JP4599315B2 - Bioartificial organ - Google Patents

Description

  The present invention relates to a bioartificial organ filled with animal cells and a method for producing the bioartificial organ.

  In recent years, bioartificial organs that combine artificial materials and living cells and take advantage of these advantages have been studied. For example, a bioartificial liver combining hepatocytes and artificial materials (for example, see Non-patent Document 1), a bioartificial pancreas combining pancreatic cells and artificial materials (for example, see Patent Document 2), kidney cells and artificial materials A bioartificial kidney in combination with (for example, see Non-Patent Document 2) is known.

  For example, in the treatment of liver failure, a plasma exchange method using a plasma separator, a plasma adsorption method using a plasma separator and a bilirubin adsorber, and the like have been performed. However, the conventional blood purification method using only artificial materials such as plasma separation membranes and adsorbents has a low lifesaving rate for patients, and expectations for a bioartificial liver have been gathered.

  Bioartificial organs are used with cells filled in a bioartificial organ module. Conventionally, cells to be filled are grown outside the module, and then the cells are collected and filled into the module. Commonly used as an organ.

  Conventionally, various cells have been studied as cells filled in bioartificial organs. For example, in the case of a bioartificial liver, primary porcine hepatocytes (see, for example, Non-Patent Document 1), human hepatocytes derived from liver cancer (for example, see Non-Patent Document 3, and Patent Document 1), reversible immortalized human hepatocytes ( For example, a non-patent document 4), a cell produced by excising a reversible immortalized gene from a reversibly immortalized human hepatocyte (for example, refer to patent document 3), and the like are known.

  In the case of primary porcine hepatocytes, after removing the pig's fresh liver, it is separated into cells using a proteolytic enzyme such as collagenase or trypsin and filled into a module.

In the case of hepatoma-derived human hepatocytes, reversible immortalized human hepatocytes, and cells generated by excising a reversible immortalized gene from reversible immortalized human hepatocytes, a proteolytic enzyme is used as described above. Then, the cells are detached from the culture dish, and the collected cells are filled into the module. Here, in the cells generated by excising the reversible immortalized gene from the reversible immortalized human hepatocytes, the reversible immortalized human hepatocytes are seeded in a culture dish or the like containing the medium and cultured in an incubator or the like. Then, the reversible immortalized gene is excised, and then peeled and filled in the module. In the case of human hepatocytes derived from liver cancer, an attempt has been made to use an incubated liver cell after filling into a module.
Japanese National Patent Publication No. 5-508312 JP-A-7-246211 Japanese Patent No. 3454818 Demetrio et al., Anals of Surgary, 239, 660-670 (2004). H. D. Humes et al., Nature Biotechnology, Vol. 17, 451-455 (1999). N. L. Sussman et al., Artificial Organs, 18, 390-396 (1994) N. Kobayashi et al. Artif. Organs, Vol. 6, pages 236-244 (2003)

Conventionally, generally used bioartificial organ production methods cannot avoid the death of some cells during cell recovery using proteolytic enzymes. Can't be high. This is an obstacle to enhancing the function of the obtained bioartificial organ. On the other hand, bioartificial liver manufactured by incubating liver cells derived from liver cancer in the module increases the survival rate of the cells in the module, but the bioartificial organ filled with immortalized cells dispels safety concerns. Can not do it.
In one aspect, the present invention shows a high cell survival rate when used as a bioartificial organ, maintains a high function of cells when used as a bioartificial organ, It is to provide a highly safe bioartificial organ that solves at least one of sufficiently exhibiting the function of the above. In another aspect, the present invention is substantially free from cell loss during production, substantially free from cell function degradation during production, easily manufactured, and in a bioartificial organ. It is an object of the present invention to provide a highly safe method for producing a bioartificial organ that solves at least one of maintaining sufficient cells to function and reducing the time required for production.

That is, the gist of the present invention is as follows.
[1] (A) a module for a bioartificial organ;
(B) animal cells;
And (A) the bioartificial organ module comprises (c) a cell fixing substrate, and the animal cell (B) comprises (c) a cell fixing group in the bioartificial organ module (A). An animal-derived reversible immortalized cell that has been immobilized on a material and the animal cell (B) is immobilized in the bioartificial organ module (A) A bioartificial organ that is a cell produced by proliferating in A) and then excising the reversible immortalization gene from the cell in the bioartificial organ module (A), and
[2] (a) a chamber having a body fluid inlet, a body fluid outlet, and at least one cell inlet;
(B) a semi-permeable separation membrane;
(C) a cell fixing substrate;
At least, and
(1) The separation membrane (b) and the cell fixing substrate (c) are accommodated in the chamber (a),
(2) The separation membrane (b) is arranged so that spaces (I) and (II) are formed in the chamber (a),
(3) The space (I) communicates with the body fluid introduction port and the body fluid introduction port, and forms a body fluid flow path in which the body fluid introduced from the body fluid introduction port is led out from the body fluid delivery port. And
(4) The space (II) communicates with the cell introduction port and houses the cell fixing substrate (c).
Into the bioartificial organ module (A) having a structure, animal-derived reversible immortalized cells are introduced from the cell introduction port, immobilized on the cell fixing substrate (c), proliferated, and then reversible. The present invention relates to a method for producing a bioartificial organ characterized by cutting out a reversible immortalized gene from a sex immortalized cell in the bioartificial organ module (A).

