WO2013005780A1

WO2013005780A1 - Matrix for recovering cell fragments for therapeutic use and method for producing cell fragments for therapeutic use using same, and matrix for recovering cell fragments for subculture and method for producing cell fragments for subculture using same

Info

Publication number: WO2013005780A1
Application number: PCT/JP2012/067119
Authority: WO
Inventors: 正敏黒田; 真佐美奈良; 岡野　光夫; 大和　雅之; 清水　達也; 広也渡邊; 恵介芦葉
Original assignee: 大日本印刷株式会社; 学校法人東京女子医科大学; 株式会社セルシード
Priority date: 2011-07-04
Filing date: 2012-07-04
Publication date: 2013-01-10
Also published as: JP2013013368A

Abstract

The main purpose of the present invention is to provide a matrix for recovering cell fragments for therapeutic use whereby cell fragments capable of exerting a therapeutic effect at a high reproducibility can be obtained. The matrix for recovering cell fragments for therapeutic use, which comprises a stimulus-responsive area showing a change in cell adhesiveness due to a stimulus and a cell non-adhesive area having cell non-adhesiveness, characterized in that the circumference of the stimulus-responsive area is surrounded by the cell non-adhesive area. The above-said purpose can be achieved by providing this matrix.

Description

Substrates for recovering therapeutic cell fragments, methods for producing therapeutic cell fragments using the same, and substrates for recovering cell fragments for subculture and methods for producing subcellular fragments using the same

The present invention provides a cell fragment recovery base material capable of obtaining a therapeutic cell fragment capable of exerting a therapeutic effect with high reproducibility, and easily obtaining a subculture cell fragment capable of stably culturing cells during subculture operations. It is about.

In recent years, cell fragments are often used in regenerative medicine and the like.
Here, the cell fragment is a cell aggregate in which cells are connected to each other in at least a single layer by intercellular bonding.

As a method for producing such a cell fragment, a proteolytic enzyme such as trypsin is allowed to act on a cell sheet formed by seeding and culturing cells on a support, thereby binding between the support and cells and between cells and cells. A method for recovering cell fragments that have been cut and a method for dividing the cell sheet into a predetermined size by pressing a metal net against a cell sheet formed by seeding and culturing cells on a support. (Non-Patent Document 1).
However, when the cell fragment obtained by such a method is used for transplantation to an experimental animal in preclinical research, it is not only invasive because it physically breaks the cell-cell junction, but also its size. However, there was a problem that only the irregular fragments were obtained and the experimental results varied.

Moreover, at the time of cell passage operation, a cell is detached from a support such as a culture vessel using a proteolytic enzyme, and an operation of planting the detached cells on a new support is performed.
However, such a method using a proteolytic enzyme requires problems such as dripping the proteolytic enzyme and stopping the reaction, and the work is complicated, and the cell is damaged by the proteolytic enzyme and the subsequent growth There were problems such as adverse effects on In addition, when the concentration of proteolytic enzyme and the treatment time cannot be strictly controlled, there is a problem that the cell growth rate varies for each passage operation.

The present invention has been made in view of the above problems, and it is easy to obtain a cell fragment capable of exerting a therapeutic effect with good reproducibility and to stably subculture cell fragments during passage operation. The main object is to provide a cell fragment collection substrate that can be obtained.

As a result of repeated studies to solve the above problems, the present inventors have found that cell fragments with irregular sizes and shapes are not obtained by conventional methods using a proteolytic enzyme such as trypsin or by dividing a cell sheet with a wire mesh or the like. Therefore, when cell fragments with irregular sizes and shapes are used, the cell fragments are physically broken when used for transplantation to experimental animals in preclinical studies. It was found that not only invasive fragments but also fragments of irregular sizes were obtained, and the experimental results varied.
In addition, the cell-cell bond is cleaved by a proteolytic enzyme such as trypsin at the time of subculture, but the size and shape of the cell fragments formed by such a treatment are not uniform, which prevents subsequent cell growth. It was found that there was variation, and the size of the cell fragment in which cells can efficiently grow, that is, the number of cells contained in one cell fragment was different for each cell, and the present invention was completed. It is.

That is, the present invention has a stimulus-responsive region in which cell adhesion is changed by stimulation, and a cell non-adhesive region having cell non-adhesiveness, and the stimulus-responsive region is surrounded by the cell non-adhesive region. A therapeutic cell fragment recovery substrate is provided.

According to the present invention, the therapeutic cell fragment is controlled to have an arbitrary size and shape by applying a stimulus to the stimulus responsive region by having the stimulus responsive region surrounded by the cell non-adhesive region. Can be easily recovered.
In addition, since the size and shape are controlled, the therapeutic effect by transplantation using such therapeutic cell fragments can be exhibited with good reproducibility.

The present invention has a stimulus-responsive region in which cell adhesiveness is changed by stimulation, and a cell non-adhesive region having cell non-adhesiveness, and the stimulus-responsive region is surrounded by the cell non-adhesive region. A substrate for recovering cell fragments for passage is provided.

According to the present invention, the cell fragment for subculture that has a stimulus-responsive region surrounded by the cell non-adhesion region, and is controlled to have an arbitrary size and shape by applying a stimulus to the stimulus-responsive region. Can be easily recovered. For this reason, passage operation can be made simple. Moreover, since the use of a proteolytic enzyme such as trypsin can be eliminated, the cells can be stably grown.
In addition, since the size and shape are controlled, when the cells are passaged using such passage cell fragments, the proliferation of the cells can be controlled with high accuracy and can be stably cultured.
For this reason, for example, when such a cell fragment collection substrate for passage is used in an automatic culture apparatus, cells can be stably proliferated and cultured.

In the present invention, it is preferable that the stimulus-responsive region includes a temperature-responsive material whose cell adhesiveness changes due to a temperature change. This is because it is easy to apply a stimulus.

