JP7655022B2 - Freestanding low protein adsorption container - Google Patents

Description

The present invention relates to a free-standing low protein adsorption container.

Biochemical containers used for protein analysis, cell culture, storage, etc. are often made of resin materials. However, biochemical containers made of resin materials can easily have proteins and cells adsorbed to their inner surfaces. In clinical tests, if proteins contained in collected urine, blood, etc. are adsorbed to the inner surface of the container, the test value and recovery amount of the protein may be lower than the actual value, which may lead to an erroneous diagnosis. In addition, if cells are adsorbed to the inner surface of the container during culture or storage, it may become difficult to recover the cells from the container, and the recovery rate, etc. may decrease.

To solve these problems, a method is known in which a treatment is performed on the inner surface of a biochemical container to make it difficult for proteins and cells to adsorb (low-adsorption treatment method). For example, Patent Document 1 discloses a method in which a polystyrene member is placed in a reaction container, ozone gas is passed through, oxygen is introduced to the surface to make it hydrophilic, and protein adsorption to the surface of the polystyrene member is reduced.

Japanese Patent Application Publication No. 10-101820

However, it is known that the hydrophilic polar groups formed by oxygen and introduced to the surface of the resin layer by the method described in Patent Document 1 penetrate into the interior of the resin layer over time due to contact with the gas phase, and there is a problem in that the effect decreases over time.

Furthermore, since freestanding containers usually have a freestanding surface (flat bottom) with a certain area or more, even if the inner surface of the container has been subjected to the above-mentioned low-adsorption treatment, there is still room for further improvement in terms of protein and cell recovery loss, and there is particularly room for improvement in large-capacity freestanding containers.

The present invention was made in consideration of the above problems, and aims to provide a free-standing low-protein adsorption container that has low adsorption of proteins and cells to the inner surface of the container that forms the storage area.

The free-standing low protein adsorption container according to the present invention has a planar bottom and sidewalls made of a resin material, is free-standing with the bottom placed on a support surface, the inner surface of the bottom and the inner surface of the sidewalls form a storage area for storing a liquid containing proteins or cells, and further, the inner surface of the bottom and the inner surface of the sidewalls are formed with a coating layer made of a water-soluble resin having a hydrophilic functional group in the side chain, and the water-soluble resin is cross-linked between itself and between the water-soluble resin and the inner surface of the bottom and the inner surface of the sidewalls.

According to the present invention, it is possible to obtain a free-standing low-protein adsorption container that has low adsorption of proteins and cells to the inner surface of the container that forms the storage area, thereby reducing recovery losses of these proteins and cells.

FIG. 1 is a schematic perspective view of a self-supporting low protein adsorption container according to an embodiment of the present invention. FIG. 2 is a schematic perspective view showing a state in which a sample (liquid containing proteins or cells) is being collected by a pipette from the free-standing low protein adsorption container according to the present embodiment. 1 is a schematic enlarged cross-sectional view showing an enlarged view of the inner surface of the container in the storage area of the free-standing low protein adsorption container according to the present embodiment. FIG. 1 is a schematic cross-sectional view showing the vicinity of a storage area of a free-standing low protein adsorption container according to the present embodiment. 1 is a schematic cross-sectional view showing the vicinity of the storage area of a modified example of a free-standing low protein adsorption container according to the present embodiment. FIG.

Hereinafter, an embodiment of a self-supporting low protein adsorption container according to the present invention will be described with reference to the drawings.
The embodiment described below is merely an example for facilitating understanding of the present invention, and is not intended to limit the present invention. In other words, the shapes, dimensions, arrangements, and other aspects of the components described below may be modified or improved without departing from the spirit of the present invention, and the present invention naturally includes equivalents thereof.
In addition, in all drawings, similar components are given the same reference numerals, and duplicate explanations are omitted as appropriate. Note that in all drawings, there are some parts that are not given reference numerals (omitted) for convenience. Furthermore, the dimensional ratios of each member shown in the drawings may differ from the actual dimensional ratios in order to facilitate understanding of the invention.

＜Overview＞
First, an overview of the free-standing low protein-adsorption container according to this embodiment will be described in detail with reference to Fig. 1 and Fig. 2. Fig. 1 is a perspective view that typically shows a free-standing low protein-adsorption container 100 according to this embodiment. Fig. 2 is a perspective view that typically shows a state in which a liquid 43 (sample) containing proteins or cells is being collected by a pipette 41 from the free-standing low protein-adsorption container 100 according to this embodiment.

