Method for producing pancreatic islet-containing capsule

Abstract

To provide an improved method for producing a capsule containing pancreatic islet.SOLUTION: (a) preparing a sodium alginate solution A containing pancreatic islets at a concentration of 10,000 I E Q / mL or more, (b) adding the sodium alginate solution dropwise to a divalent cation solution, and collecting gelled particles, (c) adding the particles collected in the step (b) to a poly-L-ornithine solution A, stirring, and then collecting particles, (d) adding the particles collected in the step (c) to a sodium alginate solution B, stirring, and then collecting particles, and (e) adding the particles collected in the step (d) to a sodium citrate solution, A step of collecting the particles after the stirring.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Techniques for encapsulating pancreatic islets are disclosed. [Background technology]

[0002] Pancreatic islet transplantation is a relatively low-impact treatment for diabetes, but strong immunosuppression may be required for long-term survival of the transplanted islets. A method for transplanting a large number of microencapsulated islets into the abdominal cavity has been investigated, which does not require immunosuppression, but satisfactory therapeutic results have not yet been obtained. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2001 / 052871 [Non-patent literature]

[0004] [Non-Patent Document 1] METHODS IN ENZYMOLOGY, VOL. 137, 575-580, 1988 [Non-patent document 2] TRANSPLANTATION, Vol. 53, 1180-1183, No. 6, June 1992 [Non-patent document 3] Bioartificial Pancreas, Vol., 98, No. 6, 1996, 1417-1422 [Non-patent document 4] Drug Delivery System, Vol. 12, No. 2, 1997 [Non-Patent Document 5] JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS, 2013 VOL., 101B, ISSUE 2, 258-268 Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object to provide an improved method for producing pancreatic islet-containing capsules. [Means for solving the problem]

[0006] As a means for solving such problems, the invention includes the following. Section 1. (a) preparing a sodium alginate solution A containing pancreatic islets at a concentration of 10,000 IEQ / mL or more; (b) adding the sodium alginate solution dropwise to a divalent cation solution and recovering the gelled particles; (c) adding the particles recovered in step (b) to poly-L-ornithine solution A, stirring, and then recovering the particles; (d) adding the particles collected in step (c) to sodium alginate solution B, stirring, and then collecting the particles; and (e) adding the particles collected in step (d) to a sodium citrate solution, stirring, and then collecting the particles; A method for producing a pancreatic islet-containing capsule, comprising: Section 2. Item 1. The method according to Item 1, wherein the pancreatic islets are obtained from a 1- to 3-week-old young pig. Section 3. Item 3. The method according to Item 1 or 2, wherein the average diameter of the islet-containing capsules is 550 to 750 μm. Section 4. Item 4. The method according to any one of Items 1 to 3, wherein the sodium alginate concentration of the sodium alginate solution A is 1 w / v % or more and 3 w / v % or less. Section 5. Item 5. The method according to any one of Items 1 to 4, wherein the poly-L-ornithine concentration in the poly-L-ornithine solution A is 0.05 w / v % or more and 3 w / v % or less. Section 6. Item 6. The method according to any one of Items 1 to 5, wherein the sodium alginate concentration of sodium alginate solution B is 0.1 w / v % or more and 0.3 w / v % or less. Section 7. Item 7. The method according to any one of Items 1 to 6, wherein in step (d), the particles recovered in step (c) are added to poly-L-ornithine solution B, stirred, recovered, and then added to sodium alginate solution B. Section 8. Item 8. The method according to Item 7, wherein the poly-L-ornithine concentration of poly-L-ornithine solution B is 0.01 w / v% or more and less than 0.1 w / v%. [Effects of the Invention]

[0007] Encapsulated pancreatic islets with improved glycoresponsiveness are provided. [Brief explanation of the drawings]

[0008] [Figure 1] Micrographs of islets immediately after encapsulation and those cultured for 25 days after encapsulation are shown. a-d are immediately after encapsulation, and e-h are after 25 days of culture. a and e were prepared using 8,000 IEQ / mL islets, b and f were prepared using 12,000 IEQ / mL islets, c and g were prepared using 16,000 IEQ / mL islets, and d and h were prepared using 20,000 IEQ / mL islets. [Figure 2] The graph shows the results of measuring the glucose responsiveness of pancreatic islets cultured for 25 days after encapsulation. [Figure 3] The glucose responsiveness measured for islets cultured for 25 days after encapsulation is shown as the stimulation index (SI). [Figure 4] 1 shows the results of measuring the sugar responsiveness of pancreatic islet-containing capsules of different sizes. [Figure 5] The results of measuring the SI for islet-containing capsules of different sizes are shown. DETAILED DESCRIPTION OF THE INVENTION

