JPH04316479A - Incubator for invertebrate nerve tissue and culture - Google Patents

Abstract

PURPOSE:To put an incubator applicable to invertebrate nerve tissue in an incubator of nerve tissue and a method of culture. CONSTITUTION:A bonded substrate coated with a thin film of collagen is provided with a culture solution comprising L-15 medium, inactivated horse serum, glucose and an antibiotic, the culture solution is permeated into the thin film of collagen to form an incubator of invertebrate nerve tissue, invertebrate nerve tissue is placed in a culture solution of the incubator and cultured to constitute culture of invertebrate.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an incubator for culturing invertebrate neural tissue and a method for culturing the same. It has been revealed that various as yet unknown neuropeptides exist in the neural tissues of invertebrates such as earthworms and slugs. (For example, “Nervous System
ms in Invertebrates” M.A.
Ali, p105-132, (1986) Peptides are defined by two or more amino acids forming a peptide bond (-CO-
A general term for compounds formed by bonding through NH-).

[0002] Therefore, it is necessary from the viewpoint of drug development to search for unknown neuropeptides and find ones that are effective on the functions of the human brain and nervous system. Note that as a method of obtaining neuropeptides, it is far more efficient to culture invertebrate neural tissues and extract the resulting neuropeptides than to extract the invertebrates by dissecting them.

0003

BACKGROUND OF THE INVENTION Compared to vertebrates such as mammals, less is known about the culture conditions for neural tissues of invertebrates.

[0004] Namely, some insects, leeches, slugs,
Although the culture medium conditions for Aplysia and the like are determined, it is necessary to add Aplysia serum to culture Aplysia, and slug brain extract to culture slug.

However, it is difficult to obtain such additives in large quantities. In addition, invertebrate nerve cells have poor adhesion to commercially available cell culture vessels;
It is difficult to improve adhesion even if a culture container coating agent, such as poly-L-lysine, which is commonly used for culturing neural tissue is used.

[0006] Therefore, in order to search for novel neuropeptides, it is necessary to find a medium that has good adhesion to invertebrate nerve tissue and allows for good culture.

[0007]

[Problem to be solved by the invention] Searching for neuropeptides in invertebrates is useful in developing pharmaceuticals that act on the human brain and nervous system, but when it comes to culturing nerve cells,
Conventional culture vessels cannot be used due to poor adhesion.

[0008] Therefore, the challenge is to find a culture vessel and medium suitable for culturing nerve cells.

[0009]

[Means for solving the problem] The above problem was solved by adding a culture solution consisting of L-15 medium, inactivated horse serum, glucose, and antibiotics to an adhesive substrate coated with a thin film of collagen. The problem is solved by configuring an invertebrate culture method characterized by forming a culture vessel for invertebrate nerve tissue by infiltration, and culturing by placing the invertebrate nerve tissue in the culture solution of this culture vessel. be able to.

0010

[Effect] In order to obtain neuropeptides by culturing invertebrate nerve tissue, ■ Use an incubator with good adhesion of nerve cells. ■ Use a medium that allows nerve fibers to unfold and elongate. is necessary.

[0011]As a method to satisfy the requirement (2), the inventor conducted various research and found that collagen (Collagen)
It has been found that adhesion can be improved by coating the substrate on which the culture is performed.

[0012] Collagen is also called collagen,
It is a fibrous protein that together with elastin constitutes the connective tissue of animals. That is, by using a culture vessel coated with collagen gel impregnated with a culture solution, it was possible for nerve cells to adhere to the substrate.

[0013] According to the results of experiments, it has been found that in addition to neurons, non-neuronal cells released from ganglia can also be cultured. As a method to satisfy the requirement (2), the inventor selected L-15 medium as the basic medium as a result of experiments, and used this commercially available culture solution to which inactivated horse serum was added.
Good results were obtained by using glucose as a nutrient source and adding antibiotics to it.