The free-standing low protein adsorption container 100 according to this embodiment has a planar bottom 11 and sidewalls 13 made of a resin material as shown in Figs. 1 and 2, and can stand on its own when the bottom 11 is placed on a mounting surface. The inner surface 11a of the bottom 11 and the inner surface 13a of the sidewalls 13 form a storage area 21 for storing a liquid 43 containing proteins or cells. Furthermore, the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewalls 13 are provided with a coating layer 51 made of a water-soluble resin having a hydrophilic functional group in the side chain, and the water-soluble resin is cross-linked between the water-soluble resins and between the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewalls 13. The "inner surface" refers to the surface on the inside of the container (the storage area 21 side) of each member of the free-standing low protein adsorption container 100 according to this embodiment.

The self-supporting low protein adsorption container 100 according to this embodiment may have at least the bottom 11 and the side wall 13 made of a resin material. Here, "made of a resin material" means that the resin material is contained as the main material (the resin material is contained in an amount of 70% by mass or more, more preferably 80% by mass or more, and even more preferably 90% by mass or more of the total mass of the member), and may contain materials other than the resin material, such as additives, within a range that does not affect the effects of the present invention. The self-supporting low protein adsorption container 100 according to this embodiment may be entirely made of a resin material (all of the members provided) or at least a part of the members other than the bottom 11 and the side wall 13 may be made of a material other than a resin material (for example, glass, etc.), but it is more preferable that the entire self-supporting low protein adsorption container 100 according to this embodiment is made of a resin material (particularly the same resin material). This is because the self-supporting low protein adsorption container 100 according to this embodiment is easy to manufacture (by blow molding, injection molding, etc.).

Examples of resin materials that can be used include polyolefin resins or cyclic polyolefin resins such as polypropylene resin, polyethylene resin, polymethylpentene resin, and ethylene-propylene copolymer, polystyrene resins such as acrylonitrile-butadiene-styrene resin, polyacrylic resins such as polyethylene terephthalate resin and polybutylene terephthalate resin, methacrylic resins such as polymethyl methacrylate resin, fluorine resins such as polytetrafluoroethylene, acrylic resins such as polyacrylonitrile, cellulose resins such as propionate resin, polycarbonate resin, vinyl chloride resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, and polyetherimide resin. These may be used alone or in combination of two or more. Among these, polyethylene terephthalate resin is particularly preferred from the viewpoint of moldability, transparency, radiation resistance (sterility), and the like required for biochemical containers.

The weight average molecular weight of the resin material is not particularly limited, but is preferably 10,000 to 500,000, and more preferably 20,000 to 100,000. If the weight average molecular weight of the resin material used is within this range, the moldability will be superior.

The weight average molecular weight is a value measured by size exclusion chromatography (Gel Permeation Chromatography system, Shodex KF-800 column, both manufactured by Showa Denko KK, elution solvent: tetrahydrofuran).

Using such a resin material as the main material, the bottom 11 and side wall 13 of the free-standing low protein adsorption container 100 according to this embodiment can be formed, for example, by blow molding, injection molding, injection blow molding, vacuum molding, etc., and further, the entire free-standing low protein adsorption container 100 according to this embodiment can also be formed.

The shape of the free-standing low protein adsorption container 100 according to this embodiment is not limited as long as it has a planar (approximately planar) bottom 11 and a side wall 13 connected to the bottom 11, can stand on its own when the planar bottom 11 is placed on a mounting surface, and the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 form a storage area 21 for storing a liquid 43. For example, it may be a substantially square bottle shape having a planar bottom 11 and side wall 13 as shown in Figures 1 and 2, or may be a substantially cylindrical bottle shape in which the side wall 13 is configured by a curved surface. In addition, it may be a flask shape in which the bottom 11 is planar (such as an Erlenmeyer flask shape in which the side wall 13 tapers from the mounting surface side to the upper part), a petri dish shape, or the like.
In addition, the storage area 21 of the free-standing low protein adsorption container 100 in this embodiment may be formed not only by the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13, but also by the inner surface of another member (e.g., the inner surface of the top portion).

The free-standing low protein adsorption container 100 according to this embodiment may be provided with one or more openings 31 that can serve as an inlet or outlet for a sample or the like, a degassing port, etc. The openings 31 may have a structure that allows them to be repeatedly opened and closed, and may have a structure in which the openings 31 are provided with a screw thread and can be sealed with a cap 33 or the like, as shown in Figures 1 and 2. Furthermore, the free-standing low protein adsorption container 100 according to this embodiment may be provided with other members (for example, a gripping portion, etc.).

