WO2020075841A1

WO2020075841A1 - Cell culturing substrate containing collagen derived from barramundi scales

Info

Publication number: WO2020075841A1
Application number: PCT/JP2019/040219
Authority: WO
Inventors: 松田　博幸; 雅莉門; 俊志本元; 務長谷阪; 俊二山森; 田口　秀典
Original assignee: 日本電熱株式会社
Priority date: 2018-10-12
Filing date: 2019-10-11
Publication date: 2020-04-16
Also published as: CN112840013A; JP2020058323A; TW202028454A

Abstract

The present invention pertains to a cell culturing substrate containing collagen derived from barramundi scales.

Description

Cell culture substrate containing collagen from barramundi scale

The present invention broadly relates to a cell culture substrate containing collagen fibers derived from barramundy scales.

In the field of regenerative medicine, collagen gel is widely used as a raw material for cell culture substrates. Most of its raw materials depend on the dermis of vertebrates such as cattle and pigs, which have high resource abundance and high yield, but the use of these collagens is not without problems. For example, in some parts of Southeast Asia, there is a problem that culturally restricted handling of products of mammalian origin.

Also, it is necessary to avoid infectious diseases from pathogens such as BSE (bovine spongiform encephalopathy), which became a problem in the early 2000s, and the search for collagen raw materials to replace cattle and pigs has become an industrial issue.

Therefore, the use of fish-derived collagen, which is easier to build a mass production system by aquaculture and has a lower risk of infection than vertebrates, has been investigated.However, most fibrous gels prepared from conventional fish-derived collagen are derived from homeothermic animals. It is insufficient from the viewpoint of heat stability in comparison with the above-mentioned one, and cannot form a mechanically stable fibrous gel such as collagen gel derived from cattle or pigs.

In recent years, there are examples of using fish collagen for cell culture. For example, it has been reported that collagen monomers extracted from tilapia (Japanese name Izumidai) living in warm regions such as Southeast Asia have a high denaturation temperature.

Regarding tilapia, JP-A-2014-218453 and JP-A-2012-126681 disclose collagen fiber gels made from collagen extracted from the skins and scales of tilapia and its use as a cell culture substrate. ing.

JP, 2014-218453, A JP 2012-126681 A

However, it is unclear whether the collagen aggregates that have undergone fibril formation are thermally stable even at 37 ° C, even if the temperature stability in the monomer state is high, such as collagen derived from Tilapia scales. In fact, Japanese Patent Laid-Open No. 2014-218453 describes that collagen derived from fish scales is preferable because of its fast fiber formation rate and good adhesiveness to cells, but cells of collagen gel prepared from scales of tilapia The suitability as a culture substrate has not been investigated.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell culture substrate containing fish-derived collagen that enables three-dimensional cell culture at 37 ° C.

As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that collagen derived from barramundy scales is remarkably suitable as a cell culture substrate compared to that derived from tilapia scales. The present invention has been completed.

That is, the present invention includes the following inventions.
[1] A cell culture substrate containing collagen derived from barramundi scale.
[2] The cell culture substrate according to [1], wherein in the collagen derived from barramundi scale, hydroxyproline per 1000 amino acid residues is 80 residues or more.
[3] The cell culture substrate according to [1] or [2], wherein the collagen derived from the barramundi scale has a difference in molecular weight between the α ₁ chain and the α ₂ chain of 5 kDa or less.
[4] A kit for cell culture, comprising the cell culture substrate according to any one of [1] to [3].

According to the present invention, the use of collagen derived from barramundi scales makes it possible to provide a cell culture substrate having high cell adhesiveness and cell spreading activity. Collagen fiber gel prepared from barramundi scale has characteristics comparable to the cell culture substrate that was conventionally made from bovine or porcine dermis collagen, so it is a substitute material for culture substrate particularly suitable for three-dimensional culture environment. Can be.

As collagen derived from barramundi, collagen derived from its skin is known (Japanese Unexamined Patent Publication No. 2015-180622). However, collagen derived from barramundi skin differs from that derived from scale in that the publication suggests that the hydroxyproline content is too low to be detected, and the thermal stability of collagen fiber gel prepared therefrom is also high. Are also inferior to those derived from scales.

