JP3204675B2 - Method for producing hybrid artificial pancreas - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

2. Description of the Related Art At present, insulin preparations are used for treating diabetes. That is, a method is used in which a required amount of an insulin preparation is administered in accordance with a previously measured blood glucose concentration of a patient. However, insulin cannot be administered in response to a continuous change in the blood glucose concentration of the patient, so that the glucose metabolism of the patient cannot be physiologically controlled. Therefore, it is difficult to prevent complications of diabetes such as vascular disorders and kidney disorders.

[0003] As a new treatment, a hybrid artificial pancreas has attracted attention. A hybrid artificial pancreas is a tissue that secretes insulin, that is, pancreatic islets of Langerhans (hereinafter, abbreviated as pancreatic islets), is immobilized in a semipermeable membrane and isolated from the patient's defense system (immune system, etc.). It is used after transplanting. Various materials have been studied as materials for encapsulating pancreatic islets in a hybrid artificial pancreas. For example, Sun et al. Entrap and immobilize pancreatic islets with a material consisting of a three-layered structure of an alginate-polylysine-alginate polyion complex (GMO'SHEA and AMSu).
n, DIABETES, 35, 943 (1986)).

On the other hand, Iwata et al. Have comprehensively fixed pancreatic islets with agarose and agarose degradation products [Hirio Iwata, Hiroshi Amemiya, Takehisa Matsuda, Ryosuke Hayashi, Hisaki Takano, Tetsuzo Akutsu, Artificial Organ, 16, 1263 ( 1987) and JP-A-62-215530].

[0005]

When allogeneic transplantation was performed using the hybrid artificial pancreas as described above, long-term engraftment was observed. Even if an artificial pancreas is used, one that is excellent in reproducibility and functions as an artificial pancreas for a long period of time cannot be obtained, and a patient may die due to hypoglycemia or be rejected due to an immune reaction or the like.

An object of the present invention is to solve the above-mentioned problem of the conventional hybrid artificial pancreas. Even if xenotransplantation is performed, blood glucose levels can be normalized over a long period of time. An object of the present invention is to provide a method for producing a hybrid artificial pancreas capable of preventing death or the like due to hypoglycemia.

[0007]

According to the present invention, there is provided a hybrid wherein the pancreatic islets are inclusively immobilized with an immobilizing material comprising a natural and / or synthetic polymer, and then cultured in vitro for 5 days or more. This is a method for producing an artificial pancreas. In the present invention, the pancreatic islets are entrapped and immobilized on an immobilization material comprising a natural and / or synthetic polymer. The immobilization material composed of a natural and / or synthetic polymer used in the present invention needs to allow nutrients such as glucose and insulin secreted from the pancreatic islets to pass therethrough, and also makes the pancreatic islets immune during transplantation. Sufficient mechanical strength is required to prevent contact with the cell in charge.

[0008] Examples of the immobilization material comprising such a natural polymer include agarose, agarose decomposition product, κ
-Carrageenan, collagen, gelatin, gellan gum, gum arabic, xanthan gum, alginate, pectin, agar, mannan, fibrin, chitosan, and the like.
Examples include photocrosslinkable polyvinyl alcohol, isopropylacrylamide copolymer, and polyacrylamide. Further, examples of the immobilizing material composed of natural and synthetic polymers include polyion complexes such as alginic acid-polylysine and carboxymethylcellulose-polylysine. The immobilizing material made of a natural and / or synthetic polymer as described above may be any material that can immobilize pancreatic islets in a living state. It is also possible to use two or more of these fixing materials in combination. The immobilized material will be further described. For example, agarose is usually contained in agar, and an agarose decomposition product can be obtained by decomposing the agarose with an acid or the like. The photocrosslinkable polyvinyl alcohol is obtained by bonding a styrylpyridinium group or a styrylquinolium group, which is a photosensitive functional group, to polyvinyl alcohol, and is already commercially available, and may be obtained from, for example, Toyo Gosei Kogyo. it can.

In the present invention, the pancreatic islet is a human,
Those isolated from cattle, pigs, hamsters or mice can be used as appropriate. The isolation method involves digesting the isolated pancreas with collagenase for a certain period of time,
The pancreatic islets are collected by specific gravity centrifugation. In the present invention, the collected pancreatic islets are comprehensively immobilized with an immobilizing material comprising a natural and / or synthetic polymer.

