an actin filament interfering agent is applied to the cell membrane fenestra. Production of a bioartificial glomerulus characterized by increasing the number, expanding the pore diameter to 0.5-2 times the phenestral pore diameter of glomerular endothelial cells, or promoting blood filtration by the effects of both Method.   As an applied amount of the actin filament interfering agent, the agent expands the pore diameter of the plasma membrane fenestra of the endothelial cells by 0.5 to 2 times the phenestral pore diameter of the glomerular endothelial cells, and the expansion of the pore diameter continues at least. The method for producing a bioartificial glomerulus according to claim 9, wherein the amount is sufficient to continue for 7 days.