Further, in WO2017 / 002767, gelatin prepared from barramundi scale is disclosed, but gelatin is different from collagen fiber gel in that the three-dimensional structure and molecular weight of collagen are changed by heat denaturation. For example, gelatin gel has a structure in which gelatin molecules having a random coil structure are cross-linked between molecules, whereas collagen fiber gel has inter-molecular cross-linking in which collagen fibers having a triple helix structure are self-assembled. Having that structure. Particularly, the collagen fiber gel derived from barramundy scale is different from the gelatin described in WO2017 / 002767 in that G'shows a value of 90 Pa or more even after fibrosis at 37 ° C and after heating to 50 ° C. ing.

The results obtained by subjecting collagen derived from barramundi scale to SDS-PAGE are shown (PC-S: pig skin, BC: barramundi scale, BC-S: barramundi skin, TC: tilapia scale (cell campus AQ-3LE), CMT. : Pig tendon (Cellmatrix Type-IP)). The staining result of the viability of the cells in the gel is shown. The microscope image of the cytoskeleton of the cell in a gel is shown.

Hereinafter, a mode for carrying out the present invention (hereinafter, simply referred to as “the present embodiment”) will be described in detail with reference to the drawings as necessary, but the present invention is limited to the present embodiment described below. Not a thing. The present invention can be variously modified without departing from the gist thereof.

In a first embodiment, the present invention provides a cell culture substrate containing collagen derived from barramundi scales.

Collagen at least partially has a helical structure (collagen helix) composed of three α chains having a molecular weight of about 100,000. Among the α chains, α ₁ chain and α ₂ chain are roughly classified according to slight differences in amino acid composition. Most of the collagen present in the body forms a heterotrimer (α ₁ × 2, α ₂ × 1), but inside it is a homotrimer composed of 3 α ₁ chains. There is also collagen with. A glycine residue appears every 3rd monomer in each monomer, and a proline residue and a hydroxyproline residue as other amino acid residues appear frequently. Among the amino acids that make up collagen, proline and hydroxyproline are known to contribute to the thermal stability of the triple helix structure.

-19 types of collagen molecules have been reported to exist due to differences in structure, and there are cases where several different types of collagen are also classified into the same type.

Among them, type I, II, III and type IV collagen are mainly used as raw materials for biomaterials. Type I is present in most connective tissues and constitutes the extracellular matrix. It is the most abundant collagen type in the body. Especially in tendons, dermis and bone, industrially collagen is often extracted from these sites. Type II is collagen that forms cartilage. Type III is often present at a similar site as type I, albeit in small amounts. Type IV is collagen that forms the basement membrane. The types I, II and III exist in the body as collagen fibers and mainly play a role of maintaining the strength of tissues or organs. Type IV does not have a fibrogenic ability, but forms a mesh-like aggregate composed of four molecules and is considered to be involved in cell differentiation in basement membrane.

The collagen derived from the scales of Barramundi (lates calcarifer) used in the present invention has a molecular weight distribution similar to that of type III collagen, but may contain any type of collagen unless otherwise specified.

As used herein, collagen fiber means a fiber of collagen that can be extracted from scales of barramundi and has at least a triple helix structure.

From the viewpoint of thermal stability, the collagen fiber preferably has 80 or more hydroxyproline residues per 1000 amino acid residues. The content of hydroxyproline in collagen derived from barramundi scale is as high as 8% or more, which is preferable. Although this value does not reach the hydroxyproline content of collagen derived from pig skin, it can be said that the content is extremely high for fish collagen.

When measured by SDS-PAGE, collagen usually shows one β chain band around ₂₀₀ kDa and _two bands of α ₁ chain and α ₂ chain around 100 kDa. On the other hand, gelatin shows a wide distribution below 300 kDa. In addition, gelatin has many low molecular weight components in which the α chain is decomposed.

The collagen in the present embodiment is characterized in that the difference in molecular weight between the α ₁ chain and the α ₂ chain constituting the fiber is 5 kDa or less. While the molecular weight of α ₁ chain and α ₂ chain in general type I collagen such as type I collagen derived from pig skin is 10 kDa or more, collagen derived from barramundy scales has the same results as shown in Examples. , Α ₁ chain and α ₂ chain have a molecular weight close to about 3.9 kDa. Although not intending to be bound by theory, collagen derived from barramundy scales, in which the α ₁ and α ₂ chains have similar molecular weights, is a type III in which fibrosis is more active than type I of the heterotrimer. It is considered to have a structure close to a homozygote such as collagen.

The difference in molecular weight between α ₁ chain and α ₂ chain can be confirmed by using a method suitable for protein separation based on molecular weight, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Specifically, the difference in molecular weight can be calculated by creating a calibration curve from the relationship between the mobility of each band of the molecular weight marker and the molecular weight, and applying the mobility of each α chain to the calibration curve.

The gel in the present embodiment is characterized in that the denaturation temperature as a monomer constituting the gel is high, that is, the collagen fibers maintain a triple helix structure at 37.0 ° C. For example, the denaturation temperature of Tilapia scale-derived collagen is about 35.7, while the denaturation temperature of Barramundi scale-derived collagen is about 36.1 ° C. The denaturation temperature can be confirmed by means known to those skilled in the art such as circular dichroism spectrum (CD).

When measuring collagen with CD, a positive peak at 221 nm due to the triple helix can be confirmed, but no such peak is seen with gelatin.

In addition to the high denaturation temperature, the gel of the present embodiment is superior to that derived from tilapia scale in terms of storage elastic modulus (G ′). The storage elastic modulus can be determined using a device capable of evaluating dynamic viscoelastic properties such as a rheometer. As used herein, the storage modulus (G ′) is 10 on a rotary rheometer MCR302 (manufactured by Anton-Paar) with an aqueous solution of pH 7 containing 0.27% by weight of collagen unless otherwise specified. It means the value when the temperature is raised from 37 ° C to 37 ° C. When measured under these conditions, the storage elastic modulus G'of the collagen gel made of barramundi scale as a raw material is 90 Pa or more. When the storage elastic modulus of the collagen gel made from barramundi scale as a raw material is measured under the same conditions, the softening temperature in the denaturation process due to temperature increase is 45 ° C. or higher. The "softening temperature" is the temperature at which the average value of G'reached for the last 10 minutes when the temperature was kept constant at 37 ° C. decreased by 5% as the temperature increased.

The concentration of collagen derived from barramundi scale can be appropriately changed depending on the application. For example, when used as a medium for culturing cells and the like, the concentration of collagen may be in the range of 0.20% to 0.53%. When the collagen concentration is less than 0.20%, it is difficult to obtain the hardness that resists the traction force of cells. When the collagen concentration is 0.53% or more, the viscosity of the collagen aqueous solution used for gel preparation is high, so that mixing with a buffer solution or the like is insufficient and the homogeneity of the gel is impaired. Since the hardness of the gel affects the differentiation of stem cells (Even-Ram et al., Cell, 126, 645-647 (2006)), nonuniform gel hardness is not preferable as a cell culture substrate.

Collagen fiber gel derived from barramundi scale can be produced by a method of cross-linking collagen molecules constituting collagen fiber, and collagen can be effectively increased in elastic modulus of collagen fiber gel. The method of causing a cross-linking reaction during the fibrosis of is preferably used. As a specific method, for example, a collagen aqueous solution having a concentration of 3% or less and having a pH of 1 to 5, preferably about 3, and a sodium phosphate buffer having a pH of 6 to 10, preferably about 7, is used as a raw material for denaturing collagen. Examples include a method of mixing at the following temperature (for example, about 10 ° C.) to obtain a solution having a predetermined collagen concentration, and then allowing it to stand. The buffer may optionally contain a cross-linking agent.

The aqueous collagen solution can be produced, for example, from scales by a known extraction method using an acid such as acetic acid and an enzyme such as pepsin. The concentration of the aqueous collagen solution can be adjusted by the weight ratio of collagen / solvent when dissolving the once-precipitated or dried collagen. If the collagen concentration exceeds 3%, the viscosity becomes high, and a non-uniform gel may occur due to insufficient mixing with the sodium hydrogen phosphate buffer solution, which is not preferable. The preferred concentration is 1% or less.

The concentration of the cross-linking agent in the buffer solution is not particularly limited. However, the concentration of the cross-linking agent, rather than the concentration in the sodium hydrogen phosphate buffer, influences the success or failure of gel formation and the physical properties of the gel after being mixed with the aqueous collagen solution (concentration in gel). The concentration of the cross-linking agent in the collagen fiber gel is preferably in the range of 5-80 mM. When the concentration of the cross-linking agent in the collagen fiber gel is less than 5 mM, cross-linking is difficult to be introduced into collagen molecules, and the elastic modulus of the gel may be insufficient, which is not preferable. On the other hand, if the concentration of the cross-linking agent exceeds 80 mM, the gelation rate after mixing the collagen aqueous solution and the sodium phosphate buffer is too fast, which may deteriorate the moldability, which is not preferable. More preferably, it is in the range of 10 to 30 mM.

If the gelation speed is too fast and moldability is poor, an inorganic salt such as sodium chloride can be added as a fibrosis inhibitor, if necessary. The concentration of the added inorganic salt is preferably in the range of 30 to 150 mM as the concentration in the collagen fiber gel. When the concentration of the inorganic salt in the gel is less than 30 mM, the fibrosis of collagen is too fast, the collagen fiber network does not develop, and the gel may become weak. When the concentration of the inorganic salt in the gel exceeds 150 mM, collagen fibrosis is suppressed and the gel may become weak. More preferably, it is in the range of 50 to 130 mM.

In the collagen fiber gel derived from barramundi scale, the cross-linking agent used for cross-linking collagen molecules is not particularly limited as long as it can cross-link proteins and has water solubility. The protein cross-linking agent is described in detail in the literature (Biomaterials, 18, p.95-105 (1997)). Among them, aldehyde-based crosslinking agents such as glutaraldehyde, carbodiimide-based crosslinking agents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide / hydrochloride (EDC), isocyanate-based crosslinking agents such as hexamethylene diisocyanate, ethylene glycol A polyepoxy-based crosslinking agent such as diethyl ether is preferably used from the viewpoints of economy, safety and operability. In particular, it is preferable to use a solution of a water-soluble carbodiimide such as EDC or 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide sulfonate in a phosphate buffer having a pH of 6 to 10.

On the other hand, gelatin molecules change due to thermal denaturation to form a random coil structure, but upon cooling, the helical structure recovers, and neighboring molecules share a helical structure portion and associate / aggregate to form a crosslinked gel. You can

The cell culture substrate containing the above collagen fiber gel serves as a scaffold for cultured cells and can be used in any medium. Collagen fiber gel is preferably used as a medium for three-dimensional culture. The cell culture substrate may be provided alone, or may be provided as a medium or a kit containing components such as serum necessary for cell culture. The cell culture substrate and medium can be contained in the cell culture container. The type of cell culture container is not particularly limited, but a plastic dish or plate that is widely used as a consumable product for cell culture is preferably used. Plastic has a low affinity for collagen and is easier to peel off gel than glass.

Cell can be adhered / proliferated or differentiated as needed in a medium containing collagen fiber gel derived from barramundi scale. The cultured cells are not particularly limited and can be used, for example, somatic cells, germ cells, stem cells and the like. Examples of stem cells include stem cell embryonic stem cells, induced pluripotent stem cells, and somatic stem cells such as mesenchymal stem cells. Collagen fiber gel derived from barramundi scale is particularly suitable for culturing cells, such as chondrocytes, which are easily dedifferentiated in flat culture and difficult to be subcultured for a long time.

The collagen can be extracted from barramundi scales using a known method. For example, a method of solubilizing collagen with an enzyme using fish scales as a raw material is known to those skilled in the art, and is described in, for example, Japanese Patent Nos. 4863433 and 5692770.

For example, as an enzyme used for extraction, an enzyme capable of hydrolyzing scales to extract collagen, such as pepsin, can be preferably used. The amount of enzyme added is not particularly limited, but is preferably 1 to 15% by weight based on the dry weight of the fish scale.

Prior to the extraction step, it is preferable to remove inorganic substances such as calcium phosphate contained in fish scales, and then perform deashing treatment to improve extraction efficiency. In addition, the scales may be washed with an aqueous solution containing a specific acid or an organic solvent in order to remove unnecessary proteins and lipids contained in the scales.

The method for obtaining fish scale-derived collagen includes a step of decalcifying fish scale, a step of treating the decalcified fish scale with an protease in an acidic aqueous solution in a temperature range of 15 ° C to 35 ° C, and recovering solubilized collagen. It may include a step of It is preferable that the fish scale used as a raw material is refrigerated or frozen after collection to prevent spoilage. The collected fish scales have a considerable amount of contaminants attached, for example, dorsal fins, caudal fins, etc., or surplus proteins attached to the surface thereof. Therefore, for the purpose of removing these before storage, it is preferable to wash the raw fish scales as a pretreatment after they are collected.

Specifically, the contaminants may be removed by washing with water, and the surplus protein adhering to the surface is, for example, 1 to 15% by weight, preferably 5 to 10% by weight of an aqueous sodium chloride solution, or 0.01 to It may be removed by washing with 0.5 M, preferably 0.05 to 0.2 M aqueous sodium hydroxide solution for 10 to 72 hours, preferably 24 to 48 hours (alkali treatment). The washing is preferably repeated by exchanging the washing solution. Particularly, when removing the surplus protein adhering to the surface, the washing solution is washed to such an extent that the waste solution does not become cloudy, preferably about 3 to 5 times. It is good to replace the product and wash it repeatedly.

The solvent of the aqueous protease solution used for the protease treatment is not particularly limited as long as the pH is in the range of 2 to 5, but inexpensive, easy-to-handle hydrochloric acid, acetic acid, and phosphoric acid are preferably used. The acid concentration is defined by the pH.

In the production of collagen fiber gel, for example, the protease treatment may be performed in an acidic aqueous solution in the temperature range of 15 ° C to 35 ° C. As a result, the extraction efficiency of collagen can be improved without causing denaturation of collagen, and a high yield can be realized. If the temperature is lower than 15 ° C., the protease activity may decrease and the collagen extraction efficiency may decrease, which is not preferable. When the temperature is 35 ° C. or higher, protease activity increases, but collagen degeneration may occur, which is not preferable. Therefore, the protease treatment is carried out in a temperature range of 15 ° C to 35 ° C, more preferably 20 ° C to 30 ° C.

The protease treatment may be completed when the collagen solubilizing ability is reduced due to protease inactivation, and for example, the treatment time may be about 12 to 48 hours. Further, the treatment liquid may be stirred using a stirring blade or the like for the purpose of enhancing the extraction efficiency.

The collagen dissolved in the acidic aqueous solution may be recovered by, for example, an ordinary physical separation means conventionally used for preparing collagen derived from mammals, etc., and the method is not particularly limited. For example, after the protease treatment, sodium chloride or the like is added to the collagen aqueous solution separated from the fish scale residue by means such as centrifugation or filtration to increase the salt concentration, or sodium hydroxide or the like is added to adjust the pH to near neutral. By doing so, the collagen may be fibrillated, and the fibrillated collagen may be separated and collected by, for example, a centrifugation method.

Thereafter, if necessary, for example, it can be purified by redissolving it in purified water and repeating the fibrosis-recovery operation once or plural times by the above-mentioned method. In order to suppress the denaturation of collagen, it is preferable to carry out the steps of the above-mentioned pretreatment, recovery, purification and the like at room temperature or below as much as possible in the preparation of collagen.

In the second embodiment, the cell culture substrate is provided as a cell culture kit.

In such a kit, for example, the cell culture substrate can be contained in a container. The type of such container is not particularly limited, but a widely used plastic dish or plate is preferable. Plastic has a low affinity with collagen and has the advantage that it is easier to peel off the gel than glass.

The container may contain a medium suitable for the cells to be cultured. The composition of the medium is not intended to be limited because it varies depending on the composition to be cultured, but usually it includes inorganic salts, various amino acids, sugars, vitamins, and further serum.

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

<Experiment>
1. Extraction of soluble collagen from fish scales (1) Pretreatment of fish scales (alkali treatment and decalcification treatment)
Barramundy scales (obtained from AO KINGDOM INTERNATIONAL CO., LTD) were thoroughly washed with water to remove impurities such as fins, and then air-dried. The washed dried fish scale was immersed in a 0.1 M aqueous sodium hydroxide solution, and gently stirred using a stirring blade for 24 hours. The fish scales were filtered with a wire net and washed repeatedly with water until the pH became neutral. Further, the fish scale was immersed in dilute hydrochloric acid having a pH of 2 and gently stirred for 24 hours for decalcification. The fish scales were filtered with a wire net, washed repeatedly with water until the pH became neutral, and freeze-dried. The freeze-dried scale thus obtained was stored in a freezer and subjected to collagen extraction.

(2) Extraction of collagen from fish scales 350 g of freeze-dried scales was added to 3000 mL of 0.5 M acetic acid aqueous solution, and gently stirred at room temperature for 3 days using a stirring blade. This aqueous solution was centrifuged (10000 × g, 20 minutes) to precipitate fish scales. The supernatant was collected and suction-filtered using a glass fiber filter (Merck KK) and a membrane filter (Merck KK; 0.65 μm followed by 0.45 μm) to obtain acid solubility. The ingredients were recovered.

Next, the fish scales were filtered with a wire mesh and washed repeatedly with water until the pH became neutral. Add fish scales to 1000mL of 0.5M acetic acid aqueous solution containing 7g of pepsin (Fujifilm Wako Pure Chemical Industries, which has the activity of hydrolyzing 100g of protein to 1g of pepsin), and gently using a stirring blade for 3 days at room temperature. It was stirred. This aqueous solution was centrifuged (10000 × g, 20 minutes) to precipitate fish scales. The supernatant was collected, suction filtered using a glass fiber filter (Merck KK) and a membrane filter (Merck KK, 0.65 μm followed by 0.45 μm), and treated with pepsin. The ingredients were recovered.

(3) Purification of extracted collagen To the above-mentioned acid-soluble component and pepsin-treated component, an aqueous sodium chloride solution was added so that the final concentration was 0.9 M, and the mixture was mixed with a glass rod and allowed to stand still at 4 ° C for 24 hours. It was placed and salted out. This was centrifuged (10000 xg, 20 minutes), and the precipitate was dissolved in 300 ml of 0.5 M acetic acid aqueous solution. This salting-out step was repeated three times, and an aqueous solution of collagen in acetic acid was placed in a cellulose tube, dialyzed against distilled water, and freeze-dried. The yield of collagen extraction was calculated from the weight of the freeze-dried product after extraction with respect to the dry weight of decalcified scale. The freeze-dried product was dissolved in dilute hydrochloric acid having a pH of 3 at an arbitrary concentration to prepare acid-soluble collagen and pepsin-extracted collagen.

2. Structural Analysis of Collagen (1) Confirmation of Primary Structure by Amino Acid Composition To investigate the primary structure of collagen (BC) derived from barramundi scale, pepsin-extracted collagen was subjected to automatic amino acid analysis. As controls, pepsin-extracted collagen (TC) derived from tilapia scales and pepsin-extracted collagen (PC-S) derived from pig skin prepared by the same extraction method as described above (manufactured by Nitta Gelatin Co., Ltd., trade name "Collagen BM"). It was used. The measurement was requested to the Japan Food Analysis Center, and each amino acid composition was calculated for 1000 amino acid residues. The results are shown in the table below.

(2) Identification of collagen by SDS-PAGE The molecular weight distribution of pepsin-extracted collagen of barramundi scales extracted and purified in (1) to (3) was examined by electrophoresis (SDS-PAGE method). A 3 to 8% trisacetate gel (Thermo Fisher Scientific) was used as the electrophoresis gel. A collagen solution having a concentration of 0.06% was applied in an amount of 10 μL per well, and electrophoresis was performed at a constant current of 20 mA per gel. As controls, barramundi skin collagen (BC-S), cell campus AQ-03LE (TC) (tilapia scale-derived collagen, manufactured by Taki Chemical Co., Ltd.), Cellmatrix Type IP (CMT) (porcine tendon-derived collagen, Nitta) Gelatin Co., Ltd.) was used.

Spectra Multicolor High Range Protein Ladder (Thermo Fisher Scientific) was used as the molecular weight marker, and Quick-CBB plus (Fujifilm Wako Pure Chemical Co., Ltd.) was used as the gel after electrophoresis.

The results are shown in Figure 1.

As shown in FIG. 1, the collagen derived from pig skin (PC-S), the collagen derived from barramundi scale (BC), and the collagen derived from barramundi skin (BC-S) were α chains near 120 kDa and α near 120 kDa. A band, a β-chain near 240 kDa, and a migration band showing a γ-chain at a position of 270 kDa or more were obtained.

The migration patterns of the α chains of BC and BC-S were suggested to have a heterotrimer structure in which the distance between the α ₁ chain and the α ₂ chain is close in both scale and skin. The close molecular weight band distance between the α ₁ chain and the α ₂ chain suggests that collagen derived from barramundy scales has a structure close to a homozygote such as type III collagen.

The molecular weight difference shown in FIG. 1 is about 4.0 kDa for barramundi scale-derived collagen (BC), about 5.5 kDa for barramandi skin-derived collagen (BC-S), and about 5.5 for tilapia scale-derived collagen (TC). The collagen derived from pig skin was about 10.0 kDa.

As described above, the collagen derived from barramundi scale has a molecular weight difference of 10 kDa or more as compared with general type I collagen derived from pig skin or tilapia scale, regardless of the treatment method, while the molecular weight difference is 5.0 kDa or less. It turns out that

3. Evaluation by gel-embedded cell culture (1) Gel-embedded cell culture Pallacin-extracted collagen (0.3%) derived from barramundi scales extracted and purified in (1) to (3), 5 times concentrated DME culture solution (5xDME, manufactured by Nitta Gelatin Co., Ltd.) and reconstitution buffer (Manufactured by Nitta Gelatin Co., Ltd.) was mixed at a ratio of 7: 2: 1 to prepare a collagen solution. Thereafter, NIH3T3 cells, which are mouse-derived fibroblasts, were dispersed in the collagen solution at a concentration of 4.2 × 10 Cell / ml, dispensed into a 24-well plate by 500 μl per well, and gelled in a 37 ° C. incubator for 1 hour. . After gelation, DMEM (manufactured by Sigma Aldrich) containing 10% calf serum (CS) was added, and the culture was started at 37 ° C. As controls, barramundi skin collagen (BC-S), cell campus AQ-03A (TC) (tilapia scale-derived collagen, manufactured by Taki Chemical Co., Ltd.), Cellmatrix Type IA (CMT) (porcine tendon-derived collagen, Nitta) The same culture was performed using Gelatin Co., Ltd.).

(2) Judgment of life and death of cells in gel by staining The gel in which the cells that had been cultured in (1) for 7 days were embedded was washed with DPBS (-) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then Cellstein (registered trademark) -Double Staining Kit (manufactured by Dojindo Laboratories). Staining was carried out by incubating at 37 ° C. for 15 minutes. Then, the distribution of live and dead cells in the gel was observed using a confocal laser scanning microscope.

The results are shown in Figure 2. In this double staining method, the cytoplasm of living cells is stained green by calcein AM, and the cell nucleus of dead cells is stained red. Cells cultured with barramundi scale-derived collagen (BC), barramundi skin-derived collagen (BC-S), tilapia scale-derived collagen (TC), and porcine tendon-derived collagen (CMT) were all stained green and survived. You can see that

(3) Observation of cytoskeleton of cells in gel by staining The gel in which the cells cultured in (1) for 7 days were embedded was washed with DPBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.). Then, 0.1% Triton X100 (manufactured by Sigma Aldrich) was added, and the mixture was allowed to stand at room temperature for 30 minutes and then washed again with DPBS (−). 100 mM Acti-Stain 488 (manufactured by Cytoskeleton) was added, and the mixture was allowed to stand in the dark and at room temperature for 60 minutes, washed with DPBS (-), and stained. Then, the shape of the cytoskeleton inside the gel was observed using a confocal laser microscope.

The results are shown in Figure 3. In this staining method, the fiber (f-actin), which is the cytoskeleton, is stained green, and the cell morphology can be observed. Cells cultured with barramundi scale-derived collagen (BC) and porcine tendon-derived collagen (CMT) spread largely in a spindle shape, whereas barramandi skin-derived collagen (BC-S) and tilapia scale-derived collagen (TC) The cells cultured in No. 2 did not show much spreading, the actin fibers supporting the cell morphology were scattered, and the cell morphology was round. This indicates that collagen derived from barramundi scale specifically has the effect of enhancing cell adhesion and maintaining cell morphology.

Claims

Cell culture substrate containing collagen derived from barramundi scales.
The cell culture substrate according to claim 1, wherein in the collagen derived from barramundi scale, hydroxyproline per 1000 amino acid residues is 80 residues or more.
The cell culture substrate according to claim 1 or 2, wherein the collagen derived from barramundi scale has a difference in molecular weight between α 1 chain and α 2 chain of 5 kDa or less.
A kit for cell culture, comprising the cell culture substrate according to any one of claims 1 to 3.