[0014] Here, the reason why L-15 medium was selected as the basic medium is because of the carbon dioxide gas (CO2) incubator.
This is because this medium can maintain a constant pH even without using ator. Inactivated horse serum is obtained by inactivating purchased serum by boiling it in hot water at 56° C. for 30 minutes.

Next, the inventor determined the optimum conditions for horse serum concentration and glucose concentration as follows. Experiment 1: (Determination of optimal concentration of inactivated horse serum) A glucose concentration of 1 was applied to an adhesive substrate with a thin film of collagen gel.
Culture solutions with a fixed concentration of 5 mg/ml and horse serum concentrations were prepared, and the ventral spinal cord ganglia of earthworms were placed on these, and the percentage of adhesion of the ganglia and the number of free cells indicating the state of fiber expansion were examined. The following results were obtained.

[0016]
Table 1 Serum concentration 0%
5% 10% 15
% 20% 25% adherent ganglia
16% 0% 20%
100% 20% 3
3% free cell count − −
− +++
- - From the results in this table, we selected 15% as the serum concentration, fixed this value and prepared a culture solution with varying glucose concentrations, placed the earthworm's celiac ganglion on it, and calculated the adhesion % of this ganglion. When we investigated the number of free cells showing the state of fiber expansion, we obtained the following results.

[0017]
Table 2 Glucose concentration (mg/ml)
0 5 10
15 20 Adhesive ganglia 16%
25% 16% 100%
33% free cell count
− − ++
+++ + From this table, the best results were obtained when the glucose concentration was added at 15 mg/ml.

[0018]

[Example] A medium mixed with the following reagents and adjusted to pH 7.4 was used as a medium for permeating collagen gel. Culture solution ■ Leuboeitz
) L-15 medium (manufactured by Sigma)...85% V/
V ■ Inactivated horse serum (UBT
company) …15
% V/V antibiotic ■ Gentamicin (Sigma
company) …2
0mg/ml ■ Fagi Zone
(Manufactured by Sigma)
...5μg/ml nutrient source ■ Glucose (Wako Pure Chemical, special grade)
...15mg/ml Here, gentamicin and phagizone are antibiotics to suppress the proliferation of bacteria.

Next, the ventral spinal cord ganglion of an earthworm was used as a sample. That is, an earthworm (thick worm) purchased from a fishing gear store was dissected in physiological saline for earthworms supplemented with antibiotics, and the celiac ganglion was removed.

[0020] The extracted ganglion was cut into individual segments, transferred to the above-mentioned culture vessel using a Pasteur pipette, and left at room temperature (22-23°C) for about 24 hours. As a result, after about 24 hours, cells with projections were observed to be released from the ganglion placed in the culture vessel.
In addition, the ganglia were strongly adhered to the culture vessel.

[0021] Furthermore, from about 10 days after the start of culture, it was observed that long fibers, which were thought to be nerve fibers, began to elongate, and some free cells not only spread on the surface of the collagen gel, but also appeared on the gel. It was also observed that it had invaded the inside of the .

[0022] Furthermore, when we performed morphological observation and cell sorting by immunofluorescence on the cells released from the ganglia, we found that the cultured cells were other than nerve cells.
It has been found that cells other than nerve cells that constitute the nervous system can also be cultured using this culture method.

[0023]

[Effects of the Invention] By carrying out the present invention, it becomes possible to culture invertebrate ganglia, which facilitates the search for neuropeptides and nerve growth factors and the study of neural networks.

Claims (3)

[Claims] 1. A culture solution consisting of L-15 medium, inactivated horse serum, glucose, and antibiotics is added to an adhesive substrate coated with a thin collagen film, and the culture solution is allowed to penetrate into the collagen thin film. A culture vessel for invertebrate nerve tissue. 2. A method for culturing invertebrate neural tissue, which comprises placing invertebrate neural tissue in a culture solution of the culture vessel. 3. The invertebrate according to claim 1, wherein the inactivated horse serum constituting the culture solution has a serum concentration of 10 to 20% and a glucose concentration of 10 to 20 mg/ml. Animal nerve tissue incubator.