ARTIFICIAL AXON BUNDLE

Abstract

The present invention relates to an axon bundle obtained by a method of culturing a nerve cell aggregation having one spheroid and an axon bundle extending from the spheroid wherein the nerve cell aggregation consisting of a plurality of neurons having a cell body and an axon obtained by a method comprising the following step: supplying a culture medium to one first chamber, one second chamber and one channel having the length of at least 1 mm, the width of 100 to 150 μm and the height of 100 to 200 μm, which connecting said first chamber and second chamber, wherein said first chamber, second chamber, and channel are contained in one of the modules disposed in a culture plate, seeding the first chamber with a nerve cell derived from a stem cell or a spheroid of primary cultured nerve cells; and culturing said nerve cell, thereby growing an axon bundle from said spheroid and extending them into said channel, wherein the length of the axon bundle obtained is 1 mm or more, and the diameter of the axon bundles is 50 μm or more.

Claims

1. An axon bundle obtained by a method of culturing a nerve cell aggregation having one spheroid and an axon bundle extending from the spheroid wherein the nerve cell aggregation consisting of a plurality of neurons having a cell body and an axon obtained by a method comprising the following step: supplying a culture medium to one first chamber, one second chamber and one channel having the length of at least 1 mm, the width of 100 to 150 μm and the height of 100 to 200 μm, which connecting said first chamber and second chamber, wherein said first chamber, second chamber, and channel are contained in one of the modules disposed in a culture plate, seeding the first chamber with a nerve cell derived from a stem cell or a spheroid of primary cultured nerve cells; and culturing said nerve cell, thereby growing an axon bundle from said spheroid and extending them into said channel, wherein the length of the axon bundle obtained is 1 mm or more, and the diameter of the axon bundles is 50 μm or more.

2. The axon bundle according to claim 1, wherein the stem cells are pluripotent stem cells or iPS derived from neural stem cell.

3. A bundle of axon bundles bundling of two or more axon bundles according to claim 1 by using tissue adhesive.

4. An artificial nerve comprising a bundle of axon bundles according to claim 3.

5. The bundle of axon bundles according to claim 3, wherein the diameter is 1 mm or more.

6. The artificial nerve according to claim 4, for transplantation into a living body.

7. The artificial nerve according to claim 4, for promoting regeneration of a cut nerve.

8. A nerve graft, wherein the artificial nerve according to claim 4 is introduced into a medically acceptable hollow tubular structure.

9. The artificial nerve according to claim 4, wherein the diameter is 1 mm or more.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1 represents a culture plate.

[0035] FIG. 2 is an illustration of a nerve cell.

[0036] FIG. 3 shows histological evaluation by observation of regenerated nerve tissue. The horizontal axis represents, from the left, the collagen gel-implanted group (n=7), the group of 6 axonal bundles (n=6), and the group of 12 axonal bundles (n=6). The lower part is an enlarged view of the upper part. Scale bars are 10 μm each. In all samples of the axon graft transplantation group, a larger number of thicker axons were regenerated than in the control group.

[0037] FIG. 4 represents histological evaluation by quantitative analysis of regenerating axonal area. The figure on the left shows the number of regenerating axons. The vertical axis is the number of axons, and the horizontal axis is 12 bundles of artificial axons, 6 bundles of artificial axons, and NC from the left. The figure on the right is the total area of the regenerating axonal area. The vertical axis is the total axonal area (μm.sup.2), and the horizontal axis is 12 bundles of artificial axons, 6 bundles of artificial axons, and NC from the left.

[0038] FIG. 5 represents histological evaluation by quantitative analysis of regenerating axonal area. The vertical axis is the total axon area (μm.sup.2) per number of axons, and the horizontal axis is 12 bundles of artificial axons, 6 bundles of artificial axons, and NC from the left. The mean axonal area of the axonal graft army was significantly larger than that of the control group, and thick nerves were regenerated (p<0.05, Kruskal-Wallis test, followed by Steeldwas test).

DESCRIPTION OF EMBODIMENTS

[0039] The invention is illustrated in detail by the following examples.