As an entrapping immobilization method, for example, in the case of agarose, agarose decomposed product, κ-carrageenan, isopropylacrylamide copolymer, etc., pancreatic islets are dispersed in an immobilizing material solution and gelled by temperature change to entrap immobilization. Become Hereinafter, this comprehensive immobilization method will be described with specific examples. When the pancreatic islets are comprehensively immobilized with agarose or agarose hydrolyzate, the agarose or agarose hydrolyzate is preferably contained in an aqueous solution containing a suitable medium component such as Eagle's MEM medium, preferably in an amount of 1 to 30% by weight, More preferably, it is dispersed so as to be in the range of 2 to 20% by weight. After heating and melting this dispersion, the temperature was 40 ° C.
Cool to the vicinity and disperse the recovered pancreatic islets into it.
A hydrophobic liquid that does not mix with water, such as liquid paraffin, is added to the obtained dispersion, and the mixture is sufficiently stirred to suspend the agarose or the agarose decomposition product solution. Next, by cooling the suspension, the agarose or the agarose decomposition product solution is gelled. In this case, the suspension is cooled with stirring to prevent aggregation. Next, after adding a buffer having a pH around neutrality such as Hanks' solution, centrifugation was performed,
The microcapsules of agarose or agarose hydrolyzate settled in the lower layer are collected. In this manner, a hybrid artificial pancreas in the form of microcapsules having a particle size of about several tens of μm to several mm in which pancreatic islets are comprehensively immobilized can be obtained.

In the case of alginate, chitosan, and the like, pancreatic islets are dispersed in an immobilizing material solution and gelled by ionic cross-linking to be inclusively immobilized. In the case of photocrosslinkable polyvinyl alcohol, the degree of polymerization of the photocrosslinkable polyvinyl alcohol is preferably 100 to 5000 or less, and the pancreatic islets are dispersed in an aqueous solution thereof. Next, the photocrosslinkable polyvinyl alcohol aqueous solution in which the pancreatic islets are dispersed is photocrosslinked and gelled to thereby comprehensively fix the pancreatic islets. Hereinafter, this comprehensive immobilization method will be specifically described. The photo-crosslinkable polyvinyl alcohol is used at a polymer concentration of preferably 1 to 50% by weight, more preferably 2 to 50% by weight.
It is dissolved in an aqueous solution containing an appropriate medium component such as Eagle's MEM medium so as to have a concentration of 30% by weight. At this time, if it does not dissolve at room temperature, it is dissolved by heating. The recovered pancreatic islets are dispersed in this aqueous solution.
A hydrophobic liquid that does not mix with water, such as liquid paraffin, is added to the obtained dispersion and stirred sufficiently to suspend the photocrosslinkable polyvinyl alcohol solution. Next, the photocrosslinkable polyvinyl alcohol solution is gelled by irradiating light. In this case, light irradiation is performed while stirring to prevent aggregation of the suspension. Next, a buffer solution having a pH around neutral, such as Hanks' solution, is added and centrifuged to collect photocrosslinkable polyvinyl alcohol microcapsules settled in the lower layer. In this way, it is possible to obtain a hybrid artificial pancreas in the form of microcapsules of several tens μm to several mm in which pancreatic islets are comprehensively immobilized.

In the case of an ion complex such as alginic acid-polylysine, the complex is immobilized on a negatively charged water-soluble substance (in this case, alginate) capable of gelling pancreatic islets, and has a durable surrounding area. A positively charged film (in this case, polylysine) is formed and entrapped and immobilized. Hereinafter, this comprehensive immobilization method will be described with specific examples. The recovered pancreatic islets are dispersed in a sodium alginate solution. This is dropped into a calcium chloride solution to recover gelled calcium alginate particles. Further, the gelled particles are treated with a molecular weight of about 15,000 to about 25,000.
The gel particles are immersed in a physiological saline solution containing polylysine to form a polylysine film on the surface of the gel particles. Further, the particles are immersed in a sodium alginate solution to form an alginate film on the surface of the polylysine film. Next, the particles were treated with a sodium citrate buffer to dissolve the calcium alginate therein, and several tens of
It is possible to obtain a microcapsule-shaped hybrid artificial pancreas having a particle size of about μm to several mm.

The shape to be comprehensively immobilized is from several tens μm to several mm in which one or several pancreatic islets are comprehensively immobilized.
Any shape may be used, such as a microcapsule type having a particle size of about a degree, a tablet type in which dozens to thousands of pancreatic islets are fixed together, or a thread-shaped one. In the present invention, the hybrid artificial pancreas obtained as described above is cultured in vitro for 5 days or more. For example, a hybrid artificial pancreas is put in Eagle's MEM-5-10% fetal bovine serum or RPMI-1640-5-10% fetal bovine serum, and 5% CO ₂ at 0-41 ° C.
Under 5 days or more, preferably at 20-40 ° C. with 5% CO 2
_Incubate under ₂ for more than 20 days. Finally, collect only the hybrid artificial pancreas including the islet in good condition,
A hybrid artificial pancreas suitable for transplantation can be obtained.