[0009] The method for producing the islet-containing capsule preferably comprises the following steps (a) to (e): (a) preparing a sodium alginate solution A containing pancreatic islets at a concentration of 10,000 IEQ / mL or more; (b) adding the sodium alginate solution dropwise to a divalent cation solution and recovering the gelled particles; (c) adding the particles collected in step (b) to poly-L-ornithine solution A, stirring, and then collecting the particles; (d) adding the particles collected in step (c) to sodium alginate solution B, stirring, and then collecting; and (e) adding the particles collected in step (d) to a sodium citrate solution, stirring, and then collecting the particles.

[0010] The pancreatic islets used in step (a) preferably contain insulin-producing β cells, glucagon-containing α cells, somatostatin-secreting delta cells, and pancreatic polypeptide-containing cells (PP cells). Preferably, the majority of the pancreatic islets are insulin-producing β cells.

[0011] The origin of the pancreatic islets is not particularly limited, but is preferably human, pig, mouse, rat, monkey, or dog. In one embodiment, the pancreatic islets are preferably derived from pigs, and are preferably pancreatic islets from young pigs (e.g., 3 days to 4 weeks old or 7 days to 3 weeks old). Pancreatic islets can be obtained by any method known in the art. For example, a method using Liberase as a digestive enzyme (TJ Cavanagh et al., Transplantation Proceedings, 30, 367 (1998)) can be preferably used.

[0012] The size of the pancreatic islets is preferably 50 μm or more and 400 μm or less. The size of the pancreatic islets can be measured using a micrometer under a microscope. A characteristic of the pancreatic islets is that they contain 10% or more β cells. There is no particular upper limit to the proportion of β cells, but it is, for example, 80%.

[0013] The physical properties of the sodium alginate constituting the sodium alginate solution A are not particularly limited.

[0014] The sodium alginate concentration of the sodium alginate solution A is not particularly limited, but is preferably, for example, 1 w / v % or more and 3 w / v % or less.

[0015] Sodium alginate solution A preferably contains pancreatic islets at a concentration of 10,000 IEQ / mL or higher, from the viewpoint of improving the glucose responsiveness of the pancreatic islets after encapsulation. Here, IEQ refers to the number of pancreatic islets per 150 μm diameter. The pancreatic islet concentration is preferably 11,000 IEQ / mL or higher, or 12,000 IEQ / mL or higher. There is no particular upper limit to the pancreatic islet concentration, but it can be, for example, 30,000 IEQ / mL or lower, or 25,000 IEQ / mL or lower.

[0016] There are no specific limitations on the method for preparing sodium alginate solution A, as long as it is prepared to contain the pancreatic islets at the above-mentioned concentration. For example, sodium alginate solution A can be obtained by dissolving an appropriate amount of sodium alginate in physiological saline, adding an appropriate amount of pancreatic islets thereto, and stirring as necessary.

[0017] The divalent cation solution used in step (b) is not limited as long as gelling particles (capsules) encapsulating pancreatic islets can be obtained by adding sodium alginate solution A dropwise thereto. Examples of divalent cations constituting such a divalent cation solution include salts that liberate divalent metal ions in aqueous solution (e.g., calcium chloride, calcium lactate, barium chloride, strontium chloride, etc.). In one embodiment, a preferred salt is calcium chloride, and a preferred divalent cation solution is a calcium chloride solution.

[0018] The concentration of the divalent cation in the divalent cation solution is not particularly limited and can be set in the range of, for example, 50 to 500 mM.

[0019] The manner in which sodium alginate solution A is added dropwise to the divalent cation solution is not limited as long as gelling particles containing pancreatic islets are obtained. In one embodiment, sodium alginate solution A is preferably added dropwise to the divalent cation solution through a needle of an appropriate size.

[0020] The gelling particles can be optionally recovered, for example, by leaving the divalent cation solution in which the gelling particles have been formed to stand for a certain period of time and removing the supernatant, or by repeating this procedure.

[0021] The poly-L-ornithine solution A used in step (c) is not particularly limited as long as it can coat the gelling particles formed in step (b). For example, it can be prepared by dissolving poly-L-ornithine in physiological saline.

