JPH0541976A - Test base material for cell cultivation and its preparation - Google Patents

Abstract

PURPOSE: To obtain a substrate for cell culture test, enabling to simply judge the adhesivity, proliferativity and peelability of a wide range of cells including cells inferior in the peelability by including a temperature-sensitive polymeric compound having LCST and a specific cell-adhesive substance.

CONSTITUTION: This substrate for cell culture tests comprises (A) a temperature- sensitive polymeric compound having LCST and (B) a cell-adhesive substance. The component B has crosslinked portions and non-crosslinked portions in a sea-island structure, sea-sea structure or laminar structure state, and the substrate comprises plural portions different in the structure or regularities of the crosslinked portions and the non-crosslinked portions.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture test base material capable of easily determining cell adhesion, proliferation and detachability. If the cell culture test substrate of the present invention is used, the cultured cells can be separated from the substrate and collected by changing the temperature of the culture solution. The present invention also relates to a method for producing such a cell culture test substrate.

[0002]

2. Description of the Related Art Today's cell culture technology is used for 1) production of cell products, 2) prostheses for lesions and defects of living bodies, 3) simulators for evaluating toxicity and pharmacological activity of drugs. ing.

The following is an overview of the prior art, focusing on the above-mentioned application fields.

Animal cells used for cell culture today are classified into two types. That is, adhesion-independent cells (anch
orage independent cells and adhesion-dependent cells (anchora
gedependent cells). The former adhesion-independent cell is a cell in which cell functions such as survival, proliferation, and substance-producing ability are normally expressed even in the absence of a substrate that is a scaffold of the cell. Typical examples include hybridomas formed from myeloma cells, lymphoma cells, and the like.

On the other hand, the latter adhesion-dependent cells are cells whose cell functions such as survival, proliferation, and substance-producing ability are not normally expressed without the presence of a substrate that is a scaffold of the cells. Most of the normal diploid cells including the primary culture cells are adhesion-dependent. Furthermore, many established cell lines capable of infinite proliferation are known to exhibit adhesion dependency. For example, established cells that produce useful cell products such as cytokines such as interferon and interleukin, erythropoietin, colony stimulating factor, various differentiated growth hormones such as thrombopoietin, tissue plasminogen activator, and vaccine. Many systems are known to exhibit adhesion dependence. Therefore, establishment of an adhesion-dependent cell culture technique is also very important for the production of these useful cell products.

In general, when cells are used for substance production, it is important to culture the cells in a large amount and a high density while maintaining a high function. However, compared to microbial cells, animal cells are more susceptible to the accumulation of waste products and insufficient supply of nutrients such as oxygen, and it is extremely difficult to maintain their function when culturing in high density and in large quantities. is there.

In the case of adhesion-independent cells, the suspension culture method is considered to be the most suitable. This is because if the suspension culture method is carried out with stirring, it is possible to quickly remove cellular waste products and efficiently supply nutrients, and it is easy to scale up the apparatus for the purpose of increasing the volume and density. However, in the case of adhesion-dependent cells, the suspension culture method cannot be applied because a substrate as a scaffold for the cells is required. Therefore, conventionally, various incubators for adhesion-dependent cells have been developed. For example, when culturing a small amount of cells in a laboratory, cell culture devices such as a dish type, a flask type, and a plate type are generally widely used. But,
These vessels are unsuitable for culturing a large amount of cells efficiently. Therefore, in order to enable large-scale culture by increasing the area of the substrate to which cells adhere to the entire volume, the following measures have been taken.

Roller bottle method in which glass bottles for cell culture are rotated to grow cells on the entire wall surface: Multi-tray method in which plates for cell adhesion are arranged in parallel in the culture solution and the culture solution is circulated therebetween: A plastic film is swirled A coil culture method in which a shaped product is inserted into a cylinder, cells are adhered while rotating horizontally, and then a culture solution is circulated between films.

A hollow fiber that uses a semipermeable hollow fiber membrane to adhere cells to the outer surface of the hollow fiber and circulate a culture solution inside to replenish nutrients from the hollow fiber membrane and remove waste products. Culturing method: Adhere cells to filled glass beads and circulate the culture solution in the gap Filled glass beads method: Suspend microbeads in the culture solution and attach cells to the surface of microbeads, and perform culture under agitation It is a floating culture method (microbead culture method).