[0014]

[Effects] When transplantation is performed immediately after isolation and comprehensive fixation of pancreatic islets, pancreatic islets damaged by these operations are also transplanted. As a result, a large amount of insulin and immunogenic substances are released from the damaged pancreatic islets, and the transplanted animal may be hypoglycemic and die, or the pancreatic islets may be immunorejected. In contrast, in the present invention, the in-fill operation is performed for 5 days or more.
Since the culture is performed in vitro, the damaged pancreatic islets are repaired or dropped off by this culture operation.
Therefore, by culturing in vitro for 5 days or more, a hybrid artificial pancreas containing only a normally functioning pancreatic islet can be collected. By transplanting the hybrid artificial pancreas, The problem based on the damaged pancreatic islets can be solved.

[0015]

Thus, according to the present invention, pancreatic islets are comprehensively immobilized with an immobilizing material composed of a natural and / or synthetic polymer and cultured in vitro for 5 days or more. It is possible to solve the problems caused by the pancreatic islets, prevent the transplanted animal from falling into a hypoglycemic state, and prevent the recipient from inducing immunity. Therefore, it is possible to provide a hybrid artificial pancreas that can survive for a long period of time.

[0016]

Description of the Examples The artificial pancreas of Examples 1 to 5 and Comparative Examples 1 to 5 were prepared as follows, and evaluated by a transplantation test and a complement-dependent cytotoxicity test. Example 1 0.15 g of agarose was added to 3.0 ml of Eagle's MEM solution to make the polymer concentration 5% by weight, and the agarose was dissolved simultaneously with heat sterilization at 121 ° C. for 20 minutes in an autoclave. Temperature.
Into this agarose solution, about 1000 pancreatic islets isolated from the hamster pancreas by the collagenase treatment method were added to Eag.
Le's MEM solution dispersed in 0.1 ml was added, stirred, and further heated to 40 ° C.
0 ml was added and stirred to disperse agarose. Next, this dispersion was cooled on ice with stirring to gel the agarose, and about 30 ml of Hank's solution was added thereto, and 2000 rpm × 1
The precipitated microcapsules were collected by centrifugation under the condition of 0 minutes. This microcapsule has a particle size of about 50-80.
At 0 μm, one to several pancreatic islets were inclusively immobilized per capsule. Next, the microcapsules were placed in Ea
gl's MEM-5% fetal calf serum, 37 ° C, 5%
After culturing under CO ₂ for 30 days, the microcapsules containing pancreatic islets in a good state were collected, and the hybrid artificial pancreas of Example 1 was produced.

Example 2 Instead of agarose, 5 g of agarose was dissolved in 50 ml of water, 1.5 ml of acetic acid was added thereto, and the resulting mixture was hydrolyzed at 100 ° C. for 10 minutes. The hybrid artificial pancreas of Example 2 was produced in the same manner as in Example 1 except that the amount was changed to 6% by weight and the number of culture days was changed to 6 days.

[0018] Example 3 Culture Conditions a 20 ° C., except that a 5% CO ₂ 20 days under, to produce a hybrid artificial pancreas of Example 3 in the same manner as in Example 1. Example 4 Photocrosslinkable polyvinyl alcohol (degree of polymerization: 1700, degree of saponification: 88, ratio of styrylpyridinium substituent:
1.3 mol%) was added to 3.0 ml of Eagle's MEM culture solution so that the polymer concentration became 10% by weight, and the photo-crosslinkable polyvinyl alcohol was simultaneously sterilized with heat in an autoclave at 121 ° C. for 20 minutes. Dissolved.
The solution was cooled to 37 ° C., and about 1,000 pancreatic islets isolated from the hamster pancreas by a collagenase treatment method were dispersed in 0.1 ml of Eagle's MEM solution, and the mixture was stirred. 20 ml was added and stirred to disperse the photocrosslinkable polyvinyl alcohol.

Next, this dispersion was stirred and irradiated with light for about 15 minutes using a slide projector equipped with a halogen lamp of 300 W to gel the photo-crosslinkable polyvinyl alcohol, and about 30 ml of Hanks solution was added. In addition, the mixture was centrifuged under the conditions of 2000 rpm × 10 minutes, and the precipitated microcapsules were collected. This microcapsule had a particle size of about 50 to 800 μm, and one to several pancreatic islets were inclusively immobilized per capsule.