In the present invention, the temperature-responsive material is preferably polyisopropylacrylamide. This is because the cell fragment can be stably recovered because it has cell adhesion at a temperature suitable for cell culture and exhibits cell non-adhesion at a temperature with little damage to the cell.

The present invention provides the above-mentioned substrate for cell fragment collection for treatment, seeding and culturing cells on the stimulus-responsive region in a state where the stimulus-responsive region expresses cell adhesion, A cell fragment forming step of forming a therapeutic cell fragment comprising at least one cell adhered to a stimulus responsive region; a stimulus applying step of stimulating the stimulus responsive region to express cell non-adhesiveness; and the stimulus And a recovery step of recovering the therapeutic cell fragment peeled from the responsive region. A method for producing the therapeutic cell fragment is provided.
In addition, the present invention provides the above-described substrate for recovering cell fragments for passage, and seeding and culturing cells on the stimulus-responsive region in a state where the stimulus-responsive region expresses cell adhesion. A cell fragment forming step of forming a cell fragment for passage consisting of at least one or more cells adhered to the stimulus responsive region, and a stimulus applying step of stimulating the stimulus responsive region to express cell non-adhesiveness, And a recovery step of recovering the passage cell fragment exfoliated from the stimulus-responsive region. A method for producing a passage cell fragment is provided.

According to the present invention, the therapeutic cell fragment and the passage cell fragment having a controlled size and shape can be easily produced by using the therapeutic cell fragment collection substrate and the passage cell fragment collection substrate. can do.

The present invention provides a cell fragment recovery base material capable of obtaining a therapeutic cell fragment capable of exerting a therapeutic effect with high reproducibility, and easily obtaining a subculture cell fragment capable of stably culturing cells during subculture operations. There is an effect that can be provided.

It is a schematic plan view which shows an example of the base material for cell fragment collection | recovery of this invention. FIG. 2 is a sectional view taken along line AA in FIG. 1. It is a schematic plan view which shows the other example of the base material for cell fragment collection | recovery of this invention. It is a schematic plan view which shows the other example of the base material for cell fragment collection | recovery of this invention. It is a schematic sectional drawing which shows the other example of the base material for cell fragment collection | recovery of this invention. It is process drawing which shows an example of the manufacturing method of the cell fragment for a treatment of this invention.

TECHNICAL FIELD The present invention relates to a therapeutic cell fragment collection substrate and a method for producing a therapeutic cell fragment using the same, and a passage cell fragment collection substrate and a method for producing a passage cell fragment using the same. is there.
The therapeutic cell fragment collection substrate, passage cell fragment collection substrate, treatment cell fragment production method, and passage cell fragment production method of the present invention are described in detail below.

A. First, the base material for cell fragment collection for treatment of the present invention will be described.
The substrate for cell fragment collection for treatment of the present invention has a stimulus responsive region in which cell adhesion changes by stimulation, and a cell non-adhesive region having cell non-adhesiveness. It is characterized by being surrounded by the cell non-adhesion region.

Such a therapeutic cell fragment collection substrate of the present invention will be described with reference to the drawings. FIG. 1 is a schematic plan view showing an example of the therapeutic cell fragment collection substrate of the present invention, and FIG. 2 is a cross-sectional view taken along line AA of FIG.
As illustrated in FIGS. 1 and 2, the therapeutic cell fragment collection substrate 10 of the present invention includes a stimulus-responsive layer 1 containing a stimulus-responsive material whose cell adhesiveness changes upon stimulation, and cell non-adhesiveness. A non-cell-adhesive layer 2 containing a non-cell-adhesive material, and a non-adhesive region in which the stimulus-responsive region 11 that is the exposed surface of the stimulus-responsive layer 1 is the exposed surface of the non-cell-adhesive layer 12 is surrounded by 12. Moreover, it has the base material 3 which supports the said stimulus responsive layer 1 and the cell non-adhesive layer 2.

According to the present invention, by having a stimulus-responsive region surrounded by the cell non-adhesive region, after seeding cells in the stimulus-responsive region and culturing to form a therapeutic cell fragment, By applying a stimulus to the stimulus-responsive region, the therapeutic cell fragment controlled to have an arbitrary size and shape can be easily detached from the stimulus-responsive region and collected.
In addition, because the size and shape are controlled, the use of such therapeutic cell fragments is compared to the case of using conventional proteolytic enzymes such as trypsin or cell fragments obtained by pressing a wire mesh. In addition, fragments having uniform sizes can be obtained at the time of transplantation to an experimental animal in preclinical research, so that the effect of improving the reproducibility of the experiment can be obtained.

The therapeutic cell fragment collection substrate of the present invention has at least a stimulus-responsive region and a cell non-adhesive region.
Hereinafter, each configuration of the therapeutic cell fragment collection substrate of the present invention will be described in detail.

1. Stimulus responsive region The stimulus responsive region in the present invention is an exposed range of the surface of the stimulus responsive layer containing the stimulus responsive material, and is surrounded by the cell non-adhesive region.

(1) Stimulus responsive region The stimulus responsive region in the present invention is surrounded by the cell non-adherent region, and adheres and detaches cells to be cultured by changing cell adhesiveness by stimulation. It is something that can be done.

