JPH07246211A - Hybrid type artificial pancreas - Google Patents

Abstract

PURPOSE:To provide hybrid type artificial pancreas capable of maintaining the transplanted cells having the physiological function adequate for a living body under an optimum cytobiological environment over a long period. CONSTITUTION:The artificial pancreas are formed by sealing three-dimensionally cultured intrapancreatic secretory cells together with a culture substrate into a diffusion chamber having a high-polymer semipermeable membrane. The intrapancreatic secretory cells are cell mass consisting of the islet cell mass of swine varying in generation, the pancreatic beta cell strains derived from the insulinoma of transgenic animals holding extrogenous tumor genes or a mixture composed thereof. The culture substrate contains at least a glycochain-contg. methacrylic acid polymer, agarose, collagen matrix and nicotine amide.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hybrid artificial pancreas. More specifically, the present invention relates to a hybrid type (implantation type) artificial pancreas useful as a root treatment method for diabetes or as an alternative organ for a patient who has had a pancreas removed due to pancreatic cancer or an accident.

[0002]

2. Description of the Related Art Due to the remarkable development of genetic engineering in recent years, the elucidation of the causative factors of many diseases and the diagnostic technology have been remarkably progressing. There are many points that are still insufficient.
For example, the diagnosis and elucidation of the onset factors have been gradually clarified even for diabetes mellitus showing various pathological conditions, but on the other hand, the root treatment method for diabetes having complicated and diverse pathological conditions is , It is in a very difficult situation due to the complexity of its pathology.

[0003] It is known that one of the etiological factors of diabetes is impaired insulin secretion from the pancreas (insulin deficiency), and recently, the administration of a small amount of insulin from the initial onset of the disease has led to the diabetic disease. It has also been shown that the progress of the period can be prevented. However, since insulin also affects gluconeogenesis and degradation in the liver, administration of an excessive amount of insulin also causes hypoglycemia. Also, having to regularly receive insulin injections over a long period of time puts a great burden on patients.

For these reasons, in recent years, tissue-cultured pancreatic endocrine cells of a different kind of living body are enclosed in a chamber, a tube, a membrane or the like and transplanted into a human body, and a necessary amount of insulin is blood-treated from these cells for a long period of time. The treatment method of supplying inside is being considered. This is a hybrid artificial pancreas,
Until now, toward the practical application, for example, the establishment of "immuno-isolation means" to protect pancreatic cells derived from different living organisms from the human body's immune system (immune cells, antibodies, etc.), and to efficiently produce insulin suitable for the human body Various proposals and improvements have been made on selection of cells to be produced, cell morphology for allowing transplanted cells to express their functions for a long period of time, and devising of culture method therefor.

Considering the above points, one of the inventors of the present invention considers the above points, and in particular, in order to maintain the function (insulin secretion) of transplanted cells for a long period of time, the cells are three-dimensionally cultured to form aggregates. Has been found to be essential, and invented a hybrid artificial pancreas in which a three-dimensional aggregate of pancreatic islet cells isolated from a newborn pig was enclosed in a chamber having a polymeric immunoisolation membrane, and a patent has already been applied for. (JP-A-1-
247082 publication. Hereinafter, it may be described as a prior invention).

[0006]

DISCLOSURE OF THE INVENTION The inventors of the present invention have diligently studied toward the development of a more practical hybrid type artificial pancreas after the invention of the above-mentioned prior application, and as a result, the pancreatic islet cells of the living body are obtained. We obtained new important knowledge about the selection of transplanted cells with physiological function similar to that of and the culture method therefor.

The present invention has been made on the basis of such new knowledge, and further develops and improves the invention of the prior application to obtain transplanted cells having physiological functions suitable for living bodies, which are optimal cells for a long period of time. The purpose is to provide a hybrid artificial pancreas that can be maintained in a biological environment.

[0008]

In order to solve the above-mentioned problems, the present invention provides an artificial pancreas in which three-dimensionally cultured pancreatic endocrine cells are enclosed together with a culture substrate in a diffusion chamber having a polymer semipermeable membrane. (1) The pancreatic endocrine cell is a pancreatic islet cell mass of different generations, a pancreatic β cell line derived from an insulinoma of a transgenic animal carrying an exogenous oncogene, or a cell mass composed of a mixture thereof. And (2) a hybrid artificial pancreas characterized in that the culture substrate contains at least a sugar chain-containing methacrylic acid polymer, agarose, a collagen matrix and nicotinamide.