[0022] The concentration of poly-L-ornithine in poly-L-ornithine solution A is not particularly limited, but can be, for example, prepared to be 0.05 w / v% or more and 3.0 w / v% or less. In one embodiment, the concentration of poly-L-ornithine in poly-L-ornithine solution A is preferably 0.1 w / v% or more and 2.0 w / v% or less.

[0023] The phrase "adding the particles recovered in step (b) to poly-L-ornithine solution A" in step (c) also encompasses an embodiment in which poly-L-ornithine solution A is added to the gelled particles recovered in step (b). The stirring speed and time in step (c) are optional, and are preferably, for example, 20 to 300 rpm for 1 to 20 minutes.

[0024] The gelling particles can be collected in any manner in step (c), for example, by leaving the poly-L-ornithine solution A to stand for a certain period of time and removing the supernatant. The collected gelling particles preferably have a structure in which the surface of the alginic acid gelling layer is coated with poly-L-ornithine.

[0025] The sodium alginate solution B used in step (d) may have the same concentration as or a different concentration from the sodium alginate solution A used in step (a). In one embodiment, the sodium alginate concentration of sodium alginate solution B is preferably lower than that of sodium alginate solution A, from the viewpoint of ease of coating operation. For example, when the sodium alginate concentration of sodium alginate solution A is 1 w / v% or more and 3 w / v% or less, the sodium alginate concentration of sodium alginate solution B is preferably 0.1 w / v% or more and 0.3 w / v% or less.

[0026] The phrase "adding the particles recovered in step (c) to sodium alginate solution B" in step (d) also encompasses an embodiment in which sodium alginate solution B is added to the gelled particles recovered in step (c). The stirring speed and time in step (d) are optional, and it is preferable to stir at 20 to 300 rpm for 1 to 15 minutes, for example.

[0027] The gelling particles can be collected in any manner in step (d), for example, by leaving sodium alginate solution B to stand for a certain period of time and removing the supernatant. The collected gelling particles preferably have a gelling layer of alginic acid coated on the surface with poly-L-ornithine, and further have a gelling layer of alginic acid thereon.

[0028] In one embodiment, it is preferable that the particles recovered in step (c) are added to poly-L-ornithine solution B before being added to sodium alginate solution B, and after stirring, the particles are recovered and added to sodium alginate solution B in step (d). Here, poly-L-ornithine solution B may have the same or a different concentration as poly-L-ornithine solution A. In one embodiment, the poly-L-ornithine concentration of poly-L-ornithine solution B is preferably lower than that of poly-L-ornithine solution A. For example, the poly-L-ornithine concentration of poly-L-ornithine solution B is preferably 0.01 w / v% or more and less than 0.1 w / v%.

[0029] The sodium citrate solution used in step (e) preferably contains sodium citrate at a concentration of 0.5 to 3 w / v %. The sodium citrate solution is added to chelate the cations in the gelling particles, thereby converting them from a gel to a liquid state and promoting insulin release.

[0030] The phrase "adding the particles recovered in step (d) to a sodium citrate solution" in step (e) also encompasses an embodiment in which a sodium citrate solution is added to the gelled particles recovered in step (d). The stirring speed and time in step (e) are optional, and are preferably, for example, 20 to 300 rpm for 1 to 15 minutes.

[0031] The gelling particles can be collected in any manner in step (e), for example, by leaving the sodium citrate solution to stand for a certain period of time and then removing the supernatant. The collected gelling particles are considered to have an interior that is closer to a liquid state.

[0032] The islet-containing capsules obtained in this manner have excellent glucose responsiveness and stably maintain this property. In one embodiment, the gelling particles (islet-containing capsules) recovered in step (e) preferably have an average diameter of 2000 μm or less, more preferably 1500 μm or less or 1000 μm or less, in order to exhibit good glucose responsiveness. The lower limit of the size of the islet-containing capsules is not particularly limited, but may be, for example, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more. The average diameter is a value calculated by measuring the size of 20 to 50 islet-containing capsules using a micrometer under a microscope and averaging the measurements. [Example]

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0034] 1. Preparation of Encapsulated Pancreatic Islets Pancreatic islets isolated from 14-day-old pigs were suspended in 1.7 w / v% sodium alginate solution (Pronova) at concentrations of 8,000 IEQ / mL, 12,000 IEQ / mL, 16,000 IEQ / mL, or 20,000 IEQ / mL. The suspension was passed through a needle and dripped into a 109 mM calcium chloride solution while being cut by airflow. The solidified alginate beads were recovered from the calcium chloride solution and added to a 0.075 w / v% poly-L-ornithine (PLO) (molecular weight 5,000-15,000) solution and stirred for 10 minutes. The beads were collected, added to a 0.038 w / v% PLO solution, stirred for 6 minutes, collected, added to a 0.17 w / v% sodium alginate solution, stirred for 6 minutes, and finally added to a 1.6% sodium citrate solution, stirred for 2 minutes, to obtain encapsulated islets. The resulting encapsulated islets were cultured at 37°C in a CO2 incubator for 25 days.