The stimuli-responsive region in the present invention changes from a state in which cell adhesion has good cell adhesion by specific stimulation to a state in which cell non-adhesion, which is a property of low cell adhesion, is expressed. It can change.
Here, the stimulus-responsive region expressing cell adhesion means that the cells are easy to adhere and spread on the stimulus-responsive region and have a high cell adhesion extension rate. is there. In the present invention, specifically, such a state where the cell adhesion extension rate is high can be a state where the cell adhesion extension rate is 60% or more.
In the present invention, it is preferably 80% or more. This is because cells can be efficiently cultured to form therapeutic cell fragments.
In the present invention, the cell adhesion spreading rate is such that bovine vascular endothelial cells are seeded in a seeding density range of 4000 cells / cm ² or more and less than 30000 cells / cm ² and stored in a 37 ° C. incubator (CO ₂ concentration 5%). It represents the percentage of cells that have passed for 3 hours or more and have spread and adhered before the doubling time ({(number of cells adhered) / (number of cells seeded)} × 100 (%)).
The cells are seeded by suspending them in a DMEM medium containing 10% FBS (serum) and seeding them on a culture substrate, and then the culture medium on which the cells are seeded so that the cells are distributed as uniformly as possible. This is done by shaking the material slowly.
In addition, the cell adhesion spread rate is measured after exchanging the medium immediately before the measurement to remove non-adhered cells. The cell adhesion spread rate is measured at a location where the density of cells tends to be specific. The measurement is performed excluding (for example, the center of the predetermined region where the existence density tends to be high and the periphery of the predetermined region where the existence density tends to be low).

In addition, the case where the stimulus-responsive region expresses cell non-adhesiveness means that the cells are difficult to adhere and spread on the stimulus-responsive region and the cell adhesion extension rate is low. is there. In the present invention, specifically, such a state where the cell adhesion extension rate is low can be a state where the cell adhesion extension rate is 5% or less. In the present invention, the content is preferably 2% or less.
Therefore, when cells are seeded in the stimulus-responsive region, cells can adhere to the stimulus-responsive region when cell adhesion is expressed, and can easily form therapeutic cell fragments. Since cells are inhibited from adhering to the stimulus-responsive area when expressing adhesiveness, the therapeutic cell fragments adhering to the stimulus-responsive area should be easily detached and recovered. Can do it.

The shape of the stimulus-responsive region used in the present invention is not particularly limited as long as it can stably recover the therapeutic cell fragment. For example, as shown in FIG. A circular shape, an elliptical shape, a polygonal shape as illustrated in FIG. 3, a line shape, a lattice shape, a star shape, or the like may be used. Further, a combination of a plurality of shapes may be used.
In the present invention, in particular, a circular shape or a hexagonal or more polygonal shape is preferable, and a circular shape is particularly preferable. This is because when the stimulus is applied to the stimulus responsive region, the therapeutic cell fragment can be stably detached from the stimulus responsive region.

The area per stimulus-responsive region used in the present invention is particularly limited as long as at least one or more cells can adhere and the therapeutic cell fragment can be stably recovered. but it not, specifically, is preferably in the range of 200 [mu] m ² ~ 100000 ^2, preferably in the range of 1000 .mu.m ² ~ 50000 ^2, preferably in the range of 10000μm ^² ~ 40000μm ² . This is because the therapeutic cell fragment can be stably detached and collected by applying a stimulus to the stimulus-responsive region.

The arrangement of the stimulus-responsive region used in the present invention is not particularly limited as long as the therapeutic cell fragment can be stably recovered, but the therapeutic cell fragment can be stably added. It is preferable that the largest number of therapeutic cell fragments can be formed within the range obtained. This is because the therapeutic cell fragment can be efficiently obtained by a single operation.

The ratio of the stimulus-responsive region used in the present invention is particularly limited as long as it is at least 50% or more when the bottom surface in the therapeutic cell fragment collection substrate of the present invention is 100%. However, it is preferably in the range of 50% to 95%, and more preferably in the range of 70% to 90%.
This is because the effect of the present invention that a therapeutic cell fragment having a controlled size and shape can be obtained can be more effectively exhibited.

The type of stimulus-responsive region used in the present invention may be only one type or may include a plurality of stimulus-responsive regions that respond to different stimuli.

The stimulation-responsive region used in the present invention is surrounded by the cell non-adhesive region, that is, the cell non-adhesive region is arranged around the stimulus responsive region, and the boundary with the cell non-adhesive region on the outer periphery It has a non-adhesive boundary.
Here, the non-adhesion boundary is capable of separating the therapeutic cell fragment from the stimulation-responsive region along the non-adhesion boundary when the stimulus-responsive region is stimulated. There is no particular limitation.
Such a non-adhesion boundary is usually in contact with the cell non-adhesion region in plan view. For example, the stimulus responsive region is stimulated between the stimulus responsive region and the cell non-adhesion region. In this case, it also includes locations where other regions such as a cell adhesion region formed with such a width that the therapeutic cell fragment can be peeled off along the non-adhesion boundary.
In the present invention, in particular, it is a place that is in contact with the cell non-adhesion region in plan view, that is, a location that does not include other regions between the stimulus-responsive region and the cell non-adhesion region. preferable. This is because the therapeutic cell fragment can be stably formed.

The position of the non-adhesion boundary formed on the outer periphery of the stimulus-responsive region in the present invention is that the therapeutic cell fragment is stably detached from the stimulus-responsive region when the stimulus-responsive region is stimulated. Any material can be used, and it can be formed on at least a part of the outer periphery of the stimulus-responsive region.
In the present invention, in particular, it is preferably formed on the entire outer periphery of the stimulus responsive region. This is because the therapeutic cell fragment can be stably peeled in the shape of the stimulus-responsive region.

The height of the stimulus-responsive region used in the present invention with respect to the cell non-adhesion region is not particularly limited as long as the cell fragment can be stably formed. The adhesion regions may be formed at the same height, that is, on the same plane, or may be different.

(2) Stimulus responsive layer The stimulus responsive layer used in the present invention includes the stimulus responsive material and has a stimulus responsive region that is an exposed surface in plan view.

(A) Stimulus-responsive material The stimulus-responsive material used in the present invention includes a stimulus-responsive material that changes cell adhesion depending on the presence or absence of stimulation and can adhere and detach cells to be cultured. There is no particular limitation as long as it is, for example, a temperature responsive material, a photoresponsive material, a pH responsive material, a potential responsive material in which cell adhesion changes according to temperature, light, pH, potential and magnetic force, and Examples thereof include a magnetic responsive material.
In the present invention, among them, a temperature responsive material is preferable. This is because it is easy to apply a stimulus.