In the hybrid artificial pancreas of the present invention, the porcine islet cell mass is a cell mass formed by mixing porcine fetal, neonatal and adult pancreatic islet cells, and the pancreatic β cell line is A pancreatic β cell established from an insulinoma of a transgenic mouse having a fusion gene in which an SV40 T antigen gene is bound to an insulin promoter, or a pancreatic β cell established by introducing a human insulin gene into the established cell line, Alternatively, it is a pancreatic β cell established from an insulinoma of a mouse obtained by mating a transgenic mouse having a human insulin gene with the transgenic mouse,
It is also a preferred embodiment that the diffusion chamber comprises a polymer that releases nicotinamide in a sustained manner.

The structure of the present invention will be described in more detail below. That is, the hybrid-type artificial pancreas of the present invention has the same basic structure as the invention of the prior application, and three-dimensionally cultured pancreatic endocrine cells are enclosed together with a culture substrate in a diffusion chamber having a polymer semipermeable membrane. Has the following features for each constituent element. (1) Diffusion chamber The diffusion chamber is a disc type in which the upper and lower surfaces of a silicon ring or the like are sealed with a polymer semipermeable membrane and the cells and tissue pieces to be transplanted are enclosed between the membranes, as in the above-mentioned prior invention. Alternatively, the transplanted cells may be enclosed in a polymer semipermeable membrane having a tubular shape, a flat plate shape, a needle shape, or the like. The polymer semipermeable membrane is, for example, a porous membrane made of polyester, polycarbon, silicon or the like, and allows only the secretory product (insulin etc.) of the transplanted cells enclosed in the chamber to pass through, while immune cells in the living body and antibodies etc. It is used as an immunoisolation means to prevent invasion. By encapsulating with a polymer membrane having excellent selective permeability, it is possible to suppress the immune reaction of the living body to transplanted cells and to transplant an artificial pancreas in vivo without using an immunosuppressant or the like. .

Also, the silicon ring may be coated with polyurethane urea which is an antithrombotic agent.
Furthermore, it is also preferable that the diffusion chamber of the present invention is provided with a polymer that continuously releases nicotinamide. Since this nicotinamide has a function of repairing the function of pancreatic cells as described below, it becomes possible to maintain the cell function after transplantation for a long period of time. (2) Pancreatic endocrine cells The pancreatic endocrine cells enclosed in the above-mentioned chamber are, for example, insulin-secreting cells and / or glucagon-secreting cells of pancreatic islets. Using a pancreatic β-cell line derived from an insulinoma of a transgenic animal possessed, or a cell mass composed of a mixture of these, three-dimensional embedding culture is performed using the culture substrate and culture medium described below, and enclosed in a chamber. To do.

[0012] Among them, as for the porcine islet cell mass, the conventional artificial pancreas used only cells isolated from a neonatal pig, whereas in the present invention, pigs of different generations (for example,
Cells isolated from each pancreas (fetal, neonatal and mature) are used in admixture. By mixing three types of cells of different generations, self-regulation due to interaction is promoted, and a function similar to that of the original pancreatic islet cell can be expressed. That is, the third-generation mixed cells produce not only insulin but also hormones secreted from islet cells such as glucagon that plays an important role in sugar metabolism and somatostatin necessary for maintaining homeostasis in the living body without excess or deficiency. .

These pancreatic islet cells can be isolated by a known procedure, suspended in a culture medium and reaggregated to collect them. On the other hand, as a pancreatic β cell line derived from insulinoma, a pancreatic β cell line (MIN6, MIN) already developed by one of the inventors of the present invention.
7: Endocrinology, vol127, p.126, 1990) can be used. This cell line is a pancreatic β cell established from an insulinoma of a transgenic mouse produced by introducing a fusion gene in which the SV40 T antigen gene is linked to the promoter of human insulin gene into a fertilized egg, and stable insulin production. It has the ability to grow in three-dimensional culture. Moreover, the normal established pancreatic β-cells lose the glucose sensitivity of the original pancreatic β-cells, but these cell lines (especially MIN6) secrete insulin depending on glucose concentration and are transplanted in vivo. Even in such a case, it has an excellent feature that it exhibits a function only during hyperglycemia.

Further, in the present invention, the above-mentioned cell line (MIN6, MI is used as the transplanted cells to be enclosed in the chamber.
N7) Pancreatic β cells established by introducing the human insulin gene into the cell line, or obtained by mating transgenic mice carrying the human insulin gene with transgenic mice carrying the above-mentioned foreign oncogene It is also possible to use pancreatic β cells established from mouse insulinoma. These cell lines stably express human insulin in addition to mouse insulin.