As described above, in order to efficiently produce a large amount of useful cell products by culturing cells at a high density in a large amount, development has been conventionally conducted with emphasis on the shapes of the culture device and the culture substrate. Has been. However, in recent years, it has been found that the properties of the culture substrate are significantly related to the activity or function maintenance and expression of the adhesion-dependent cells, and as described above, the nutrients such as oxygen are changed depending on the shape of the culture substrate. It has been found that it is almost impossible to culture cells for a long period of time while maintaining their activity and specific functions only by efficient supply or efficient removal of cell waste products. Therefore, attempts have recently been actively made to improve the activity and function maintenance of cells by variously changing the properties of the culture substrate. Currently, polystyrene is widely used as a material for cell culture vessels in terms of optical transparency, nontoxicity, good mechanical properties and moldability, and low cost. However, the surface of polystyrene is highly hydrophobic and has a serious drawback in that cell adhesion is significantly inhibited in order to maintain cell activity. Therefore, a cell culture substrate has been developed and widely used in which the polystyrene surface is subjected to corona discharge treatment to introduce anion groups only to the surface to impart hydrophilicity to improve cell adhesion and proliferation. However, it has been found that it is difficult to express and maintain the specific function of cells with this degree of improvement, and recently, by making the environment of the cultured cells as close as possible to the state where the cells exist in the living body, Studies have been conducted to improve functions such as cell adhesion, proliferation, differentiation, and substance production ability. That is, it is a method of combining an extracellular matrix with a cell substrate.

By the way, in recent years, research on the function of the extracellular matrix in vivo has been rapidly advanced, and not only the conventionally known simple passive role of supporting and immobilizing cells but also the function of cells has been investigated. It has been found that it also has a function of actively controlling.

For example, it has been discovered that there are more than 10 types of collagen, which is the main component of extracellular matrix, and each collagen is synthesized by certain cells, localized in a certain tissue, and has different cell functions. It is becoming clear that it has a role of controlling Further, it has been found that even with the same type of collagen, cell function is affected by denaturation of higher-order structure or modification such as introduction of various functional groups.

The second component of the extracellular matrix, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, etc., has a specific binding site for collagen and cell membrane, and has a specific binding site to the cell matrix. Plays an important role in adhesion. In addition,
Adhesive proteins specifically bind to certain types of cells, collagen. For example, fibronectin has binding sites mainly for fibroblasts and type I and type II collagen, and laminin has binding sites specific for epithelium, endothelial cells and type IV collagen. Further, in addition to the above extracellular matrix, gelatin that is a heat-denatured product of collagen, lectin that specifically binds to sugar chains on the cell membrane, adhesive oligo that is a binding site for fibronectin, etc. have a great influence on cell functions. Peptides, cell adhesion proteins obtained from mussels and the like are known. An example of combining the above-mentioned factors that control cell adhesion and proliferation with a culture substrate is a collagen-coated culture substrate (K.Yoshiz
ato.et al..Annals of Plastic Surgery.13.9.1984),
The base material coated with fibronectin (F.Grin
nell.Expl.Cell Res.102.51.1976) and a cell adhesion protein-coated substrate (PTPicc) obtained from mussels.
iano.et all..In Vitro Cellular and Developmental Bi
ology 22 (3) .24A.1986) has been developed and improved cell adhesion and proliferation effects have been observed. Furthermore, instead of the extracellular matrix of biological origin, a substrate coated with polystyrene having a galactose terminal on the side chain (Takahiro Akaike et al.,
An artificial organ, 17.227.1988) has been developed, and in particular, it has been observed that hepatocyte adhesion is improved and viability is maintained. Conventionally,
It has become possible to cultivate cells, which are difficult to adhere due to severe culture conditions, by using the culture substrate whose surface is treated by the above-mentioned method. As mentioned above,
Development of cell culture method capable of efficient supplementation of nutrients and efficient removal of waste products, development of culture substrate containing cell adhesion and growth factors for the purpose of maintaining specific cell functions However, the following serious problems still remain in the current technology.