EXAMPLES

[0040] Motor neurons differentiated from human iPS cells are seeded in one or two cylindrical recesses in the culture chamber shown in FIG. It was cultured in an incubator at 37° C. and 5 vol % carbon dioxide for months. The composition of the culture solution was prepared by adding GDNF 20 ng/ml, BDNF 50 ng/ml, B27 Plus supplement 1:50, and Penicillin-Streptomycin (Thermofisher Scientific) to the basal medium, Neurobasal plus medium (Thermofisher Scientific).

[0041] Neurites extended from the structure of the culture chamber into channels extending from within the culture chamber. The cell bodies remained in the culture chamber, forming neuroaxon bundles with neurites longer than 2 cm but without cell bodies in the channels. The single axon bundle contained at least several hundred axons, depending greatly on the distance from the cell body and the number of culture days.

[0042] Nerve axon bundles were cultured by changing the composition of the culture medium in the range of 1 to 200 ng/ml for GDNF and 1 to 200 ng/ml for BDNF. The length and diameter of the obtained nerve axon bundle were measured as follows.

[0043] The axon bundle formed in the channel 13 was imaged at 5 times magnification with an optical microscope (Leica microsystems) while being cultured in the channel 13. The diameter of the nerve axon bundle in the captured image was measured as the thickness of the thinnest part of the nerve axon bundle. Also, the length of the nerve axon bundle on the captured image was measured. 10 nerve axon bundles were randomly selected from the nerve axon bundles cultured above, imaged, the diameter and length were calculated, and the average was calculated. As a result, the average diameter of the nerve axon bundle was 80 μm, and the average length was 5 mm. The minimum length of the axonal nerve bundle cultured above was 0.5 mm, the maximum length was 6 mm, the minimum diameter was 10 μm, and the maximum diameter was 150 μm.

[0044] Multiple (2-6) axonal bundles were enclosed in a silicone tube (1.5 mm inner diameter, 2.5 mm outer diameter, manufactured by Taiyo Kogyo Co., Ltd.) using collagen gel (Nitta Gelatin), and the ischium of mice and rats. It was implanted into the nerve and evaluated for its functional and histological recovery.

[0045] The sciatic nerve injury model was prepared by immunodeficient NOD-SCID mice, wild-type c57bl6j mice (6-week-old, male, Sankyo Lab) and immunodeficient Nude rats and wild-type SD rats (7-week-old, male, Sankyo Lab). used. All mice were deeply anesthetized with a mixed solution of ketamine (100 mg/kg; Sankyo) and xylazine (10 mg/kg; Bayer) by intraperitoneal injection.

[0046] Eight weeks after implantation, a longitudinal skin incision was made on the dorsum of the thigh and the sciatic nerve was exposed by incising the thigh and dividing the thigh muscle with a scalpel. Experimental animals were divided into three groups each time: (1) axonal bundle artificial nerve transplantation group;) A comparison was made between a control negative control group consisting of a collagen gel without axonal bundles and a silicone tube.

[0047] In (1) axonal bundle artificial nerve transplantation group and (2) target negative control group, a sciatic nerve defect of about 6 mm was created in mice, and multiple axonal bundles were transplanted with a silicone tube length of 8 mm. The nerve stump was secured 1 mm inside the tube stump using a 9-0 monofilament nylon suture. In rats, a sciatic nerve defect of about 12 mm was created, the length of the silicone tube was 14 mm, and 9-0 monofilament nylon suture was used at both ends to fix the nerve stump 2 mm inside the tube stump.

[0048] As a result, (1) the mice and rats in the axonal bundle artificial nerve transplantation group showed functionally excellent motor function recovery compared to the mice and rats in the (3) negative control group. Histologically, a wider area of new nerve tissue regeneration was observed. Since this experiment was evaluated 8 weeks after the transplantation, further effects are expected if more time passes after the transplantation.

INDUSTRIAL APPLICABILITY

[0049] The artificial axon bundle, the bundle of artificial axon bundles, the artificial nerve, and the nerve graft of the present invention can be used for functional reconstruction of damaged nerves.