The change in cell adhesion of the stimulus-responsive material used in the present invention is not particularly limited as long as it can be changed from a state having cell adhesion to a state having cell non-adhesion. However, it may be reversibly changed, or may be changed irreversibly, and is selected depending on the application.

The content of the stimulus-responsive material used in the present invention in the stimulus-responsive layer is such that a desired adhesive change can be obtained in the stimulus-responsive region that is the surface of the stimulus-responsive layer by stimulation. If it is not specifically limited, it will differ according to the kind of the said material.

In the present invention, the kind of the stimulus responsive material contained in the stimulus responsive layer may be one kind or two or more kinds.

The temperature-responsive material used in the present invention is not particularly limited as long as the cell adhesiveness changes due to a temperature change.
In the present invention, the temperature range in which the above-mentioned temperature-responsive material exhibits cell adhesion is preferably in the range of 10 ° C. to 45 ° C., and more preferably in the range of 33 ° C. to 40 ° C. preferable. This is because the cells can be stably cultured when the temperature region is within the above range.

In the present invention, the temperature range in which the non-adhesive property of the temperature-responsive material is exhibited is preferably in the range of 1 ° C. to 36 ° C., and more preferably in the range of 4 ° C. to 32 ° C. This is because when the temperature range is within the above range, damage to cells can be reduced.

Specific examples of the temperature-responsive material used in the present invention include poly-N-isopropylacrylamide (PIPAAm), poly-Nn-propylacrylamide, poly-Nn-propylmethacrylamide, poly -N-ethoxyethyl acrylamide, poly-N-tetrahydrofurfuryl acrylamide, poly-N-tetrahydrofurfuryl methacrylamide, poly-N, N-diethyl acrylamide, etc. can be mentioned, among them PIPAAm, poly-N- n-Propylmethacrylamide and poly-N, N-diethylacrylamide can be preferably used, and PIPAAm can be particularly preferably used. This is because the cell fragment can be stably recovered because it has cell adhesion at a temperature suitable for cell culture and exhibits cell non-adhesion at a temperature with little damage to the cell.
In the present invention, the temperature-responsive material may be composed of only one type or may contain two or more types. Further, in order to adjust the temperature region, a material copolymerized with the above temperature-responsive materials and / or other polymers may be used.

The photoresponsive material used in the present invention is not particularly limited as long as cell adhesion changes depending on the presence or absence of light irradiation. For example, as disclosed in JP-A-2005-210936 , Photocatalysts, and those containing photoresponsive components such as azobenzene, diarylethene, spiropyran, spirooxazine, fulgide and leuco dyes can be used.

The potential responsive material used in the present invention is not particularly limited as long as the cell adhesiveness is changed by application of a potential. For example, as disclosed in Japanese Patent Application Laid-Open No. 2008-295382. And an electrode and a spacer substance such as alkanethiol, cysteine, and alkanedisulfide having a cell adhesive portion such as a peptide containing an RGD sequence and binding to the electrode surface via thiolate.

The magnetic force responsive material used in the present invention is not particularly limited as long as the cell adhesiveness is changed by the application / removal of magnetic force. For example, as disclosed in JP-A-2005-312386. An example is a positively charged liposome encapsulating magnetic particles in which magnetic particles such as ferrite are encapsulated in positively charged liposomes.

(B) Stimulus responsive layer The stimulus responsive layer used in the present invention contains at least the stimulus responsive material.
In the present invention, additives such as leveling agents, plasticizers, surfactants, antifoaming agents, sensitizers, polyvinyl alcohol, unsaturated polyesters, etc., as necessary, within a range that does not inhibit the stimulus responsiveness. , Acrylic resin, polyethylene, diallyl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin, polycarbonate, polyvinyl chloride, polyamide, polyimide, styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyacetic acid Vinyl, nylon, polyester, polybutadiene, polybenzimidazole, polyacrylonitrile, epichlorohydrin, polysulfide, polyisoprene, polyethylene glycol, MPC polymer (trade name) And they comprise a binder resin zwitterionic polymers such like may.

The thickness of the stimulus responsive layer used in the present invention is not particularly limited as long as it can exhibit stimulus responsiveness, and specifically, within a range of 0.5 nm to 300 nm. It is preferable that it is within a range of 1 nm to 100 nm.

The method for forming the stimulus-responsive layer used in the present invention is not particularly limited as long as it can be formed in a desired pattern.
Specifically, the stimuli-responsive material composition containing the stimuli-responsive material is applied using a known application method such as spin coating, and patterned by photolithography, gravure printing, flexographic printing, screen printing, The method of apply | coating the said stimulus responsive material composition in pattern shape using well-known pattern application | coating methods, such as the inkjet method, can be mentioned.
In addition, when the stimulus-responsive layer is made of only an inorganic substance such as a photocatalyst as the stimulus-responsive material, a method of using a vacuum film-forming method such as a sputtering method, a CVD method, or a vacuum evaporation method can be cited. it can. This is because a layer having a uniform film thickness can be obtained.

2. Cell non-adhesion region The cell non-adhesion region used in the present invention is an exposed range of the surface of the cell non-adhesion layer containing the cell non-adhesion material, and has cell non-adhesion.

(1) Cell non-adhesion region The cell non-adhesion region used in the present invention includes at least the region surrounding the stimulation-responsive region.

The degree of cell adhesiveness of such a cell non-adhesive region is not particularly limited as long as it exhibits a desired cell non-adhesive property, and the stimulation-responsive region expresses cell non-adhesive property. It can be the same as the cell adhesiveness in the case where it is present.

The formation site of the cell non-adhesion region used in the present invention is not particularly limited as long as it includes at least a portion surrounding the stimulation-responsive region. For example, as illustrated in FIG. It may be formed in the responsive region.
In addition, about the code | symbol in FIG. 4, since it shows the same member as the thing of FIG. 1, description here is abbreviate | omitted.

