JP2016067330A - Production method for frozen cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method for frozen cells which freezes cells while adhering the cells on a cell-culturing substrate, and which has a high cell survival rate after freezing and defrosting.SOLUTION: The invention provides a production method for frozen cells comprising freezing cells adhering on a wall surface of a microchip in a condition where the cells are adhered on a wall surface of the microchip.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing frozen cells using a microchip.

  Cells that are not used in experiments are often stored frozen. At that time, if the cells are frozen and thawed while adhered to a substrate such as a conventional culture dish or flask, the cells may be damaged and killed even if immersed in an appropriate storage solution. Therefore, before cryopreservation, cells are generally detached from the substrate using trypsin or the like, suspended in a preservation solution, transferred to a tube and frozen. However, the frozen cells can be used for experiments only after thawing, seeding and adhering to a substrate such as a culture dish, and further culturing them to an appropriate cell density. This defrosting process usually takes several days.

  Non-Patent Document 1 reports a method of wrapping cells on a gel as a technique for freezing and thawing with cells adhered to a substrate. Patent Document 1 discloses an animal cell cryopreservation carrier provided with a sheet in which a plurality of openings having substantially the same shape are formed at a constant pitch, and at least a part of an inner wall forming the opening. Describes a carrier for cryopreservation of animal cells having a curved surface portion and further satisfying a predetermined condition of the survival rate of animal cells or the survival cell density of animal cells.

  Studies have also been reported in which cells are cryopreserved with a microfluidic chip (Non-patent Documents 2 and 3). These techniques are for freezing suspended cells, not for cryopreserving adherent cells.

R. Malpique, F. Ehrhart, A. Katsen-Globa, H. Zimmermann, P. M. Alves, Tissue Engineering: Part C, 15, 373-386, (2009) E. Berthier et al., Lab Chip, 13, 424-431 (2013) Y. S. Song, et al., Lab Chip, 9, 1874-1881 (2009)

Japanese Patent No. 5140155

  The techniques described in Non-Patent Document 1 and Patent Document 1 have not been widely spread. Although the problems of these techniques are unknown, it is thought that the reason is that the culture environment is too different from the conventional one and there is anxiety about data compatibility and that it is difficult to observe three-dimensionally under a microscope.

  An object of the present invention is to provide a method for producing a frozen cell in which cells are frozen while adhered to a culture substrate, and has a high cell survival rate after freezing and thawing.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention greatly improve the cell viability when the cells adhered to the bottom surface of the microchip are frozen and thawed as compared with the case without the microchip. I found out. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A method for producing frozen cells, comprising a step of freezing cells adhered on one wall surface of the microchip in a state where the cells are adhered on one wall surface of the microchip.
(2) The method for producing frozen cells according to (1), wherein a microspace in which the microchip is sealed is filled with cells and a cell culture medium, and the cells are adhered to one wall surface of the microchip.
(3) The method for producing frozen cells according to (1) or (2), wherein the height of the microspace of the microchip is 10 to 100 μm.
(4) The method according to any one of (1) to (3), wherein a micropocket for capturing a single cell is provided on a wall surface to which the cells of the microchip adhere.
(5) The step of freezing the cells adhered on one wall surface of the microchip with the cells adhered on the one wall surface of the microchip is 30% to 70% confluent on the one wall surface of the microchip. The method according to any one of (1) to (4), wherein the medium is replaced with a medium containing a cryoprotective agent, and then the cells are frozen after culturing until.
(6) A frozen cell comprising a microchip obtained by the method according to any one of (1) to (5) and a frozen cell adhered on one wall surface of the microchip.
(7) (i) a step of producing frozen cells by the method according to any one of (1) to (5); and (ii) a step of thawing the frozen cells:
A method for freezing and thawing cells.
(8) (i) a step of producing frozen cells by the method according to any one of (1) to (5);
(Ii) thawing the frozen cells;
(Iii) contacting the thawed cells with a test substance; and (iv) detecting the action of the test substance on the thawed cells:
A test method for a test substance, comprising:

  According to the present invention, cells can be frozen and thawed while adhered to a substrate, and a high cell survival rate can be achieved after freezing and thawing. In particular, according to the present invention, it is possible to cryopreserve cells while the cells are adhered to the culture surface using the surface of a cell substrate made of plastic that has been widely used as a culture surface.