[0035] Immediately after encapsulation and after 25 days of culture, a small amount of encapsulated islets was removed from the flask and transferred to a 35 mm dish. The dish was observed under an inverted microscope (4x magnification), and photographs of the encapsulated islets were taken (Figure 1). Figure 1 shows images of the islets immediately after encapsulation (a-d), and images of the islets after 25 days of culture (e-h). Images a and e were prepared using 8,000 IEQ / mL islets, images b and f were prepared using 12,000 IEQ / mL islets, images c and g were prepared using 16,000 IEQ / mL islets, and images d and h were prepared using 20,000 IEQ / mL islets. The average capsule diameter was approximately 600 μm.

[0036] 2. Evaluation of sugar response After encapsulation, 500 IEQ of encapsulated islets were cultured in RPMI 1640 medium supplemented with 2% porcine serum, 10 mM nicotinamide, and antibiotics for 25 days and then placed in a 74 μm netwell insert (Corning). After washing, the inserts containing the islets were placed in 500 mg / L low-glucose medium for 1 hour, after which the medium was collected. The inserts containing the encapsulated islets were then placed in 3750 mg / L high-glucose medium for 1 hour, after which the medium was collected. The insulin concentration in the collected medium was measured using ELISA (Figure 2). The ratio of insulin concentration in high-glucose medium to insulin concentration in low-glucose medium was calculated as the stimulation index (SI) (Figure 3). As shown in Figure 2, capsules prepared using a higher concentration of islets exhibited higher glucose responsiveness than capsules prepared using 8,000 IEQ / mL islets.

[0037] 3. Capsule size and sugar responsiveness Using the same procedure as in 1. above, encapsulated islets with diameters of approximately 500 to 1800 μm were prepared by adjusting the airflow speed. 3000 μm encapsulated islets were prepared by dripping them into an 18G Surflow syringe. Islet-containing capsules were prepared using a suspension containing 16,000 IEQ / mL of islets derived from young pigs.

[0038] The sugar responsiveness of the obtained islet-containing capsules was evaluated in the same manner as in 2 above, and the results are shown in Figures 4 and 5. These results indicated that capsule diameters of 2000 μm or less, 1800 μm or less, and 1500 μm or less are preferable for sugar responsiveness.

Claims

1. (a) preparing a sodium alginate solution A containing pancreatic islets at a concentration of 10,000 IEQ / mL or more; (b) adding the sodium alginate solution dropwise to a divalent cation solution and recovering the gelled particles; (c) adding the particles recovered in step (b) to poly-L-ornithine solution A, stirring, and then recovering the particles; (d) adding the particles recovered in step (c) to sodium alginate solution B, stirring, and then recovering the particles; and (e) adding the particles collected in step (d) to a sodium citrate solution, stirring, and then collecting the particles; A method for producing a pancreatic islet-containing capsule, comprising:

2. The method according to claim 1, wherein the pancreatic islets are obtained from a young pig aged 1 to 3 weeks.

3. 3. The method according to claim 1, wherein the average diameter of the islet-containing capsules is 550 to 750 μm.

4. The method according to any one of claims 1 to 3, wherein the sodium alginate concentration of the sodium alginate solution A is 1 w / v % or more and 3 w / v % or less.

5. The method according to any one of claims 1 to 4, wherein the poly-L-ornithine concentration of the poly-L-ornithine solution A is 0.05 w / v% or more and 3 w / v% or less.

6. The method according to any one of claims 1 to 5, wherein the sodium alginate concentration of the sodium alginate solution B is 0.1 w / v % or more and 0.3 w / v % or less.

7. The method according to any one of claims 1 to 6, wherein in step (d), the particles recovered in step (c) are added to poly-L-ornithine solution B, stirred, recovered, and then added to sodium alginate solution B.

8. The method according to claim 7, wherein the poly-L-ornithine concentration of the poly-L-ornithine solution B is 0.01 w / v% or more and less than 0.1 w / v%.

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