The type of the cell non-adhesion region used in the present invention may be only one type, or may include a plurality of cell non-adhesion regions including different cell non-adhesion materials.

The width of the cell non-adhesion region used in the present invention from the non-adhesion boundary with the stimulus-responsive region is not particularly limited as long as cells can be adhered and cultured in the shape of the stimulus-responsive region. It is preferably 5 μm or more, and particularly preferably 10 μm or more. This is because, when the width is in the above-described range, it is possible to stably suppress cell growth on the cell non-adhesion region.

(2) Cell non-adhesion layer The cell non-adhesion layer used in the present invention contains a cell non-adhesion material.
As such a cell non-adhesive material, any material having non-adhesiveness with cells may be used. For example, a material having water repellency or oil repellency, or a material having super hydrophilicity can also be used. Specifically, ethylene glycol materials using diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, polyethylene glycol diacrylate, polyethylene glycol methacrylate, etc., long chain alkyl materials such as decylmethoxysilane, fluoroalkylsilane, etc. Fluorine-based materials, water-repellent materials such as silicon, hydrophilic materials such as polyvinyl alcohol (PVA) and polyacrylamide, phospholipid materials such as MPC polymer, and BSA protein.
In the present invention, among these, an ethylene glycol-based material can be preferably used. This is because the therapeutic cell fragment can be formed with high pattern accuracy by exhibiting excellent cell non-adhesiveness and cytotoxicity with cells.

The additive and binder resin that can be contained in the cell non-adhesive layer used in the present invention, the film thickness, and the formation method can be the same as those described in the above section “1. Stimulus responsive region”. .

3. Therapeutic cell fragment The therapeutic cell fragment formed on the stimulus-responsive region by the base material for recovering the therapeutic cell fragment of the present invention is one in which the cells are bound to each other, from the stimulus-responsive region. Even if it peels, the state which the said cells couple | bonded can be maintained.
In addition, as a cell constituting such a therapeutic cell fragment, any tissue existing in a living body and cells derived therefrom can be used as long as it is other than a non-adherent cell such as a blood cell line or a cell having a weak intercellular bond.
Specifically, epithelial cells and endothelial cells that constitute each tissue and organ in the living body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiomyocytes, neurons that constitute the nervous system, glial cells, fibroblasts, Periodontal ligament cells, endometrial derived cells, vascular cells, chondrocytes, bone cells, liver parenchymal cells related to metabolism in the living body, non-hepatic parenchymal cells and fat cells, cancer cells, various cells with differentiation potential Stem cells present in the tissue, as well as bone marrow cells, ES cells, iPS cells, stem cells, various progenitor cells, various types of cells induced to differentiate from ES cells or iPS cells, and the like can be used.
Among them, it is preferable to use human-derived mesenchymal stem cells. This is because human-derived mesenchymal stem cells are often used as standard model cells for regenerative medicine.

4). The base material for cell fragment collection | recovery for treatment The base material for cell fragment collection | recovery of this invention contains the said stimulation responsive area | region and a cell non-adhesion area | region at least.
In the present invention, as shown in FIG. 2 already described, the stimuli-responsive layer and the non-cell-adhering region are composed of the stimuli-responsive layer and the base material supporting the cell-non-adhering layer. It may be.

Such a substrate is not particularly limited as long as it can support the stimulus-responsive layer, the cell non-adhesive layer, and the like.
In the present invention, the material of the substrate is polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE). , Medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, urethane acrylate and other acrylic materials, cellulose, glass and the like. It may be a biodegradable polymer such as polylactic acid, polyglycolic acid, polycaprolactan, or a copolymer thereof. Further, the substrate may be porous.
In the present invention, among these, polyethylene terephthalate, polystyrene, and polycarbonate can be preferably used, and polyethylene terephthalate can be particularly preferably used. This is because it excels in properties such as transparency, dimensional stability, mechanical properties, electrical properties, and chemical resistance.

The film thickness of the substrate in the present invention is not particularly limited as long as it can stably support the stimulus-responsive layer and the like. For example, it is in the range of 10 μm to 1000 μm, preferably , In the range of 50 μm to 200 μm.

In addition, the shape of the base material in the present invention may be a plate shape or a concave shape such as a Petri dish or a multiwell plate.

In addition, as illustrated in FIG. 5, the stimulation-responsive layer or the cell non-adhesion layer is used as a base material for supporting the cell non-adhesion layer 2. It may be a substrate that supports the layer.

Furthermore, if necessary, other layers that do not have an exposed surface such as an adhesive layer that bonds the above-mentioned layers, and other layers that have an exposed surface that becomes another region may be included.

The method for producing the therapeutic cell fragment recovery base material of the present invention is not particularly limited as long as it can stably form the stimulus-responsive region and the like. A method of forming a stimulus-responsive layer and a cell non-adhesive layer in this order can be mentioned.

The therapeutic cell fragment recovery substrate of the present invention is used for the production of cell fragments for treatment such as transplantation, and in particular, the therapeutic cell fragments required to be controlled in size and shape. It can use suitably for manufacture of.

B. Substrates for cell fragment collection for passage Next, the substrate for cell fragment collection for passage of the present invention will be described.
The substrate for cell fragment collection for passage of the present invention has a stimulus responsive region in which cell adhesion changes by stimulation, and a cell non-adhesive region having cell non-adhesive properties, and the periphery of the stimulus responsive region Is surrounded by the cell non-adhering region.

Such a cell fragment collection substrate for passage will be described with reference to the drawings. The base material for cell fragment collection for passage of the present invention can be the same as that shown in FIGS.

The substrate for cell fragment collection for passage of the present invention includes at least the stimulation-responsive region and the cell non-adhesion region.
The stimulation-responsive region, cell non-adhesion region, obtained cell fragment for passage, other constitution, production method and the like are described in the above section “A. Base material for cell fragment collection for treatment”. Since it can be the same as that of the contents, description thereof is omitted here.