Furthermore, each of the above pancreatic endocrine cells, that is, the three-generation mixed cells derived from porcine pancreas or the pancreatic β cell line derived from insulinoma, can be used alone as a transplant cell, but more preferably, any of them can be used. Use by mixing at a ratio. By doing so, the physiological function of the transplanted cells (secretion of insulin, glucagon, somatostatin, etc.) can be approximated to the original pancreas. (3) Culture Substrate In the artificial pancreas of the present invention, the above pancreatic endocrine cells are embedded and cultured, and enclosed in a diffusion chamber as a three-dimensional structure. By doing so, the original cell biological environment can be imparted to the cultured cells, and their differentiation ability and function expressing ability can be approximated to those of pancreatic islet cells in vivo.

Such three-dimensional culture can be performed by a known method in which cells are embedded in a solid or semi-fluid substrate (eg, collagen gel) and suspended or layered in a culture solution. In the invention, a culture substrate containing at least a sugar chain-containing methacrylic acid polymer, agarose, a collagen matrix and nicotinamide is used. Specifically, the sugar chain-containing methacrylic acid polymer is a random polymer of a polystyrene derivative having a sugar chain in its side chain and hydroxyethyl methacrylate, which has a function as a signal for cell recognition and various information transmission. The polymer can be prepared, for example, by adding a cross-linking agent to a polystyrene derivative having maltose (for example, polyvinyl maltoamido galactone: PVMA) and polyhydroxyethyl methacrylate (PHEMA), and radically polymerizing the gel. it can. The polymer is then mixed with agarose gel and collagen matrix to form a hydrogel.

When a hydrogel consisting of such a sugar chain-containing methacrylic acid polymer and agarose is used as a culture substrate, compared to the conventional method using a collagen gel alone, cells in culture are The degree of lysis and recontraction is small, and the cell mass is similar to the original pancreatic islet cells in terms of morphology and physiological function. On the other hand, nicotinamide is known to functionally repair chemically or physically damaged pancreatic cells (β cells) (Mo
lecular biology. Cambridge University Press: p.209
-231, 1990). Therefore, by culturing islet cells damaged by a mechanical operation during separation / collection in a culture substrate containing this nicotinamide, the physiological function of cells can be restored. By encapsulating in a chamber and transplanting, the functional expression can be maintained for a long period of time.

Due to the above-mentioned structural features, such as the type and morphology of pancreatic endocrine cells enclosed in the diffusion chamber, the composition of the culture substrate for three-dimensionally culturing these cells, and the like, the hybrid artificial The pancreas is an extremely effective therapeutic means for diabetes because transplanted cells maintain their physiological functions (secretion of insulin, glucagon, somatostatin, etc.) for a long period of time. It can also be used as an alternative organ for the pancreas.

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following examples.

[0020]

Example 1 Pig fetal, neonatal, and adult pancreatic islet cells were mixed and three-dimensionally cultured, and the cell mass was sealed together with a culture substrate in a diffusion chamber to prepare a hybrid artificial pancreas. 1) Separation and collection of pancreatic islet cell mass The pancreas was extracted from each of a pig fetus, a newborn baby (7 to 10 days old) and maturation, cut into pieces each having a size of about 1 to 2 mm, and then placed in a well-cooled RPMI1640 solution. It was washed by shaking (3 minutes × 3 times). P.
EDTA-Dispase solution adjusted to H7.4 (0.005% EDTA, 150
prtotease unit Dispase / ml, RPMI 1640 with 3% FBS)
, And then culture under the condition of 37 ° C-5% CO ₂ ,
The cell mass (reaggregated cells) was collected. Then, the islet cells of different generations collected in this manner were treated with 4.0% BSA-R.
The PMI1640 solution was gently overlaid and spun at 600 rpm for 5 minutes (50 G) for 10 minutes to obtain a mixed cell mass. 2) Three-dimensional culture of cells and encapsulation of cultured cells First, as a culture substrate, agarose and collagen gel were added to a polymer of PVMA and PHEMA to prepare a hydrogel. Then, the culture solution containing nicotinamide was mixed with the above hydrogel, and the islet cell mass of the third generation mixture was gently mixed therein, and then immediately enclosed in the diffusion chamber.

As shown in the exploded perspective view of FIG. 1, the diffusion chamber is made by attaching a semipermeable membrane (2) made of polyester or polycarbon to both upper and lower surfaces of an O-ring (1) made of silicon and attaching them to each other. A disk type chamber fixed with a ring holder (3) was used. As the semipermeable membrane (2), a porous membrane having a pore diameter of 0.2 μm or less was used.