An important feature of the adhesion-dependent cells is that when the cells adhere to the culture substrate and proliferate and completely cover the substrate surface, a function called contact inhibition works and further cell proliferation occurs. It has the property of stopping. Therefore, in order to further proliferate the cells, a subculturing operation for transplanting the cells to a new culture substrate is required.

Heretofore, proteolytic enzymes such as trypsin and collagenase, and EDTA which is a complex compound of calcium ion have been generally used for substituting to adhere cells to the surface of the substrate and detach the grown cells. However, these cell releasing agents cause serious obstacles to cell function and cell culture process as described below.

1) Conventional cell releasing agents not only break the bonds between the culture substrate and the cells, but also break all bonds between the cells. It is known that there are three types of binding modes called tight junctions, gap junctions, and desmosomes between cells,
When cells are aggregated to form tight junctions, they act as a barrier against permeation of substances as cell aggregates and maintain a specific environment inside the living body. When a gap junction is formed, information and substances are exchanged between adjacent cells through the bond. On the other hand, desmosomes mechanically retain cell aggregates so that tight junctions and gap junctions are maintained. Therefore, it is difficult for a cell to be active and express its function by itself, and it is considered that the function is expressed only after forming a society by the above-mentioned intercellular bond (B. Alberts. Et al. “Molecular Bioloby of t
heCell ″ .3rd edn.Garland Publishing.Inc..New York & L
ondon P.673.1983). Therefore, the conventional cell detachment agent causes a fatal damage at the time of subculture even to cells cultured in a state in which the function is highly maintained.

2) Receptors for various signal substances such as hormones and neurotransmitters are present on the cell membrane, and cells are remotely controlled by the specific reaction between these signal substances and the receptors. It is known that conventional cell detachment agents such as proteolytic enzymes destroy these cell membrane receptors (C.Sung. Et al .. BiochemPha
rmocol.38.696.1989). Therefore, it is almost impossible to control the function of cells passaged by the conventional technique with a signal substance having a specific physiological activity.

3) Serum is generally added as a nutrient to the cell culture medium, and a strong trypsin inhibitor is present in the serum. Therefore, it is necessary to wash the cells sufficiently with a buffer solution to remove the trypsin inhibitor at the time of subculture of the trypsin treatment. This washing step not only complicates the operation, but also becomes a serious cause of fatal contamination for cell culture. As mentioned above, despite the progress of the culture substrate using factors that promote the expression of functions such as cell adhesion, proliferation, and differentiation such as extracellular matrix, the conventional methods for recovering and subculturing cells have been adopted. If the cell detachment agent is used, the high functionality of the cells that have been maintained will be significantly impaired due to the reasons (1) and (2) described above. This problem is a major obstacle to the technology for culturing cells that maintain high functionality.

On the other hand, the conventional cell detachment operation brings a fatal drawback to the mass culture technique. That is, it is said that if the initial cell concentration in the culture medium of the adhesion-dependent cells is too low, the cells cannot sufficiently exhibit their growth and substance-producing functions even if they adhere to the substrate. Especially in the case of primary culture cells or normal diploid cells that are difficult to collect, culturing in a large volume of culture solution from the beginning will result in too low cell concentration, so use a small-volume culture device to gradually It is necessary to increase the capacity by repeating cell passage. That is, normally
During large-scale culture, several cell recovery and passage operations are required. Therefore, due to the reason (3), the conventional cell detachment method leads to complication of the operating device and a great risk of contamination, which is a fatal gap especially in large-scale culture.

[0020]

In order to address the above problems of the conventional cell culture technique, the present inventors have developed a cell culture technique using a temperature-sensitive polymer compound. . However, as a result of the subsequent study by the present inventors, the conditions of the base material which are very good in the adhesiveness and the proliferative property of the cells and which are suitable for efficiently collecting the proliferated cells are:
It was revealed that the cell types differ slightly. Therefore, the present inventors have developed a cell culture test substrate consisting of a plurality of parts with different conditions, and made it possible to easily determine the adhesion, proliferation, and detachability of various cells. However, subsequent studies have revealed that even with such a substrate, the properties of a small number of cells with poor detachability cannot be accurately determined. It is an object of the present invention to provide a substrate for cell culture test, which can easily judge the adhesion, proliferation and detachability of a wide range of cells including such cells with poor detachability.