FIG. 1 shows the design of the microchip. A is a photograph of the entire microchip, B is a photograph in the vertical direction, C is an outline in the horizontal direction, D is an enlarged view of the X portion, and E is an enlarged view of the micro pocket. FIG. 2 shows a schematic diagram of a chrome mask design. A indicates the first layer and B indicates the second layer. FIG. 3 shows a microdevice fabrication chart. FIG. 4 is a graph showing cell adhesion rate and survival rate. The vertical axis represents the percentage (%) of the adhesion rate (black bar) and the survival rate (gray bar), and the horizontal axis represents the microchip (Chip) or the culture dish (Dish), the cell type (HeLa, NIH3T3, MCF- 7, PC12), indicates the presence or absence of collagen coating. The lower figure shows the experimental results using HeLa cells and a microchip. When micropockets are present (channel depth is 18 μm) and when there are no micropockets (channel depths are 25 μm and 60 μm). ) Result. FIG. 5 shows immunostaining of PC12 cells. The left of FIG. 5 shows the case where neuronal differentiation of PC12 cells was induced by adding NGF after cryopreservation and thawing on a microchip, and the center of FIG. 5 shows a negative control in which NGF treatment is not performed after cryopreservation. The right side of FIG. 5 shows a positive control in which cells not cryopreserved were treated with NGF on a culture dish. FIG. 6 shows the results of the cytotoxicity assay. The upper part of FIG. 6 shows the result when a 96-well plate is used, the middle part of FIG. 6 shows the result when the microchip is not frozen, and the lower part of FIG. 6 shows the result after the freeze-thaw using the microchip. The vertical axis shows the percentage (%) of the survival rate, and the horizontal axis shows the cisplatin concentration (μM).

Hereinafter, the present invention will be described more specifically.
The method for producing frozen cells according to the present invention includes a step of freezing cells adhered on one wall surface of the microchip in a state where the cells are adhered on one wall surface of the microchip. In the present invention, the operation of thawing the cryopreserved cells, seeding them on a substrate such as a culture dish and adhering them, and further culturing them to an appropriate cell density is unnecessary. About 30 minutes after thawing the adhered cells, the intended experiment using the cells can be started.

  In the microchip used in the present invention, a minute space (preferably a flow path) is provided inside. Preferably, the bottom surface, the side surface, and the top surface of the minute space are surrounded by the wall surface of the container. That is, the minute space is preferably a sealed minute space except that it has an inlet and an outlet. In addition, the number of microspaces should just be one or more, and one or two or more microspaces can be installed.

  In the present invention, the cells adhere to the one wall surface by culturing the cells on one wall surface of the bottom surface, the side surface, and the top surface of the minute space. Hereinafter, the wall surface to which the cells adhere is also referred to as the culture surface. If necessary, a coating (for example, collagen) may be applied to the surface to which the cells adhere. In the present invention, the cells are mainly intended to adhere on at least one of the bottom surface, the side surface, and the top surface, but the cells may adhere on the other wall surfaces. Particularly preferably, the cells can be cultured and adhered on the bottom surface of the minute space.

