JP2006136339A - Primary culture medium for insect cell, extracellular matrix, and method for creating insect culture cell strain in short period by using them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new cell culture medium suitable for the primary culture of insect cells, an insect-originating water-soluble chitin subjected only to deacetylation as chemical modification, and a method for creating an insect culture cell strain in a short period using the primary culture medium for insect cells and the insect-originating water-soluble chitin. <P>SOLUTION: Disclosed are the primary culture medium for insect cells comprising a lactalbumin hydrolysate, a yeast extract and a tryptose phosphate broth as protein extracts and polyvinylpyrrolidone as a viscous auxiliary agent, and the insect-originating water-soluble chitin subjected only to deacetylation as chemical modification. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a technique for producing insect cultured cell lines.

  There are two stages to creating cultured animal cell lines, including insects. The first stage is called primary culture, and the next stage is called subculture. When producing a cultured cell line, the target tissue is aseptically removed from the insect to be produced, and this tissue is placed in a culture flask together with a cell culture medium. Then, the cells that have emerged from the tissue section to the flask surface are divided. The culture is continued for about one year until a sufficient number is reached. The sufficiently expanded cells are transferred to a new flask and subcultured. The culture of the produced cell line is subculture.

  Conventionally, various insect cell culture media have been available. For example, Grace medium, IPL-41 medium, Schreider's Drosophila medium, Sf900II, TC-100 medium, Sf-9 cell medium, Sf-21 cell medium, Express Five medium, EX-400 medium, and the like. In addition, Minoru Mitsuhashi et al. Produced a medium such as the MGM-strain with reference to the results of insect body fluid analysis (MGM-443; see Non-Patent Document 1, MGM-448; see Non-Patent Document 2, and MGM- 450; see Non-Patent Document 3, MGM-464; see Non-Patent Document 4, MGM-443, MGM-448, MGM-450, and MGM-464 are medium names).

  Furthermore, the present inventors have produced MM-8 SF medium and the like (see Non-Patent Document 5). However, these media are not suitable for primary culture, and have been used exclusively for the passage of the cultured cell line produced, that is, for the maintenance of the cells after the cell line has been produced. When primary culture was performed using these media, cell growth was not good, and the primary culture required a long period of about one year as described above. Thus, there has not been a medium suitable for primary culture that can transfer cells to subculture at an early stage. In addition, many of these media have been used exclusively for specific species of insects. For example, the culture medium for Schreider's Drosophila was intended for cultivating Diptera insects, particularly Drosophila cells, and the medium for Sf900II, Sf-9 cells, and cells for Sf-21 cells were intended for lepidopterous insects, especially hornworm cells. There has been no medium suitable for the growth of a wide range of insect cells.

  On the other hand, flasks generally used for cell culture are made of plastic, and collagen I, II, III, IV or V, fibronectin, gelatin, laminin, poly-L-lysine, matrigel, EHS-Natricks on the plastic surface. It is considered that the extracellular matrix such as is coated. Although there are few detailed reports on these compositions, for example, chitin and chitosan have been reported on extracellular matrix extracted from crustacean shrimp and crabs. These chitin and chitosan were subjected to various chemical modifications. Extracellular matrix has the effect of adhering tissue to a cell culture container and attaching cells that have migrated from the tissue to the surface of the container to divide and proliferate. A high extracellular matrix was desired.

Mitsuhashi, J. (1980) In: Kurstak, E., Maramorosch, K. and Dubendorfer, A. (eds) Invertebrate Systems In Vitro Elsevier / North Holland Biomedical, Amsterdam, pp47-58 Mitsuhashi, J. (1984) Zool.Sci. 1,415-419 Mitshuhashi, J. and Inoue, H (1988) Appl. Entomol. Zool. 23, 488-490 Mitsuhashi, J. (2001) In Vitro Cell. Dev. Biol. 37A, 330-337 Shigeo Imanishi (1994) "Development of insect function experiment system and insect cell culture system" research result 295, Agriculture, Forestry and Fisheries Technology Conference Secretariat, 74-84

  An object of the present invention is to provide a novel cell culture medium suitable for primary culture of insect cells. Another object of the present invention is to provide an insect-derived water-soluble chitin that is only deacetylated as a chemical modification. It is another object of the present invention to provide a method for producing an insect culture cell line in a short period of time using the insect cell primary culture medium and the insect-derived water-soluble chitin.