As a use of the cell fragment collection substrate for passage of the present invention, it is used for culturing and proliferating cells, and in particular, suitable for culturing and proliferating cells that require simplicity and stability, automatic culture apparatuses, etc. Used for.

C. Next, a method for producing a therapeutic cell fragment of the present invention will be described.
The method for producing a therapeutic cell fragment of the present invention comprises preparing the above-mentioned therapeutic cell fragment collection substrate, and in the state where the stimulus-responsive region expresses cell adhesion, on the stimulus-responsive region. Cell fragment formation step of seeding and culturing cells to form a therapeutic cell fragment consisting of at least one cell adhered to the stimulus-responsive region, and stimulating the stimulus-responsive region to express cell non-adhesiveness And a recovery step of recovering the therapeutic cell fragment peeled from the stimulus-responsive region.

Such a method for producing a therapeutic cell fragment of the present invention will be described with reference to the drawings. FIG. 6 is a process diagram showing an example of a method for producing a therapeutic cell fragment of the present invention. As illustrated in FIG. 6, in the method for producing a therapeutic cell fragment of the present invention, the therapeutic cell fragment collection substrate 10 is prepared, and the stimulus responsive region which is the exposed surface of the stimulus responsive layer 1 is prepared. In a state in which cells are expressing cell adhesion, cells are seeded and cultured on the stimulus-responsive region (FIG. 6 (a)), and used for treatment comprising at least one cell adhered to the stimulus-responsive region The therapeutic cell which formed the cell fragment 21 (FIG. 6 (b)), stimulates the stimulus-responsive region to express cell non-adhesiveness (FIG. 6 (c)), and is detached from the stimulus-responsive region. The fragment 21 is recovered (FIG. 6 (d)).
In this example, FIGS. 6A and 6B are cell fragment forming steps, FIG. 6C is a stimulus applying step, and FIG. 6D is a collecting step.

According to the present invention, a therapeutic cell fragment having a controlled size and shape can be easily obtained by using the therapeutic cell fragment collection substrate. Accordingly, it is possible to exert effects such as treatment with good reproducibility.

The method for producing a therapeutic cell fragment of the present invention comprises at least a cell fragment formation step, a stimulus application step, and a recovery step.
Hereinafter, each step of the method for producing a therapeutic cell fragment of the present invention will be described in detail.

1. Cell Fragment Formation Step The cell fragment formation step in the present invention comprises preparing the above-mentioned therapeutic cell fragment collection base material, and in the state where the stimulus responsive region expresses cell adhesiveness, on the stimulus responsive region. Cells are seeded and cultured to form a therapeutic cell fragment comprising at least one cell adhered to the stimulus-responsive region.

In this step, the method for expressing cell adhesion in the stimulus responsive region is different depending on the type of the stimulus responsive material constituting the stimulus responsive region, and the above-mentioned cell adhesion can be expressed. It is not particularly limited as long as it can be performed.
Specifically, when the stimulus-responsive material is PIPAAm, there is a method of setting the stimulus-responsive region to a temperature condition higher than 32 ° C.

The method for seeding the cells in this step is not particularly limited as long as the cells are uniformly seeded on the stimulus-responsive region, and a general seeding method can be used. For example, the method of seed | inoculating the said cell in the state which immersed the said base material for cell fragment collection | recovery for treatment in the culture medium can be mentioned.

The method for culturing the cells in this step is not particularly limited as long as it is a method capable of forming a therapeutic cell fragment composed of one or more cells adhered to the stimulus-responsive region. Any culture method can be used.

The therapeutic cell fragment formed in this step can be the same as that described in the above section “A. Base material for recovery of therapeutic cell fragment”.

2. Stimulus imparting step The stimulus imparting step in the present invention is a step in which the stimulus responsive region is stimulated to express cell non-adhesiveness.

In this step, the stimulation-responsive region is stimulated to express cell non-adhesiveness by expressing cell non-adhesiveness in the stimulus-responsive region, and the therapeutic cell fragment is removed from the stimulus-responsive region. Any method can be used as long as it can be peeled off, and it depends on the type of the stimulus-responsive region.
Specifically, when the stimulus-responsive material is PIPAAm, the therapeutic cell fragment collection substrate to which the therapeutic cell fragment is attached is left in an incubator set at a temperature of 32 ° C. or lower. Or the like can be used.

In this step, after applying a stimulus to the stimulus responsive region, a process of applying external stress such as pipetting to the stimulus responsive region may be performed. This is because the therapeutic cell fragment can be more stably detached.

3. Collection step The collection step in the present invention is a step of collecting the therapeutic cell fragment peeled from the stimulus-responsive region.

In this step, the method for recovering the therapeutic cell fragment may be any method that can separate the therapeutic cell fragment recovery base material and the therapeutic cell fragment. Examples thereof include a method of adhering the therapeutic cell fragment peeled from the stimulus-responsive region to a recovery base material to which the therapeutic cell fragment can adhere, a method of sucking the therapeutic cell fragment together with a medium, and the like.
In the present invention, the method of aspirating the therapeutic cell fragment together with the medium is preferable. This is because it can be recovered as a suspension suspended in a medium, and thus can be easily used for treatment such as transplantation.

4). Others The method for producing a therapeutic cell fragment of the present invention includes at least a cell fragment formation step, a stimulus applying step, and a recovery step.

D. Next, a method for producing a cell fragment for passage will be described.
In the method for producing a cell fragment for passage of the present invention, the substrate for cell fragment collection for passage is prepared, and the stimulus-responsive region is expressing cell adhesiveness on the stimulus-responsive region. Cell fragment formation step of seeding and culturing cells to form a cell fragment for passage consisting of at least one cell adhered to the stimulus-responsive region, and stimulating the stimulus-responsive region to express cell non-adhesiveness And a recovery step of recovering the passage cell fragment peeled from the stimulus-responsive region.