  The height of the micro space when the culture surface of the microchip is the bottom surface (that is, the distance between the bottom surface and the top surface opposite to the bottom surface; if the microchip is a flow channel, the height of the flow channel) is Although not particularly limited as long as the effect of the present invention can be achieved, it is generally 10 μm to less than 1 mm, preferably 10 to 100 μm, more preferably 10 to 60 μm, and further preferably 15 to 60 μm. More preferably, it is 15-30 micrometers, Most preferably, it is 18-25 micrometers. If the height of the micro space is less than 10 μm, it is not preferable as a space where cells exist, and if the height of the micro space is 1 mm or more, it is not preferable because it is difficult to achieve the effect of the present invention to improve the cell viability. .

When the minute space is a rectangular parallelepiped composed of a bottom surface, a side surface, and an upper surface, the size of the bottom surface is not particularly limited, but generally the lateral width is about 100 μm to 2 mm, preferably about 200 μm to 1 mm ( The vertical width is about 500 μm to 50 mm, preferably about 1 mm to 5 mm (3 mm in FIG. 1).
Alternatively, the size of the microspace is preferably large enough for cells to adhere and small enough to increase the survival rate of the cells after freeze-thawing. The cell viability after freeze-thawing is, for example, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The cell viability after freeze-thawing is, for example, 1.5 times, 2 times, 4 times, 6 times compared to the cell viability after freeze-thawing on a normal culture dish having no microspace. 8 times, or 10 times higher.

  Preferably, the microchip is provided with a micro pocket for capturing a single cell (one cell) on a culture surface (wall surface to which the cell adheres). The structure of the micropocket is not particularly limited, but preferably has a structure and size suitable for capturing one cell when the cell is flowed on the culture surface, and is used in the examples of the present specification. A micro pocket structure (see D and E in FIG. 1) provided on the microchip is preferable. Specifically, a structure having a width of 5 to 25 μm (10 μm in FIG. 1E), a vertical width of 20 to 40 μm (30 μm in FIG. 1E), and a height of 3 to 10 μm (5 μm in FIG. 1E), This is a structure in which a semi-cylinder having a diameter of 10 to 20 μm (15 μm in FIG. 1E) is removed from the side surface.

  The raw material of the microchip is not particularly limited as long as it has cold resistance (−80 ° C. or lower), and for example, a polymer material, glass, or silicon can be used. For example, rubber, which is one of the polymer materials, has a very small solid microstructure and a considerably large degree of freedom of movement of solid molecules, so that gas can easily enter into the solid, and there are many It is a solid material in which gas dissolves. Thus, it is preferable to form the microchip with a polymer material such as rubber, which is a solid material in which so many gases can be dissolved. As a specific example of rubber, silicone rubber such as polydimethylsiloxane (PDMS) can be used.

A method for manufacturing a microchip used in the present invention will be described.
In order to form a reversal pattern for forming a microchannel with a predetermined height, an ultra-thick film photoresist (SU-8; manufactured by Microchem, USA) is spin-coated on a silicon substrate, and the manufacturer Process according to instructions. Thereafter, the reverse pattern is exposed and developed.

  Further, in the case of providing a micro pocket, a photoresist is spin-coated on the structure manufactured as described above and processed according to the manufacturer's instructions. Thereafter, the reverse pattern is exposed and developed. Thus, a photoresist structure having two kinds of heights can be obtained.

After development, in order to strengthen the adhesion, it is baked in an oven for 4 minutes at 150 ° C. and then slowly cooled to room temperature over 1-2 hours. In order to improve mold separation, the silicon substrate was subjected to a fluorocarbon layer polymerized by CHF ₃ plasma for 2 minutes in a reactive ion etching (RIE) machine (RIE-10NR; manufactured by Samco International Laboratories, Japan). Form a film. The conditions at that time can be, for example, a CHF ₃ gas flow rate of 50 sccm, a pressure of 20 Pa, and a power of 200 W.

  Further, an unpolymerized liquid of PDMS (eg, Sylgard 184; manufactured by Dow Corning, USA) is poured onto the silicon substrate using a mold that holds the solution. On the other hand, the 1st hardening process for 1 hour at 65 degreeC and the 2nd hardening process for 1 hour at 100 degreeC are performed. The cured PDMS chip is peeled from the silicon substrate. When the PDMS chip thus manufactured is placed on the bottom of the culture dish, the two adhere to each other due to the adhesiveness of the PDMS surface. Thereby, the microchip used by this invention can be produced.