  As described above, cell division and proliferation are limited to cells suitable for primary culture of insect cells, that is, cells that have started cell culture and have emerged from the cut tissue pieces to the bottom of the culture flask (primary cells). Insect cell culture media that facilitates and allows early transfer to subculture have been anxious.

  The present inventors have intensively studied a medium suitable for primary culture that promotes divisional proliferation of cells obtained from tissue pieces and enables early transfer to subculture. The inventors have found that a novel medium containing an agent is suitable for primary culture, and have completed the present invention.

  In addition, the present inventors have intensively studied a novel extracellular matrix coated on a culture vessel suitable for culturing a novel insect cell, and have a water-solubility without chemical modification other than insect-derived deacetylation. When cell culture is performed by coating a cell culture vessel using chitin as an extracellular matrix, the tissue adheres well to the culture vessel, and cells that have migrated from the tissue also adhere to the vessel surface and divide. -It has been found that it has proliferated, and the present invention has been completed. Furthermore, the present inventors can perform primary culture more efficiently by using a cell culture vessel coated with an extracellular matrix of water-soluble chitin that is not chemically modified from insects and a medium suitable for primary culture. And the present invention has been completed.

That is, the present invention is as follows.
(1) a medium for primary culture of insect cells containing lactalbumin hydrolyzate, yeast extract and tryptose phosphate broth as protein extracts, and polyvinylpyrrolidone as a viscosity aid;
(2) Lactalbumin hydrolyzate: 1000-3000 mg / L, yeast extract: 1000-3000 mg / L, tryptose phosphate broth: 1000-3000 mg / L, polyvinylpyrrolidone: 200-500 mg / L, (1) Insect cell primary culture medium,
(3) The medium for primary culture of insect cells according to (1) or (2), wherein the polyvinylpyrrolidone is polyvinylpyrrolidone K-90,
(4) Insect-derived water-soluble chitin that is only deacetylated as a chemical modification,
(5) Water-soluble chitin derived from an insect of (4), which is derived from a silkworm,
(6) Water-soluble chitin derived from insects of (5), which is derived from silkworm husks,
(7) an extracellular matrix containing water-soluble chitin derived from the insect of any one of (4) to (6),
(8) An extracellular matrix solution for coating a culture vessel containing 0.001% to 1% of the water-soluble chitin derived from the insect of any one of (4) to (6),
(9) An insect cell culture container coated with the insect-derived water-soluble chitin according to any one of (4) to (6),
(10) The method for producing water-soluble chitin derived from an insect according to (6), which comprises extracting chitin from silkworm husk and deacetylating the chitin,
(11) Production of an insect cultured cell line in a short period of time using the insect primary culture medium of any one of (1) to (3) and the water-soluble chitin derived from the insect of any one of (4) to (6) And the insect cell in the insect primary culture medium of any one of (1) to (3) using a container coated with the water-soluble chitin derived from the insect of any one of (12) (4) to (6) (11) A method for producing an insect culture cell line in a short period of time.

  The insect cell primary culture medium of the present invention is a novel insect cell primary culture medium containing at least lactalbumin hydrolyzate, yeast extract and tryptose phosphate broth as protein extracts and polyvinylpyrrolidone as a viscosity auxiliary. Yes, by using this medium, a cultured cell line can be established from each insect tissue efficiently in a short period of time.

  In addition, the insect-derived water-soluble chitin that is only deacetylated as a chemical modification of the present invention can be used for coating a cell culture container, and the coating allows the cells to adhere to the surface of the culture container and efficiently proliferate / Can split. Furthermore, by using the insect cell primary culture medium of the present invention and water-soluble chitin derived from insects that are only deacetylated as a chemical modification, a cell line can be efficiently established in a shorter period of time by performing primary culture. Can do.

Hereinafter, the present invention will be described in detail.
1. Composition of Insect Cell Primary Culture Medium of the Present Invention The insect cell primary culture medium of the present invention contains at least a protein extract and a viscosity auxiliary agent. The primary cell culture medium of the present invention may further contain an inorganic salt mixture composition, a saccharide composition, an amino acid mixture composition, and a vitamin mixture composition.