  According to the bioartificial organ of the present invention, when used as a bioartificial organ, it exhibits a high cell survival rate and maintains the function of the cell at a high level. It has the excellent effect of being able to exert its function as a bioartificial organ sufficiently and being highly safe. In addition, according to the method for producing a bioartificial organ of the present invention, the number of cells sufficient to exert a function in the bioartificial organ without substantially losing cells at the time of production, reducing the function of the cells, etc. The bioartificial organ can be produced easily and with substantially reduced time required for production.

  In one aspect, the present invention comprises (A) a bioartificial organ module and (B) an animal cell, and the animal cell (B) is immobilized in the bioartificial organ module (A). And the reversible immortalized gene is transferred from the animal-derived reversible immortalized cell in which the animal cell (B) is immobilized in the bioartificial organ module (A). (A) It is related with the bioartificial organ which is the cell produced by cutting out.

  The bioartificial organ of the present invention is an animal cell immobilized in a bioartificial organ module, and the animal-derived reversible immortalized cell immobilized in the bioartificial organ module is reversible immortalized. One major feature is the use of cells produced by excising the activating gene.

  Therefore, according to the bioartificial organ of the present invention, the cells can be sufficiently proliferated in the bioartificial organ module at the time of manufacture, so that a sufficient number of cells can be maintained for use as an artificial organ. .

  According to the bioartificial organ of the present invention, since the animal cell is used, the reversible immortalized gene is excised after sufficiently growing the reversible immortalized cell in the bioartificial organ module. Can be manufactured. Therefore, according to the bioartificial organ of the present invention, an excellent effect is exhibited that it is not necessary to perform an operation of filling the grown cells into a container for use in the artificial organ immediately before use. Furthermore, according to the bioartificial organ of the present invention, it is possible to exhibit an excellent effect that it is not necessary to substantially perform the treatment of the cell with a proteolytic enzyme such as trypsin or collagenase after completion of the growth of the animal cell. Therefore, the bioartificial organ of the present invention is highly convenient and can hold a sufficient amount of cells for use as an artificial organ in a treatment or the like.

  In addition, the bioartificial organ of the present invention does not require substantial treatment of the cells with a proteolytic enzyme such as trypsin or collagenase after the completion of the growth of the animal cells at the time of manufacture. Exhibits an excellent effect that does not occur. Therefore, according to the bioartificial organ of the present invention, an excellent effect that the function of a cell suitable for use as an artificial organ can be sufficiently exhibited in a therapeutic application or the like.

  That is, in the bioartificial organ of the present invention, the animal cell preferably includes a reversible immortalized cell grown in the bioartificial organ module.

  In the present invention, after a reversibly immortalized cell is filled in a module for a bioartificial organ, an operation for growing the reversible immortalized cell in the module and a reversible immortalized gene from the propagated reversible immortalized cell are performed. The bioartificial organ using the module that holds the “cells from which the reversible immortalized gene has been excised” obtained by performing the excision operation is maintained with high cell viability and function when used as a bioartificial organ. This is based on the knowledge of the present inventor.

  “Bioartificial organ” as used in the present invention refers to, for example, those that metabolize and detoxify harmful substances in blood or plasma, those that produce and supply albumin, blood coagulation factors, etc., and produce physiologically active substances such as insulin, What is necessary is just what to supply, such as a bioartificial liver, a bioartificial kidney, a bioartificial pancreas, a bioartificial immune system, and a bioartificial endocrine system.

  The term “reversible immortalized cell” as used in the present invention means that a nucleic acid encoding a factor that can cause immortalization (hereinafter also referred to as “reversible immortalized gene”) is artificially retained or removed from the nucleic acid. A cell that retains the function of an expression product in a deficient manner. More specifically, the “reversible immortalized cell” is constructed by, for example, constructing a cell that can proliferate by temporarily introducing the reversible immortalized gene into a normal cell using genetic engineering technology, Thereafter, it refers to a cell that can be returned to a normal cell by specifically excising the integrated reversible immortalizing gene.

  In the present specification, the “reversible immortalizing gene” refers to a gene having an action of promoting cell division and proliferation. The reversible immortalizing gene is not particularly limited, and for example, simian virus 40 large T antigen gene (SV40Tag), ras gene, myc gene, human telomerase reverse transcriptase (hTERT) and the like can be used. Among these, the hTERT is a gene normally expressed in stem / progenitor cells of organs such as human blood, skin, intestinal mucosa, and uterine mucosa that are repeatedly regenerated in the life of humans, and is preferably used. .

  The reversible immortalized cell is preferably a reversible immortalized human cell when a bioartificial organ is used for human.

  The reversible immortalized cell can be prepared, for example, by a technique using a Cre / loxP site-specific recombination reaction. Examples of reversible immortalized cells produced by such a technique include reversible immortalized human cells. The reversible immortalized human cells are not particularly limited. For example, reversible immortalized human hepatocytes [for example, N. Kobayashi et al., Science, 287, 1258 to 1262 (2000). And human immortalized hepatocyte cell line TTNT-1 cells (accession number: FERM BP-7498)] described in Japanese Patent No. 3454818 (Patent Document 3), reversible immortalized human hepatic stellate cells [for example, Watanabe (See T. Watanabe) et al., Transplantation, Vol. 75, pages 1873 to 1880 (2003)], reversibly immortalized human mesenchymal bone marrow cells [e.g., Japanese Patent Application Laid-Open No. 2003-174870; Yoshiaki Shiba Et al., Chemical Engineering Papers, Vol. 27, pages 134 to 136]. Further, in the present invention, depending on the purpose of use of the bioartificial organ, an appropriate cell can be obtained by reversibly immortalizing a suitable cell by a technique using the Cre / loxP site-specific recombination reaction as appropriate. Cells may be used.

The bioartificial organ module is not particularly limited as long as it can be filled with reversible immortalized cells, then proliferate the cells in the module, and then cut out the reversible immortalized gene from the cells. Often,
(A) a chamber having a body fluid inlet, a body fluid outlet, and at least one cell inlet;
(B) a semi-permeable separation membrane;
(C) a cell fixing substrate;
It is preferable that at least be provided.

  The “body fluid” as used in the present invention is a liquid containing blood, plasma and serum of an individual to which a bioartificial organ is applied, for example, a patient (patient) to be treated, and is obtained by subjecting the blood to a plasma separator. It may be plasma.

  The shape of the “body fluid inlet”, “body fluid outlet”, and “cell inlet” is not particularly limited, but preferably has a shape that can be sealed with a stopper, a three-way stopcock, or the like.

  The “chamber” can be made of an existing polymer material, but it is preferable that the “chamber” has transparency that allows the inside to be observed from the viewpoint of facilitating observation of the passage state of cells and body fluids inside. Examples of the polymer material include polycarbonate, polysulfone, polymethyl methacrylate, polyethylene, and polypropylene. The chamber (a) preferably contains the separation membrane (b) and the cell fixing substrate (c).

  The “semi-permeable separation membrane” as used in the present invention is a “semi-permeable separation membrane” that does not allow the cells in the module for bioartificial organs to pass through, but has the permeability of a substance necessary as a bioartificial organ. Any film that exhibits “permeability” may be used.

  The separation membrane may be a separation membrane having a property of allowing the metabolite such as ammonia to permeate when the bioartificial organ is, for example, a bioartificial liver. In the case of a bioartificial pancreas, the separation membrane is secreted from a cell. Any separation membrane having the property of permeating insulin or the like may be used.

  The material of the separation membrane may be any material suitable for cell maintenance and function expression. For example, regenerated cellulose, cellulose diacetate, cellulose triacetate, polymethyl methacrylate, polysulfone, polyvinyl alcohol, ethylene-vinyl alcohol copolymer Coalescence etc. can be used conveniently.

  The separation membrane may be added with an additive imparting hydrophilicity such as polybilylpyrrolidone or polyethylene glycol.

  The membrane pore size of the separation membrane may be selected according to the purpose of the bioartificial organ, as long as a substance necessary as a bioartificial organ can pass through the separation membrane, but the cell may have a pore size that does not pass through the separation membrane. However, from the viewpoint of appropriately performing substance exchange between the blood, plasma, etc. of the patient (patient) and the cells through the separation membrane, it is preferably 1 nm or more, more preferably 5 nm or more, and through the separation membrane. From the viewpoint of suppressing cell leakage, it is preferably 1 μm or less, more preferably 400 nm or less.

  In the present invention, the form of the separation membrane is not particularly limited, and a flat membrane, a hollow fiber membrane or the like can be used. From the viewpoint of increasing the cell packing density and producing a small bioartificial organ, a hollow fiber membrane type separation membrane is more preferable.

  The thickness of the hollow fiber membrane is not particularly limited, but is preferably 5 μm or more, more preferably 10 μm or more from the viewpoint of sufficiently obtaining the pressure resistance performance of the hollow fiber membrane, and from the viewpoint of increasing the cell packing density. The thickness is preferably 300 μm or less, more preferably 100 μm or less.

  Further, the inner diameter of the hollow fiber membrane is not particularly limited, but is preferably 50 μm or more, more preferably 100 μm or more from the viewpoint of fluid permeability such as body fluid in the hollow fiber membrane, and the inside and outside of the hollow fiber membrane. From the viewpoint of permeation and diffusion of substances, it is preferably 500 μm or less, more preferably 200 μm or less.

  Space (I) and (II) are formed in the chamber (a) by arranging the separation membrane (b) to connect the body fluid inlet and the body fluid outlet in the chamber (a). Can be done. In such a case, the space (I) forms a body fluid flow path in which the body fluid introduced from the body fluid introduction port is led out from the body fluid outlet, and the space (II) communicates with the cell introduction port and the cell A stationary substrate (c) can be accommodated. When the separation membrane (b) is a hollow fiber membrane from the viewpoint of allowing the body fluid to pass through without drift and improving the efficiency of material exchange between the spaces (I) and (II), It is preferable that the space (I) is formed and the space (II) is formed outside the hollow fiber membrane.

  The cell-fixing substrate (c) can be inserted into the space (II) (for example, the space outside the hollow fiber membrane), and a cell-fixing group that can function by adhering reversible immortalized cells to the surface. Any material may be used, and from the viewpoint of culturing cells at high density, a cell fixing substrate made of fibers is more preferable. For example, a nonwoven fabric or a cotton-like fiber aggregate can be suitably used as the cell fixing substrate.

  In the aspect of the present invention, the “space (II)” may be a space outside the hollow fiber membrane in the chamber (a).

  As the material for the cell fixing substrate, animal cells, for example, human cells can be used as long as they adhere and are suitable for the growth of the cells. For example, polyester, polypropylene, polyethylene, polytetrafluoroethylene, polymethyl methacrylate, Examples thereof include ethylene-vinyl alcohol copolymer.

  As the cell fixing substrate used in the present invention, a cell fixing substrate including a fiber having at least a surface portion made of polyamino acid urethane is more preferable from the viewpoint of adhesion to cells.

  The polyamino acid urethane referred to in the present invention refers to a copolymer having a polyamino acid segment and a polyurethane segment, and more specifically, an average of four or more amino acid units that are continuously bonded and an average of polyamino acid and urethane. Refers to a copolymer (see, for example, Yoshiaki Shiba et al., Chemical Engineering Papers, Vol. 27, pages 134-136; JP 2001-136960 A).

  In addition, since the module for bioartificial organs is mainly used for the human body, the material of the module is safety (for example, low elution, sterilization resistance, etc.), body fluid compatibility, in particular body fluid, For example, it is preferable that the material has excellent compatibility in a portion that comes into contact with blood, plasma, or the like.

  The bioartificial organ of the present invention can be used for the production of reversible immortalized cells and excision of the reversible immortalized gene in the module for bioartificial organs during production. Particularly suitable for certain bioartificial livers. That is, the bioartificial organ of the present invention having human cells produced by cutting out a reversible immortalized gene from reversible immortalized hepatocytes as a cell, that is, the bioartificial liver maintains the high survival rate and function. It is obtained as a bioartificial liver filled with a large amount of cells and is suitable for the treatment of liver failure.

  The bioartificial organ of the present invention can be used as an in vivo bioartificial organ to be implanted in the body or as an extracorporeal bioartificial organ attached outside the body.

  According to the present invention, animal-derived reversible immortalized cells are introduced into the bioartificial organ module from the cell introduction port, immobilized on the cell-fixing substrate, proliferated, and then reversible immortalized. There is also provided a method for producing a bioartificial organ, characterized in that a reversible immortalized gene is cut out from the transformed cell in the bioartificial organ module.

  In many conventional manufacturing methods, immediately before using a bioartificial organ for treatment, the proteolytic enzyme is allowed to act on the cultured cells to cut the adhesion between the cells and the culture dish or between the cells. And the subsequent operation of filling the obtained cells into the bioartificial organ module. On the other hand, in the production method of the present invention, there is an excellent effect that neither the cutting operation nor the filling operation immediately before using the bioartificial organ for treatment is required. In addition, according to the production method of the present invention, since there is no cutting operation and filling operation for cells after completion of proliferation, the survival rate of the cells and the reduced function of the cells associated with the cutting operation and filling operations are reduced. An excellent effect of eliminating or reducing the adverse effect on the cells maintained in the cell is also exhibited. Therefore, according to the bioartificial organ obtained by the production method of the present invention, cells can be maintained with a sufficiently high survival rate to be used as an artificial organ even when used as an artificial organ in therapeutic applications. The excellent effect of expressing the high cell function suitable for use as an artificial organ is exhibited.

  In the production method of the present invention, the reversible immortalized cells can be introduced into the bioartificial organ module, for example, by introducing the reversible immortalized cells from the cell introduction port. Here, at the time of introduction, it is desirable that the reversible immortalized cells are suspended in a medium or the like in a state where the cells are dissociated from the viewpoint of evenly existing in the bioartificial organ module. In addition, as a bioartificial organ module into which cells are introduced, a sterilized one is usually used.

  After the reversible immortalized cells are introduced into the bioartificial organ module, the cell inlet may be sealed with a stopper, a three-way stopcock, or the like.

  The reversible immortalized cells introduced into the bioartificial organ module are subjected to appropriate immobilization conditions according to the type of cell, the type of cell fixation substrate, etc. By immobilizing (or adhering) to a cell fixing substrate, and further maintaining the immobilized reversible immortalized cells under appropriate culture conditions according to the cell type and the like, Growth of reversible immortalized cells can be performed. From the viewpoint of expressing a high cell function, it is preferable that the reversibly immortalized cells are preferably proliferated in a state in which several to several hundreds of cell clusters are formed in the bioartificial organ module. .

  The immobilization conditions are not particularly limited. For example, after the cells are filled in the bioartificial organ module, the method of fixing the module while shaking, the method of fixing the module while standing, the method using these in combination, etc. Can be adopted. Among them, a method of fixing the cells in a stationary state after the cells are uniformly filled in the module is preferable. Conditions such as temperature and time for fixation are not particularly limited, and can be appropriately selected in consideration of the characteristics of the cells used. Although not necessarily limited, in general, the temperature is preferably around 37 ° C., and the time is preferably from 10 minutes to 12 hours. Moreover, you may supply the air and a nutrient containing a carbon dioxide gas in a system as needed.

  The culture conditions are not particularly limited. In the case of reversibly immortalized human cells, for example, the culture is performed in a medium containing nutrients necessary for culture in air containing 5% by volume of carbon dioxide at 37 ° C. Is mentioned.

  The excision of the reversible immortalized gene from the reversible immortalized cell can be performed by, for example, a technique using the Cre / loxP site-specific recombination reaction. In addition, the reversible immortalized gene is excised from the reversible immortalized cell, for example, between a pair of LoxP sequences, together with the reversible immortalized gene, a nucleic acid encoding a marker protein, green fluorescent protein (GFP By using a system in which a nucleic acid encoding) is inserted, it can be confirmed as an indicator that a marker protein is not expressed.

  The bioartificial organ of the present invention can be evaluated, for example, in the case of a bioartificial liver, for example, by circulating a medium through the bioartificial liver in an in vitro experiment, or in the animal experiment or clinical test, blood or plasma of an animal or patient. Can be circulated through the bioartificial liver, and in these circulations, ammonia metabolism, urea synthesis, lidocaine metabolism, albumin synthesis, etc. can be measured over time.

  As an example (mode 1) of the form of the bioartificial organ of the present invention, a flow path for blood or the like is formed by a hollow portion of a semipermeable hollow fiber membrane, and a non-woven fabric or the like is formed in a region on the outer peripheral side of the hollow fiber membrane. And a bioartificial organ in which the hollow fiber membrane and the cell fixing substrate are housed in a chamber. In the bioartificial organ of aspect 1, the hollow fiber membrane has one end for introducing a bodily fluid such as blood into the body fluid inlet and the other end for deriving the treated body fluid such as blood after treatment. Connected to the body fluid outlet. Further, the chamber is provided with at least one, preferably two, cell introduction ports for seeding reversible immortalized cells on the cell fixing substrate occupying the cell accommodating portion.

  For example, a specific example of aspect 1 includes the bioartificial organ shown in FIG. Hereinafter, from the viewpoint of ease of explanation, the bioartificial organ of FIG. 1 will be described as an example.

The bioartificial organ of FIG. 1 includes a bioartificial organ module and animal cells 7 immobilized in the bioartificial organ module. Here, the animal cell 7 is a cell generated by excising a reversible immortalized gene from an animal-derived reversible immortalized cell immobilized in the bioartificial organ module in the bioartificial organ module. is there. On the other hand, the module for bioartificial organs is
A chamber 6 having a body fluid inlet 2, a body fluid outlet 3 and at least one cell inlet 5,
A semipermeable separation membrane (for example, a hollow fiber membrane) 1;
A cell fixing substrate 4;
At least, and
The separation membrane 1 and the cell fixing substrate 4 are accommodated in the chamber 6;
The separation membrane 1 is arranged so that spaces (I) and (II) are formed in the chamber 6;
The space (I) communicates with the body fluid inlet 2 and the body fluid outlet 3, and the body fluid flow path through which the body fluid introduced from the body fluid inlet 2 is led out from the body fluid outlet 3. Forming,
The space (II) communicates with the cell introduction port 5 and accommodates the cell fixing substrate 4 (cell accommodating portion).
It has a structure. Here, the cell introduction port 5 is arranged so that the cells can move from the outside of the chamber 6 to the cell fixing substrate 4. The body fluid inlet 2 and body fluid outlet 3 and the separation membrane 1 are connected by, for example, polyurethane.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention further more concretely, this invention is not limited to these Examples.

[Preparation Example of Reversible Immortalized Cells]
A human immortalized hepatocyte cell line TTNT-1 cell (accession number: FERM BP-7498) (provided by Dr. Naoya Kobayashi, Department of Gastroenterological Oncology, Okayama University Graduate School of Medical and Dental Sciences) was used. Final concentration 10wt% Fetal calf serum (Sanko Junyaku), final concentration 100U / mL penicillin, final concentration 0.1mg / mL Streptomycin [Penicillin-streptomycin solution (trade name: P071, Sigma)] glucose concentration 450mg / DL DMEM medium (Dulbecco's modified Eagle medium) (trade name: D5790, manufactured by Sigma) was placed in a roller bottle type incubator (catalog number: 480853, Corning, culture area 1700 cm ² ). The roller bottle type incubator was seeded with 3.0 × 10 ⁷ TTNT-1 cells and cultured in an incubator at 37 ° C. and 5% carbon dioxide concentration for 72 hours. After culture, the cells were detached from the culture vessel with a trypsin EDTA solution (trade name: T4049, manufactured by Sigma), and the cells were collected by centrifugation at 300 × g for 5 minutes. The obtained cells were dispersed in DMEM medium (liquid) and subjected to precipitation by centrifugation at 300 × g for 5 minutes to remove trypsin. The obtained TTNT-1 cells were suspended in 20 ml of DMEM medium to obtain a TTNT-1 cell suspension.