For example, the method for producing a cell fragment for passage of the present invention can be the same as that described in FIG.

According to the present invention, it is possible to easily obtain a cell fragment for passage having a controlled size and shape by using the substrate for cell fragment collection for passage.

The method for producing a cell fragment for passage of the present invention comprises at least a cell fragment forming step, a stimulus applying step, and a recovery step.
In addition, each step included in the method for producing a cell fragment for passage of the present invention is the same as the contents described in the above-mentioned section “C. Method for producing a cell fragment for treatment”, and thus description thereof is omitted here. To do.

Note that the present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

Hereinafter, the present invention will be specifically described with reference to examples and comparative examples.

[Example 1]
(1) Production of temperature-responsive film N-isopropylacrylamide was dissolved in isopropyl alcohol (IPA) to a final concentration of 20% by weight. A commercially available easy-adhesive polyethylene terephthalate film (obtained from Sanko Sangyo Co., Ltd., transparent 50-F Sepa 1090) (hereinafter sometimes abbreviated as “easily-adhesive PET”) was procured and cut into 10 cm squares. The solution was spread on the easy-adhesion surface of easy-adhesion PET and coated with a Miya bar. The sample was irradiated with an electron beam using an electron beam irradiation apparatus (manufactured by Iwasaki Electric Co., Ltd.), and the solution was graft polymerized. The electron beam irradiation dose at this time was 300 kGy.

(2) Formation of Cell Non-Adhesion Layer Methacryloxysilane (obtained from Momentive Performance Materials, TSL8370) was prepared and diluted with isopropyl alcohol (IPA) to 0.1% by weight.
The temperature-responsive film obtained above was fixed to glass with a tape, coated with methacryloxysilane using a spinner (manufactured by Mikasa), and subjected to silane treatment.

Next, polyethylene glycol diacrylate (molecular weight 300, obtained from Aldrich) is diluted with pure water so that the final concentration is 50% by weight, and 2-hydroxy-4 ′-(2- Hydroxyethoxy) -2-methylpropiophenone (obtained from Aldrich) was added to a final concentration of 1% by weight. This solution was stirred with a stirrer for 15 minutes.
1 mL of this was developed on a silane-treated temperature-responsive film, and spin coating was performed under the conditions shown in the following table. Then, it processed with the energy of 150 mJ with the exposure apparatus using the photomask. As the photomask design, a stimulus responsive region (temperature responsive region) designed so that patches of 200 μm square were arranged at intervals of 200 μm was used.
Thereafter, it was visually confirmed that polyerylene glycol diacrylate was immobilized on the silane-treated substrate as a cell non-adhesive layer.

(3) Production of substrate for cell fragment recovery The culture support tool thus obtained was cut into 20 mmφ, the adhesive layer was exposed, and affixed to the bottom of a 35 mmφ polystyrene dish (Becton Dickinson). It was set as the base material for collection | recovery.

(4) Formation of cell fragments The cell fragment collection substrate obtained in the examples was sterilized with 70% ethanol. The sterilization time was 1 hour.
Thereafter, human-derived mesenchymal stem cells (hMSC) were seeded at 4 × 10 ⁵ cells / cm ² and cultured for 7 days. Seven days later, when observed with a phase contrast microscope, it was confirmed that cell fragments composed of a plurality of cells of 200 μm square were obtained at intervals of 200 μm. In addition, when a phase contrast image was obtained at a magnification of 10 times the eyepiece and 4 times the objective so that 16 200 μm square cell fragments were contained in one visual field, and the number of cells contained in each fragment was measured, an average of 7. There were 5 (standard deviation 0.9).

(5) Cell Fragment Recovery Thereafter, the above-mentioned substrate for cell fragment recovery is transferred from a 37 ° incubator to a low temperature incubator at 25 ° C., left to stand for 25 minutes, and then pipetting is performed about 10 times, thereby causing the above-mentioned stimulus responsiveness. The area (temperature responsive area) was stimulated and the cell fragment was recovered. As a result of measuring the obtained cell fragment using a phase-contrast microscope, it was confirmed that the fragment size was 30 μmφ, and cell fragments having almost the same size and shape could be recovered.

[Comparative Example 1]
(1) Production of temperature-responsive film N-isopropylacrylamide was dissolved in isopropyl alcohol (IPA) to a final concentration of 20% by weight. A commercially available easy-adhesive polyethylene terephthalate film (obtained from Sanko Sangyo Co., Ltd., transparent 50-F Sepa 1090) (hereinafter sometimes abbreviated as “easily-adhesive PET”) was procured and cut into 10 cm squares. The solution was spread on the easy-adhesion surface of easy-adhesion PET and coated with a Miya bar. The sample was irradiated with an electron beam using an electron beam irradiation apparatus (manufactured by Iwasaki Electric Co., Ltd.), and the solution was graft polymerized. The electron beam irradiation dose at this time was 300 kGy.

(2) Production of Cell Fragment Recovery Base Material The culture support tool thus obtained was cut into 20 mmφ, the adhesive layer was exposed, and affixed to the bottom of a 35 mmφ polystyrene dish (Becton Dickinson). It was set as the base material for collection | recovery.

(3) Formation of cell sheet The substrate for cell fragment recovery obtained in the examples was sterilized with 70% ethanol. The sterilization time was 1 hour.
Thereafter, human-derived mesenchymal stem cells (hMSC) were seeded at 4 × 10 ⁵ cells / cm ² and cultured for 7 days. Seven days later, when observed with a phase-contrast microscope, it was confirmed that hMSC was obtained in sheet form.

(4) Cell fragment recovery In a clean bench, the culture solution is aspirated and a sterilized wire mesh is pressed onto the hMSC sheet. After that, the culture solution is added and left at room temperature for 25 minutes, and then pipetting is performed 10 times. Thus, stimulation was applied to the stimulation-responsive region (temperature-responsive region), and the cell fragment was recovered. As a result of measuring the obtained cell fragment using a phase contrast microscope, the size of the fragment was 15 μm to 150 μmφ, and the size and shape of the cell fragment were uneven.

[Comparative Example 2]
(1) Production of temperature-responsive film N-isopropylacrylamide was dissolved in isopropyl alcohol (IPA) to a final concentration of 20% by weight. A commercially available easy-adhesive polyethylene terephthalate film (obtained from Sanko Sangyo Co., Ltd., transparent 50-F Sepa 1090) (hereinafter sometimes abbreviated as “easily-adhesive PET”) was procured and cut into 10 cm squares. The solution was spread on the easy-adhesion surface of easy-adhesion PET and coated with a Miya bar. The sample was irradiated with an electron beam using an electron beam irradiation apparatus (manufactured by Iwasaki Electric Co., Ltd.), and the solution was graft polymerized. The electron beam irradiation dose at this time was 300 kGy.

(2) Formation of Cell Non-Adhesion Layer Methacryloxysilane (obtained from Momentive Performance Materials, TSL8370) was prepared and diluted with isopropyl alcohol (IPA) to 0.1% by weight.
The temperature-responsive film obtained above was fixed to glass with a tape, coated with methacryloxysilane using a spinner (manufactured by Mikasa), and subjected to silane treatment.
Next, polyethylene glycol diacrylate (molecular weight 300, obtained from Aldrich) is diluted with pure water so that the final concentration is 50% by weight, and 2-hydroxy-4 ′-(2- Hydroxyethoxy) -2-methylpropiophenone (obtained from Aldrich) was added to a final concentration of 1% by weight. This solution was stirred with a stirrer for 15 minutes.
1 mL of this was developed on a silane-treated temperature-responsive film, and spin coating was performed under the conditions shown in the following table. Then, it processed with the energy of 150 mJ with the exposure apparatus using the photomask. As the photomask design, a stimulus responsive region (temperature responsive region) designed so that patches of 200 μm square were arranged at intervals of 200 μm was used.
Thereafter, it was visually confirmed that polyerylene glycol diacrylate was immobilized on the silane-treated substrate as a cell non-adhesive layer.

(4) Formation of cell sheet The cell fragment collection substrate obtained in the examples was sterilized with 70% ethanol. The sterilization time was 1 hour.
Thereafter, human-derived mesenchymal stem cells (hMSC) were seeded at 4 × 10 ⁵ cells / cm ² and cultured for 7 days. Seven days later, when observed with a phase contrast microscope, it was confirmed that cell fragments composed of a plurality of cells of 200 μm square were obtained at intervals of 200 μm. In addition, when a phase contrast image was obtained at a magnification of 10 times the eyepiece and 4 times the objective so that 16 200 μm square cell fragments were contained in one visual field, and the number of cells contained in each fragment was measured, an average of 7. There were 5 (standard deviation 0.9).

(5) Recovery of cell fragments In a clean bench, the culture solution was aspirated, and a 0.25% trypsin-EDTA solution was added and treated at room temperature for 5 minutes. Subsequent observation using a phase-contrast microscope revealed that hMSC was broken in cell-cell adhesion and suspended in a trypsin solution as a single cell, and cell fragments of uniform size and shape could not be obtained. It was.

DESCRIPTION OF SYMBOLS 1 ... Stimulus response layer 2 ... Cell non-adhesion layer 3 ... Base material 10 ... Cell fragment collection base material 11 ... Stimulus response region 12 ... Cell non-adhesion region 21 ... Cell fragment

Claims

A stimulus-responsive region in which cell adhesion changes by stimulation;
A cell non-adhesion region having cell non-adhesion;
Have
The therapeutic cell fragment collection substrate, wherein the stimulus-responsive region is surrounded by the cell non-adhesion region.
2. The therapeutic cell fragment collection substrate according to claim 1, wherein the stimulus-responsive material is a temperature-responsive material that exhibits cell detachability by temperature change.
3. The therapeutic cell fragment collection substrate according to claim 2, wherein the temperature-responsive material is poly-N-isopropylacrylamide (PIPAAm).
A stimulus-responsive region in which cell adhesion changes by stimulation;
A cell non-adhesion region having cell non-adhesion;
Have
The substrate for cell fragment collection for passage, wherein the stimulus-responsive region is surrounded by the cell non-adhesion region.
5. The cell fragment collection substrate for passage according to claim 4, wherein the stimulus-responsive material is a temperature-responsive material that exhibits cell detachability by temperature change.
The base material for recovering cell fragments for passage according to claim 5, wherein the temperature-responsive material is poly-N-isopropylacrylamide (PIPAAm).
Preparing a substrate for cell fragment collection for treatment according to any one of claims 1 to 3,
A therapeutic cell fragment comprising at least one or more cells that are seeded and cultured on the stimulus responsive region in a state where the stimulus responsive region expresses cell adhesion, and adheres to the stimulus responsive region. A cell fragment forming step to form
Providing a stimulus to the stimulus-responsive region to develop cell non-adhesiveness; and
A recovery step of recovering the therapeutic cell fragment detached from the stimulus-responsive region;
A method for producing a therapeutic cell fragment, comprising:
Preparing a cell fragment collection substrate for passage according to any one of claims 4 to 6,
A cell fragment for subculture consisting of at least one or more cells adhering to the stimulus-responsive region after seeding and culturing cells on the stimulus-responsive region in a state where the stimulus-responsive region expresses cell adhesion A cell fragment forming step to form
Providing a stimulus to the stimulus-responsive region to develop cell non-adhesiveness; and
A recovery step of recovering the passage cell fragment detached from the stimulus-responsive region;
A method for producing a cell fragment for passage, comprising: