JP3059127B2 - Artificial organs using tissue engineering - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an extracorporeal circulation type artificial organ having a hollow fiber.

[0002]

2. Description of the Related Art The liver is responsible for most of metabolism and regulation of metabolism, such as metabolism of carbohydrates, proteins, lipids, nucleic acids, vitamins, hormones, etc., and detoxification and excretion of endogenous and exogenous substances. It is an organ that performs complex and enormous functions necessary for living organisms. Acute liver failure such as fulminant hepatitis or postoperative liver failure
If the liver function is assisted for a certain period of time until the liver regeneration, life saving is considered possible. However, since the liver is an organ having a complicated and enormous function as described above, it is difficult to assist the liver function with an artificial liver using a purely artificial technique. On the other hand, a hybrid artificial liver equipped with hollow fibers having hepatocytes, which are the main functions of liver function, attached to the surface has been proposed, which is advantageous for application to extracorporeal circulation (Shinichi Kasai, Masayuki Sawa et al., Abdominal care 11, 827, 1
991, Tobe Seishiro, Takei Yuka et al., Artificial Organs 21, 1045, 19
92, Kazuyoshi Takeshita, Haruaki Ishibashi et al., Artificial Organ 22 (1), 153, 199
3, see JP-A-9-56814).

[0003] Similarly, hollow fiber artificial kidneys have been proposed which are used to remove waste products to be excreted by the kidneys from blood when the biological function is reduced due to renal failure or the like.

[0004] By the way, chemotherapy by administering a drug, such as administering an anticancer antibiotic to a cancer patient, has been actively performed. It may be better to discharge to Further, there is a case where it is desired to selectively excrete the toxic substance outside the body when the toxic substance is accidentally ingested for a purpose other than the purpose of treatment. Therefore, development of a system capable of selectively discharging such drugs and poisons is desired.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a hollow fiber type artificial organ capable of selectively discharging a drug or a toxic substance.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, adhered a transformed cell into which a gene relating to transport of a specific drug was introduced to the outer surface of a hollow fiber. Then, when a solution containing the drug was perfused into the hollow fiber, the drug was found to be selectively transported by the cells, thereby completing the present invention.

The present invention relates to an extracorporeal circulation type artificial organ comprising hollow fibers to which cells are attached, wherein the cells are transformed cells containing a recombinant vector containing a gene related to drug transport. is there.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, as a gene relating to drug transport, for example, a gene relating to an organic anion transporter (for cloning of this gene, see Kanai N., Lu R., Satriano J.
A., Bao Y., Wolkoff, AWand Schuster VL, "Ident
ification and characterizaion of a prostaglandin t
ransporter ", Science (Wash) 268: 866-9 (1995)),
Genes related to organic cation transporters (For cloning of this gene, see Lopez-Nieto-CE, You-G, Bush-KT, Ba
rros-EJ, Beier-DR and Nigma-SK, "Molecular cloning
and characterization of NKT, a gene product relate
d to the organic cation transporter family that is
almost exclusively expressed in the kidney ", J. B
iol. Chem. 272 (10), 6471-8 (1997)), a peptide transporter gene (for this gene, refer to Boll-M, He
rget-M, Wagener-M, Weber-WM, Markovich-D, Biber-J,
Clauss-W, Murer-H and Daniel-H, "Expression cloni
ng and functional characterization of the kidney c
ortex high-affinity proton-coupled peptide transpo
rter ", Proc. Natl. Acad. Sci. USA, 1996 Jan 9,
93 (1), 284-9). Other genes related to water channels (Fushimi-K, Uchida-S, Hara-Y, Hirata-Y, Marumo-Fa
nd Sasaki-S, "Cloning and expression of apical mem
brane water channel of rat kidney collecting tubul
e ", Nature, 361 (6412), 549-52 (1993)), a gene related to cytochrome P450 (for this gene, Emi-Y, Chi
jiiwa-C and Omura-TA, `` different cytochrome P450 f
orm is induced in primary cultures of rat hepatocy
tes ", Proc. Natl. Acad. Sci. USA, 1990Dec, 87 (24),
9746-50) can also be used. Specifically, the human multidrug resistance gene MDR-1 related to the transport of digoxin and / or doxorubicin (Kiona N., Tsubota J., Kakehi Y., Komano T., Go
ttesman MM., Pastan I. and Ueda K., "P-glycoprotei
n carries Gly185 with an altered patteren of multi
dtug resistance ", Biochem. Biophys. Res. Commun.,
162, 224-231 (1989)).

The preparation of a recombinant vector containing the above gene and the preparation of a transformed cell using the recombinant vector can be performed, for example, as follows. Examples of the vector incorporating the gene include, for example, an expression plasmid p-
HIR) and pbluescript (Strategene). The recombinant vector is pHaMAIRESneo (Mets etal Vir
Ueda et al. (1996) 217: 230-241) incorporated MDR-1 (Taguchi et al., Biochemistry 36: 8).
883-8889 (1997)).

In the present invention, the host cells include hepatocytes, kidney cells and the like, and fibroblasts, cardiomyocytes and the like can also be used. SV4 in these cells in primary culture
Created by immortalizing with 0large Tantigen. The method of introduction is by electroporation or lipofection.

In the present invention, as the hollow fiber to which the transformed cells are adhered, porous fibers conventionally used as artificial kidneys, artificial livers and the like can be used. As the material of the hollow fiber, for example,
Hydrophilic polyolefin, cupra ammonium rayon and the like can be mentioned. The thickness of the hollow fiber is
Usually, it is 5 to 70 μm, preferably 10 to 50 μm. Also, the inner diameter of the membrane is usually 100-400 μm,
Preferably it is 180 to 330 µm. Further, the pore size is usually from 0.01 to 3 μm, preferably from 0.1 to 0.3 μm. Membrane area hollow fibers in an artificial organ of the present invention is usually from 0.99 to 300 cm ^2, preferably 160
Is ~200 cm ^2.

FIG. 1 shows an embodiment of the artificial organ according to the present invention. In FIG. 1, an upstream lid 2 and a downstream lid 3 are provided at both open ends of a cylindrical housing 1. The upstream lid 2 is provided with a blood inlet 4, and the downstream lid 3 is provided with a blood outlet 5.
The housing 1 has a liquid inlet 6 and a liquid outlet 7.
Is provided. Further, inside the housing 1,
Many hollow fibers are provided. Both ends of these hollow fibers are combined into one, and are connected to the blood inlet 4 and the blood inlet 5. A space inside the housing 1 and outside the hollow fiber (outside fiber space) communicates with a liquid inlet 6 and a liquid outlet 7 provided in the housing 1.

FIG. 2 is a cross-sectional view of an enlarged portion of one hollow fiber among hollow fibers having transforming cells 8 attached to the outer surface, which are provided in a large number in the housing 1. A large number of transformed cells 8 adhere to the outer surface of the hollow fiber 9. According to the artificial organ of the present invention illustrated in FIG. 1, a specific drug in the blood flowing into the fiber passes through the pore of the fiber, is selectively transported by the transformed cells 8, and moves to the outer space of the fiber. I do. Also, at this time, by moving a phosphate buffer or the like into the outer space of the fiber, the transferred drug can be discharged out of the housing.

FIG. 3 is a diagram schematically showing the entire apparatus for producing the artificial organ of the present invention shown in FIGS. 1 and 2. This apparatus has a hollow fiber type module 10 containing hollow fibers similar to that shown in FIG. 1 except that no transformed cells are attached, and a bottle 11 containing a medium or the like.
, A peristaltic pump 12, a syringe 13 for injecting a cell suspension or the like, and an empty syringe 14. As the hollow fiber type module,
For example, a high-density cell culture system Cultureflo G (manufactured by Asahi Medical Co., Ltd.) can be used. In preparing this device, a high-density cell culture unit Cultureflo TC-50 (manufactured by Asahi Medical Co., Ltd.) in which a hollow fiber type module and a tube or the like are integrated can also be used.

The production of the artificial organ of the present invention can be performed, for example, by the method shown in FIG.
The device is placed in an incubator as shown in (1). First, a culture solution is put into a bottle 11, and the culture solution is circulated using a peristaltic pump 12. Then
Set the syringe 13 containing the cell suspension, inject the cell suspension into the outer space of the fiber, and use the syringe 12 and the syringe 13
Press alternately to disperse the cells into the voids. The culture solution
Usually, the culture is carried out while circulating at a rate of 0.5 to 10 ml / min, preferably 0.5 to 2 ml / min. The cultivation is usually performed at a temperature of 36 to 38 ° C. The culturing time is usually 24-240 hours.

As described above, the artificial organ of the present invention comprising a hollow fiber having a transformed cell adhered to the outer surface can be produced. In the case of hepatocytes, the density of the cells in the outer space of the fiber is usually 1 × 10 ⁷ to 1
× 10 ¹¹ cells / ml, preferably 1 × 10 ⁹ to 1 × 10 ¹⁰ cells / ml. In the case of kidney cells, usually 1 × 10 ⁸ to 1 × 10 ¹⁰
Particles / ml, preferably 1 × 10 ^{9 to} 5 × 10 ⁹ particles / ml. In dialysis and blood adsorption using a general dialysis membrane, there is a major problem that excretion of necessary (for example, albumin) occurs in a living body. However, this system has the great advantage that you can freely choose only what you want to remove. Further, by introducing antisense, it is also possible not to transport what is transported by this cell (for example, PAH).

The present invention is not limited to artificial organs having hollow fibers having transformed cells attached to the outer surface, but also includes artificial organs having hollow fibers having transformed cells attached to the inner surface. In this case, blood can be perfused into the hollow fiber outer space, and a drug to be discharged can be moved into the fiber and discharged together with a phosphate buffer or the like.

[0018]

[Example 1]

Preparation of Artificial Organ with Hollow Fiber Attached to Transformed Cells Containing Recombinant Vector Containing Human Multidrug Resistance Gene (1) Preparation of Rabbit Proximal Tubule Cultured Cell PTCL Male or Female with 1-2 kg Body Weight Japanese White rabbi
After anesthetizing t (Japanese white rabbit) with pentobarbitol or ether, the kidney was removed. Then, several proximal tubules (about 1 mm) were isolated from the kidney using tweezers under a stereoscopic microscope, and the medium (Dulbec 1: 1 (v / v)) was isolated.
o's modified Eagle medium, HAM F-12, and 10% fetal bovine serum). This was cultured in an incubator at 37 ° C. and 5% CO ₂ until the number of cells reached about 1 × 10 ⁶ to obtain primary cultured cells. Next, a fetal bovine serum-free medium was added to the obtained primary cultured cells instead of the above medium, and plasmid p-SVneo3 (1 μg / ml, Bethesda lob) containing SV40 large T antigen and neomycin resistance gene was added.
oratory) (5 μl) and Lipofectin (GIBC) (5 μl)
And cultured for 24-48 hours. Then, the medium was changed to a medium containing 10% fetal bovine serum and 40 μg / ml neomycin, and the culture was continued as it was. Thus, a neomycin-resistant cell line PTCL was prepared.

(2) Introduction of human multidrug resistance gene (MDR-1) into PTCL Human MDR-1 was inserted into the multicloning site of the expression plasmid p-HIR, and selected by adding neomycin.
Thus, a plasmid vector p-HIRMDR1 containing MDR-1 was prepared. That is, this plasmid vector p-HIRMDR1 has pH
Ueda et al. incorporated MDR-1 based on aMAIRESneo (Mets et al. Virology (1996) 217: 230-241) (Taguchi
etal, Biochemistry 36: 8883-8889 (1997)).

The obtained plasmid vector p-HIRMDR1 and the PTCL obtained above were placed in an electroporation container, and p-HIRMDR1 was introduced into PTCL as described below.
Mix 2 × 10 ⁶ PCTLs / 30 μl medium with 10 μg / 10 μl of pHIRHORI and use GeneRulser from BioRad to apply a voltage of 500 V for 30 μsec. 300μ mix it again to DMEM / HAMF ^'-1210% FBS
Culture with g / ml colchicine. As a result, only cells expressing colchicine resistance, that is, MDR-1 well expressed, can be cultured. The transformed cell PTCL (pH
The IRMDR1), was mixed with medium ^{(DMEM / HAMF '-12 10%} FBS), were cultured to 1 × 10 ⁵ cells at 37 ° C.. This was used as a cell suspension.

(3) Cultivation of PTCL (p-HIRMDR1) in hollow fiber PTCL (p-HIRMDR1) was cultured using the apparatus shown in FIG. This apparatus is a high density cell culture unit Cultureflo T / C-50 manufactured by Asahi Medical Co., Ltd. (material of hollow fiber: New aqueous polyolefin, thickness of hollow fiber: 50 μm, inner diameter of hollow fiber membrane: 330
μm, pore size: 0.3 μm, effective length of hollow fiber: 80
mm, the number of hollow fibers: 150, the membrane area of hollow fibers: 160 cm ² , the outer volume of hollow fibers: 2 ml, and the inner volume of hollow fibers: 0.1 ml).

First, a bottle containing a phosphate buffer was connected as the bottle 11, and the phosphate buffer was circulated using the peristaltic pump 12 at a flow rate of 10 ml / min for 1 hour. Further, a syringe containing a phosphate buffer was connected as the syringe 13, and 10 ml of the phosphate buffer was injected into the space outside the fiber. Next, the device was placed in an incubator, and the phosphate buffer bottle was placed in a medium (DMEM / HAMF ^' -1).
2 Replace the bottle with 10% FBS)
After circulating for 48 hours at / min, it was confirmed that there was no contamination. Further, the medium outside the fiber was replaced with a phosphate buffer and a medium was added, and the medium was replaced several times.

Next, the circulation of the medium was stopped, a syringe containing the transformed cell suspension obtained in the above (2) was connected as the syringe 13, and the cell suspension was injected into the outer space of the fiber. The cells were dispersed in the voids by pushing the syringe 12 and the syringe 13 alternately. Without circulating the medium,
Leave at 30 ° C. for 30 minutes. Thereafter, the pump 12 is operated from an extremely low speed, and the flow rate is increased to 2 ml / min over 6 hours, and the pump 12 is operated at 48-72.
Culture was performed for hours. In this way, an artificial organ as shown in FIG. 1 was provided, which was provided with a hollow fiber having a large number of transformed cells attached to the outer surface of the hollow fiber.

[Example 2] Evaluation of drug transport ability of the artificial organ of the present invention in vitro The transport ability of digoxin was evaluated using the artificial organ of the present invention shown in FIG. 1 and prepared in (3) above. . FIG. 4 shows a test apparatus used in this example. 1: 1 (v / v) Dulbeco's
A modified Eagle medium, a medium containing HAM F-12 and 10% fetal bovine serum, and a predetermined concentration of digoxin (7.25 ng / ml, 5.
85 ng / ml and 4.05 ng / ml), 2 μg / ml of paraaminohippuric acid and 1 μm of inulin.
The container 15 containing 50 ml of the mixed solution with g / ml was connected to the blood inlet 4 of the artificial organ 10 via a syringe pump. The mixed solution was perfused into the artificial organ using the syringe pump 16 at a flow rate of 1 ml / hour. Then, the perfusate was collected in the collection container 17, and the amount of each drug discharged for 30 minutes was measured. Also, 50 hours later, verapamil, which is an MDR-1 blocker, was added to the container 15 and perfused, and the concentration of each drug in the perfusate collected in the collection container 17 was measured.

As a comparative example, a test similar to the above was conducted using an artificial organ having hollow fibers to which renal cells not transfected with the gene MDR-1 were attached (MD
R (-)).

FIG. 5A shows the change in the transport amount J _digoxin (ng / 30 min) of digoxin in the perfusate from the inside to the outside of the hollow fiber in each test. Also, when the concentration of digoxin in the mixed solution of 4.05 ng / ml, the transport volume J _PHA (μg / 30min) to the outside from the hollow fiber para hippuric acid in the perfusate, inulin concentrations J _inulin ( (g / 30 min) are shown in FIGS. 5 (B) and (C), respectively. From these results, PTCL (pH
It turns out that IRMDR1) selectively transports digoxin.

Example 3 A test was performed in the same manner as in Example 2 except that 6 μg / ml of doxorubicin (ADM) was used instead of digoxin. FIG. 6A shows the change in the transport amount J _ADM (ng / 30 min) of ADM in the perfusate in each test. In addition, the transport amount of paraaminohippuric acid J _PHA (μg / 30 min) in the perfusate and the concentration of inulin J
The change in the concentration of _inulin (μg / 30min) was
These are shown in (B) and (C). From these results, PTCL (p-HIRMDR
It can be seen that 1) selectively transports ADM.

[0028]

According to the artificial organ of the present invention, specific drugs and toxic substances can be selectively discharged.

[Brief description of the drawings]

FIG. 1 is a view showing one embodiment of an artificial organ according to the present invention.

FIG. 2 is a cross-sectional view of an enlarged portion of a hollow fiber having a transformed cell adhered to an outer surface.

FIG. 3 is a view schematically showing an entire apparatus for manufacturing an artificial organ according to the present invention.

FIG. 4 is a view showing an apparatus for testing an artificial organ according to the present invention used in an example.

FIG. 5 shows the transport amount J of digoxin in Example 2.
_digoxin, is a diagram showing changes in transport volume J _inulin transportation amount J _PHA and inulin para hippuric acid.

FIG. 6 shows the transport amount J _{ADM of ADM} , the transport amount J _PHA of para-aminohippuric acid and the transport amount J of inulin in Example 3.
It is a figure which shows the change of an _insulin .

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Housing, 2 ... Upstream lid, 3 ... Downstream lid, 4
... blood inlet, 5 ... blood outlet, 6 ... liquid inlet, 7 ...
Liquid inlet, 8: Transformed cells, 9: hollow fiber, 10: artificial organ, 11: bottle, 12: pump, 13: syringe, 14: syringe, 15: container, 16: pump, 17: collection container

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12N 15/09 C12N 5/00 B (58) Investigated field (Int.Cl. ⁷ , DB name) A61M 1/14-1 / 36 C12M 3/00 C12N 5/10 C12N 15/09

Claims (5)

(57) [Claims] 1. An extracorporeal circulation type artificial organ comprising hollow fibers to which cells are attached, wherein said cells are transformed cells containing a recombinant vector containing a gene related to drug transport. 2. The artificial organ according to claim 1, wherein the host cell is a hepatocyte. 3. The artificial organ according to claim 1, wherein the host cell is a kidney cell. 4. The artificial organ according to claim 1, wherein the gene is involved in transport of digoxin and / or doxorubicin. 5. The artificial organ according to claim 1, wherein the gene is a multidrug resistance gene.