Further, the microcapsules were placed in Eagl.
e's MEM-5% fetal calf serum, 37 ° C, 5% CO
_After culturing for 20 days under the same conditions, microcapsules containing pancreatic islets in good condition were collected, and the hybrid artificial pancreas of Example 4 was produced. Example 5 0.045 g of sodium alginate was added to 3 ml of physiological saline to a polymer concentration of 1.5% by weight, and about 1,000 pancreatic islets isolated from the hamster pancreas by collagenase treatment were physiologically added to the solution. About 0.1m
1 was added and stirred to disperse the pancreatic islets. This dispersion was dropped from a 22 G air jet nozzle into a beaker containing 100 ml of a 1.5% by weight calcium chloride solution, and gelled calcium alginate particles were collected. The particles were dissolved in a buffer solution (pH 8.0) containing 0.6% by weight of sodium chloride and 2% by weight of 2-cyclohexylaminoethanesulfonic acid.
1) parts by weight of 2) and 2 parts by weight of a 1% by weight calcium chloride solution
The mixture was washed successively with a solution (hereinafter abbreviated as CHES solution) mixed with 0 parts by weight and a 1.1% by weight calcium chloride solution. Further gelled calcium alginate particles,
After immersing in 50 ml of physiological saline containing 0.5% of polylysine having a molecular weight of 18,000 for 6 minutes to form a polylysine film, the microcapsules were taken out and the CHES solution, 1.1
Washing was performed sequentially with a weight% calcium chloride solution and physiological saline.

Further, this microcapsule was added to 0.15
It was immersed in 20 ml of a 20% sodium alginate solution for 4 minutes to form an alginate film on the surface of the polylysine film. Wash the obtained microcapsules with saline,
Further, the resultant was treated with a 0.05 M sodium citrate buffer for 6 minutes to dissolve the calcium alginate inside, and then washed again with physiological saline to collect microcapsules. This microcapsule has a particle size of about 50-800.
At μm, one to several pancreatic islets were encapsulated and immobilized per capsule.

Next, the microcapsules were placed in Eagle's
s The cells were cultured in MEM-5% fetal calf serum at 37 ° C. under 5% CO ₂ for 30 days, and microcapsules containing pancreatic islets were collected in good condition to produce the hybrid artificial pancreas of Example 5. Comparative Example 1 A hybrid artificial pancreas of Comparative Example 1 was produced in the same manner as in Example 1 except that the number of culture days was changed to 4 days.

[0023] except that the Comparative Example 2 culture days to 1 day in the same manner as in Example 2, was produced hybrid artificial pancreas of Comparative Example 2. Comparative Example 3 A hybrid artificial pancreas of Comparative Example 3 was produced in the same manner as in Example 4 except that the number of culture days was changed to 3 days.

[0024] except that the Comparative Example 4 culture days and one day, the same procedure as in Example 5, were produced hybrid artificial pancreas of Comparative Example 4. Comparative Example 5 A hamster pancreatic islet itself was prepared as Comparative Example 5 instead of the hybrid artificial pancreas. (Transplantation Test) 200 mg / kg streptozotocin was intraperitoneally administered to 6-week-old male BALB / C mice to make them diabetic. About 1,000 hybrid artificial pancreas were transplanted intraperitoneally into the diabetic mouse. Table 1 shows the blood glucose levels before and after the transplantation.

[0025]

[Table 1]

As shown in Table 1, the blood glucose level before transplantation was 500
mg / dl or more, which is a typical diabetes.
In Nos. To 5, the blood sugar level was maintained at 200 mg / dl or less, which is the normal blood sugar level of the mouse, even after 109 days, and it functions as a hybrid artificial pancreas.
On the other hand, in Comparative Examples 1 to 5, it can be seen that all the mice returned to the diabetic state, and the mice died. (Complement-dependent cytotoxicity test) Serum before and after transplantation was
A 5-fold dilution with (−), 0.1 ml of the solution was taken, and hamster lashima was used as an antigen and dispersed in single cells by a dispase treatment method (5 × 10 ⁵ cells / ml).
1 ml and normal serum of guinea pig as a complement in PBS
0.2 ml of the solution diluted 10-fold with (−) was added, and 3
Incubated for 1 hour at 7 ° C. Thereafter, 0.4 ml of a 0.5% trypan blue PBS (-) solution was added, and the number of live cells and dead cells was counted to determine the cytotoxicity. The results are shown in Table 2.

[0027]

[Table 2]

As shown in Table 2, no change in the cytotoxicity rate was observed in Examples 1 to 5, but in Comparative Examples 1 to 5, the cytotoxicity rate increased with an increase in blood sugar level. That is, in Comparative Examples 1 to 5, it is considered that hamster / pancreatic islet antibodies were induced in recipient mice.

Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12N 5/00 A61K 35/39 // A61K 35/39 A61P 3/10 A61P 3/10 C12N 5/00 (58) Fields surveyed (Int. Cl ^7, DB name) A61K 9/00 -. 9/72 A61K 47/00 - 47/48 A61L 27/00 - 27/60 A61M 1/36 563 - 567 C12M 5/00 - 5/28 CA (STN) MEDLINE (STN)

Claims (1)

(57) [Claims] 1. A method for producing a hybrid artificial pancreas, wherein pancreatic islets of Langerhans are entrapped and immobilized with an immobilizing material composed of a natural and / or synthetic polymer, and then cultured in vitro for 5 days or more.