The islet cell mass mixed in the culture substrate is
As shown in FIG. 2, a syringe (4) was used to inject into the O-ring (1) of the diffusion chubber, where the gel matrix was cured to create a hybrid artificial pancreas. This artificial pancreas contains RPMI164 containing FBS and nicotinamide.
0 solution and continued to culture the encapsulated cells. Example 2 A pancreatic β cell line derived from an insulinoma of a transgenic mouse carrying an exogenous oncogene was three-dimensionally cultured and enclosed in a diffusion chamber together with a culture substrate to prepare a hybrid artificial pancreas. 1) Preparation of pancreatic β cell line As the pancreatic β cell line, the pancreatic β cell line MIN6 which one of the inventors of the present invention had already established was used. This cell line was isolated from an insulinoma of a transgenic mouse produced by introducing a fusion gene in which the SV40 T antigen gene was linked to the promoter of the human insulin gene into a fertilized egg and established as a cell line.

The cells were prepared from Dulbecco's modified Eagle medium (2
The cells subcultured with 5 mmol / l glucose, 20% fetal bovine serum, containing penicillin-streptomycin) were used to recover cells that reached confluence by culturing at 37 ° C. and 5% CO ₂ . 2) Three-dimensional cell culture and encapsulation of cultured cells First, agarose and collagen gel were mixed with a polymer of agarose and PVMA, and a nicotinamide-containing culture solution was added thereto to obtain a culture substrate.

Then, MIN6 cells were gently mixed in this culture substrate and immediately injected into the same diffusion chamber as in Example 1 to cure the gel substrate to prepare a hybrid artificial pancreas. This artificial pancreas contains RPMI16 containing D-glucose, FBS and nicotinamide.
40 solution was added and the culturing of the encapsulated cells was continued. 3) Transplantation of artificial pancreas and confirmation of its effect The artificial pancreas prepared in 3) above was transplanted into the abdominal cavity of a diabetic model rat by streptozotocin, and changes in blood glucose level were measured over time.

As a result, the blood glucose level of diabetic rats (459
± 30 mg / dl) is 97 days 7 days after the artificial pancreas transplantation.
It fell to ± 12 mg / dl, which persisted for at least 4 months. Example 3 Porcine islet cell masses of different generations and an insulinoma-derived pancreatic β-cell line were three-dimensionally mixed and cultured, and enclosed in a diffusion chamber together with a culture substrate to prepare a hybrid artificial pancreas.

That is, the porcine islet cells (mixed mass of fetal, neonatal and mature islet cells) separated and collected in 1) of Example 1 and the MIN6 cells of Example 2 were
It was cultured three-dimensionally in the same manner as in 2) of Example 1, and this was cultured in FIG.
The artificial pancreas was prepared by enclosing it in the diffusion chamber of.

[0027]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a hybrid artificial pancreas capable of maintaining transplanted cells having physiological functions suitable for living bodies in an optimal cell biological environment for a long period of time. To be done. This will open up new ways to develop root cures for diabetes and alternative organs for the pancreas.

[Brief description of drawings]

FIG. 1 is an exploded perspective view showing an embodiment of a diffusion chamber used in the hybrid type artificial pancreas of the present invention.

FIG. 2 is a cross-sectional perspective view of essential parts showing a method for encapsulating cells in the diffusion chamber of FIG.

[Explanation of symbols]

 1 O-ring 2 Semi-permeable membrane 3 Ring holder 4 Syringe

Claims (5)

[Claims] 1. A diffusion chamber having a polymer semipermeable membrane,
An artificial pancreas obtained by encapsulating three-dimensionally cultured pancreatic endocrine cells together with a culture substrate, wherein (1) the pancreatic endocrine cells are insulinoma of a transgenic animal having a pancreatic islet cell mass of a different generation and an exogenous oncogene. Is a pancreatic β cell line derived from, or a cell mass composed of a mixture of these,
(2) A hybrid artificial pancreas characterized in that the culture substrate contains at least a sugar chain-containing methacrylic acid polymer, agarose, a collagen matrix and nicotinamide. 2. The hybrid type artificial pancreas according to claim 1, wherein the porcine islet cell mass is a cell mass obtained by mixing and culturing fetal, neonatal and mature porcine islet cells. 3. A pancreatic β cell line established from an insulinoma of a transgenic mouse having a fusion gene in which an SV40 T antigen gene is linked to an insulin promoter, or a human insulin gene in this established cell line. The pancreatic β cell introduced and established, or the pancreatic β cell established from an insulinoma of a mouse obtained by mating a transgenic mouse having a human insulin gene with the transgenic mouse. Hybrid artificial pancreas. 4. The hybrid artificial pancreas according to claim 1, wherein the sugar chain-containing methacrylic acid polymer is a polymer of a sugar chain-containing polystyrene derivative and hydroxyethyl methacrylate. 5. The hybrid artificial pancreas according to claim 1, wherein the diffusion chamber comprises a polymer that continuously releases nicotinamide.