[0021]

[Means for Solving the Problems]
In a base material for cell culture test comprising a temperature-sensitive polymer compound and a cell-adhesive substance having: a cell-adhesive substance in the base material for cell-culture test and a portion not cross-linked are sea islands. A structure, a sea-sea structure, or a layered structure, and;
The present invention has been achieved by the present invention, which provides a substrate for cell culture test, which comprises a plurality of portions having different structures or regularities between a crosslinked portion and a non-crosslinked portion of a cell adhesive substance.

The cell-adhesive substance used in the cell culture test substrate of the present invention contains at least one substance that adheres to the sample cells without denaturation, and contains 50 collagen.
% Means more than 100%. Examples of substances that adhere to cells without denaturing cells include extracellular matrix components such as collagen, fibronectin, vitronectin, laminin, proteoglycan, and glycosaminoglycan,
Gelatin, which is a heat-denatured product of collagen, and other collagen derivatives, lectins such as Concanavalin A (Con A) that have an affinity for sugar chains on cell membranes, mussel-derived adhesion proteins, and adhesion that corresponds to the adhesion site between fibronectin and cells And the like. on the other hand,
The type of collagen that can be contained in the cell adhesive substance is not particularly limited. Therefore, various types of collagen alone or a mixture thereof can be widely used.

L used for the substrate for cell culture test of the present invention
The temperature-sensitive polymer having CST is a polymer compound having a negative temperature coefficient of solubility in water, and is a hydrate (oxonium oxonium) that depends on the hydrogen bond between the polymer compound formed at low temperature and the water molecule. Hydroxide) is characterized in that it decomposes at high temperature and dehydrates, causing the polymer compounds to aggregate and precipitate. LCST (Lower Critical Solution)
Temperature) refers to the transition temperature of hydration and dehydration of such a temperature-sensitive polymer compound (see, for example, J. Macromol, Sci.-Chem., A2 of M. Haskins et al.
(8), 1441 (1968)). Therefore, the temperature-sensitive polymer compound is water-insoluble and in a solid state at a temperature higher than LCST, and becomes reversibly water-soluble by lowering the temperature lower than LCST.

In the present invention, a temperature-sensitive polymer compound having an LCST lower than the culture temperature is used. Such a polymer compound can be widely used regardless of its structure. Since the temperature-sensitive polymer compound is in a solid state insoluble in water at the cell culture temperature, cells can adhere and proliferate using the layer composed of the cell-adhesive substance and the temperature-sensitive polymer compound as a scaffold. In addition, the temperature is LC
Since the polymer compound becomes soluble in water by adjusting the amount to be equal to or less than ST, the proliferated cells lose the scaffold, and the proliferating cells can be recovered without impairing the cell-cell bond and damaging the protein on the cell membrane surface. Therefore, by using the substrate of the present invention composed of a cell adhesive substance and a temperature-sensitive polymer compound, cells can be easily peeled and collected without impairing the function without using a release agent such as trypsin or EDTA. can do. Examples of the temperature-sensitive polymer compound that can be used in the present invention include poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives and copolymers thereof, polyvinyl methyl ether, polyethylene oxide, etherified methyl cellulose, polyvinyl alcohol moieties. Examples include acetic acid. Particularly preferred are poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives or copolymers thereof, polyvinyl methyl ether, and polyvinyl alcohol partial acetyl chloride.

The preferred polymer compounds are listed below in order of increasing LCST.

Poly-N-acryloylpiperidine; poly-N-n-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; poly-N- Acryloylpyrrolidine; poly-N, N-ethylmethyl acrylamide; poly-N-cyclopropyl methacrylamide; poly-N-ethyl acrylamide Even if the above polymer is obtained by polymerizing a single monomer, other polymer It may be a copolymer with the body. The monomer to be copolymerized may be either a hydrophilic monomer or a hydrophobic monomer. Generally, LCST increases when copolymerized with a hydrophilic monomer and LST increases when copolymerized with a hydrophobic monomer.
CST goes down. Therefore, a polymer compound having a desired LCST can be obtained by appropriately selecting these.

Examples of the hydrophilic monomer include N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, and acidic groups. Having acrylic acid, methacrylic acid and salts thereof, vinyl sulfonic acid, still sulfonic acid and salts thereof, vinyl sulfonic acid, still sulfonic acid, etc., and basic N, N-dimethylaminoethyl methacrylate, N, N -Diethylaminoethyl methacrylate, N, N-dimethylaminopropyl acrylamide and salts thereof, and the like,
It is not limited to these.

On the other hand, as the hydrophobic monomer, acrylate and methacrylate derivatives such as ethyl acrylate, methyl methacrylate and glycidyl methacrylate, N-substituted alkyl methacrylamide derivatives such as Nn-butyl methacrylamide, vinyl chloride, acrylonitrile, Examples thereof include, but are not limited to, styrene and vinyl acetate.

The temperature-sensitive polymer compound used in the present invention preferably has a molecular weight of 1.0 × 10 ⁵ or more. More preferably, it is 1.0 × 10 ⁶ or more. When the molecular weight is small, a part of the layer containing the cell adhesive substance and the temperature-sensitive polymer compound is exfoliated when the cells are seeded, so that the cells cannot be sufficiently detached. The molecular weight as used herein means the average molecular weight obtained from the viscosity. For example, the relationship between the average molecular weight of poly-N-isopropylacrylamide and the intrinsic viscosity is represented by the following formula.

[0030]

The feature of the substrate for cell culture test of the present invention is that the portion where the cell adhesive substance in the substrate is crosslinked and the portion where it is not crosslinked are in a sea-island structure, a sea-sea structure or a layered structure. In addition, the base material is composed of a plurality of portions having different structure or regularity between the crosslinked portion and the non-crosslinked portion of the cell adhesive substance. It is known that collagen, which is a typical example of a cell adhesive substance, generally forms a fiber in a culture state and has a structure similar to that in a living body. The collagen thus formed with fibers hardly dissolves in the culture solution.
However, in a system in which a temperature-sensitive polymer compound and collagen coexist, it is not possible to sufficiently exhibit the original fibrogenic ability of collagen. Part elutes in the culture solution. As a result, the cell adhesiveness and proliferation may be deteriorated and the utility of the system may be impaired. In order to cope with such a situation, a crosslink is partially introduced into the collagen of the cell culture substrate of the present invention. By introducing the cross-linking, the collagen becomes insoluble in the culture medium, so that the cells can be sufficiently adhered to the substrate.

On the other hand, cells proliferate by adhering to the cell culture substrate by the binding sites existing in a mosaic pattern on the cell membrane surface. Since this adhesion mode is multi-point adhesion, the larger the number of adhesion points, the stronger the adhesive strength of cells and the more difficult it is to peel off. Therefore, when culturing cells having poor detachability from the cell culture substrate, it is advantageous to reduce such adhesion points to some extent. Therefore, if the cell culture substrate for which the portion where the crosslinks are introduced into the cell adhesive substance and the portion where the crosslink is not introduced is present in the sea-island structure, sea-sea structure or layered structure, It becomes easy to control the peelability by controlling the number. However, the optimum structure of the base material having a structure in which the crosslinked portions are not introduced and a portion in which the crosslinked portions are not introduced depends on the cell type.
Therefore, in order to easily judge the adhesiveness, proliferation and detachability of cells, the crosslinked portion and the non-crosslinked portion have a sea-island structure, a sea-sea structure or a layered structure, And, the base material for cell culture test of the present invention is extremely effective, in which the base material is composed of a plurality of parts having different structure or regularity between the crosslinked part and the non-crosslinked part of the cell adhesive substance. I can say.

The substrate for cell culture test of the present invention may be composed of a plurality of portions having different regularity of any one structure of sea-island structure, sea-sea structure or layered structure,
It may be composed of a plurality of portions having different regularities of two or three types of structures. A substrate having one sea-island structure, one sea-sea structure, and one layered structure is also included in the cell culture test substrate of the present invention. In addition, since the difference in regularity between multiple parts is not particularly limited, not only when the distance between islands, the size of islands, and the size of the cycle are different, but the relationship between islands and the sea is reversed. The case is also included in the scope of the present invention. It is general that a plurality of portions having different structures or regularities are present on the same plane. In addition, the number of portions having different structures or regularity may be two or more, and the upper limit of the number is not limited. Usually 2-1
It is typically about 0 and 4 to 8.

In the portion having a sea-island structure in the base material for cell culture test of the present invention, the portion where the cross-link is introduced may be either the sea or the island. It is desirable that the size of the islands and the distance between the islands be less than or equal to the size of the cells to be cultured, regardless of whether the portion in which the bridge is introduced is the island or the sea. The average of both sizes is preferably 50 μm or less, and more preferably 20 μm or less. The lower limit is usually 0.1 μm.

The sea-sea structure or layer-structured portion preferably has a cycle of not more than twice the size of the cells to be cultured. Here, the cycle means an average value of distances between two adjacent portions (a crosslinked portion and a non-crosslinked portion) when the cell culture substrate is cut along a certain line. The period is preferably 100 μm or less, more preferably 40 μm or less.

Of the cell adhesive substances contained in the cell culture test substrate of the present invention, the total area of the crosslinked substances is preferably 50% or less of the cell growth area. Further, it is more preferable if it is 30% or less.

The method for introducing a crosslink into a cell adhesive substance is as follows:
The method is not particularly limited, and any method commonly used may be used. For example, a chemical treatment method using glutaraldehyde, an ozone treatment method, an ultraviolet ray treatment method, an electron beam treatment method, a plasma treatment method or the like can be used. The ultraviolet treatment method, the electron beam treatment method, and the plasma treatment method are suitable for forming a sea-island structure, a sea-sea structure, or a layered structure in the crosslinked portion and the non-crosslinked portion as in the present invention. I think that the. The weight ratio of the cell-adhesive substance to the temperature-sensitive polymer compound contained in the cell culture test substrate of the present invention is 1: 9 to 1:49.
It is preferable for it to be within the range since good cell adhesion, proliferation and exfoliation are achieved. A more preferable range is 1: 9 to 1:
39. Further, in the cell culture test substrate of the present invention, the structures of the cell adhesive substance and the temperature-sensitive polymer compound are not particularly limited, and may have any structure.

The cell culture test substrate of the present invention is generally formed of a coating layer comprising the above-mentioned cell adhesive substance and a temperature-sensitive polymer compound and a support. However, substrates not coated on the support are also included in the scope of the present invention. The thickness of the coating layer after drying is preferably 0.5 μm or more, more preferably 1.0 μm or more. The thickness of the coating layer is calculated from the concentration of the mixture solution and the thickness of the coating layer before drying, assuming that the density of the substance forming the substrate is 1.0. When the thickness of the coating layer is 0.5 μm or less, the detachability of cells becomes poor, and the time until detachment becomes extremely long, resulting in unstable performance. The coating of the layer comprising the cell-adhesive substance and the temperature-sensitive polymer compound may be over the entire surface of the support or may be a part thereof.

In the present invention, the support to be coated is preferably transparent or translucent. Especially,
It is preferable that the cells have sufficient transparency to be observed under a microscope. As the material, a transparent or translucent material such as glass, polystyrene, polypropylene, polyethylene, polyester, polyamide, polyvinyl chloride, polyvinylidene chloride, polymethylmethacrylate, and acrylic resin can be widely used. It is also preferable to use a membrane having substance permeability. Examples of such membranes, regenerated cellulose, cellulose acetate, collagen, or semi-permeable membrane, such as chitosan, polypropylene, cellulose acetate, Teflon ^TM, there may be mentioned a porous membrane such as polyvinylidene fluoride, with these It is not limited. The shape of the support is generally, but not limited to, a dish, plate, bottle, tube, flask, film and the like. The cell culture substrate of the present invention can be produced by a commonly used method obvious to those skilled in the art, and the method is not particularly limited. For example, if the solvent casting method or the dip method is used, it can be easily produced.

In the above-mentioned cell culture substrate, (a) a step of forming a layer containing a cell adhesive substance and a temperature-sensitive polymer compound; (b) a structure of a portion through which ultraviolet rays penetrate is a sea-island structure. Irradiating the layer formed in step (a) with ultraviolet rays by using a plurality of ultraviolet ray transmitting masks having a sea-sea structure or a layered structure and having different structures or regularities from each other. By the method described above, a cell culture test substrate having stable performance can be manufactured very efficiently.

In the step (a), a method capable of forming a layer such as a casting method or a dipping method can be widely used. Therefore, it is possible to adopt a method in which the layers of the cell adhesive substance and the temperature-sensitive polymer compound are separately formed.

The ultraviolet ray transmitting mask used in the step (b) has a sea-island structure, a sea-sea structure or a layered structure in the portion where the ultraviolet ray is transmitted, and the object of the present invention can be effectively achieved. There is no particular limitation on the type and material of the material. Therefore, a commercially available mesh or a porous film may be substituted, and the material may be quartz glass.

In the step (b), a plurality of masks having different structures or regularities of ultraviolet ray transmitting portions may be used, or a single mask composed of a plurality of portions having different structures or regularity of ultraviolet ray transmitting portions may be used. A mask may be used. When the former mask is used, it is necessary to change the mask and perform ultraviolet irradiation a plurality of times, and when the latter mask is used, the ultraviolet irradiation can be performed once. Of course, the latter mask can also be manufactured by using it several times while changing the ultraviolet intensity. Further, it can be manufactured by irradiating ultraviolet rays using two or more kinds of masks each having a plurality of portions. All of these aspects are included in the method of the invention.

Irradiation amount of ultraviolet rays (irradiation intensity x time: 254
nm) is preferably set to ^{100~10,000J / m 2, 500~8,000J / m} 2 Tosureba more preferred. 1
If it is less than 00 J / m ² , there is almost no effect of UV irradiation,
Also, at 10,000 J / m ² or more, the cells are hardly detached. When ultraviolet light having a wavelength other than 254 nm is used, the amount of cross-linking is the same as that of irradiation of ultraviolet light of 254 nm in the above range.

[0045]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is determined by the scope of the claims and is not limited by the following examples.

(Example 1 and Comparative Example 1) 0.5% (w / v) bovine dermis pepsin solubilized type I collagen solution (pH 3) (KOKEN CELLGEN I-PC, manufactured by Koken Co., Ltd.)
And 0.5% (w / v) poly-N-isopropylacrylamide (average molecular weight 3.5 × 10 ⁶ , PNIPAAm) aqueous solution (adjusted to pH 3 with hydrochloric acid, this solution was redissolved after autoclaving) , Collagen and PNIPAAm were mixed at a mixing ratio of 1: 9 to prepare a casting solution. 2.4 ml of this casting solution
It was dispensed in a 90 mm dish (Falcon # 3003, manufactured by Nippon Becton), and spread quickly and uniformly to coat. Then, it was dried in an incubator at 10 ° C. for about 10 hours. The thickness of the coating layer after drying is about 2 μm. A line was preliminarily drawn on the back surface of this dish so that the area was equally divided into four.

Next, a mask (see FIG. 1) having a window through which ultraviolet rays can be transmitted only at one of the four divided areas is made, and a mesh of the size shown in Table 1 is put on this window portion, and this is dished. Place it on the UV irradiation device (XX-
100, wavelength 254 nm, made by Funakoshi) was irradiated with ultraviolet rays. The energy of all ultraviolet rays at this time is 2,000
Unified to J / m ² . The coating and irradiation with ultraviolet rays were performed in a sterile environment. Culture evaluation was performed using this coated dish.

In addition, as a comparative example, a cell that was irradiated with ultraviolet rays without a mesh was also evaluated for culturing at the same time.

The contents of the culture evaluation experiment are shown below. Human dermis-derived fibroblasts were suspended in a Dulbecco's modified Eagle medium (D-MEM, containing 10% fetal bovine serum, manufactured by GIBCO) so that the final cell concentration was about 2 × 10 ⁵ cells / ml. Was prepared and kept at 37 ° C. Also, the dish is a plate that has been kept warm at 37 ° C in advance (Micro warm plate, made by Kitasato Supply)
12 ml of the cell suspension was injected by placing it on the plate and keeping it warm. This was rapidly transferred into a carbon dioxide gas incubator (5% carbon dioxide gas) at 37 ° C. and cultured for 3 days.

Regarding cell adhesion, proliferation and detachability,
Observing under a phase contrast microscope, the adhesion, proliferation rate and peeling rate were calculated according to the following formulas. The observation of adhesion and proliferation was carried out at 37 ° C., and the observation of peelability was carried out at room temperature after the dish was placed on ice and left for about 3 minutes.

[0051]

The results are shown in Table 1. In the system that did not use a mesh, although the cell adhesion and proliferation were good, the cells were hardly detached. On the other hand, in the system using the mesh, cell adhesion, proliferation and detachability were changed depending on the structure of the mesh. It was revealed that the No. 1 mesh was suitable for this cell.

[0053]

(Example 2 and Comparative Example 2) Coating and cell evaluation were carried out in the same manner as in the method shown in Example 1 and Comparative Example 1. However, instead of the mesh, a photomask (see FIG. 2 and Table 2) in which four kinds of lattice-shaped patterns were engraved on quartz glass was used. Collagen and P
The mixing ratio of NIPAAm was 1:19, and the ultraviolet irradiation energy was 6,000 J / m ² . The cells are IMR-90
(Human fetal lung-derived fibroblast cell line) was used. Further, as a comparative example, a sample in which the ultraviolet irradiation energy was changed under the conditions shown in Table 3 without using a mask was used.

The results are shown in Tables 2 and 3. In the comparative example, it was not possible to find good conditions for cell adhesion, proliferation and exfoliation, but in the system using the photomask, cell adhesion, proliferation and exfoliation were observed due to the structure of the mask pattern. Has changed. N for this cell
It became clear that the o.1 pattern is suitable.

[0056]

(Example 3 and Comparative Example 3) Coating and cell evaluation were carried out in the same manner as in the method shown in Example 1 and Comparative Example 1. However, a photomask (see FIG. 3 and Table 4) in which four kinds of layered patterns were engraved on quartz glass was used instead of the mesh. Collagen and PN
The mixing ratio of IPAAm was 1: 9, and the ultraviolet irradiation energy was 6,000 J / m ² . Human chorion-derived fibroblasts were used as cells, and Eagle medium was used as the medium.
Further, as a comparative example, a sample in which the ultraviolet irradiation energy was changed under the conditions shown in Table 5 without using a mask was used.

The results are shown in Tables 4 and 5. In the comparative example, it was not possible to find good conditions for cell adhesion, proliferation and exfoliation, but in the system using the photomask, cell adhesion, proliferation and exfoliation were observed due to the structure of the mask pattern. Has changed. N for this cell
It became clear that the o.2 pattern is suitable.

[0059]

[0060]

EFFECTS OF THE INVENTION By using the substrate for cell culture test of the present invention, even if it is difficult to judge the adhesion, proliferation and peelability by the conventional method, the adhesion, proliferation and peelability can be easily and reproducibly obtained. It became possible to judge.

By using the production method of the present invention, a cell culture test substrate having stable performance can be produced by a simple method.

[Brief description of drawings]

FIG. 1 is a view showing one mode of a photomask, in which a indicates an ultraviolet ray transmitting portion and b indicates an ultraviolet ray non-transmitting portion.

FIG. 2 shows one mode of a photomask, in which c indicates a pattern portion (lattice shape) and d indicates an ultraviolet ray opaque portion.

FIG. 3 is a view showing one mode of a photomask, in which e indicates a pattern portion (layer) and f indicates an ultraviolet ray opaque portion.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Michiko Tsuchida 671-1 Tomuro, Atsugi City, Kanagawa Prefecture SATII I ATSUGI 306 (72) Inventor Yuichi Mori 1642-212 B-4, Kamariya-cho, Kanazawa-ku, Yokohama, Kanagawa Prefecture B-4

Claims (2)

[Claims] 1. A cell culture test substrate comprising a temperature-sensitive polymer compound having LCST and a cell adhesive substance, wherein the cell adhesive test substance in the cell culture test substrate is crosslinked with a crosslinked portion. Having a sea-island structure, a sea-sea structure or a layered structure, and the base material for cell culture test, which is not cross-linked with the cross-linked portion of the cell adhesive substance A cell culture test substrate comprising a plurality of parts having different structures or regularities. 2. The step (a) of forming a layer containing a cell adhesive substance and a temperature-sensitive polymer compound. (B) The structure of the portion through which ultraviolet rays penetrate is a sea-island structure, a sea-sea structure or a layered structure. And a step of irradiating the layer formed in step (a) with ultraviolet rays using a plurality of ultraviolet ray transmitting masks having different structures or regularities from each other. Production method.