In addition, the freestanding low protein adsorption container 100 of this embodiment may have a capacity in its storage area 21 that can store more than 50 mL, or even more than 100 mL, or even more than 200 mL of liquid 43 containing protein or cells. In other words, the storage area 21 of the freestanding low protein adsorption container 100 of this embodiment may have a capacity that can store more than 50 mL of a sample liquid containing protein or a liquid culture medium containing cells, and store, react, culture, etc. in this storage area 21. Therefore, for example, if the opening 31 has a structure that can be sealed, the volume of the storage area 21 itself may be more than 50 mL, and if the opening 31 has a structure that cannot be sealed, the volume may be such that the storage area 21 does not interfere with subsequent work when it contains more than 50 mL of liquid 43 (a volume that does not easily spill out of the opening 31 during processing, etc.). The freestanding low protein adsorption container 100 of this embodiment is characterized in that, even though it is such a large-capacity container, there is little adsorption of proteins and cells to the inner surface of the container that forms the storage area 21, and the liquid 43 is easily recovered from the storage area 21, resulting in little recovery loss.

<Configuration of the inner surface of the container forming the storage area>
Next, the configuration of the container inner surface forming the storage area 21 of the self-supporting low protein-adsorption container 100 according to this embodiment will be described in detail with reference to Fig. 3 to Fig. 5. Fig. 3 is a schematic enlarged cross-sectional view showing the container inner surface (the inner surface 11a of the bottom 11 or the inner surface 13a of the side wall 13) in the storage area of the self-supporting low protein-adsorption container 100 according to this embodiment. Figs. 4 and 5 are schematic cross-sectional views showing the vicinity of the storage area 21 in two different embodiments of the self-supporting low protein-adsorption container 100 according to this embodiment.

In the freestanding low protein adsorption container 100 according to this embodiment, a coating layer 51 made of a water-soluble resin having a hydrophilic functional group in the side chain is formed at least on the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 that form the storage area 21. The water-soluble resins are cross-linked between each other, that is, between the molecules of the water-soluble resin in the coating layer 51, between the water-soluble resin (the water-soluble resin molecules in the coating layer 51) and the inner surface 11a of the bottom 11, and between the water-soluble resin and the inner surface 13a of the side wall 13. By forming such a coating layer 51, the surface layer of the inner surface of the container that forms the storage area 21 contains hydrophilic functional groups, and proteins and cells contained in the liquid 43 contained in the storage area 21 are less likely to be adsorbed to the inner surface of the container that forms the storage area 21.

In this embodiment, the "water-soluble resin" refers to a resin that is hydrated by ionic or hydrogen bonds with water molecules and dissolves in water, and is capable of dissolving at least 1.0 g in 100 g of water at 25°C. The "coating layer 51 made of a water-soluble resin" refers to a coating layer 51 that contains the above-mentioned water-soluble resin as a main component (e.g., 80% by mass or more, or even 90% by mass or more), and also includes coating layers 51 that have been modified to be water-insoluble by crosslinking and hardening of the water-soluble resin contained in the coating layer 51. In other words, as long as the coating layer 51 is formed using a material that contains a water-soluble resin as a main component, it may be crosslinked and hardened to be water-insoluble.

Examples of water-soluble resins having hydrophilic functional groups in the side chain include saponified polyvinyl acetate, polyvinylpyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate, copolymers of the monomers that constitute them, and copolymers of 2-methacryloyloxyethyl phosphorylcholine and other monomers (such as butyl methacrylate). Some of these water-soluble resins also include those to which another hydrophilic functional group has been added as a side chain. Among these, one or more selected from saponified polyvinyl acetate, polyvinylpyrrolidone, and polyethylene glycol are more preferred. This is because it is possible to suppress irritation to proteins and cells and further improve their quality. The average degree of polymerization of this water-soluble resin is not particularly limited, but is preferably 100 to 10,000, more preferably 200 to 5,000, because this makes it easier to form a uniform coating on the inner surface of the container that forms the storage area 21 and improves workability.

Examples of saponified polyvinyl acetate include polyvinyl alcohol or copolymers of vinyl alcohol with other compounds, and saponified vinyl acetate modified with hydrophilic groups, hydrophobic groups, anions, cations, amide groups, or reactive groups such as acetoacetyl groups, and vinyl alcohol. The degree of saponification of the saponified polyvinyl acetate is not particularly limited, but is preferably 20 to 100 mol % of the total polyvinyl acetate, and more preferably 50 to 95 mol %.

The water-soluble resin constituting the coating layer 51 has hydrophilic functional groups in its side chains, but may also have functional groups for crosslinking and curing, or the functional groups for crosslinking and curing may be hydrophilic functional groups or parts thereof. Examples of such functional groups include functional groups containing hydroxyl groups and carbonyl groups (carboxy groups, aldehyde groups, ketone groups, ester groups, amide groups, etc.), and functional groups that are reactive to radiation, photosensitive, or thermally reactive. Furthermore, functional groups containing photosensitive hydrophilic functional groups such as diazo groups, azide groups, and diazide groups are more preferred.