  Protein extracts include at least lactalbumin hydrolysate, yeast extract (Yeastlate) and tryptose phosphate broth (Tryptose Phosphate Broth), and fetuin, cytochrome C, inosine, bovine plasma albumin V, etc. May be included. Lactalbumin, yeast extract, and tryptose phosphate broth can be obtained by known techniques. Fetuin can be derived from, for example, fetal calf serum, and cytochrome C can be derived from, for example, horse heart. These protein extracts can all be commercially available. For example, lactoalbumin hydrolyzate can be manufactured by DIFCO (No. 5996), and yeast extract prepared for cell culture can be used (TC (Tissue Culture) -yeast extract). DIFCO's (No. 5577) can be used, and tryptose phosphate broth can be used as DIFCO's (No. 0060). Further, fetuin can be manufactured by Sigma (No. F2379), cytochrome C can be manufactured by Sigma (No. C2506), and inosine can be used by Wako Pure Chemical Industries, Ltd.

  The content of lactalbumin hydrolyzate, yeast extract and tryptose phosphate broth in the medium is preferably 500 to 3,000 mg, more preferably 1,000 to 3,000 mg, and particularly preferably 1,000 to 2,000 mg per liter of the medium. . The fetuin content is preferably 1 to 100 mg per liter of medium, more preferably 1 to 50 mg, particularly preferably 5 to 15 mg, and the cytochrome C content is preferably 1 to 500 mg, more preferably 1 to 100 mg, per liter of medium. Particularly preferred is 10 to 100 mg. The content of inosine is preferably 1 to 500 mg, more preferably 1 to 200 mg, particularly preferably 10 to 200 mg, and the content of bovine plasma albumin V is preferably 1000 to 10,000 mg, more preferably 100 to 10,000. mg, particularly preferably 1,000 to 10,000 mg.

  Polyvinyl pyrrolidone is used as a viscosity adjuvant contained in the primary cell culture medium of the present invention. Examples of polyvinyl pyrrolidone include polyvinyl pyrrolidone K-25, polyvinyl pyrrolidone K-30, and polyvinyl pyrrolidone K-90, and any of these can be used. Polyvinyl pyrrolidone K-90 is preferably used and manufactured by Wako Pure Chemical Industries, Ltd. (Catalog number 168-03115 of the company) can be exemplified. The content of polyvinylpyrrolidone is preferably from 100 to 1,000 mg, more preferably from 100 to 500 mg, particularly preferably from 200 to 500 mg per liter of the medium.

As the inorganic salt mixed composition, saccharide composition, amino acid mixed composition, and vitamin mixed composition, those that can be generally added to animal cell culture media may be added. For example, NaH ₂ PO ₄ , NaHCO ₃ , KCl, CaCl ₂ , CuCl ₂ , CoCl, FeSO ₄ , MgCl ₂ , MgSO ₄ , MnCl ₂ , NaCl, NaH ₂ PO ₄ , (NH ₄ ) ₆ as an inorganic salt mixed composition And (Mo ₇ O ₂₄ · 4H ₂ O), a composition containing ZnCl ₂ . Examples of the saccharide composition include a composition containing glucose, fructose, sucrose, malic acid, α-ketoglutaric acid, succinic acid, fumaric acid, and maltose. The amino acid mixed composition includes α-alanine, β-alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, hydroxyproline, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan. , Tyrosine, and an amino acid composition containing valine. Examples of the vitamin mixed composition include vitamin mixed compositions containing biotin, calcium D-pantothenate, choline chloride, folic acid, i-inositol, nicotinic acid, pyridoxine, riboflavin, thiamine, vitamin B ₁₂ , and paraaminobenzoic acid. These inorganic salt mixed composition, saccharide composition, amino acid mixed composition, and vitamin mixed composition desirably contain all the above-mentioned substances, but may lack some of the substances. Other substances may be added. As these, all commercially available products can be used. In addition, commercially available inorganic salt compositions, saccharide compositions, amino acid compositions, and vitamin compositions for medium addition may be used. You may add and use a protein extract and a viscosity adjuvant. In this case, known media include known insect cell culture media such as Grace media and Shneider's Drosophila media. Furthermore, antibiotics such as penicillin and streptomycin, glutathione and the like may be added to the medium. In short, as long as the medium contains at least lactalbumin hydrolysate, yeast extract (Yeastlate) and tryptose phosphate broth and polyvinylpyrrolidone, it is included in the medium of the present invention. Furthermore, when culturing using the culture medium of the present invention, it is preferable to add animal serum such as fetal bovine serum, insect lymph and the like. The addition amount of serum and lymph is several to 30%.

  Insect cell primary culture medium of the present invention comprises lactalbumin hydrolysate (Lactoalbumine Hydrolysate), yeast extract (Yeastlate) and tryptose phosphate broth (Tryptose Phoaphate Broth), additives for the medium containing polyvinylpyrrolidone, and other proteins It can be obtained by mixing an extract mixture composition, an inorganic salt mixture composition, a saccharide composition, an amino acid mixture composition, and a vitamin mixture composition. Therefore, the medium additive containing at least lactalbumin hydrolysate, yeast extract (Yeastlate) and tryptose phosphate broth and polyvinylpyrrolidone is also included in the scope of the present invention.

  Examples of the primary cell culture medium of the present invention include MX medium. The primary cell culture medium of the present invention can be prepared by dissolving the above-mentioned protein extract, viscosity auxiliary, inorganic salt mixed composition, saccharide composition, amino acid mixed composition and vitamin mixed composition in pure water. it can. Further, a necessary amount of the above-mentioned animal serum or insect lymph is added. If necessary, the pH is adjusted with an acidic solution such as hydrochloric acid or a basic solution such as sodium hydroxide. The pH is preferably 6.0 to 7.0, particularly preferably 6.2 to 6.5.

2. Use of the medium of the present invention for primary cell culture The medium of the present invention can be used for primary culture of all insect cells, particularly insects belonging to Lepidoptera, Diptera, Coleoptera, Hemiptera, etc. Suitable for In addition, cultured cell lines can be produced in a short period of time from all tissues of these insects. In particular, it is suitable for culturing from embryonic tissue, fat body tissue, testicular or ovarian reproductive tissue, digestive system tissue, nervous system tissue, and muscle system tissue. That is, by using the culture medium of the present invention, it is possible to produce a cultured cell line in a short period of time from a tissue such as the testis, which has been conventionally difficult.

  Tissues from which insect cell cultures are to be produced are aseptically removed and placed in a suitable cell culture container such as a cell culture flask together with the medium of the present invention, and the medium is replaced and supplemented as appropriate for 2 to 3 months. By culturing, the expanded cell population can be replanted into a new flask in a shorter period of time than before. At this time, normal culture conditions for insect cells may be adopted as the culture conditions. For example, culture may be performed in an incubator at 20 to 28 ° C. The cultured cell line produced can be transferred to a known subculture medium such as MGM-464 medium, IPL-41 medium, Grace medium, EXCELL400 system medium, Sf900II medium, Schneider medium, etc. By using the medium of the present invention as compared with the above medium, it is possible to quickly shift to subculture. In addition, it may be cultured in the medium of the present invention as it is, or by proliferating / dividing well by reducing the proportion of the serum content added to the medium of the present invention, long-term subculture is possible. Is possible.

3. Extracellular matrix of the present invention In the present specification, the extracellular matrix refers to a matrix, a substrate, and a carrier to which cells can adhere and divide and proliferate in cell culture. Preferably, the extracellular matrix is used to coat the cell culture surface of the culture vessel. The extracellular matrix of the present invention contains, as a main component, insect-derived water-soluble chitin that has undergone only deacetylation as a chemical modification. As the water-soluble chitin, those derived from all insects can be used. Further, those extracted from the molting shells of insect moths are preferable, and those extracted from the molting shells of silkworm moths are particularly preferable.

  The water-soluble chitin of the present invention can be obtained as follows. １ Add 1N hydrochloric acid solution to the molting shell and treat it at 100 ℃ for 20 minutes in an environment filled with nitrogen gas, then remove protein in 1N sodium hydroxide solution at 80 ℃ for 36 hours to prepare chitin . Water-soluble chitin can be prepared by dissolving chitin in a concentrated alkaline aqueous solution at room temperature, and leaving the high-viscosity alkaline chitin aqueous solution at room temperature for a long time to perform random deacetylation. At this time, water solubility is exhibited only when the degree of deacetylation is 45 to 55%.

For the preparation of water-soluble chitin used in the extracellular matrix of the present invention, it is desirable to select chitin having a high affinity for water. The affinity of chitin for water is determined by the method of Brunauer et al. (J. Amer. Chem. Soc 62.1723 -1732 (1940)). That is, the moisture absorption amount at each relative humidity can be measured by an indirect method of a saturated salt solution, and the BET theoretical formula can be applied. In the preparation of chitin suitable as the extracellular matrix of the present invention, the internal surface area of chitin as a raw material is desirably 180 m ² / g or more.

  In other words, the chitin extracted from insects is evaluated for its affinity for water, and those evaluated as having high affinity for water can be used as the extracellular matrix of the present invention. In this respect, water-soluble chitin derived from the above-mentioned insect wings, particularly silkworm husks of silkworms, is excellent. Chitin prepared from silkworm husks has a large internal surface area and more water molecules adsorbed than other silkworm-derived cuticles. Also, the heat of adsorption is high, and the affinity for water is greater than that of Tensan, Saksan, and Semi. Moreover, chitin derived from the silkworm cocoon shell has a similar affinity for water compared to chitin derived from crabs and shrimps. As the silkworm, for example, wide-ranging rice cake varieties No. 601, No. 602 × No. 602, Naka No. 603 (nickname Shin Asagiri) can be used. The insect-derived water-soluble chitin of the present invention can be obtained by deacetylating the chitin thus obtained as follows.

  Deacetylation refers to removal of the acetyl group of chitin, and can be performed by treating the extracted water-soluble chitin with an alkali or acid, preferably concentrated ant potassium or acid. At this time, heating may be performed. It can also be carried out by a reaction such as Clementen reduction. However, deacetylation can be carried out in a homogeneous system by treating with concentrated alkali utilizing the fact that chitin dissolves in concentrated alkali as described later.

  The chitin of the present invention is used after deacetylation, that is, by removing the N-acetyl group. Whereas the conventionally used crustacean-derived chitin has undergone various chemical modifications such as acylation, tosylation and carboxylmethylation, the water-soluble chitin of the present invention only needs to be deacetylated. . Deacetylation is desirably performed in a homogeneous system utilizing the fact that chitin dissolves in a concentrated alkaline solution. In the reaction in a heterogeneous system, N-acetyl groups are preferentially removed from the chitin molecule surface and the amorphous part, so that the distribution of N-acetyl groups is biased. On the other hand, deacetylation in a homogeneous system proceeds at random, and the distribution of remaining N-acetyl groups is ubiquitous as scattered. As a result, hydrogen bonds between chitin molecules are weakened, so that water solubility is developed. That is, it is reported that partially deacetylated chitin is soluble in water when the degree of deacetylation is in the range of 40-60% (Kramer. KJ et al., Insect Biochem. 14 (3), 293-298 ( 1984)).

According to this method, deacetylation in a homogeneous reaction system is performed using a high-viscosity alkaline solution to which chitin is added, and the reaction conditions may be set so that the degree of deacetylation is 45 to 55%. Identification of partially deacetylated chitin can be examined from FT-IR (Fourier transform infrared spectroscopy) spectra. The degree of deacetylation can be easily measured by using an IR (infrared light) spectrum method. When chitin is deacetylated to become partially deacetylated chitin, absorption due to an amide group peculiar to the N-acetyl group decreases, and therefore the degree of deacetylation can be estimated from the IR spectrum. Specifically, the quantitative characteristic bands of the amide II band at 1560 cm ^-1 in the IR spectrum, a band of 1070 cm ^-1 or 1039cm ^-1 as an internal standard, measuring the ratio of absorbance A ₁₅₆₀ / A ₁₀₇₀ Can do. The degree of deacetylation can be determined using a calibration curve prepared in advance using a deacetylated chitin preparation as a control. Therefore, the water-soluble chitin derived from insects that is only deacetylated as a chemical modification of the present invention is chitin having a high affinity for water extracted from insects, and has a degree of deacetylation of 40 to 60%, preferably 45-50% chitin.

  The thus obtained water-soluble chitin derived from insects that is only deacetylated as a chemical modification has a structure of 40 to 60% chitin and the remaining 40 to 60% has a chitosan structure. That is, insect-derived water-soluble chitin that is only deacetylated as a chemical modification is chitin with high affinity for water extracted from insects, of which 40-60% exists as chitin, and the rest 40-60% is a substance present as chitosan. Since the surface of the cell membrane is negatively charged, the cell can adhere to the positively charged amino group of chitosan and divide and proliferate. This water-soluble chitin can be used as an extracellular matrix. Further, the water adsorption amount of this water-soluble partially deacetylated chitin is improved by about 6 times that before the treatment and exhibits high hygroscopicity, and can be used as a moisturizing agent and a hygroscopic agent in the food and cosmetic fields.

4). Use of extracellular matrix of the present invention The water-soluble chitin of the present invention is dissolved in water at a concentration (W / V) of 0.001% to 1%, poured so as to cover the cell attachment surface of the cell culture container, and air-dried. The cell culture container can be coated by adhering to the surface of the culture container. For example, a good coating can be achieved by placing 0.5 mL of a chitin aqueous solution per 200 mm ^{2 of} the cell culture flask surface and air-drying it.

  Accordingly, culture containers such as culture flasks, petri dishes, plates, etc., in which a part or all of the inner surface of the container is coated with the extracellular matrix of the present invention are also included in the scope of the present invention. In this way, when insect cells are cultured using the cell culture vessel coated with the extracellular matrix of the present invention, the cells adhere to the culture vessel and grow and divide well.

5. Production of insect cell line using the insect cell primary culture medium of the present invention and the deacetylated insect-derived chitin of the present invention A container coated with the deacetylated insect-derived chitin of the present invention By using the insect cell primary culture medium of the present invention, an insect culture cell line can be efficiently produced.

  For primary culture of insect cells of the present invention in which fetal bovine serum is added to a container obtained by aseptically removing a tissue from an insect and coating the tissue with the deacetylated insect-derived chitin of the present invention as an extracellular matrix Incubate with medium and start culturing. At this time, it is desirable to wash the container coated with the extracellular matrix once with sterile physiological saline. Any insect can be used as the insect, but insects belonging to the order Lepidoptera, Diptera, Coleoptera, Hemiptera, etc. are suitable. Any tissue can be used, but embryonic tissue, fat body tissue, testicular or ovarian reproductive tissue, digestive system tissue, nervous system tissue, and muscle system tissue are suitable. The culture is continued until the cells that have emerged from the tissue section on the surface of the culture vessel have reached a sufficient number of divisions. The culture conditions may be normal culture conditions for insect cells. In the meantime, cell migration from the tissue can be activated by replacing half of the medium in the culture vessel with a fresh medium, for example, every 1-2 weeks. When cell migration started and a large number of cell populations were formed on the bottom of the culture vessel, and the population was greatly enlarged, the content of fetal calf serum in the primary culture medium added with fetal calf serum was gradually reduced. Go. For example, 30% fetal bovine serum may be added at the start of the culture, and 20% fetal bovine serum may be added when the cell population is enlarged. Next, subculture is performed by replacing half of the medium with a known subculture medium such as MGM-464 medium, IPL-41 medium, Grace medium, EXCELL400 system medium, Sf900II medium, or Schneider medium. The medium can be finally cultured in the desired subculture medium by exchanging half of the medium at an appropriate interval.

The details of the production process of the silkworm intestinal tissue-derived cell line are shown as an example.
1. The body surface of aseptically bred silkworms is washed 3 times with sterile Carlson solution (100,000 units of penicillin / 100 mL with 0.05% gentamicin and 0.05% antiformin added).
2. Immerse in the last Carlson solution for 1-2 minutes to stop silkworm movement.
3. Cut the silkworm tail. Peel the skin of the chest with tweezers. Grab the head with tweezers and drag out the intestinal tissue. Immerse intestinal tissue in Carlson's solution in a Petri dish.
4). The pericardium is removed with tweezers.
5. Place in an Eppendorf tube.
6). Place collagenase I solution in an Eppendorf tube (4,000 U / 0.5 g tissue weight / collagenase mL).
7. Leave at 27 ° C for 2 hours.
8). Centrifuge for 10 seconds in a small table centrifuge (such as Chibitan ™).
9. Discard the supernatant and wash twice with balanced salt solution.
10. Pipet strongly.
11. Take into a 15 mL disposable centrifuge tube and centrifuge at 500-800 rpm for 1 minute.
12 Discard the supernatant. Add balanced salt solution again and discard the re-centrifuged supernatant at 1,000 rpm for 1 minute. A collection of cells is obtained as a pellet.
13. Add MX30 medium, transfer to 24-multiwell plate and culture. At this time, a plate whose culture surface is coated with 0.01% (W / V) chitin in advance is used (particularly in the case of primary culture of blood cell line, ovary, testis and embryonic tissue).
14. Incubate at 25 ° C.
15. Half of the medium (0.5-0.7 mL) is changed every 2 weeks.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Preparation of insect cell primary culture medium A medium was prepared by dissolving the following substances in pure water so that the amount per 1 L of the medium was as shown below. This medium was designated as MX medium.

Inorganic salt mixture composition
NaH ₂ PO ₄・ 2H ₂ O 507mg
NaHCO ₃ 300mg
KCl 1720mg
CaCl ₂・ 2H ₂ O 750mg
CuCl ₂・ 2H ₂ O 0.1mg
CoCl ・ 6H ₂ O 0.03mg
FeSO ₄・ 7H ₂ O 0.28mg
MgCl ₂・ 4H ₂ O 1140mg
MgSO ₄・ 7H ₂ O 3269mg
MnCl ₂・ 4H ₂ O 0.01mg
NaCl 1425mg
NaH ₂ PO ₄・ H ₂ O 580mg
(NH ₄ ) ₆ (Mo ₇ O ₂₄・ 4H ₂ O) 0.02mg
ZnCl ₂ 0.02mg

Sugar composition
D-glucose 2917mg
Fructose 20.9mg
Sucrose 11865mg
Malic acid 306mg
α-ketoglutaric acid 169mg
Succinic acid 27.4mg
Fumaric acid 25.2mg
Maltose 500mg

Amino acid mixture composition
L-α Alanine 131.5mg
β-alanine 234mg
L-Arginine ・ HCl 692mg
L-asparagine 797mg
L-Aspartic acid 797mg
L-cystine 10.5mg
L-glutamic acid 1000mg
L-glutamine 750mg
Glycine 371mg
L-histidine 1142mg
L-isoleucine 396mg
L-leucine 157mg
L-cystine ・ 2Na 60mg
L-Hydroxyproline 400mg
L-Lysine / HCl 610mg
L-methionine 521mg
L-Phenylalanine 562mg
L-proline 396mg
DL-serine 559mg
L-threonine 173mg
L-tryptophan 91.5mg
L-tyrosine 21mg
L-Tyrosine ・ 2Na 180mg
L-valine 292mg
L-histidine 1142mg

Vitamin mix composition <br/> Biotin 0.123mg
D-Calcium pantothenate 0.089mg
Choline chloride 10.85mg
Folic acid 0.125mg
i-Inositol 0.285mg
Nicotinic acid 0.165mg
Pyridoxine / HCl 0.285mg
Riboflavin 0.125mg
Thiamine ・ HCl 0.125mg
Vitamin B ₁₂ 0.12mg
Paraaminobenzoic acid 0.245mg

Protein extract composition <br/> Lactalbumin hydrolyzate 1500mg
TC-yeast extract 1500mg
Tryptophosphate broth 1500mg
Fetuin 10mg
Cytochrome C 50mg
Inosine 100mg
Bovine plasma albumin V 5000mg

Viscosity aid <br/> Polyvinylpyrrolidone K-90 250mg
(mg content per 1000mL medium)

  Further, a medium supplemented with 20% fetal bovine serum (FBS) (referred to as MX20 medium) and a medium supplemented with 30% (referred to as MX30 medium) were prepared. The prepared medium was adjusted to pH 6.3 with potassium hydroxide. The prepared medium was sterilized and stored in a Sterilup (trademark) (manufactured by Millipore, model number SCGV05012) filter sterilization container.

[Example 2] Preparation of water-soluble chitin derived from deacetylated insects Preparation of water-soluble chitin from persimmon shells (1) Preparation method 5g of persimmon shells of wide-ranging persimmon varieties No. 601 ・ Day No.602 × Naka 602 ・ Naka 603 (nickname Shin Asagiri) in 300mL of 1N hydrochloric acid solution Then, in an environment filled with nitrogen gas, it was treated at 100 ° C. for 20 minutes, washed with warm water and distilled water until the husks were neutral and dried in vacuo. Next, the dried cocoon shell was immersed in 300 mL of 1N sodium hydroxide solution, and the protein of the cocoon shell was removed while stirring at 80 ° C. for 36 hours. As a result, 0.9 g of chitin was obtained.

(2) Evaluation of affinity for water The hygroscopic analysis of the obtained chitin was measured according to the method of Yano, Bull, Mellon, Ashpole et al. A theoretical formula was applied. As a result, the chitin obtained had a large internal surface area and a high adsorption heat, and thus was evaluated as having a high affinity for water.

(3) Deacetylation of chitin Deacetylation was performed in a homogeneous solution system by adding 3 g of chitin obtained from the above-mentioned cocoon dehulled shell to a 40% sodium hydroxide solution and allowing to stand at 25 ° C. for 70 hours. . Partially deacetylated chitin was synthesized with a yield of 74%. When the obtained deacetylated chitin was analyzed by IR spectroscopy, the degree of deacetylation was 45 to 48%. This partially deacetylated chitin showed water solubility at room temperature. The amount of water adsorbed at the saturated water vapor pressure of the water-soluble partially deacetylated chitin increased 6.3 times before the treatment, and exhibited high hygroscopicity. This deacetylated chitin was used as the extracellular matrix.

[Example 3] Production of cell culture strain Multiplate for culture (product number MS-8024R, manufactured by Sumitomo Bakelite Co., Ltd.) with 0.5 mL of 0.01-0.1% aqueous solution of water-soluble chitin derived from the above-mentioned deacetylated husk shell After pouring into each culture hole and allowing it to reach the entire bottom, it was air dried at room temperature to evaporate the liquid components. Thereafter, the culture surface was washed once with sterile physiological saline.

  Aseptically extracting fat pads from larvae larvae, in the holes of the multiplate containing 1.5 mL (1.5 mL per 200 mm square hole bottom area) of the MX30 medium prepared in Example 1 after the above treatment. The culture was started. Half of the medium was changed every 14 days. After 30 days, cell migration from the tissue became active, and after 2 to 3 months, a large number of cell populations were formed on the culture surface, and the population was enlarged. At this time, it was observed by microscopic observation that the cells were firmly attached to the bottom of the flask. Therefore, half of the medium was replaced with MX20 medium. In addition, change the MX20 medium half every 14 days, and after several changes, make sure that the cells have grown sufficiently, and use a culture Pasteur pipette to spray the medium strongly onto the cell population and culture the cells. It was suspended from the surface and planted in a new culture flask (Falcon, Traditional Type Flask No. 3018). Furthermore, the culture was continued, and the operation of replanting with a new flask after the number of cells increased was repeated. During this period, the medium was gradually replaced with a medium supplemented with 10% FBS for subculture, and then the subculture was repeated as an established cell line to maintain the cells.

Claims (7)

Containing lactalbumin hydrolyzate, yeast extract and tryptose phosphate broth as protein extracts, polyvinylpyrrolidone as a viscosity aid, cystine 2Na, hydroxyproline, isoleucine, serine, tyrosine 2Na, CaCl ₂ , CuCl ₂ Insect cell primary culture for the production of insect cell lines containing, CoCl ₂ , FeSO ₄ , MnCl ₂ , NaCl, NaH ₂ PO ₄ , (NH ₄ ) ₆ (Mo ₇ O ₂₄ · 4H ₂ O) and ZnCl ₂ Culture medium. Lactalbumin hydrolyzate: 1000-3000 mg / L, yeast extract: 1000-3000 mg / L, tryptose phosphate broth: 1000-3000 mg / L, polyvinylpyrrolidone: 200-500 mg / L, cystine 2Na: 60 mg / L, Hydroxyproline: 400 mg / L, Isoleucine: 396 mg / L, Serine: 559 mg / L, Tyrosine 2Na: 180 mg / L, CaCl ₂ · 2H ₂ O: 750 mg / L, CuCl ₂ · 2H ₂ O: 0.1 mg / L, CoCl ₂ · 6H ₂ O: 0.03 mg / L, FeSO ₄ · 7H ₂ O: 0.28 mg / L, MnCl ₂ · 4H ₂ O: 0.01 mg / L, NaCl: 1425 mg / L, NaH ₂ PO ₄ · The medium for primary culture of insect cells according to claim 1, comprising H ₂ O: 580 mg / L, (NH ₄ ) ₆ (Mo ₇ O ₂₄ · 4H ₂ O): 0.02 mg / L and ZnCl ₂ : 0.02 mg / L.   The medium for primary culture of insect cells according to claim 1 or 2, wherein the polyvinylpyrrolidone is polyvinylpyrrolidone K-90.   Insect cell culture cells containing water-soluble chitin with a deacetylation degree of 45-55%, deacetylated by dissolving silkworm-derived chitin in concentrated alkaline aqueous solution and leaving the high viscosity alkaline chitin aqueous solution at room temperature Outside matrix.   An extracellular matrix solution for container coating for insect cell culture, comprising 0.001% to 1% of the water-soluble chitin derived from silkworm cocoons according to claim 4.   An insect cell culture container coated with the silkworm cocoon-derived water-soluble chitin according to claim 4 or 5.   An insect in a short period of time, wherein the insect cell is cultured in the insect cell primary culture medium according to any one of claims 1 to 3, using the container coated with the insect-derived water-soluble chitin according to claim 6. Production method of cultured cell lines.