[Production of modules for bioartificial organs]
Using a polysulfone hollow fiber membrane as a separation membrane and a non-woven sheet made of polytetrafluoroethylene fibers surface-coated with polyamino acid urethane as a cell fixing substrate, a module A having the same form as in FIG. 1 was produced. The polysulfone hollow fiber membrane is produced according to the method described in Example 2 described in JP-A-6-165926. Moreover, the nonwoven fabric sheet which consists of the polytetrafluoroethylene fiber surface-coated with the polyamino acid urethane uses the nonwoven fabric sheet made from a polytetrafluoroethylene fiber [Co., Ltd., Yodogawa Paper Mill, Tommy Filek F, R-125], It was produced by coating the fiber surface with polyamino acid urethane according to the method described in Example 1 described in JP-A-2001-136960. The number of hollow fiber membranes in module A was 500, the effective length of the hollow fiber membranes was 10 cm, and the surface area of the cell fixing substrate was 140 cm ² (10 cm × 14 cm). The module A was filled with distilled water for injection as a filling liquid, and the module A was sterilized at 121 ° C. for 20 minutes by an autoclave sterilizer. The sterilized module A was primed with sterilized physiological saline so that cells could be filled.

[Example 1]
TTNT-1 cell suspension (number of cells: 2.3 × 10 ⁸ cells) obtained by the method described in the above preparation example was injected and filled from the cell inlet 5 in module A, and the cell inlet 5 was three-way. Sealed with a stopcock. This cell filling operation was performed aseptically in a clean bench. The body fluid introduction port 2 and the body fluid outlet port 3 were also closed with the attached stopcocks, and left in that state in an incubator at 37 ° C. for 4 hours to adhere TTNT-1 cells to the cell fixing substrate 4. Thereafter, a tube (trade name: JMS extension tube ET-3, inner diameter: 3.5 mm, manufactured by JMS Co., Ltd.) is inserted into the body fluid inlet 2 and body fluid outlet 3 in module A filled with TTNT-1 cells. Each tube was connected, and the tip of each tube was immersed in a container containing 500 mL of DMEM medium. A peristaltic pump was installed in the middle of the tube so that the medium could be circulated to module A.

  The module A, the tube, the container containing the DMEM medium, and the peristaltic pump are placed in an incubator with a carbon dioxide concentration of 5% and a temperature of 37 ° C., and the DMEM medium is circulated through the module A at a flow rate of 20 mL / min. TTNT-1 cells were grown.

  The medium in the container was changed every 24 hours, and the circulation of the medium was continued. The glucose concentration in the exchanged medium was measured by a trade name: GLU2 L type Wako (manufactured by Wako Pure Chemical Industries, Ltd.) and used as an index of cell proliferation.

  Four days later, an adenovector AxCANCre (RIKEN Genebank, RDB No. 1748) MOI = 50 capable of specifically excising a reversible immortalizing gene (hTERT) in the genome of TTNT-1 cells was introduced from the cell inlet 5. The reversible immortalized gene was excised specifically from the genome of TTNT-1 cells by further adding the same incubation for 3 days. In addition, glucose consumption was also measured as an index of the number of cells for these 3 days. In addition, excision of the reversible immortalized gene from TTNT-1 cells was confirmed by the disappearance of fluorescence from the cells.

  Thereafter, the module A was disassembled, and the cell fixing substrate 4 to which the cells adhered was taken out and transferred into 50 mL of the DMEM medium. A part of the cell fixing substrate 4 taken out was transferred into the medium, and then the survival rate of the cells adhering to the cell fixing substrate 4 was measured by trypan blue staining. Further, cells are recovered from a part of the cell fixing substrate 4 taken out using a trypsin solution (manufactured by Sigma), and adhered to the cell fixing substrate 4 by observing the number of recovered cells with a microscope. The number of cells ("fixed cell number") was measured.

[Comparative Example 1]
The TTNT-1 cell suspension obtained by the method of the above preparation example was incubated in a roller bottle type incubator similar to the above preparation example under the same operating conditions of 5% carbon dioxide gas and 37 ° C. as in Example 1. The culture was performed while changing the medium every 24 hours. Four days later, the adenovector AxCANCre was added to the roller bottle type culture container, and the culture was further continued for 3 days to excise the reversible immortalized gene from the TTNT-1 cells. Thereafter, the cells were treated with collagenase (manufactured by Sigma), and the cells were taken out of the roller bottle type incubator to obtain TTNT-1 cells from which the reversible immortalizing gene was excised.

4.1 × 10 ⁸ TTNT-1 cells obtained after excision of the reversible immortalized gene thus obtained were injected and filled into module A, and the cell inlet 5 was sealed with a three-way cock. The body fluid inlet and outlet were also closed with the attached stopcock, and in that state, the cells were allowed to adhere to the cell-fixing substrate by allowing them to stand in a 37 ° C. incubator for 4 hours. Thereafter, the module A was immediately disassembled and the cell fixing substrate to which the cells adhered was taken out and transferred into the medium, and then the cell viability and the number of fixed cells were measured in the same manner as in Example 1.

[Comparative Example 2]
Primary porcine hepatocytes were collected by a conventional collagenase method. That is, an EDTA 0.02% by volume solution was injected into a pig under anesthesia weighing 18 kg from a catheter inserted into the portal vein to perfuse the liver, and then 1000 mL of collagenase solution (manufactured by Sigma) was applied to the liver for 20 minutes. The liver was digested by perfusion. Thereafter, the liver was removed, the hepatocytes were released, and dispersed in 500 ml of Hank's solution at 4 ° C. The obtained dispersion was filtered through a mesh, centrifuged at 300 × g for 5 minutes, and the supernatant was discarded. Collagenase was removed by repeating the operation of adding 500 ml of Hanks' solution to the precipitated cells, dispersing the mixture, centrifuging at 300 × g for 5 minutes, and discarding the supernatant twice. The obtained primary pig hepatocytes were dispersed in Hank's solution to prepare a primary pig hepatocyte suspension. The survival rate of the obtained primary porcine hepatocytes was measured by trypan blue staining. As a result, the survival rate of primary porcine hepatocytes was 88%.

The obtained primary porcine hepatocytes (number of cells: 4 × 10 ⁸ cells) were filled in module A and allowed to stand at 37 ° C. for 4 hours to adhere the primary porcine hepatocytes to the cell fixing substrate. Thereafter, the module was immediately disassembled and the cell fixing substrate to which the primary porcine hepatocytes adhered was taken out and transferred into the culture solution, and then the cell viability and the number of fixed cells were measured in the same manner as in Example 1.

[Test Example 1]
Table 1 shows the results of the number of cells and the cell viability that were immobilized on the cell fixation substrate in module A in Example 1, Comparative Example 1 and Comparative Example 2.

  The results of Example 1 are as follows: the number of cells and the cell viability immediately before use as a bioartificial organ for a bioartificial liver obtained by expanding reversible immortalized human hepatocytes and cutting out an immortalized gene in module A. Indicates. The results of Comparative Example 1 correspond to the number of cells and the cell viability immediately before use of the bioartificial organ filled with reversible immortalized human hepatocytes in which the reversible immortalized gene was excised outside module A. . The result of Comparative Example 2 corresponds to the number of cells and the cell viability immediately before use of the bioartificial organ filled with primary porcine hepatocytes.

As a result, as shown in Table 1, in Example 1, the number of cells 7 days after filling was 3.9 × 10 ⁸ . The initial number of cells loaded was 2.3 × 10 ⁸ , indicating that reversibly immortalized human hepatocytes grew approximately 1.7 times in the module in 7 days.

  The cell viability of Example 1 is higher than the cell viability of Comparative Example 1 and Comparative Example 2, and the reversible immortalized cells are proliferated in the module, and the reversible immortalized cell in the module It is shown that a good bioartificial organ with high cell viability can be provided by cutting out the reversible immortalized gene. That is, unlike Comparative Example 1 and Comparative Example 2, since the trypsin treatment and the collagenase treatment are not performed several hours before use as a bioartificial organ, the bioartificial organ of Example 1 has a high cell survival rate. It was maintained.

[Example 2]
In the module A, the bioartificial organ A prepared by preparing the TTNT-1 cells in which the reversible immortalizing gene was excised by performing the same operation as in Example 1 until the completion of the incubation for 7 days in total was completed. Produced.

  To the obtained bioartificial organ A, 500 mL of a DMEM medium prepared by adding ammonium chloride to an ammonium ion concentration of 500 μg / dL was placed in an incubator with a carbon dioxide concentration of 5% and a temperature of 37 ° C. using a perista pump. Circulation was performed at a flow rate of 20 mL / min for 24 hours.

  The concentration of ammonia in the culture medium before and after the 24-hour circulation was measured using an ammonia test Wako (Wako Pure Chemical Industries, Ltd., colorimetric method modified by Fujii / Okuda method), which is a clinical test kit. The ammonia treatment function was evaluated.

  Further, the bioartificial organ A after circulation is decomposed to take out the cell fixing substrate, and using a trypsin solution (manufactured by Sigma), TTNT-1 cells from which the reversible immortalizing gene has been excised are recovered, and the cell fixing group is recovered. The number of cells adhering to the material (“number of fixed cells”) was counted. In addition, the number of cells in the module that had not adhered to the cell-fixing substrate was also measured, and the sum of the number of cells and the number of fixed cells was recorded as “total number of cells in the module”.

Example 3
A bioartificial organ B was prepared in the same manner as in Example 2 except that a cell fixing substrate not coated with polyamino acid urethane was used. For bioartificial organ B, the ammonia treatment function and the number of cells (number of fixed cells and total number of cells in the module) were measured in the same manner as in Example 2.

[Comparative Example 3]
In the same manner as in Comparative Example 1, TTNT-1 cells were expanded and reversible immortalized genes were excised in a roller bottle type incubator, and further treated with trypsin to remove the reversible immortalized genes. Obtained. The obtained cells were filled into the module A, left for 4 hours, and the same procedure as in Comparative Example 1 was performed until the cells were allowed to adhere to the cell fixing substrate, thereby producing a bioartificial organ C.

  For bioartificial organ C, the ammonia treatment function was evaluated and the number of cells (the number of fixed cells and the total number of cells in the module) was measured in the same manner as in Example 2.

[Test Example 2]
Table 2 shows the evaluation results of the ammonia treatment function of Example 2, Example 3 and Comparative Example 3, and the measured number of cells (number of fixed cells and total number of cells in the module).

  As a result, as shown in Table 2, when a medium supplemented with ammonium chloride at a concentration of 500 μg / dL was circulated through the bioartificial organ A of Example 2 for 24 hours, the ammonium ion concentration in the medium after the circulation Decreased to 245 μg / dL, and the ammonia concentration reduction rate showed an excellent reduction rate of 51%.

  Moreover, as shown in Table 2, the ammonia concentration reduction rate in the case of the bioartificial organ B in Example 3 is 39%, which is slightly inferior to the bioartificial organ A in Example 2, but excellent ammonia treatment. The function was shown.

  On the other hand, in the bioartificial organ C of Comparative Example 3, the ammonia reduction rate was only 11% and did not have a sufficient ammonia treatment function.

  From these results, bioartificial organs A and B produced by excising the reversible immortalized gene from reversibly immortalized human hepatocytes in the module do not require trypsin treatment immediately before use. These functions were maintained at a high level, indicating that they have a high ammonia treatment function. In addition, it was shown that a bioartificial organ that is superior in function as an organ can be produced by using a cell-fixing base material containing a fiber whose surface is composed of polyamino acid urethane.

  According to the present invention, for example, a bioartificial organ such as a bioartificial liver that can be suitably used as a therapeutic tool for patients with liver failure and a bioartificial pancreas that can be suitably used as a therapeutic tool for diabetic patients is provided.

FIG. 1 is a schematic diagram showing an example of a bioartificial organ according to the present invention.

Explanation of symbols

1. Separation membrane (for example, hollow fiber membrane)
2. 2. Body fluid inlet 3. Body fluid outlet Cell fixation substrate (cell storage part)
5). 5. Cell introduction port Chamber 7. Animal cells (cells generated by reversible immortalizing genes excised from reversible immortalized cells derived from animals)

Claims (7)

(A) a module for bioartificial organs;
(B) animal cells;
And (A) the bioartificial organ module comprises (c) a cell fixing substrate, and the animal cell (B) comprises (c) a cell fixing group in the bioartificial organ module (A). An animal-derived reversible immortalized cell that has been immobilized on a material and the animal cell (B) is immobilized in the bioartificial organ module (A) A bioartificial organ which is a cell produced by proliferating in A) and then excising the reversible immortalized gene from the cell in the module for bioartificial organ (A).   The bioartificial organ according to claim 1, wherein the reversible immortalized cell is a reversible immortalized human cell. The bioartificial organ module (A)
(A) a chamber having a body fluid inlet, a body fluid outlet, and at least one cell inlet;
(B) a semi-permeable separation membrane;
(C) a cell fixing substrate;
At least, and
(1) The separation membrane (b) and the cell fixing substrate (c) are accommodated in the chamber (a),
(2) The separation membrane (b) is arranged so that spaces (I) and (II) are formed in the chamber (a),
(3) The space (I) communicates with the body fluid introduction port and the body fluid introduction port, and forms a body fluid flow path in which the body fluid introduced from the body fluid introduction port is led out from the body fluid delivery port. And
(4) The space (II) communicates with the cell introduction port and houses the cell fixing substrate (c).
The bioartificial organ according to claim 1 or 2, which has a structure.   The separation membrane (b) is a hollow fiber membrane, the space (I) is formed inside the hollow fiber membrane, and the space (II) is formed outside the hollow fiber membrane. Item 4. The bioartificial organ according to item 3. The bioartificial organ according to any one of claims 1 to 4 , wherein the cell-fixing substrate (c) includes a fiber having at least a surface portion made of a polyamino acid urethane. (A) a chamber having a body fluid inlet, a body fluid outlet, and at least one cell inlet;
(B) a semi-permeable separation membrane;
(C) a cell fixing substrate;
At least, and
(1) The separation membrane (b) and the cell fixing substrate (c) are accommodated in the chamber (a),
(2) The separation membrane (b) is arranged so that spaces (I) and (II) are formed in the chamber (a),
(3) The space (I) communicates with the body fluid introduction port and the body fluid introduction port, and forms a body fluid flow path in which the body fluid introduced from the body fluid introduction port is led out from the body fluid delivery port. And
(4) The space (II) communicates with the cell introduction port and houses the cell fixing substrate (c).
Into the bioartificial organ module (A) having a structure, animal-derived reversible immortalized cells are introduced from the cell introduction port, immobilized on the cell fixing substrate (c), proliferated, and then reversible. A method for producing a bioartificial organ, comprising cutting out a reversible immortalized gene from a sex immortalized cell in the bioartificial organ module (A).   The separation membrane (b) is a hollow fiber membrane, the space (I) is formed inside the hollow fiber membrane, and the space (II) is formed outside the hollow fiber membrane. Item 7. The manufacturing method according to Item 6.

Images