In particular, water-soluble resins having functional groups containing azide groups and/or carbonyl groups in the side chains are more preferred, water-soluble resins having functional groups containing aromatic azide groups and/or carbonyl groups in the side chains are even more preferred, and water-soluble resins represented by the following formula (Ia) or (Ib) are even more preferred, because they can be easily crosslinked and cured by irradiation with light having a wavelength of 300 to 500 nm and can further reduce the amount of adsorption of proteins, cells, etc.

In the above formula (Ia), R1 represents a hydrocarbon group having a carbonyl and an amine, R2, R3, R4, and R5 each represent hydrogen or an alkyl group, r1 represents 1 to 1000, r2 represents 40 to 4995, r3 represents 0 to 4000, and n represents 1, 2, or 3. In particular, it is more preferable that R2, R3, R4, and R5 each represent hydrogen.
In the above formula (Ib), R represents a hydrocarbon group having a carbonyl and an amine, r1 represents 1 to 1,000, r2 represents 40 to 4,995, and r3 represents 0 to 4,000.

Furthermore, the water-soluble resin represented by the above formula (Ia) is more preferably a water-soluble resin represented by the following formula (IIa).
In the following formula (IIa), R represents an alkyl group having a carbonyl and an amine, r1 represents 1 to 1000, r2 represents 40 to 4995, r3 represents 0 to 4000, and n represents 1, 2, or 3.

Using the water-soluble resin having hydrophilic functional groups in the side chains as described above, a coating layer 51 is formed on at least the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 that form the storage area 21 of the freestanding low protein adsorption container 100 according to this embodiment. Note that a coating layer 51 made of the water-soluble resin described above may also be formed on the inner surfaces of other members (such as the inner surface of the top part, etc.).

The coating layer 51 is formed by coating the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 with the water-soluble resin described above, and crosslinking and curing the molecules of the water-soluble resin contained in the coating layer 51 by light irradiation or the like. In other words, the water-soluble resin in the coating layer 51 is crosslinked with each other (for example, the crosslinked structure 61 in FIG. 3). This crosslinking is preferably formed by functional groups of the side chains of the water-soluble resin. Note that the coating layer 51 may contain some molecules of the water-soluble resin that are not crosslinked, and it is sufficient that the coating layer 51 is crosslinked and cured to the extent that the structure of the coating layer 51 can be maintained. This makes it possible to reduce the amount of elution from the coating layer 51 when the liquid 43 is contained in the storage area 21, and to obtain a coating layer 51 that is resistant to physical stimuli, and the low adsorption ability of the coating layer 51 (low adsorption to proteins and cells) is maintained for a long period of time. Here, the cross-linked structure 61 in Figure 3 is an easy-to-understand illustration of the cross-linked structure between the molecules of the water-soluble resin, and does not represent the actual size or number of the cross-linked structures.

Furthermore, in the self-supporting low protein adsorption container 100 according to this embodiment, not only the water-soluble resins constituting the coating layer 51 are cross-linked with each other, but also the water-soluble resins constituting the coating layer 51 and the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 (for example, the cross-linked structure 63 in FIG. 3). This cross-link is preferably formed by a functional group of the side chain of the water-soluble resin and a functional group of the inner surface 11a of the bottom 11 or the inner surface 13a of the side wall 13 (such as a functional group formed by a surface treatment described later). This results in a coating layer 51 with high adhesion to the inner surface of the container. In other words, the stability of the coating layer 51 is increased. Here, the cross-linked structure 63 in FIG. 3 is also an easy-to-understand representation of the cross-linked structure between the molecules of the water-soluble resin and the inner surface 11a of the bottom 11 or the inner surface 13a of the side wall 13, and does not represent the actual size or number of the cross-linked structure.

The inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 on which the coating layer 51 is formed may be subjected to a surface treatment such as hydrophilization or a surface treatment functional group formation treatment before the coating layer 51 is formed. Such a surface treatment may be, for example, a treatment to form an oxygen-containing functional group. By forming this oxygen-containing functional group on the surface, the above-mentioned crosslinking (crosslinking with the water-soluble resin of the coating layer 51, the crosslinking structure 63 in FIG. 3) is more easily formed. Examples of the oxygen-containing functional group include polar functional groups such as carbonyl groups (carboxy groups, aldehyde groups, ketone groups, ester groups, etc.), hydroxyl groups, ether groups, peroxy groups, and epoxy groups. In particular, it is more preferable to perform a treatment to form a carbonyl group as the oxygen-containing functional group. In addition, for example, plasma treatment, corona discharge treatment, excimer laser treatment, flame treatment, etc. can be adopted as the surface treatment, and in particular, plasma treatment is more preferable.