  In the present invention, the cells are frozen together with the cell culture medium in a state of being adhered to the culture surface of the microchip. Preferably, a sealed microspace of the microchip is filled with cells and a cell culture medium, and the cells adhere to one wall surface (culture surface) of the microchip. The fact that the micro space is filled with cells and cell culture medium means that 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 100% of the micro space is filled with cells and cell culture medium. It means the state that has been.

  In the present invention, the cells adhered on one wall surface of the microchip are frozen in a state where the cells are adhered on the one wall surface of the microchip. The method for freezing the cells is not particularly limited, but preferably the cells are 30-70% confluent (preferably 40-60% confluent, more preferably 45-55% confluent, eg 50% confluent) on one wall of the microchip. ), The medium can be replaced with a medium containing a cryoprotectant and the cells can then be frozen.

  The type of cell used in the present invention is not particularly limited and may be any of microorganisms, bacteria, animal cells, and plant cells, preferably animal cells, such as mammals (mouse, rat, guinea pig, hamster, rabbit, Vertebrates such as cats, dogs, sheep, pigs, cows, horses, goats, monkeys, humans), birds (chicken, ostrich, etc.), reptiles, amphibians, fish (zebrafish, medaka, etc.), insects (silkworm, moth, etc.) And non-vertebrate cells such as Drosophila). The cell is more preferably an animal cell, and particularly preferably a mammalian cell. When using animal cells, for example, cell lines of animal cells may be used, or cells derived from organs, tissues, or body fluids (including blood, lymph, and semen) collected from animals such as mammals are used. Alternatively, transformed cells obtained by genetic engineering techniques may be used. The cells may be somatic cells, germ cells, or stem cells.

  As the medium used for culturing the cells until the cells become 30 to 70% confluent, an appropriate cell culture medium can be selected and used according to the type of the cells to be used. When the cell is an animal cell, an animal cell culture medium, culture serum and / or serum substitute can be preferably used. Preferably, it is a medium for animal cell culture containing 5 to 20% of serum or serum substitute. Animal cell culture media include Eagle's minimal essential medium (EMEM), Dulbecco's modified Eagle medium (DMEM), α-Minimum Essential Medium (α-MEM), RPMI 1640 medium (GIBCO), Nutrient Mixture F-12 (F-12). Etc.) can be preferably used. Examples of the serum for culture include fetal bovine serum (FBS), fetal bovine serum (FCS), horse serum and the like. Serum substitutes include human autologous serum, human allogeneic serum, human platelet derived products, albumin / albumin substitute, insulin / insulin substitute, and transferrin alone or in combination, or knockout serum substitute (KnockOut Serum). (Replacement; KSR, manufactured by Invitrogen Life Technologies) and the like can be preferably used.

  In the present invention, it is preferable to replace the medium with a cryoprotectant-containing medium before freezing the cells. A cryoprotectant is a substance that has a high affinity for water molecules and has a high effect of suppressing the growth of ice crystals when dissolved in a medium. Examples thereof include dimethyl sulfoxide (DMSO), glycerin, trehalose, sucrose. Examples thereof include sugar, ethylene glycol, polyethylene glycol, propylene glycol, polyvinyl pyrrolidone, and carboxylated polylysine. The concentration of the cryoprotectant in the medium is preferably 5% by mass to 20% by mass, and more preferably 7% by mass to 15% by mass.

  The cell freezing method can be performed by a conventional method. That is, a microchip having cells adhered to the wall surface is placed in an ultra-low temperature freezer at -40 ° C. or lower (eg, −80 ° C., etc.), rapidly cooled with liquid nitrogen, or gradually placed in a program freezer. The method of freezing is mentioned. Examples of the method for preserving the frozen cells include keeping the cells at a frozen temperature as they are, or holding them in liquid nitrogen when they are frozen in a program freezer.

  A cell frozen product comprising a microchip obtained by the method of the present invention described above and a frozen cell adhered on one wall surface of the microchip is also within the scope of the present invention. In the cell frozen product of the present invention, preferably, the sealed microspace of the microchip is filled with cells and a cell culture medium, and the cells are in a state of being adhered to one wall surface of the microchip. .

  Furthermore, according to the present invention, there is provided a method for freezing and thawing cells, comprising the steps of producing frozen cells by the method of the present invention; and thawing the frozen cells.

  As a method for thawing cells, for example, thawing can be performed by leaving a cryopreserved microchip at room temperature.

  Furthermore, according to the present invention, a frozen cell is produced by the method of the present invention, and after the frozen cell is thawed, the test substance is detected by detecting the effect of the test substance brought into contact with the obtained thawed cell. It can be tested. The test may be any biological test, for example an assessment of efficacy and / or toxicity. That is, the cells obtained by thawing after freezing the cells according to the method of the present invention are adhered to one wall surface of the microchip. Can be subjected to physical testing. In addition, the cells obtained by thawing after freezing the cells by the method of the present invention have the same phenotype as the cells before freezing. Can be used for Therefore, according to the present invention, a ready-to-use assay microchip is provided, and a test substance can be tested using, for example, human cells.

  As a test substance that can be used in the present invention, any substance can be used. The kind of the test substance is not particularly limited, and may be an individual low molecular weight compound, a compound present in a natural product extract, or a synthetic peptide. Alternatively, the test compound can also be a compound library, a phage display library or a combinatorial library. The test substance is preferably a low molecular compound, and may be a compound library of low molecular compounds. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.

  The present invention is illustrated by the following examples, but the present invention is not limited to the examples.

Example 1: Production of microchip (with pocket and depth of 25 microns) Except for the pocket structure, Kondo et al. J. Biosci. Bioeng., Vol.118, No.3, 356-358, 2014 A microchip having the structure shown in FIG. 1 was fabricated by the following procedure in the same manner as described. See FIG. 1E for the structure of the pocket. A through hole of 5 μm × 5 μm was provided.

  The microchip does not require a perfusion device, and perfusion is achieved by gravity flow due to a head differential of several μm between the inlet and outlet (FIG. 1B). This microchip has two regions, a filter zone and a cell culture zone (FIG. 1C). The filter zone has an array of 20 μm slits to exclude large cell aggregates. Individual cells are introduced into a 500 μm wide cell culture zone with an array of micropockets to capture the cells (FIG. 1D).

(1) Fabrication of mold A photoresist structure was fabricated on a silicon substrate using two photomasks (FIG. 2).
(1-1)
Photoresist SU-85 (MicroChem, USA) was spin-coated on a silicon substrate at 3,000 rpm for 15 seconds, and then heated in an oven at 90 ° C. for 30 minutes.
(1-2)
Exposure was performed using the first mask (FIG. 3A). The exposure apparatus was PEM-800 (Union Optics) with an exposure time of 15 seconds.
(1-3)
Heated in an oven at 90 ° C. for 30 minutes.
(1-4)
Developed with SU-8 developer (MicroChem) for 5 minutes.
(1-5)
SU-825 (MicroChem) was spin-coated at 3000 rpm for 15 seconds from above the 5 micron thick structure prepared above, and then heated in an oven at 90 ° C. for 30 minutes.
(1-6)
Exposure and development (FIG. 3B, conditions are the same as (1-2) to (1-4) above) were performed using the second mask. As described above, photoresist structures having two kinds of heights of 5 microns and 25 microns were produced.

(1-7)
To strengthen the bond, it was baked at 150 ° C. for 4 minutes in an oven and then slowly cooled to room temperature over 1-2 hours. In order to improve the mold release, the silicon substrate was subjected to 2 fluorocarbon layers polymerized by CHF3 plasma in a reactive ion etching (RIE) machine (RIE-10NR: manufactured by Samco International Laboratory, Japan). The film was formed for a minute. The conditions at that time are a CHF3 gas flow rate of 50 sccm, a pressure of 20 Pa, and an electric power of 200 w.

(2) Production of PDMS (polydimethylsiloxane) chip The main component of PDMS (Sylgard 184, Dow Corning, USA) and the polymerization initiator were mixed at a weight ratio of 10: 1 and poured onto a mold. After defoaming in vacuum for about 10 minutes, the first stage curing was performed by heating in an oven at 65 ° C. for 1 hour (FIG. 3C). The PDMS chip was peeled off from the mold, unnecessary portions around it were cut off with a knife, and a through hole having a diameter of 2.5 mm was opened at the entrance using a metal pipe. A piece of silicone tube (inner diameter 5 mm, length 7 mm) serving as a reservoir was bonded to the top using uncured PDMS. The PDMS chip was heated in an oven at 100 ° C. overnight to perform the second stage curing.

(3) The PDMS chip was placed on the bottom of the culture dish, and both were adhered by the adhesiveness of the PDMS surface (FIG. 3E).

Example 2: Variation of microchip Variation 1: With pocket and depth of 18 microns A microchip was fabricated in the same manner as in Example 1 except that the rotational speed of (1-5) was 3500 rpm.

Variation 2: No pocket, depth of 25 microns A microchip was fabricated in the same manner as in Example 1 except that one-step exposure using a mask without a pocket started from (1-5) above.

Variation 3: No pockets, depth 60 microns Starting with (1-5) above in one-step exposure using a mask without pockets, and the spin coating speed of (1-5) above is 1000 rpm A microchip was manufactured in the same manner as in Example 1 except that.

Example 3 Cell Preparation Ng3T3 cells (Abe, T., et.al. Genesis, 49, 579e590 (2011)) obtained from NIH3T3 cells by stable transfection with a nuclear targeted EGFP gene were transferred to 4500 mg / The cells were cultured in Dulbecco's modified Eagle medium (Gibco) containing L glucose (Wako Pure Chemical Industries) and supplemented with 10% fetal bovine serum. HeLa and MCF-7 were cultured in Eagle's minimum essential medium (Wako Pure Chemical Industries) supplemented with 10% fetal calf serum, non-essential amino acids, 100 mg / mL penicillin and 0.1 mg / mL streptomycin (Wako Pure Chemical Industries). PC12 cells were cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% horse serum, 5% fetal calf serum, 100 mg / mL penicillin and 0.1 mg / mL streptomycin.

Example 4: Cryopreservation HeLa, PC12, NIH3T3 and MCF-7 cells were added to each microchip and cultured. At this time, the cells were allowed to adhere to the surface of the culture dish, which is the bottom of the micro space in the microchip, and cultured. At the time of 50% confluence, the medium was changed to a cryoprotectant-containing medium (medium containing 10% DMSO) by centrifugation of the microchip, and the microchip was stored in a refrigerator at −80 ° C. In addition, the cell introduction | transduction to the microchip without a pocket was performed in the following procedures. 100 μL of cell suspension was added to the inlet and allowed to stand for about 30 seconds. In order to stop the flow of liquid due to gravity, the liquid medium was placed in a 35 mm culture dish until the microchip was completely submerged. Incubation was performed in a constant temperature bath at 37 ° C. with 5% CO ₂ to adhere the cells on the surface of the culture dish, which is the bottom of the microspace in the microchip. After confirming cell adhesion, the liquid medium outside the chip was collected with an aspirator. 100 μL of liquid medium was placed at the inlet, and the cells were cultured to a desired density (50% confluent) in a 37 ° C. thermostat with 5% CO ₂ (Kondo et al. J. Biosci. Bioeng., Vol. 118, No .3, 356-358, 2014). At the time of 50% confluence, the medium was changed to a cryoprotectant-containing medium (medium containing 10% DMSO) by centrifugation of the microchip, and the microchip was stored in a refrigerator at −80 ° C.

  After 36 hours of cryopreservation at −80 ° C., 50 μl of medium was added dropwise to the inlet and outlet of the microchip, and the microchip was thawed at room temperature for 10 minutes. The medium was replaced with culture medium. After 1 hour incubation at 37 ° C. in a 5% CO 2 incubator, the viability of the cryopreserved cells was evaluated. Dead cell nuclei were stained with propidium iodide (PI). The whole cell nuclei were then stained with Hoechst 33258. Viability was calculated from the number of PI positive cells and Hoechst positive cells.

  As a control, cells on bulk cell culture dishes (dish) were stored frozen. At 50% confluence, the medium was replaced with 800 μl of cryoprotectant-containing medium. The cell culture dish was stored in a −80 ° C. refrigerator. After cryopreservation, cell culture dishes (dish) were thawed at room temperature for 10 minutes. The medium was replaced with culture medium. After incubation for 1 hour at 37 ° C. in a 5% CO 2 incubator, the viability of the cryopreserved cells was evaluated as described above.

The results of the above evaluation are shown in FIG.
As can be seen from the upper graph in FIG. 4, cells cryopreserved on the microchip showed higher survival rates than cells cryopreserved on the cell culture dish (dish). Moreover, the cells cryopreserved on the microchip showed a high survival rate regardless of whether or not the culture substrate was treated with collagen.
As can be seen from the lower graph of FIG. 4, the survival rate was high in both cases with and without pockets, but in the presence of pockets, high cell adhesion rate and high survival rate were achieved. be able to.

Example 5: PC12 cell differentiation and immunostaining To examine the effect of cryopreservation on cell phenotype, neural differentiation of PC12 cells was induced after cryopreservation. PC12 cells were cultured in medium containing 100 μg / mL nerve growth factor (NGF). After treatment with NGF for 4 days, PC12 cells were immunostained. Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes and demembraneed with 0.2% Triton X-100 for 30 minutes. After rinsing with TBST, cells were blocked with 2% goat serum albumin in TBST for 1 hour at room temperature. The cells were then incubated overnight at 4 ° C. with a primary antibody, rabbit anti-βIII tubulin antibody (abcomab 18207). After rinsing with TBST, cells were incubated with secondary antibody Alexa Fluor 488 labeled anti-rabbit IgG (Life technologies A-11008) and Hoechst 33258 for 1 hour at room temperature. The cells were rinsed with TBST and then observed with a fluorescence microscope.

  The results are shown in FIG. The left side of FIG. 5 shows that growth of tubulin-rich neurite-like structures is observed in cells after cryopreservation by the method of the present invention. This structure was also observed in the positive control (right of FIG. 5), but not in the negative control without treatment with NGF (middle of FIG. 5). This result indicates that cryopreservation on the microchip according to the present invention does not adversely affect PC12 cell neural differentiation. That is, it was shown that cryopreservation of adherent cells on a microchip can maintain the phenotype of adherent cells.

Example 6: Cytotoxicity assay The following experiment was performed using a microchip with a pocket (the height of the flow path was 25 μm).
HeLa cells (1.0 × 10 ⁶ cells / mL) were suspended in the liquid medium, and the suspension was added to the inlet and introduced into the pocket by centrifugation. The medium was changed every day, and the liquid medium was circulated to grow to 50% confluence.

After photography, the culture medium was replaced with a cryopreservation solution (cell culture solution containing 10% DMSO) by centrifugation, and left in a deep freezer at -80 ° C. After 36 hours, 50 μL of liquid medium was added dropwise to the inlet and outlet, and allowed to stand at room temperature for 10 minutes for thawing. After thawing, the inlet was replaced with 100 μL of a liquid medium, followed by recovery culture at 5% CO ₂ and 37 ° C. for 1 hour. After carrying out recovery culture, a liquid medium of 0 to 1000 μM (0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, 1000 μM) of cisplatin (Wako Pure Chemical Industries) was placed at the inlet and 5% CO _2. And incubated at 37 ° C. for 24 hours. After incubation, double staining of propidium iodide-Hoechest was performed. After washing with PBS, staining was performed with 1 μg / mL PI for 20 minutes. After fixing with 4% paraformaldehyde for 10 minutes, the cells were stained with 0.2% Triton X-100 (Wako Pure Chemical Industries), 1 μg / mL Hoechst 33258 (in PBS). After washing with PBS, the images were taken with a fluorescence microscope. Images were taken using a fluorescence microscope (ZEISS, Axio Observer). The number of viable cells was counted, and the relative cell viability at each cisplatin concentration was calculated with the average viable cell count of 0 μM cisplatin as 100%.

(96-well cytotoxicity assay)
HeLa cells were suspended and seeded at 30000 cells / well in each well of a 96-well plate. Incubated overnight at 37 ° C. with 5% CO ₂ . Subsequently, cisplatin was added to each well to a final concentration of 0 to 100 μM (0 μM, 0.1 μM, 1 μM, 10 μM, 100 μM). Incubated for 24 hours at 37 ° C. with 5% CO ₂ . After the incubation, double staining of propidium iodide-Hoechest was performed in the same manner as the microchip.

(result)
Each result is shown in FIG. Error bars represent plus or minus standard deviation (n = 3). The curve is a four-parameter logistic function y = (ad) / (1+ (x / c) ^b ) + d that best fits the measurement point
It is. Here, a, b, c, and d are fitting parameters, which are optimized by the least square method. At that time, the inverse of the variance of each measurement point was used as the weight. From this function, the cisplatin concentration at which the survival rate becomes 50%, that is, IC ₅₀ (50% inhibitory concentration), is 1.2 μM in the 96-well plate, and 8.9 μM in the condition where the microchip is not frozen. After cryopreservation and thawing according to the present invention, it became 7.3 μM. The experimental results did not show a significant effect due to cryopreservation, suggesting that the cells cryopreserved according to the present invention are useful for toxicity evaluation tests.

Claims (8)

A method for producing frozen cells, comprising a step of freezing cells adhered on one wall surface of a microchip in a state where the cells are adhered on one wall surface of the microchip. The method for producing frozen cells according to claim 1, wherein a sealed microspace of the microchip is filled with cells and a cell culture medium, and the cells adhere to one wall surface of the microchip. The manufacturing method of the frozen cell of Claim 1 or 2 whose height of the micro space of a microchip is 10-100 micrometers. The method according to any one of claims 1 to 3, wherein a micropocket for capturing a single cell is provided on a wall surface to which a cell of the microchip adheres. Until the step of freezing the cells adhering on one wall surface of the microchip with the cells adhering to one wall surface of the microchip is 30-70% confluent on the one wall surface of the microchip The method according to any one of claims 1 to 4, which is a step of, after culturing, replacing the medium with a medium containing a cryoprotectant and then freezing the cells. A frozen cell product obtained by the method according to any one of claims 1 to 5 and comprising a microchip and frozen cells adhered to one wall surface of the microchip. (I) a step of producing frozen cells by the method according to any one of claims 1 to 5; and (ii) a step of thawing the frozen cells:
A method for freezing and thawing cells. (I) a step of producing frozen cells by the method according to any one of claims 1 to 5;
(Ii) thawing the frozen cells;
(Iii) contacting the thawed cells with a test substance; and (iv) detecting the action of the test substance on the thawed cells:
A test method for a test substance, comprising: