JPH05337176A - Hybrid-type artificial pancreas - Google Patents

Abstract

PURPOSE:To make an immunosuppressant unnecessary even when implantation is performed between animals each with an antibody and to keep the value of blood sugar at an normal value by forming Langerhans's island of the pancreas with hybrid-type artificial pancreas comprehended and fixed with an agarose gel with an agarose concn. of 7-30wt.%. CONSTITUTION:A hybrid-type artificial pancreas formed by comprehending and fixing a pancreas Langerhans's island secreting insulin in a living body in a semipermeable membrane of an agarose gel with an agarose concn. of 7-30wt.%, is implanted in the body of a patient. The artificial pancreas is comprehended and fixed in an agarose gel semipermeable membrane and is separated from a living body protective system (an immunity system, etc., or the patient. It is possible thereby to keep the value of blood sugar at a normal value for a long time without any immunosuppressant even when implantation is performed between animals each with a natural antibody and/or an antibody to the pancreas Langerhans's island.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an artificial pancreas for substituting a pancreatic function for a diabetic patient whose pancreatic function is impaired or deteriorated, and more particularly to a hybrid type artificial pancreas.

[0002]

2. Description of the Related Art At present, the method of injecting an insulin preparation occupies the majority of diabetes treatment methods. That is, a method has been adopted in which the required amount of insulin preparation is administered by injection according to the blood glucose concentration of the patient measured in advance. However, a patient's blood glucose concentration changes continuously. Therefore, insulin cannot be administered in response to continuous changes in blood glucose concentration by injection of the above insulin preparation. Therefore, the method of injecting an insulin preparation cannot physiologically control glucose metabolism in a patient, and it has been very difficult to prevent complications such as vascular disorder and renal disorder.

Therefore, a hybrid type artificial pancreas has been attracting attention as one that enables administration of insulin according to a continuous change in blood glucose level of a patient. A hybrid artificial pancreas is a tissue that secretes insulin in a living body, that is, pancreatic islets of Langerhans (hereinafter,
Abbreviated as pancreatic la island. ) Is entrapped and immobilized in a semipermeable membrane and is used by being transplanted into the body after being isolated from the patient's biological defense system (immune system etc.).

As a material for comprehensively immobilizing pancreatic islets in the hybrid type artificial pancreas, as shown in the following references, 2.5-5% by weight agarose gel or 10-30% by weight is used. Agarose degradation product gels have been proposed. Reference: Hiroo Iwata, Osamu Amamiya, Takehisa Matsuda, Ryosuke Hayashi, Hisaki Takano, Tetsuzo Akutsu; Hybrid artificial pancreas, artificial organ, 16 (3), 1263 (1987). Reference: Hiroo Iwata, Kazuo Kobayashi, Hiroshi Shimizu, Osamu Amamiya, Tetsuzo Akutsu; Application of hybrid artificial pancreas, islet with agarose microbeads to allograft between mice, artificial organ, 20 (1), 213 (1991). .. References: Tatsuya Takagi, Hiroo Iwata, Osamu Amamiya; Application of microencapsulated la islets to xenotransplantation, Proceedings of the 29th Society of Artificial Organs P87. Reference: JP-A-62-215530.

In the above-mentioned literature, long-term engraftment is recognized when a hybrid artificial pancreas is prepared using agarose microcapsules having an agarose concentration of 5% by weight and allogeneic transplantation between mice is performed. However, when hamster-mouse xenotransplantation was performed, the blood glucose level was normalized for a long period of time in 2 out of 7 cases, but the blood glucose level increased within 10 days in the remaining 5 cases, and the diabetic state. Has been confirmed to return to (Reference). Therefore, when microcapsules having an agarose concentration of 5% by weight were used, it was not possible to obtain a product having excellent reproducibility and functioning as an artificial pancreas for a long period of time. In addition, when a humoral antibody such as IgG passes through, it may be rejected due to an immune reaction or the like.

Further, in the above-mentioned documents and documents, agarose hydrolyzed with an acid is used, and a hybrid artificial pancreas having a concentration of the agarose acid hydrolyzate of 11 to 20% by weight is produced. However, when hamster-mouse xenotransplantation was performed for this hybrid type artificial pancreas, the period in which the blood glucose level was normalized was 29 days in 2 cases out of 6, and 5 cases in 1 case.
It was 4 days, and the remaining 3 cases were only around 10 days. Therefore, the agarose acid decomposition product concentration is 11 to 20.
Even in the case of the hybrid type artificial pancreas of weight%, it was not possible to obtain one that is also excellent in reproducibility and can function as an artificial pancreas for a long period of time. In addition, there is also an example in which a rejection reaction involving an immune reaction is avoided by using an immunosuppressant together, and long-term engraftment is possible in hamster-mouse xenotransplantation (Reference). Toxicity due to immunosuppressants is considered to be a problem.

The experiments that have been conducted so far are transplants between animals in a relatively close relationship such as between hamster and mouse. That is, it is a transplant between animals in which the humoral antibody that the recipient has by nature against the transplanted pancreatic islets, that is, the natural antibody, does not pose a problem. On the other hand, when considering the application of the hybrid artificial pancreas to humans, since pancreatic islets of a species apart from humans such as pigs and cows are used, natural antibodies possessed by humans are It will be a problem. Furthermore, it must be taken into consideration that when the hybrid artificial pancreas is transplanted, the immune system of the recipient is activated due to the antigenicity of the transplanted pancreatic islets. Therefore, it is necessary to develop a hybrid artificial pancreas that is effective even for an animal in which antibodies against the transplanted pancreatic islets have been produced by previously immunizing with the transplanted pancreatic islets.

[0008]

The object of the present invention is to solve the problems of the hybrid type artificial pancreas using the agarose described above, and it is intended that animals having natural antibodies and / or antibodies against transplanted islets Another object of the present invention is to provide a hybrid artificial pancreas that can normalize blood glucose levels for a long period of time without using an immunosuppressant together even when used for transplantation.

[0009]

The present invention is a hybrid-type artificial pancreas characterized in that pancreatic islets are entrapped and immobilized on an agarose gel having an agarose concentration of 7 to 30% by weight. In the present invention, pancreatic islets are entrapped and immobilized on agarose gel, and this agarose is usually contained in agar. In the agarose gel used in the present invention, it is necessary that the agarose concentration, that is, the agarose polymer concentration is 7% by weight or more. This is because, in agarose gel, nutrients such as glucose and insulin secreted from pancreatic islets (molecular weight:
It is necessary to allow 6,000 to pass through, and pancreatic islets are humoral antibodies (IgG: molecular weight 16000) during transplantation.
This is because in order to prevent contact with 0), it is necessary to entrap and immobilize with a membrane through which the humoral antibody cannot pass.

The above polymer concentration means a solvent suitable for dissolving agarose (for example, Eagle's).
The agarose content (% by weight) with respect to MEM solution, physiological saline, phosphate buffered saline, etc. is shown. If the polymer concentration is less than 7% by weight, invasion of humoral antibody cannot be prevented, while if it exceeds 30% by weight, the viscosity of the agarose gel becomes too high and the pancreatic islets are encapsulated in the agarose gel. Becomes difficult. As for the above pancreatic islets,
Those isolated from humans, cows, pigs, hamsters, mice and the like can be used as appropriate. Isolation of pancreatic islets is carried out, for example, by digesting the excised pancreas with collagenase for a certain period of time and then fractionating by specific gravity centrifugation. An example of a method for isolating pancreatic islets from a hamster will be described.

The hamster was anesthetized with ether and the abdomen was opened,
Remove the pancreas. The excised pancreas is cut into small pieces with scissors to a length of about 1 mm, immersed in Hanks' solution, and washed several times. Next, collagenase was dissolved in Hank's solution to a concentration of 8 mg / ml, and 10 ml of the collagenase was added to the pieces of pancreas of 6 hamsters washed as described above, and the temperature was adjusted to 37 ° C.
Shake in water bath for 6-8 minutes to digest. After adding Hank's solution to the digest and pipetting, 120
Centrifuge for 2 minutes at 0 rpm.

The precipitate obtained by centrifugation is dispensed into six 15 ml centrifuge tubes, Hanks' solution is added, pipetting is carried out, and centrifugation is again carried out at 1200 rpm for 2 minutes. Ficoll-Conray solution (specific gravity = 1.095) was added to the obtained precipitate.
Add 5 ml to disperse. On this dispersion, a specific gravity of 1.0
Overlay two Ficoll-Conlay solutions of 72 and 1.048. Next, centrifugation is performed at 2000 rpm for 15 minutes, and the tissue pieces collected at the interface of the layers having specific gravities of 1.072 and 1.048 are collected. After washing the collected tissue pieces with Hanks' solution, Eagle's M containing 5% by weight of fetal bovine serum.
Disperse in EM culture medium and separate pancreatic islets under a stereoscopic microscope. In this way, about 2 per hamster pancreas
It is possible to collect 00 pancreatic islets.

In the hybrid type artificial pancreas of the present invention, the pancreatic islets collected as described above are entrapped and immobilized by agarose gel. When a high-concentration agarose gel is used for this comprehensive immobilization, the viscosity becomes high, and it is difficult to microencapsulate the islets of pancreatic islets. Also,
Even if microencapsulation is possible, only large beads will be formed, and there will be many pancreatic islets that are not entrapped and immobilized. Therefore, as described in the literature mentioned in the section of the prior art, it is conventionally 2.5 to 5% by weight.
An agarose gel of the following concentration was used. On the other hand, in the present invention, pancreatic islets are entrapped and immobilized in agarose gel having a high concentration of 7 to 30% by weight as described above. In addition, in this case, microencapsulation refers to immobilizing one or several pancreatic islets in a spherical shape.

The following first to third methods will be described as specific examples of the entrapping immobilization method. First, in the first method, agarose is added to Eagle's MEM culture solution, heated and melted, and the obtained solution is cooled to a temperature around 40 ° C. 100 ml of recovered pancreatic islets for 3 ml of this solution
Disperse about 0 to 3000 pieces. A hydrophobic liquid (liquid paraffin or silicone oil, etc.) that is immiscible with water and heated to a temperature around 40 ° C. is added to the resulting dispersion in an amount of about 5 to 10 times the amount of the agarose solution ( capacity)
In addition, the mixture is sufficiently stirred to suspend the agarose solution.

Next, the above-obtained agarose solution is gelated by cooling the obtained suspension. In this case, in order to prevent aggregation, it is necessary to cool with stirring. Thereafter, in order to remove the hydrophobic liquid, Hank's solution is added to the above suspension and centrifuged to collect the agarose gel microcapsules that have sunk in the lower layer. As described above, a hybrid artificial pancreas having a particle size of several tens of μm to several mm can be obtained.

In the second method, the agarose-containing Eagle's MEM culture solution in which pancreatic islets are dispersed is a hydrophobic liquid (liquid paraffin or silicone oil, etc.) which is not mixed with water from a nozzle and is 4 ° C. The microcapsules may be prepared by dropping into a cooled product in the vicinity. The method for producing microcapsules in which pancreatic islets are entrapped and fixed as described above can be performed using an agarose gel having an agarose concentration in the range of 1 to 15% by weight.

In the third method, the agarose is formed into a cylindrical shape. Specifically, after adding agarose to the solvent and heating and melting, the recovered pancreatic islets are dispersed in the agarose-containing Eagle's MEM culture solution cooled to a temperature of around 40 ° C. Next, one end of a hollow tube made of polyvinyl chloride, for example, is dipped in the obtained dispersion liquid, and the other end is inserted with a syringe or the like and suctioned, thereby filling the dispersion liquid in the tube and cooling. To gel. Next, by extruding from the tube, a columnar hybrid-type artificial pancreas can be produced.
Even in such a columnar hybrid artificial pancreas, entrapping immobilization of pancreatic islets can be reliably performed when the agarose concentration is in the range of 1 to 30% by weight.

The inventors of the present invention have further investigated the method of entrapping immobilization of pancreatic islets in a high-concentration agarose gel. As a result, the use of a water-soluble polysaccharide can reduce the solution viscosity. Therefore, it was found that pancreatic islets can be more comprehensively immobilized by a high-concentration agarose gel. For example, in the first and second methods, the solution viscosity can be reduced by adding a water-soluble polysaccharide to Eagle's MEM culture solution in addition to agarose. The polysaccharide may be removed together with the hydrophobic liquid in a later step.

As the water-soluble polysaccharide used for the entrapping immobilization method, amylose, pullulan, dextran, dextrin and the like are preferably used. Also, the amount of this polysaccharide added is 100 parts by weight of agarose,
2 to 50 parts by weight is preferable. This is because if it is less than 2 parts by weight, the effect of lowering the viscosity is not sufficient, and if it exceeds 50 parts by weight, no gel is formed. The prepared hybrid artificial pancreas is dispersed in, for example, 5% by weight of fetal calf serum-containing Eagle's MEM culture solution at 37 ° C.
The cells are cultivated under 5% carbon dioxide gas at the temperature of 1, and at the time of transplantation, they are washed with Eagle's MEM culture solution or Hank's solution to remove serum, and then used. The culturing period may be about one day, or long-term culturing may be performed to reduce the antigenicity, and further culturing may be performed at a low temperature such as 24 ° C.

[0020]

[Function] When entrapping pancreatic islets with agarose gel in a comprehensive manner, if the agarose concentration is 7% by weight or less, the ability of the agarose gel to block humoral antibodies is insufficient. It is difficult to protect the islets of the pancreas from the cellular damage involved. In the present invention, since the pancreatic islets are entrapped and fixed by the agarose gel having the agarose polymer concentration of 7% by weight or more, invasion of humoral antibody can be reliably prevented.

[0021]

EFFECTS OF THE INVENTION According to the present invention, therefore, the pancreatic islets are comprehensively fixed by agarose gel having an agarose concentration of 7 to 30% by weight, so that the pancreatic islets are protected from cell damage involving humoral antibodies. be able to. Therefore, it is possible to provide a hybrid artificial pancreas capable of engraftment for a long period of time without using an immunosuppressant in transplantation between animals having a natural antibody and / or an antibody against the transplanted pancreatic islets.

[0022]

Example 1 Agarose LGT (manufactured by Nacalai Tesque, Inc.) 225 mg
Was added to 3.0 ml of Eagle's MEM culture solution to adjust the polymer concentration to the solvent to 7.5% by weight, and sterilized by heat in an autoclave at 120 ° C. for 20 minutes, and at the same time, agarose was dissolved. The solution was kept at 40 ° C. Approximately 2000 pancreatic islets isolated from the pancreas of a hamster by the collagenase treatment method were added to this solution.
A dispersion of 0.1 ml of Eagle's MEM culture solution was added and stirred, and then about 20 ml of liquid paraffin heated to 40 ° C. was further added and stirred to disperse agarose.

The agarose dispersion obtained as described above was ice-cooled with stirring to gel the agarose, about 30 ml of Hank's solution was added, and the mixture was centrifuged at 2000 rpm for 10 minutes. Collect the precipitated microcapsules by centrifugation,
The cells were dispersed in Eagle's MEM culture medium containing 5% by weight of fetal bovine serum and cultured at 37 ° C. and 5% carbon dioxide gas for 3 days. After culturing, using a stereoscopic microscope, only the microcapsules in which pancreatic islets were entrapped and fixed were collected to produce the hybrid artificial pancreas of Example 1.

Example 2 300 mg of agarose LGT (manufactured by Nacalai Tesque)
Was added to 3.0 ml of Eagle's MEM culture solution to adjust the polymer concentration to the solvent to 10% by weight, and a hybrid artificial pancreas of Example 2 was produced in the same manner as in Example 1.

Example 3 600 mg of agarose LGT (manufactured by Nacalai Tesque)
Was added to 3.0 ml of Eagle's MEM culture solution to a polymer concentration of 20% by weight with respect to the solvent, and this solution was sterilized by heating in an autoclave at 120 ° C. for 20 minutes and at the same time dissolved agarose, and then obtained. The solution was kept at 40 ° C. About 1 ml of the above solution was dropped onto a petri dish kept at about 40 ° C., and 150 pancreatic islets isolated from the pancreas of a hamster by the collagenase treatment method were added thereto.
Add 0 pieces dispersed in about 0.05 ml of Eagle's MEM culture solution, stir, and then fill by suction using a syringe into a hollow tube (internal diameter: 1 mm) made of polyvinyl chloride, The gel was formed by leaving it at room temperature. Then, it was extruded from the tube and the columnar gel was taken out. This cylindrical gel was dispersed in Eagle's MEM culture medium containing 5% by weight of fetal bovine serum,
The cells were cultured at 37 ° C and 5% carbon dioxide gas for 3 days. After the culture, under a stereoscopic microscope, the part where the pancreatic islets were not entrapped and fixed was excised to produce the hybrid artificial pancreas of Example 3.

Example 4 360 mg of agarose LGT (manufactured by Nacalai Tesque) and pullulan (manufactured by Wako Pure Chemical Industries, Ltd., average molecular weight 50,000-)
100,000) 50 mg was added to 3.0 ml of Eagle's MEM culture solution to give a polymer concentration of 1 to the solvent.
A hybrid artificial pancreas of Example 4 was produced in the same manner as in Example 1 except that the content was 2% by weight.

Example 5 Instead of pullulan, dextran (manufactured by Nacalai Tesque,
Example 5 was carried out in the same manner as in Example 4 except that 100 mg of the average molecular weight of 50,000 to 70,000 was used.
The hybrid type artificial pancreas of

Comparative Example 1 Agarose LGT (Nakarai Tesque) 150 mg
Was added to 3.0 ml of Eagle's MEM culture solution to give a polymer concentration of 5% by weight with respect to the solvent, and a hybrid artificial pancreas of Comparative Example 1 was produced in the same manner as in Example 1. Comparative Example 2 A hybrid artificial pancreas of Comparative Example 2 was produced in the same manner as in Example 4 except that the polymer concentration was further changed to 5% by weight without adding pullulan.

Comparative Example 3 Instead of the hybrid artificial pancreas, a pancreatic islet itself of a hamster was prepared as Comparative Example 3. The performance of each of Examples 1 to 5 and Comparative Examples 1 to 3 obtained as described above was evaluated by using them in the following transplantation test.

Transplantation Test 250 mg / kg of streptozotocin was intraperitoneally administered to the mice to make them diabetic. Heparin blood was collected from the subclavian vein 1 to 3 times a week, and the glucose concentration in plasma during non-fasting was measured. Diabetic mice whose plasma glucose concentration was 400 mg / dl or more twice consecutively were used for transplantation test. Sensitization was carried out by transplanting 1000 non-encapsulated hamster pancreatic islets into the abdominal cavity of the diabetic mouse. This sensitized diabetic mouse was used as an animal model in which natural antibodies were a problem. On the 14th day after the sensitization, about 1500 hybrid artificial pancreas were re-implanted into the abdominal cavity. Blood was collected once or twice a week, and it was assumed that mice whose plasma glucose concentration during non-fasting exceeded 250 mg / dl for two consecutive times returned to diabetes, and the blood glucose level was normalized until the first day. Shown in 1.

[0031]

[Table 1]

As is clear from Table 1, when the hamster pancreatic islets were directly transplanted to sensitized diabetic mice as in Comparative Example 3, the blood glucose level did not reach the normal level.
Further, in Comparative Examples 1 and 2, although the blood glucose level normalization period is extended to 32 days, it is understood that the effect is still insufficient. On the other hand, in the hybrid type artificial pancreas of Examples 1-5, it turns out that the blood glucose level normalization period can be extended to 100 days or more by far.

Claims (1)

[Claims] 1. A hybrid artificial pancreas characterized in that pancreatic islets of Langerhans are entrapped and immobilized in an agarose gel having an agarose concentration of 7 to 30% by weight.