The amount of oxygen-containing functional groups formed by the above treatment is not particularly limited, but is preferably 0.01 to 100 nmol/cm ² , and more preferably 0.05 to 10 nmol/cm ^2. If the amount of oxygen-containing functional groups is less than this lower limit, the desired effect may not be fully exerted, and if it exceeds the upper limit, the characteristics of the bottom 11 or side wall 13 itself may change, making it impossible to meet the required performance as a biochemical container.

In particular, it is preferable that the water-soluble resin constituting the coating layer 51 has a side chain containing an aromatic azide group and a side chain containing a carbonyl group, that carbonyl groups are formed on the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13, and that crosslinks are formed between the aromatic azide group of the water-soluble resin and the carbonyl group of the water-soluble resin, and between the aromatic azide group of the water-soluble resin and the carbonyl group of the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13. This is because these crosslinks are very easy to form, and tend to result in a more stable coating layer 51.

The thickness of the coating layer 51 is not particularly limited, but is preferably 0.1 to 50 μm, and more preferably 0.5 to 20 μm. Here, the "thickness of the coating layer 51" refers to the shortest distance from the surface of the coating layer 51 (the surface on the housing area 21 side) to the surface that contacts the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewall 13. The same applies hereinafter.
The thickness of the coating layer 51 is measured by an ellipsometer.

The thickness of the coating layer 51 is preferably uniform on the inner surface 11a of the bottom 11, uniform on the inner surface 13a of the sidewall 13, and uniform on the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewall 13. If the thickness of the coating layer 51 is uniform on the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewall 13 as a whole, the difference in the low adsorption ability of proteins and cells depending on the location in the storage area 21 is very small, and chemical reactions, enzyme reactions, cell culture, etc. in the storage area 21 can be performed extremely stably. Here, "uniform thickness" means that the variation in the measured values (difference between maximum and minimum) when the thickness of the coating layer 51 is measured at 10 arbitrary points on the inner surface 11a of the bottom 11 or the inner surface 13a of the sidewall 13 is within 0.1 μm, more preferably within 0.05 μm.

For example, as shown in FIG. 4, it is very preferable that the thickness of the coating layer 51 formed on the inner surface 11a of the bottom 11 is uniform, the thickness of the coating layer 51 formed on the inner surface 13a of the sidewall 13 is also uniform, and further, the thickness of the coating layer 51 formed on the inner surface 11a of the bottom 11 and the coating layer 51 formed on the inner surface 13a of the sidewall 13 are both uniform, i.e., the thicknesses of the coating layers 51 formed on the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewall 13 are all uniform.

Alternatively, the thickness of the coating layer 51 is preferably such that on the inner surface 11a of the bottom 11, the thickness of the coating layer 51 gradually increases from the center toward the end side that contacts the side wall 13, and further, on the inner surface 13a of the side wall 13, the thickness of the coating layer 51 gradually increases from the lower side that contacts the bottom 11 toward the upper side. When the liquid 43 containing proteins and cells is sucked up and collected from the storage area 21 using a pipette 41 or the like, the proteins and cells tend to gather in the center of the bottom 11 where the coating layer 51 is relatively thin, and by sucking up the liquid 43 using a pipette 41 or the like at the center of the bottom 11 as shown in FIG. 2, the proteins and cells are more easily collected and the collection loss is reduced.

For example, as shown in FIG. 5, it is preferable that the thickness of the coating layer 51 formed on the inner surface 11a of the bottom 11 is gradually increased from the center of the bottom 11 toward the end side in contact with the side wall 13, and further, that the thickness of the coating layer 51 formed on the inner surface 13a of the side wall 13 is gradually increased from the lower side in contact with the bottom 11 toward the upper side. Also, although not limited thereto, it is more preferable that the thickness of the coating layer 51 at the end of the bottom 11 is thinner than the thickness of the coating layer 51 at the lower part of the side wall 13. In other words, it is more preferable that the thickness of the coating layer 51 is gradually increased from the center of the bottom 11 toward the upper side of the side wall 13, as in the embodiment of FIG. 5.

In the above embodiment, the same effect as above can be achieved even if the thickness of the coating layer 51 at the end of the bottom 11 is the same as the thickness of the coating layer 51 at the lower part of the side wall 13. Also, in the above embodiment, the thickness of the coating layer 51 at the lower part of the side wall 13 may be thinner than the thickness of the coating layer 51 at the end of the bottom 11. This makes it possible to prevent the upper coating layer 51 on the inner surface 13a of the side wall 13 from becoming unnecessarily thick.

On the other hand, in the free-standing low protein adsorption container 100 according to this embodiment, the thickness of the coating layer 51 on the inner surface 11a of the bottom 11 may be gradually thinner from the center toward the end side in contact with the side wall 13. In other words, the thickness of the coating layer 51 at the center of the inner surface 11a of the bottom 11 may be the thickest. Also, the thickness of the coating layer 51 on the inner surface 13a of the side wall 13 may be gradually thinner from the lower side in contact with the bottom 11 toward the upper side. In other words, the thickness of the coating layer 51 on the lower side of the inner surface 13a of the side wall 13 may be the thickest.

The embodiment of the thickness of the coating layer 51 on the inner surface 11a of the bottom 11 and the embodiment of the thickness of the coating layer 51 on the inner surface 13a of the sidewall 13 may be a combination other than the combination of the above-mentioned embodiments. For example, the thickness of the coating layer 51 formed on the inner surface 11a of the bottom 11 may be uniform, or may be gradually thinner from the center of the bottom 11 toward the end side that contacts the sidewall 13, and further, the thickness of the coating layer 51 formed on the inner surface 13a of the sidewall 13 may be gradually thicker from the lower side that contacts the bottom 11 toward the upper side.

The self-supporting low protein adsorption container 100 according to this embodiment, in which the container inner surface forming the storage area 21 is configured as described above, makes it difficult for proteins and cells to be adsorbed to the container inner surface forming the storage area 21, and also makes it easy to recover the liquid 43 containing proteins and cells from this storage area 21, thereby reducing recovery losses of proteins and cells. Another feature is that recovery is easy even if the container has a large capacity.

The protein or cell adsorption rate in the storage area 21 of the freestanding low protein adsorption container 100 according to this embodiment is not limited, but is preferably 20% or less in terms of protein adsorption rate, more preferably 10% or less, and even more preferably 5% or less (for example, 0.0001 to 10%, or even 0.0002 to 5%). If the adsorption rate is within such a range, it can be more suitably used as a specimen storage container for clinical testing, etc.

The protein adsorption rate is calculated by the following formula (1).
Adsorption rate (%) = adsorption amount / total amount of protein in solution (1)

Specifically, iodine ( ¹²⁵ I)-labeled bovine serum immunoglobulin G is diluted with phosphate buffer (pH 7.4) to concentrations of 1.0E-6 g/mL, 1.0E-7 g/mL, and 1.0E-8 g/mL to prepare each solution, and a predetermined amount of each solution is dispensed into the storage area 21 of the freestanding low protein adsorption container 100 according to this embodiment and allowed to stand for 1 hour at 37° C. Thereafter, washing is repeated three times with phosphate buffer containing 0.05 v/v % Tween 20, and measurement is performed with a gamma ray counter.
Then, the weight (adsorbed amount) of iodine ( ^125I )-labeled bovine serum immunoglobulin G remaining on the inner surface of the container is determined from a separately prepared calibration curve, and the adsorption rate at each solution concentration is calculated using the above formula (1), and the average value is calculated.

<Method of manufacturing a self-supporting low protein adsorption container>
Next, a method for producing the self-supporting low protein adsorption container 100 according to this embodiment will be described.

In the manufacturing method of the free-standing low protein adsorption container 100 according to this embodiment, first, a container having a desired shape (a shape that can stand on its own when the planar bottom 11 is placed on a mounting surface) with a planar bottom 11 and side wall 13 is formed by blow molding or the like using a material composed of the resin material described above. If necessary, a member such as an opening 31 may be provided. Then, a coating layer 51 is formed on the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 of the obtained container using a material containing a water-soluble resin as a main component as described above. Note that it is preferable to form the coating layer 51 after subjecting the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 to a surface treatment such as plasma treatment under an oxygen atmosphere.

Methods for forming the coating layer 51 made of a water-soluble resin on the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewall 13 include, for example, spin coating, dipping, and dispensing a water-soluble resin solution into the storage area 21 and then tilting the container to drain the solution. By using such a method, the inner surface 11a of the bottom 11 and the inner surface 13a of the sidewall 13 are brought into contact with a solution of a material containing a water-soluble resin as a main component, and the remaining solvent is then dried to form the coating layer 51.

In this case, the water-soluble resin is preferably used as a solution prepared using a solvent so that the viscosity at 20°C is preferably 1 mPa·s or more and 10 mPa·s or less, more preferably 2 mPa·s or more and 7 mPa·s or less. The solvent in this case is water, or a mixture of water and an organic solvent can be used to increase the solubility. If the viscosity of the water-soluble resin solution used is within the above range, a coating layer 51 with excellent low adsorption ability for proteins and cells can be easily obtained.

In addition, the remaining solvent, etc., can be dried, for example, by a method in which the opening 31 of the freestanding low protein adsorption container 100 according to this embodiment is oriented downward (in the direction of gravity). As described above, it is more preferable to perform drying while adjusting the thickness of the coating layer 51 formed on the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13 so that the thickness is uniform throughout, or the thickness of the coating layer 51 on the inner surface 13a of the bottom 11 gradually increases from the center toward the end, and the thickness of the coating layer 51 on the inner surface 13a of the side wall 13 gradually increases from the lower side toward the upper side.

Then, a process is carried out in which the water-soluble resins of the formed coating layer 51 are crosslinked and cured between each other and between the water-soluble resin and the specified inner surface of the container, thereby modifying the resin into a water-insoluble cured coating. This process can be carried out by the above-mentioned light irradiation process, heat treatment, radiation irradiation process, etc., and can be appropriately selected depending on the workability, the characteristics of the water-soluble resin constituting the coating layer 51, and the type of functional groups formed on the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall 13. Among these, light irradiation process (UV irradiation process, etc.) is a method that can quickly perform crosslinking and curing processes and can be performed with simple equipment.

The light source for crosslinking and curing the coating layer 51 by light irradiation is not particularly limited, but may be an ultra-high pressure mercury lamp with an illuminance of about 5.0 mW/ ^cm2 or a UV lamp with an illuminance of about 0.1 mW/ ^cm2 . Crosslinking and curing by light irradiation can be controlled by the illuminance and irradiation time, so when a light source with low illuminance is used, the irradiation time may be extended, and when a highly reactive photosensitive group is selected, crosslinking and curing can also be performed under a fluorescent lamp. For example, when a 5.0 mW/ ^cm2 ultra-high pressure mercury lamp is used, irradiation for 1 to 10 seconds can be used, and when a 0.1 mW/ ^cm2 UV lamp is used, irradiation for 3 to 10 minutes can be used to achieve sufficient crosslinking and curing.

In this way, by modifying the coating layer 51 into a water-insoluble cured coating, a stable coating layer 51 having hydrophilic functional groups on the surface layer can be constructed. Then, by forming the coating layer 51 in which the water-soluble resin is coated on the inner surface 11a of the bottom portion 11 and the inner surface 13a of the side wall portion 13 in advance by the method described above, the layer is crosslinked and cured to modify it into a water-insoluble cured coating, and a coating layer 51 of a predetermined thickness can be easily obtained.

After curing by crosslinking, the surface of the coating layer 51 may be washed with water or an aqueous solvent to remove at least a portion of the unreacted water-soluble resin. For example, if elution of uncrosslinked water-soluble resin or the like is confirmed, the amount of elution can be reduced by adding a washing process for the coating layer 51 after curing.

Furthermore, if necessary, the self-supporting low protein adsorption container 100 according to this embodiment on which the coating layer 51 as described above is formed is subjected to a sterilization treatment. Examples of the sterilization method include ethylene oxide gas (EOG) sterilization, dry heat sterilization, steam sterilization, and radiation sterilization. However, radiation sterilization using gamma rays or electron beams is preferred, and in the case of mass production, gamma ray sterilization is particularly preferred from the viewpoint of radiation transparency.

The absorbed dose of radiation is not particularly limited, but if the absorbed dose is too low, sterilization may not be sufficient, and if the absorbed dose is too high, the container body and coating layer 51 may deteriorate, so for example, the absorbed dose is preferably 1 kGy to 50 kGy, and more preferably 5 kGy to 30 kGy. This allows the freestanding low protein adsorption container 100 of this embodiment to be sterilized while maintaining its characteristics.

By using the above manufacturing method, it is possible to manufacture the self-supporting low protein adsorption container 100 according to this embodiment, which has low adsorption of proteins and cells to the inner surface 11a of the bottom 11 and the inner surface 13a of the side wall portion 13 that form the storage area 21.

The above embodiment encompasses the following technical ideas.
(1) A free-standing low-protein-adsorption container for containing a liquid including proteins or cells, the free-standing container having a planar bottom and side walls made of a resin material, capable of standing on its own when the bottom is placed on a support surface, the inner surfaces of the bottom and the side walls forming a storage area for holding the liquid, and further comprising a coating layer formed on the inner surface of the bottom and the inner surface of the side walls, the coating layer being made of a water-soluble resin having a hydrophilic functional group in its side chain, and the water-soluble resin being cross-linked between each other and between the water-soluble resin and the inner surface of the bottom and the inner surface of the side walls.
(2) A free-standing low-protein-adsorption container as described in (1), wherein the coating layer on the inner surface of the bottom portion has a uniform thickness, the coating layer on the inner surface of the side wall portion has a uniform thickness, and further, the coating layer on the inner surface of the bottom portion and the coating layer on the inner surface of the side wall portion have uniform thicknesses.
(3) A self-supporting low protein adsorption container as described in (1), wherein, on the inner surface of the bottom, the thickness of the coating layer gradually increases from the center toward the end side that contacts the side wall, and, on the inner surface of the side wall, the thickness of the coating layer gradually increases from the lower side that contacts the bottom toward the upper side.
(4) A free-standing low protein-adsorption container described in any one of (1) to (3), wherein the storage area has a volume capable of storing more than 50 mL of the liquid.
(5) A free-standing low-protein-adsorption container according to any one of (1) to (4), wherein the water-soluble resin constituting the coating layer has a side chain containing an aromatic azide group and a side chain containing a carbonyl group, carbonyl groups are formed on the inner surface of the bottom and the inner surface of the side wall, and the aromatic azide group of the water-soluble resin and the carbonyl group of the water-soluble resin, and the aromatic azide group of the water-soluble resin and the carbonyl group on the inner surface of the bottom and the inner surface of the side wall are crosslinked.
(6) The self-supporting low protein adsorption container described in any one of (1) to (5), wherein the water-soluble resin constituting the coating layer is a water-soluble resin represented by formula (Ia) or formula (Ib) above.

100 Self-supporting low protein adsorption container 11 Bottom 11a Bottom inner surface 13 Side wall 13a Side wall inner surface 21 Storage area 31 Opening 33 Cap 41 Pipette 43 Liquid containing protein or cells 51 Coating layer 61 Cross-linked structure (water-soluble resin - water-soluble resin)
63 Crosslinked structure (water-soluble resin - inner surface)

Claims (5)

A vessel for containing a liquid containing proteins or cells,
The container has a flat bottom and sidewalls made of a resin material,
The bottom part is self-supporting when placed on a placement surface,
an inner surface of the bottom portion and an inner surface of the side wall portion form a storage area for storing the liquid;
Furthermore, a coating layer made of a water-soluble resin having a hydrophilic functional group at a side chain is formed on the inner surface of the bottom portion and the inner surface of the side wall portion, and the water-soluble resins are crosslinked with each other and with the inner surface of the bottom portion and the inner surface of the side wall portion,
On the inner surface of the bottom portion, the thickness of the coating layer gradually increases from the center toward the end portion in contact with the side wall portion,
On the inner surface of the side wall portion, the thickness of the coating layer gradually increases from a lower side in contact with the bottom portion toward an upper side,
a thickness of the coating layer at an end of the bottom portion in contact with the side wall portion is thinner than a thickness of the coating layer at a lower portion of the side wall portion in contact with the bottom;
Freestanding low protein adsorption container. A vessel for containing a liquid containing proteins or cells,
The container has a flat bottom and sidewalls made of a resin material,
The bottom part is self-supporting when placed on a placement surface,
an inner surface of the bottom portion and an inner surface of the side wall portion form a storage area for storing the liquid;
Furthermore, a coating layer made of a water-soluble resin having a hydrophilic functional group at a side chain is formed on the inner surface of the bottom portion and the inner surface of the side wall portion, and the water-soluble resins are crosslinked with each other and with the inner surface of the bottom portion and the inner surface of the side wall portion,
On the inner surface of the bottom portion, the thickness of the coating layer gradually increases from the center toward the end portion in contact with the side wall portion,
On the inner surface of the side wall portion, the thickness of the coating layer gradually increases from a lower side in contact with the bottom portion toward an upper side,
The thickness of the coating layer at the lower part of the side wall portion in contact with the bottom is equal to the thickness of the coating layer at the end part of the bottom portion in contact with the side wall portion, or is thinner than the thickness of the coating layer at the end part of the bottom portion in contact with the side wall portion.
Freestanding low protein adsorption container. 3. The free-standing low-protein-adsorption container according to claim 1 or 2 , wherein the storage area has a volume capable of storing more than 50 mL of the liquid. the water-soluble resin constituting the coating layer has a side chain containing an aromatic azide group and a side chain containing a carbonyl group,
a carbonyl group is formed on the inner surface of the bottom portion and the inner surface of the side wall portion,
The free-standing low protein-adsorption container according to any one of claims 1 to 3, wherein the aromatic azide group of the water-soluble resin is crosslinked with the carbonyl group of the water-soluble resin, and the aromatic azide group of the water-soluble resin is crosslinked with the carbonyl group of the inner surface of the bottom and the inner surface of the side wall. The self-supporting low protein adsorption container according to any one of claims 1 to 4 , wherein the water-soluble resin constituting the coating layer is a water-soluble resin represented by the following formula (Ia) or the following formula (Ib):

(In formula (Ia), R1 represents a hydrocarbon group having a carbonyl and an amine, R2, R3, R4, and R5 each represent a hydrogen atom or an alkyl group, r1 represents 1 to 1000, r2 represents 40 to 4995, r3 represents 0 to 4000, and n represents 1, 2, or 3.)

(In formula (Ib), R represents a hydrocarbon group having a carbonyl and an amine, r1 represents 1 to 1000, r2 represents 40 to 4995, and r3 represents 0 to 4000.)

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