Cell housing container, and recovery method of implantation potential-enhanced embryo using container

Abstract

To provide a cell housing container that can house a cell while suppressing adhesion with a container, and a recovery method of an embryo for transfer which avoids a damage by a capillary using the container.SOLUTION: Provided is a cell housing container, where the container is equipped with a recess and a capillary fixing part which fixes a capillary to an upper part or inside the recess, the recess is equipped with a cell housing chamber formed by the recess, a capillary tip part and the capillary fixing part when the capillary is fixed, the cell housing chamber is equipped with a communication part communicating the outside of the container, a portion inside the cell housing chamber which contacts the cell is made of a low adhesive material, the capillary tip part does not contact the cell when the capillary is fixed, and the pressures of the cell housing chamber and the outside of the container are equalized when the communication part fixes the capillary.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell-containing container and a method for recovering an implantation-enhanced embryo using the container. [Background technology]

[0002] In recent years, the number of couples undergoing infertility treatment has been increasing in developed countries and other countries due to factors such as late marriage. For example, in 2020, one in 14 babies born in Japan was born through embryo transfer (Non-Patent Documents 1 and 2).

[0003] However, the success rate of in vitro fertilization or intracytoplasmic sperm injection (ICS) in infertility treatment is not particularly high. For example, in 2020, the success rate of in vitro fertilization in Japan (birth rate per embryo transfer) was only 14.7% when fresh embryos were used, and only 25.5% when frozen embryos were used (Non-Patent Document 1). Since the success rates for in vitro fertilization, development from fertilized eggs to blastocysts, and implantation to pregnancy are all above 50%, the aforementioned low success rate is believed to be due to the low rate of implantation of the transferred embryos into the endometrium of the mother's uterus.

[0004] Embryo implantation is achieved through the interaction of cell adhesion proteins on the surface of the embryo with receptor proteins on endometrial cells. Therefore, enhancing embryo adhesion to the endometrium is thought to be effective in improving the implantation rate of transferred embryos and increasing the success rate of in vitro fertilization, and related research is being conducted.

[0005] For example, Non-Patent Document 3 discloses a method for improving the production of fibronectin on the embryo surface by adding IGF-1 (insulin-like growth factor 1) to the culture medium in order to improve adhesion between blastocysts and uterine epithelial cells. However, because this method indirectly enhances the production of fibronectin, an adhesive protein, via IGF-1, it is difficult to control the amount of fibronectin produced or the timing of its expression. The amount of fibronectin has a direct effect on strengthening adhesion and embryo growth, and the timing of its expression is known to have a significant impact on the implantation rate during transplantation in infertility treatments, etc. Therefore, this method has the problem of being unable to improve the embryo implantation rate.

[0006] Furthermore, Non-Patent Document 4 discloses the relationship between early embryo growth and fibronectin concentration. Specifically, fibronectin was added at different concentrations to the culture medium for early embryos, and the effect of fibronectin on the embryos was evaluated. As a result, it was reported that at fibronectin concentrations of 5 μg / mL and 300 μg / mL, 0% of the samples grew from the 2-cell stage to blastocysts, and even at 50 μg / mL, only about 7% grew. This result suggests that exposing embryos to high concentrations of fibronectin for a long period of time inhibits embryo growth.

[0007] Furthermore, Non-Patent Document 5 discloses the in vitro implantation rate of blastocysts treated with fibronectin, in which 15.2±3.1 μg / mL of fibronectin was added to a culture medium containing blastocysts. This document reports that when the embryos used were blastocysts that had been developed in vivo from fertilization to the blastocyst stage, the addition of fibronectin in vitro was found to have an effect of improving implantation ability, but when the blastocysts were collected after in vivo fertilization and cultured in vitro from the 2-cell stage to the late blastocyst stage, no improvement was observed by adding fibronectin. This result suggests that the addition of low concentrations of fibronectin does not improve the implantation ability of blastocysts cultured in vitro.

[0008] As described above, previous research reports have revealed that adhesive proteins are involved in improving the rate of embryo implantation. However, there have been problems in that treatment with high concentrations of adhesive proteins damages the embryos, inhibiting their growth, and conversely, treatment with low concentrations of adhesive proteins does not improve the cell adhesion of the embryos, meaning that adhesive proteins cannot be used to improve the rate of embryo implantation. Summary of the Invention [Problem to be solved by the invention]

[0009] As a result of extensive research aimed at solving the above problems, the present inventors have found that when a high concentration of cell adhesion protein is brought into contact with a pre-implantation embryo for a short period of time in vitro, the cell adhesion of the embryo can be enhanced and damage to the embryo can be minimized. Based on these research results, the present inventors provide a method for producing an embryo with enhanced implantation ability and an embryo with enhanced implantation ability for transplantation as described below.

[0010] (1) A method for producing an embryo with enhanced implantation ability, the method comprising a contacting step of contacting the surface of a placental embryo in vitro with a solution of a polypeptide containing an RGD motif. (2) Embryos for transplantation with enhanced implantation potential, in which polypeptides containing RGD motifs are attached to the cell surface.

[0011] However, this method has a problem in that the embryos adhere to the bottom of the container containing the solution during the contact step, making it difficult to recover the implantation-enhanced embryos after the contact step.

[0012] To address the problem of cells adhering to the container, conventional techniques use a container such as a glass substrate that has low adhesiveness to cells.

[0013] However, although such containers can suppress adhesion between cells and the container, recovery of embryos from the container remains difficult due to the tendency of the cell-containing solution to spread. Furthermore, Patent Documents 1 and 2 describe methods for recovering cells by using a material with low adhesiveness to cells or by rotating the container, but even these methods cannot avoid the risk of damage to cells caused by thin tubes such as chips used to recover the cells.

[0014] Therefore, the object of the present invention is to provide a cell storage container that can store cells while suppressing adhesion to the container, and a method for recovering embryos for transplantation using such a container that avoids damage caused by tubules. [Means for solving the problem]

[0015] As a result of extensive research conducted by the present inventors to develop a solution to the above-mentioned problems, they have succeeded in easily recovering the solution and cells contained in the cell storage chamber by constructing the inner surface of a cell storage container that comes into contact with the cells from a low-adhesion material, and by providing a communication part such as a groove that communicates with the outside of the container in the cell storage chamber where the cells are stored when a thin tube such as a tip is brought into contact with or inserted into the container and the thin tube is fixed. The present invention is based on the results of this research and provides the following cell storage container and a method for recovering implantation-competence enhanced embryos using the container.

[0016] (1) A cell storage container comprising a recess and a capillary tube fixing part for fixing a capillary tube on or inside the recess, the recess comprising a cell storage chamber formed by the recess, the capillary tube tip, and the capillary tube fixing part when the capillary tube is fixed, the cell storage chamber comprising a communication part that communicates with the outside of the container, the part of the inner surface of the cell storage chamber that comes into contact with the cells being made of a low-adhesion material, the capillary tube tip does not come into contact with the cells when the capillary tube is fixed, and the communication part equalizes the pressure between the cell storage chamber and the outside of the container when the capillary tube is fixed. (2) A method for recovering embryos with enhanced implantation ability, comprising the steps of: contacting the surface of a placental embryo with a solution of a polypeptide containing an RGD motif in vitro in a container described in (1) to produce embryos with enhanced implantation ability; fixing a tubule to the container; and applying pressure to the inside of the tubule to recover the embryos with enhanced implantation ability. [Effects of the Invention]

[0017] The present invention provides a cell storage container that can store cells while suppressing adhesion to the container, and a method for recovering embryos for transplantation that uses the container and avoids damage caused by tubules.

[0018] Aspects of the present invention are as follows. (1) A cell-containing container, The container recesses, and a capillary fixing portion for fixing a capillary on or inside the recess; Equipped with The recessed portion is A cell-accommodating chamber formed by the recess, the tip end of the capillary tube, and the capillary tube fixing portion when the capillary tube is fixed Equipped with The cell-containing chamber A communication part that communicates with the outside of the container Equipped with a portion of the inner surface of the cell-containing chamber that comes into contact with the cells is made of a low-adhesion material; When the capillary tube is fixed, the tip of the capillary tube does not come into contact with the cell, the communicating portion equalizes the pressure between the cell-containing chamber and the outside of the container when the capillary is fixed. The container. (2) A container as described in (1), in which the horizontal cross-sectional area of ​​the cell storage chamber gradually decreases toward the bottom of the cell storage chamber. (3) The container according to (1) or (2), wherein the opening area of ​​the upper surface of the recess has an equivalent circle diameter of 0.1 mm to 20 mm. (4) The container according to any one of (1) to (3), wherein the cell is a single cell. (5) The container according to (4), wherein the single cell is an embryo of a placental mammal. (6) The container according to (5), wherein the placental embryo is an embryo with enhanced implantation ability. (7) A cell collection kit comprising the container according to any one of (1) to (6) and a capillary tube. (8) A method for recovering an implantation-enhanced embryo, comprising: a step of contacting the surface of a placental embryo with a solution of a polypeptide containing an RGD motif in vitro in a container according to any one of (1) to (6) to produce an embryo with enhanced implantation ability; Fixing a capillary to the container; and a step of applying suction pressure to the inside of the tubule to recover the implantation ability enhanced embryos. A method comprising: (9) The method according to (8), wherein the inner diameter of the tubule is larger than the diameter of the embryo with enhanced implantation ability. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a diagram schematically illustrating one embodiment of a cell-containing container of the present invention. [Figure 2] FIG. 10 is a diagram showing a state in which a capillary is inserted into one embodiment of a cell storage container of the present invention. [Figure 3] 1 is a photograph showing an embodiment of Example 1. [Figure 4] 1 is a photograph showing an embodiment of Comparative Example 1. [Figure 5] 1 is a photograph showing an embodiment of Example 2. [Figure 6] 10 is a photograph showing the state in which a tip 8 is inserted in Example 2. [Figure 7] FIG. 10 is a cross-sectional view schematically showing the state in which a particle suspension is added to a cell-containing container of Example 3. [Figure 8] 10 is a cross-sectional view schematically showing the state in which a particle suspension is added to a cell-containing container of Comparative Example 2. FIG. [Figure 9]This figure shows the enhancement of cell adhesiveness in embryos recovered by the method for recovering embryos with enhanced implantation ability of the present invention. In the figure, the stability score is a four-level score based on the immobility (adhesion strength) of the embryo when tapped onto the culture vessel to evaluate cell adhesiveness. A score of 4 indicates strong adhesion, a score of 3 indicates moderate adhesion, a score of 2 indicates weak adhesion, and a score of 1 indicates no adhesion. Example 4 was performed with n = 16 to 24, and Comparative Example 3 with n = 16 to 20. DETAILED DESCRIPTION OF THE INVENTION

[0020] 1. Cell container 1-1. Overview A first aspect of the present invention is a cell storage container. The cell storage container of the present invention includes a recess and a capillary tube fixing part for fixing a capillary tube to the top or inside of the recess. The recess includes a cell storage chamber formed by the recess, the capillary tube tip, and the capillary tube fixing part when the capillary tube is fixed. The cell storage chamber includes a communication part that communicates with the outside of the container. The part of the inner surface of the cell storage chamber that comes into contact with the cell is made of a low-adhesion material. When the capillary tube is fixed, the capillary tube tip does not come into contact with the cell. The communication part equalizes the pressure between the cell storage chamber and the outside of the container when the capillary tube is fixed. The cell storage container of the present invention has the following effects. First, because the part that comes into contact with the cell is made of a low-adhesion material, adhesion between the cell and the container is suppressed when the cell is stored. Second, because the capillary tube fixing part is configured to prevent excessive insertion of the capillary tube and avoid contact between the capillary tube tip and the cell, damage to the cell due to contact with the capillary tube is avoided. In addition, because the solution containing the cells comes into contact with the micro-recess, the cells are easily collected without escaping. Furthermore, because the cell-containing chamber is configured with a communicating section, the cells can be collected at the tip of the capillary or inside the capillary while minimizing damage to the cells when pressure is applied inside the capillary.

[0021] 1-2.Definition of Terms Terms used in this specification are defined below. The term "cell" refers to a building block of an organism. The cell herein may be of any species. Examples include mammals and birds, and examples of mammals and birds include primates including humans and chimpanzees, pet animals such as dogs, cats, and parakeets, livestock animals such as cows, horses, sheep, goats, and chickens, rodents such as mice and rats, and animals kept in zoos. In one embodiment, the cell is a single cell. In one embodiment, the cell is a cell mass (e.g., a spheroid or an organoid). In one embodiment, the cell is a placental embryo. In one embodiment, the cell is an embryo with enhanced implantation competence, which will be described in detail below.

[0022] "Storage" means to contain cells. Storage includes temporary storage, i.e., storage for seconds or minutes, to storage (preservation) for hours, days, weeks, months, or years.

[0023] A "container" is a vessel for containing cells. The material of the container is not limited. Examples of the material of the container include ceramics, glass, resin, and / or low-adhesion materials, which will be described in detail below. Examples of resins include polyolefin resins or cyclic polyolefin resins such as polypropylene resin, polyethylene resin, and ethylene-propylene copolymer; polystyrene resins such as acrylonitrile-butadiene-styrene resin; methacrylic resins such as polycarbonate resin, polyethylene terephthalate resin, and polymethyl methacrylate resin; vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, polyetherimide resin, and fluorine-based resins such as polytetrafluoroethylene; acrylic resins such as polymethylpentene resin and polyacrylonitrile; and cellulose-based resins such as propionate resin. In one embodiment, the resin is polystyrene resin from the viewpoints of moldability, transparency, and radiation resistance of the container. In one embodiment, the container is made of polydimethylsiloxane (PDMS). The container material may be different for each component.

[0024] The term "well" refers to a recessed portion in the container of the present invention for accommodating cells. One or more wells are present in each container of the present invention. In one embodiment, one well is present in each container of the present invention. In one embodiment, a plurality of wells are present in each container of the present invention, for example, 4, 6, 8, 12, 24, 48, 96, 384, or 1536 wells. However, the number of wells present in each container of the present invention is not limited and can be freely determined. The size of the well is not limited as long as it is large enough to accommodate the target cells. In one embodiment, one cell is accommodated per well. In one embodiment, the horizontal cross-sectional area of ​​the well gradually decreases toward the bottom of the well. The horizontal cross-sectional shape of the well is not limited. In one embodiment, the horizontal cross-sectional shape of the well is circular or approximately circular. For example, the circle-equivalent diameter of the opening area of ​​the top surface of the well is typically 0.1 mm to 20 mm, in one embodiment, 1 mm to 15 mm, and in one embodiment, 5 mm to 15 mm. The vertical cross-sectional shape of the recess is not limited. In one embodiment, the vertical cross-sectional shape of the recess is approximately U-shaped. For example, the vertical height of the recess is typically 1 mm to 10 mm, in one embodiment 1 mm to 8 mm, in one embodiment 2 mm to 6 mm, and in one embodiment 3 mm to 5 mm. The volume of the recess is not limited. For example, the volume of the recess is typically 80 μL to 500 μL, and in one embodiment 100 μL to 300 μL.

[0025] A "capillary tube" refers to a thin tube for collecting cells. Cells can be collected by applying pressure to the inside of the capillary tube. For example, cells can be collected by suctioning one end of the capillary tube, causing the cells to adhere to the other end or by sucking the cells into the capillary tube. Examples of capillaries include, but are not limited to, chips, glass tubes, plastic tubes, etc. The shape of the hole at the tip of the capillary tube is not limited, but is usually circular or approximately circular. The inner diameter of the tip of the capillary tube is not limited. In one embodiment, the inner diameter of the tip of the capillary tube (the equivalent circle diameter of the opening area at the tip of the capillary tube) is larger than the equivalent circle diameter of the opening area at the top surface of the recess, and therefore the capillary tube cannot be inserted into the recess. In one embodiment, the inner diameter of the tip of the capillary tube (the equivalent circle diameter of the opening area at the tip of the capillary tube) is smaller than the equivalent circle diameter of the opening area at the top surface of the recess, and therefore the capillary tube can be inserted into the recess. For example, the inner diameter of the tip of the capillary tube (the circle-equivalent diameter of the area of ​​the opening at the tip of the capillary tube) is usually 0.1 mm to 6 mm, and in one embodiment, 0.5 mm to 5 mm, in another embodiment, 1 mm to 4 mm, and in another embodiment, 2 mm to 3 mm. In one embodiment, the inner diameter of the tip of the capillary tube (the circle-equivalent diameter of the area of ​​the opening at the tip of the capillary tube) is larger than the diameter of a cell, for example, an implantation-competence-enhanced embryo. Note that the capillary tube is not an essential element constituting the cell-containment container of the present invention. Therefore, any capillary tube that corresponds to the cell-containment container of the present invention, i.e., that meets the usage requirements of the cell-containment container of the present invention, can be used. The present invention encompasses a kit comprising the cell-containment container of the present invention and a capillary that can be used for the cell-containment container of the present invention.

[0026] The "capillary tube fixing portion" is a portion provided on or inside the well, and fixes the capillary tube when the capillary tube is brought into contact with the well to collect cells. In one embodiment, the capillary tube fixing portion is the edge of the opening on the top surface of the well. In this case, the circle-equivalent diameter of the opening area on the top surface of the well may be smaller than the inner diameter of the capillary tube tip, preventing the capillary tube from being inserted into the well. In one embodiment, the capillary tube fixing portion is located inside the well. In this case, the capillary tube fixing portion may be a portion of the well whose circle-equivalent diameter of a horizontal cross section of the well matches the inner diameter of the capillary tube tip, and the capillary tube fixing portion may fix the capillary tube tip, preventing the capillary tube tip from moving downward beyond the capillary tube fixing portion of the well. Alternatively, the capillary tube fixing portion may be a portion of the well whose circle-equivalent diameter of a horizontal cross section of the well matches the inner diameter of any horizontal portion of the capillary tube, preventing the capillary tube from moving downward by fixing any horizontal portion of the capillary tube. Therefore, when the capillary tube and the well are fixed by the capillary tube fixing portion to collect cells, the capillary tube tip does not move downward beyond a certain height, i.e., a height from the bottom of the well that is longer than the diameter of the contained cells, so that the capillary tube tip does not come into contact with the cells contained at the bottom of the well. The height from the capillary tube tip to the bottom of the well corresponds to the vertical height of the cell-containing chamber, which will be described in detail below. Note that the capillary tube fixing portion is intended to restrict the downward movement of the capillary tube, and therefore the fixed capillary tube may move in directions other than downward. In one embodiment, the capillary tube fixing portion is a part that fixes the capillary tube so that it does not move except in the direction opposite to the direction in which it was moved to bring the capillary tube into contact with the well.

[0027] The term "cell-receiving chamber" refers to a compartment for accommodating cells present inside the recess formed by the recess, the capillary tip, and the capillary tip when the capillary is fixed to the capillary tip for cell collection. The cell-receiving chamber is located at the bottom of the tip of the recess. In one embodiment, the horizontal cross-sectional area of ​​the cell-receiving chamber gradually decreases toward the bottom of the cell-receiving chamber. In one embodiment, the cell-receiving chamber is formed by the recess itself. In one embodiment, the cell-receiving chamber is formed inside the recess. The horizontal cross-sectional shape of the cell-receiving chamber is not limited. In one embodiment, the horizontal cross-sectional shape of the cell-receiving chamber is circular or approximately circular. For example, the circle-equivalent diameter of the upper area of ​​the cell-receiving chamber is typically 0.1 mm to 20 mm, in one embodiment, 1 mm to 15 mm, and in one embodiment, 5 mm to 15 mm. The vertical cross-sectional shape of the cell-receiving chamber is not limited. In one embodiment, the vertical cross-sectional shape of the cell-receiving chamber is approximately U-shaped. For example, the vertical height of the cell-receiving chamber is typically 1 mm to 10 mm, and in one embodiment, 1 mm to 8 mm, and in one embodiment, 2 mm to 6 mm, and in one embodiment, 3 mm to 5 mm. In one embodiment, the vertical height of the cell-receiving chamber is (cell diameter + 0.1 mm to 1 mm). In one embodiment, the vertical height of the cell-receiving chamber is (cell diameter + 0.1 mm to 0.3 mm). The volume of the cell-receiving chamber is not limited. For example, the volume of the cell-receiving chamber is typically 80 μL to 500 μL, and in one embodiment, 100 μL to 300 μL. At least the inner surface portion of the cell-receiving chamber that comes into contact with the cells, i.e., the inner surface, is made of a low-adhesion material, for example, a layer made of a low-adhesion material, which will be described in detail below. In one embodiment, the cell-receiving chamber is made of a low-adhesion material.

[0028] The "outside of the container" refers to the part of the container that is in contact with the outside world. The outside of the container may be the atmosphere (atmospheric pressure) or a closed space. However, if the outside of the container is a closed space, the space must be large enough to equalize the pressure (internal pressure) applied to the cell-containing chamber when the inside of the capillary is suctioned, or it must be at positive pressure.

[0029] "Communicating" means that solids, liquids, and / or gases can flow through the communicating portion, and a "communicating portion" means a portion that spatially connects the cell-accommodating chamber and the outside of the container when the capillary is fixed, allowing solids, liquids, and / or gases to flow through the communicating portion. The communicating portion equalizes the pressure between the cell-accommodating chamber and the outside of the container when the capillary is fixed, particularly when the capillary is fixed and subjected to suction. The container of the present disclosure has a communicating portion, which can minimize damage to cells when the capillary is suctioned. Examples of communicating portions include, but are not limited to, grooves, notches, holes, and the like. The size of the communicating portion is not limited. For example, the length of the communicating portion is typically 2 mm to 20 mm, and in one embodiment, 3 mm to 10 mm; the width is typically 0.1 mm to 3 mm, and in one embodiment, 0.5 mm to 2 mm; and the depth is typically 0.1 mm to 3 mm, and in one embodiment, 0.5 mm to 2 mm.

[0030] A "low-adhesion material" is a material that has low adhesiveness to cells. Examples of low-adhesion materials include hydrophilic resins. Hydrophilic resins can be formed by modifying water-soluble resins into water-insoluble cured resins. A "water-soluble resin" is a resin that hydrates through ionic or hydrogen bonds with water molecules and, as a result, dissolves in water. A water-soluble resin has a necessary and sufficient amount of ionic or polar side chains on the main chain in the molecule to dissolve in water, and has a solubility of 1.0 g or more per 100 g of water at 25°C. The average degree of polymerization of the water-soluble resin is not limited, but is usually 100 to 10,000, and in one embodiment, 200 to 5,000. Examples of water-soluble resins include saponified polyvinyl acetate, polyvinylpyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate, copolymers of the monomers constituting these, and copolymers of 2-methacryloyloxyethyl phosphorylcholine with other monomers (e.g., butyl methacrylate). The water-soluble resin further has a functional group selected from the group consisting of a radiation-reactive functional group, a photosensitive functional group, and a heat-reactive functional group in its side chain. Examples of heat-reactive functional groups and radiation-reactive functional groups include vinyl groups and epoxy groups. Examples of photosensitive functional groups include those containing nitrogen atoms, such as azide groups, diazo groups, azide groups, and cinmonyl groups. In one embodiment, the water-soluble resin has a structure containing one or more selected from the group consisting of saponified polyvinyl acetate, polyvinylpyrrolidone, and polyethylene glycol, as well as the functional groups. The saponified polyvinyl acetate refers to, for example, polyvinyl alcohol or a copolymer of vinyl alcohol and another compound. The saponified polyvinyl acetate includes, for example, vinyl alcohol and saponified vinyl acetate modified with a hydrophilic group, a hydrophobic group, an anion group, a cation group, an amide group, or a reactive group such as an acetoacetyl group. The saponified polyvinyl acetate is typically 20 mol % to 100 mol %, and in one embodiment, 50 mol % to 95 mol % of the total polyvinyl acetate.The water-soluble resin is modified into a hydrophilic resin by curing it through irradiation with radiation, light irradiation, and / or heating, depending on the functional groups of the resin. The low-adhesion material is used as a material constituting the entire container of the present invention, the well, the cell-receiving chamber, the inner surface of the well, the inner surface of the cell-receiving chamber, or the portion of the inner surface of the cell-receiving chamber that comes into contact with cells.

[0031] 1-3.Configuration The configuration of the container of the present invention will be described with reference to FIGS. 1 and 2. FIG. 1A is a cross-sectional view of one embodiment of the cell-holding container of the present invention. FIG. 1B is a top view of one embodiment of the cell-holding container of the present invention. FIG. 2 shows the cell-holding container of FIG. 1 containing a solution containing cells and inserting a capillary tube to collect the cells. The cell-holding container 1 of the present invention shown in FIGS. 1 and 2 includes an inverted truncated cone-shaped recess 2 and a capillary tube fixing portion 3 that fixes a capillary tube 8 inside the recess 2. The recess 2 includes a cell-holding chamber 6 that is formed by the recess 2, the capillary tube tip, and the capillary tube fixing portion 3 when the capillary tube 8 is fixed. The cell-holding chamber 6 includes a communicating portion 4 that communicates with the outside of the container. Furthermore, the bottom of the inner surface of the cell-holding chamber 6 that comes into contact with the cell 7 is made of a low-adhesion material 5. Furthermore, when the capillary tube 8 is fixed, the capillary tube tip does not come into contact with the cell 7, and the communicating portion 4 equalizes the pressure between the cell-holding chamber 6 and the outside of the container when the capillary tube 5 is fixed. In the cell storage container 1 of the present invention, the bottom portion that comes into contact with the cells 7 is made of low-adhesion material 5, which prevents adhesion of the cells 7 to the container 1 when the cells 7 are stored therein. Next, the capillary fixing portion 3 is configured to avoid contact between the capillary tip and the cells 7, which prevents the cells 7 from being damaged by contact with the capillary 8. Furthermore, since the cell storage chamber 6 is configured to have a communicating portion 4, when attempting to collect the cells 7 into the capillary 8 by applying suction pressure to the inside of the capillary 8, the cells 7 can be collected into the inside of the capillary 8 while minimizing damage to the cells 7 caused by the suction pressure.

[0032] 1-4. Manufacturing method The container of the present invention can be adjusted to have the properties described above and manufactured by techniques known in the art. The container of the present invention can be manufactured by, for example, injection molding, blow molding, or injection blow molding. Furthermore, when the inner surface of the well, the inner surface of the cell-receiving chamber, or the portion of the inner surface of the cell-receiving chamber that comes into contact with the cells is composed of a layer made of a low-adhesion material, the inner surface can be formed by contacting, for example applying, the water-soluble resin described above to the inner surface and then curing it.

[0033] 2. Method for recovering embryos with enhanced implantation potential 2-1. Overview A second aspect of the present invention is a method for producing and recovering transfer embryos with enhanced implantation ability using the cell-holding container of the first aspect of the present invention. In the recovery method of the present invention, embryos with enhanced implantation ability are produced in the cell-holding container of the first aspect of the present invention by contacting the surface of the embryo with a solution of a polypeptide containing an RGD motif, a capillary tube is fixed to the container, and then the inside of the capillary tube is subjected to suction pressure to recover the embryo. According to the recovery method of the present invention, embryos with enhanced implantation ability can be produced and recovered at the desired timing in a container in which adhesion to the embryo is suppressed, in a simple and short process while minimizing damage to the embryo due to chemical or physical treatment. Furthermore, the embryos can be recovered without damage and transferred into the uterus, thereby improving pregnancy efficiency.

[0034] 2-2.Definition of Terms Terms used in this specification are defined below. The term "embryo" refers to an individual in a multicellular organism at an early developmental stage shortly after cleavage has begun after fertilization. In this specification, it particularly refers to an individual from a placental fertilized egg to the stage at which it can implant in the uterine lining, specifically the blastocyst stage. In this specification, when simply referring to an "embryo," unless otherwise specified, it refers to an embryo for transplantation.

[0035] "Placental animals" is roughly synonymous with extant mammals, and refers to a group of animals in which the embryo is implanted in the placenta and the offspring are raised in the uterus until a later stage of development when they assume a form similar to that of the parents.

[0036] As used herein, the term "embryo for transfer" refers to an embryo primarily intended for transfer into the maternal uterus. This typically refers to an embryo prepared in vitro by artificial insemination or intracytoplasmic sperm injection. However, the destination of the embryo is not necessarily limited to the uterus of a human or placental species, and may also be, for example, an in vitro region containing endometrial epithelial cells for drug discovery or clinical trials.

[0037] As used herein, the term "implanted embryo" refers to an embryo that has been implanted into the maternal uterus or onto the endometrium in vitro.

[0038] "Cell adhesion" refers to the property of adhering to or binding to the surface of other cells. This cell adhesion is typically, but not exclusively, achieved by binding between cell adhesion proteins present on the cell membrane and their receptor proteins. As used herein, "cell adhesion" primarily refers to the property of embryos to adhere to other cell surfaces, such as endometrial cells or in vitro cultured epithelial cells.

[0039] As used herein, the term "implantation ability" refers to the ability of an embryo to implant into the endometrium. This ability is proportional to the strength of the embryo's cytoadhesiveness, which in turn depends on the number and area of ​​cytoadhesive proteins present on the embryo's surface.

[0040] As used herein, the term "enhancing implantation ability" refers to strengthening the cell adhesiveness that normal embryos have, thereby increasing the implantation ability of the embryo.

[0041] As used herein, the term "implantation-enhanced transfer embryo" refers to a transfer embryo whose implantation ability has been enhanced by strengthening the adhesion of the embryo to the endometrium.

[0042] The term "RGD motif" refers to an amino acid sequence motif having cell adhesion activity, which is composed of three amino acid residues: arginine (R), glycine (G), and aspartic acid (D).

[0043] As used herein, the term "contacting" refers to bringing two substances into contact with each other.

[0044] 2-3. Method The method for recovering implantation-competence-enhanced embryos of the present invention includes a contacting step and a separation and recovery step as essential steps in the cell-holding container of the first aspect of the present invention, and a culture step as a selection step. Each step will be explained below.

[0045] 2-3-1.Culture process The "culturing step" is a step of culturing the fertilized eggs to a desired developmental stage before the contacting step. The fertilized eggs used in this step may be prepared by known methods. For example, after administering an ovulation-inducing agent to the mother, eggs are collected from the ovaries together with follicular fluid using an egg collection needle. Subsequently, fertilization is carried out using sperm collected on the day of fertilization or frozen and stored sperm. Fertilization may be carried out by either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

[0046] Fertilized eggs can be cultured using methods known in the art. There are no particular limitations on the medium, as long as it is a medium capable of culturing embryos. A general cell culture medium may be used. Cell culture media contain components necessary for cell maintenance and growth, and are therefore suitable for culturing fertilized eggs until they reach an appropriate developmental stage of embryos.

[0047] Media are classified into natural media, semi-synthetic media, synthetic media, etc. depending on their composition, and any of these media can be used. For example, but not limited to, Dulbecco's Modified Eagle's Medium (D-MEM), Ham's Nutrient Mixture F12, D-MEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), MEM medium (Minimum Essential Medium), RPMI1640 (Roswell Park Memorial Institute-1640) medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, M199 medium, High Performance Medium 199, StemPro 34 (Thermo Fisher Scientific), X-VIVO 10 (Chembrex), X-VIVO 15 (Chembrex), HPGM (Chembrex), StemSpan H3000 (STEMCELL Technologies), StemSpan SFEM (STEMCELL Technologies), Stemline II (Sigma-Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Thermo Fisher Scientific), Essential 8 (registered trademark) medium (Thermo Fisher Scientific), mTeSR1 or mTeSR2 medium (STEMCELL Technologies), ReproFF or ReproFF2 (Reprocell), PSGro hESC / iPSC medium (System Biosciences), NutriStem (registered trademark) medium (BiologicalExamples of suitable medium include Thermo Fisher Scientific (manufactured by Thermo Fisher Industries), CSTI-7 medium (manufactured by Cell Science Institute), MesenPRO RS medium (manufactured by Thermo Fisher Scientific), MF-Medium® Mesenchymal Stem Cell Growth Medium (manufactured by Toyobo Co., Ltd.), Sf-900II (manufactured by Thermo Fisher Scientific), and Opti-Pro (manufactured by Thermo Fisher Scientific). These may be used alone or in combination. In particular, the mixing ratio of DMEM / F12 medium is not particularly limited, but it is preferable to mix DMEM and F12 in a weight concentration ratio of the components ranging from 6:4 to 4:6. The specific composition of each medium is known in the art and may be prepared based on the composition described in the appropriate literature (e.g., Kaech S. and Banker G., 2006, Nat. Protoc., 1(5): 2406-15). Furthermore, commercially available media from various manufacturers are available for purchase.

[0048] The temperature for culturing the embryos is preferably in the range of 37° C.±1° C. The carbon dioxide concentration in the medium may be 2% to 5%, or 3% to 4%.

[0049] The culture period is not limited as long as it is a period during which the embryos can develop to the desired stage. Typically, it is sufficient for the embryos to develop to the blastocyst stage. For example, under the above culture conditions, this period is 3 to 5 days after fertilization.

[0050] If there is a period of time between the culture and the next contact step, the cultured embryos can be cryopreserved as needed. When used, they are completely thawed before the contact step is carried out.

[0051] After culturing or thawing from cryopreservation, the embryos may be washed as needed, for example, with a buffer solution or physiological saline.

[0052] 2-3-2. Contact process The "contacting step" is a step in which a solution of a polypeptide containing an RGD motif is contacted with the surface of an embryo in vitro. This step is a key step in the method for recovering implantation embryos with enhanced implantation ability of the present invention, and aims to attach the polypeptide containing an RGD motif to the surface of the embryo in vitro in order to enhance the cell adhesiveness of the embryo. In this specification, when the term "polypeptide" is simply used, it is intended to mean a polypeptide containing an RGD motif, unless otherwise specified.

[0053] The embryos used in this step may be in any developmental stage from early embryos (cleavage stage embryos) to blastocysts. Blastocysts are preferred. The grade of the embryos used in this step is not limited, but for example, early embryos are preferably grade 1 or 2 according to the Wieck classification, and blastocysts are preferably class 5 or 6 according to the Gardner classification.

[0054] The polypeptide used in this step is not limited as long as it contains an RGD motif and has adhesive activity to endometrial epithelial cells, but is preferably a cell adhesion protein.

[0055] "Cell adhesion proteins" are proteins that have integrin-binding activity, and examples thereof include fibronectin, vitronectin, cadherin, laminin, and collagen. The origin of any cell adhesion protein is not limited. It is preferably derived from placental organisms, and more preferably from humans.

[0056] As more specific examples, the amino acid sequence of human fibronectin is shown in SEQ ID NO: 1, the amino acid sequence of human vitronectin is shown in SEQ ID NO: 2, the amino acid sequence of human cadherin is shown in SEQ ID NO: 3, the amino acid sequence of human laminin is shown in SEQ ID NO: 4, and the amino acid sequence of human collagen is shown in SEQ ID NO: 5.

[0057] Furthermore, the peptide fragment may be a peptide fragment of the cell adhesion protein, as long as it contains an RGD motif and has integrin-binding activity. The length of the peptide is not limited. For example, the length may be 50 amino acids or more but less than the full length, 100 amino acids or more but less than the full length, 150 amino acids or more but less than the full length, or 200 amino acids or more but less than the full length.

[0058] The polynucleotide solution used in this step is a solution containing the polypeptide containing the RGD motif in an appropriate solvent. The solvent used for this solution is preferably one that has no or only minimal effect on the embryo upon contact, although there is no particular limitation. Examples include buffers such as phosphate buffer (PBS buffer), physiological saline, and culture media (particularly the media used in the culture step). The polynucleotide concentration in the polynucleotide solution may be 50 μg / mL to 2 mg / mL, 80 μg / mL to 1.5 mg / mL, or 1 mg / mL to 1.2 mg / mL. A concentration of less than 50 μg / mL is insufficient to allow a sufficient amount of polypeptide to adhere to the embryo surface, while a polypeptide solution with a concentration greater than 2 mg / mL will significantly damage the embryo.

[0059] The method for contacting an embryo with a polypeptide solution is not limited as long as it allows the polypeptide to adhere to the embryo surface. Examples include a method in which the polypeptide solution is applied or sprayed onto the embryo surface, and a method in which the entire embryo or a part of the embryo is immersed in the polypeptide solution. Considering that the enhancement of cell adhesiveness of an embryo may depend on the adhesion area of ​​the polypeptide and the number of attached polypeptides, a contact method in which the embryo is completely immersed in the polypeptide solution is preferred.

[0060] The contact time may be 5 to 90 minutes, 10 to 60 minutes, 15 to 50 minutes, 20 to 40 minutes, or 25 to 30 minutes, because a time of less than 5 minutes is too short to allow a sufficient amount of polypeptide to adhere to the embryo surface, and a time of more than 90 minutes causes significant damage to the embryo due to contact with the polypeptide solution.

[0061] In order to reduce damage to the embryos during this process, it is preferable that the contact temperature be in the range of 28 to 39°C, 30 to 38°C, 32 to 37°C, or 34 to 36°C, and that the carbon dioxide concentration be in an atmosphere of 2% to 5%, or 3% to 4%.

[0062] The embryo after this step may be directly transplanted together with a polypeptide solution into the uterus of the mother, for example, or may be used after the recovery step described below.

[0063] 2-3-3. Separation and recovery process The "separation and recovery step" is a step of recovering the embryos after the contact step. This step is a key step in the method for recovering implantation-enhanced embryos of the present invention, in which the embryos after the contact step are separated from the polypeptide solution and the treated embryos to which the polypeptide has been attached are recovered. The purpose of this step is to avoid excessive contact between the embryos and the polypeptide solution in order to avoid damaging the embryos.

[0064] The method for separating the embryo from the polypeptide solution can be carried out by fixing a capillary tube, such as a micropipette or glass tube, to the cell-holding container of the first aspect of the present invention, and then applying suction pressure to the inside of the capillary tube to recover the embryo at the tip or inside of the capillary tube. For example, if the contact step is performed by immersing the embryo in the polypeptide solution, the treated embryo can be removed from the polypeptide solution as described above.

[0065] Thereafter, the embryos may be washed to remove excess polypeptide solution adhering to the embryo surface. As in the contacting step, the liquid used as the washing liquid is not particularly limited as long as it has no or only a slight effect on the embryo. Examples include buffer solutions such as phosphate buffer (PBS buffer), physiological saline, and culture media (particularly the culture media used in the culturing step). Excess polypeptide solution can be removed by washing once or multiple times with this washing liquid. Even if the polypeptide solution is applied or sprayed onto the embryo surface in the contacting step, it can be separated and recovered by washing.

[0066] The embryos after this step can be used for intrauterine transplantation, drug discovery research, etc., depending on the intended use, together with an appropriate solvent such as a buffer solution.

[0067] The embryos with enhanced implantation ability for transplantation recovered by the present invention have a greater amount of RGD motif-containing polypeptides attached to their surface by in vitro treatment compared to normal embryos at the same developmental stage. Due to this characteristic, the embryos of the present invention have high cell adhesiveness, thereby enhancing their implantation ability.

[0068] The embryos for implantation with enhanced implantation ability recovered by the present invention are characterized by having a polypeptide containing an RGD motif attached to their cell surface. Although such a polypeptide is also present on the surface of normal blastocysts, the embryos for implantation with enhanced implantation ability produced by the present invention have a larger amount of the polypeptide than normal embryos of the same stage because the polypeptide has been artificially attached to the surface of the embryo in vitro by the recovery method described in the second aspect.

[0069] The implantation-enhanced embryos for transplantation recovered according to the present invention are in vitro embryos primarily intended for transplantation, and are stored immersed in an appropriate solvent until use. Naturally, the cell-containing container according to the first aspect of the present invention can be used as the storage container. The solvent used here is not particularly limited, as long as it has no or only a slight effect on the embryo. For example, if the storage period is short, a buffer solution such as phosphate buffer (PBS buffer) or physiological saline may be used, and if the storage period exceeds 6 hours, a culture medium is preferred. The method for storing the embryos is not limited, as long as it is a method known in the art. For example, cryopreservation in liquid nitrogen may be used.

[0070] The implantation-enhanced embryos for transplantation recovered according to the present invention can be transplanted into the uterus as embryos with high implantation efficiency, and in the case of non-human animal embryos, can be used for drug discovery, clinical research, etc. [Example]

[0071] The following examples demonstrate specific aspects and performance of the cell-containing container of the present invention. Examples 4 and onward demonstrate specific examples of producing embryos with enhanced implantation ability for transplantation in a method for recovering embryos with enhanced implantation ability. The following examples are merely examples of embodiments of the present invention, and the present invention is not limited to the following examples.

[0072] <Verification of solution recovery effect> (the purpose) We will verify whether the solution can be collected through the communication part provided in the cell storage container of the present invention.

[0073] (method) Example 1 In this example, we confirm the effect of the groove 4 as a communication section for releasing pressure generated by suctioning the tip 8 as a capillary tube. This will be explained using Figure 3. First, a planar substrate 10 was fabricated using polydimethylsiloxane (PDMS). Next, a groove 4 measuring 5 mm in length, 2 mm in width, and 2 mm in depth was fabricated on the planar substrate 10. Furthermore, a 100 μL droplet 9 was formed so as to cover part of the groove 4. Then, using a pipette equipped with the tip 8, the tip of the tip 8 was brought into close contact with the planar substrate 10 so as to include the area where the groove 4 was formed, and the droplet 9 was sucked. As a result, it was confirmed that the droplet 9 could be sucked.

[0074] (Comparative Example 1) This comparative example was carried out in the same manner as in Example 1, except that the groove 4 in Example 1 was not formed. An explanation will be given with reference to FIG. 4. First, a flat substrate 10 was made using polydimethylsiloxane (PDMS). A 100 μL droplet 9 was formed on the flat substrate 10. Thereafter, using a pipette equipped with a tip 8, the tip of the tip 8 was brought into close contact with the flat substrate 10, and the droplet 9 was sucked. As a result, it was confirmed that suction was not possible, and the droplet 9 could not be sucked.

[0075] <Verification of contact between tubules and cells in cell container> (the purpose) The contact between the tubules and the cells in the cell container of the present invention will be verified.

[0076] (method) Example 2 This will be explained using Figures 5 and 6. As shown in Figure 5, a conical microcontainer was made of PDMS. Then, a pipette equipped with a tip 8 was inserted into the microcontainer, and it was confirmed using an optical microscope whether the tip of the tip 8 could contact the bottom of the microcontainer (microwell). As a result, as shown in Figure 6, it was confirmed that the wall of the tip 8 contacted the wall of the container, i.e., the capillary fixing portion 3, and therefore the tip 8 could not be inserted toward the bottom beyond the contact site (capillary fixing portion 3), and the tip of the tip 8 could not contact the bottom. Therefore, according to this example, with the container of the present invention, contact between the tip 8 and cells that may be present at the bottom of the container can be avoided.

[0077] <Verification of solution recovery in cell container> (the purpose) Solution recovery in the cell container of the present invention will be verified.

[0078] (method) Example 3 This will be explained using Figure 7. A conical microcontainer (microwell) 1 was fabricated from PDMS. Next, a groove 4 measuring 5 mm in length, 1 mm in width, and 2 mm in depth was fabricated on the inner wall of the microcontainer 1 to release pressure. Furthermore, 100 μL of particle suspension was dispensed so as to fill the cell storage chamber 6 of the microcontainer 1. After that, a pipette equipped with a tip 8 was inserted up to the capillary fixing part 3, and the particle suspension was aspirated. As a result, it was confirmed that the particle suspension could be aspirated.

[0079] (Comparative Example 2) This comparative example was carried out in the same manner as in Example 3, except that the groove 4 was not formed. An explanation will be given using FIG. 8. A conical microcontainer (microwell) 1 was made of PDMS. 100 μL of particle suspension was dispensed so as to fill the cell storage chamber 6 of the microcontainer 1. A pipette equipped with a tip 8 was then inserted up to the capillary fixing portion 3, and the particle suspension was aspirated. As a result, it was confirmed that the particle suspension could not be aspirated.

[0080] <Verification of cell adhesion in implantation-enhanced embryos> (the purpose) It will be verified that the implantation-enhanced embryos obtained by the recovery method of the present invention have enhanced cell adhesiveness compared to untreated embryos.

[0081] (method) Example 4 The embryos used were frozen blastocyst-stage mouse fertilized eggs (Arc Resources, C57BL / 6J Jcl). As an adhesive protein, a 0.1% fibronectin solution (Sigma-Aldrich, F0895-2MG) was used. The method for preparation was to thaw the frozen mouse fertilized eggs, then culture them in 100 μL of KSOM culture medium (Ark Resources) in a 35 mm dish at 37°C under 5% CO2 for 4 days until they reached class 6 according to the Gardner classification.

[0082] Next, the embryos were removed with a glass pipette, immersed in a 0.1% fibronectin solution (Sigma-Aldrich, F0895-2MG), and incubated at 37°C under 5% CO 2 for 10 minutes to apply fibronectin to the embryo surface.

[0083] The embryos were then removed and seeded into a culture vessel containing 1 mL of DMEM medium (Thermo Fisher Scientific) pre-coated with endometrial epithelial cells (Ishikawa cells, ECACC) at 37°C under 5% CO2. Four, 24, and 48 hours after seeding, the vessel was tapped under a microscope. Embryo adhesion was assessed based on the embryo's movement and its attachment to the endometrial epithelial cells, and a score of 4 was assigned. A score of 4 represented strong adhesion, a score of 3 represented moderate adhesion, a score of 2 represented weak adhesion, and a score of 1 represented no adhesion.

[0084] (Comparative Example 3) In Example 4, the cultured embryos were seeded onto a culture vessel surface-coated with endometrial epithelial cells without immersion in a fibronectin solution, and cultured at 37°C under 5% CO. Four hours, 24 hours, and 48 hours after seeding, tapping stimulation was applied to the culture vessel under a microscope, and the embryo adhesion was evaluated using a four-point score as in Example 4.

[0085] (result) The results are shown in Figure 9. As shown in this figure, the embryos of Example 4, which were immersed in vitro for a short period of time in a high-concentration fibronectin solution of 1 mg / mL, had a significantly higher adhesion stability score than the embryos of Comparative Example 3, demonstrating that cell adhesion was enhanced.

[0086] <Verification of implantation ability based on fibronectin concentration and contact time> (the purpose) We will examine the effect of enhancing cell adhesion of embryos depending on the concentration of fibronectin and the contact time with the embryo.

[0087] (method) The fibronectin concentration conditions and contact time were varied, and the cell adhesiveness of the embryos was examined by comparing with Comparative Example 3 to see if it was enhanced by these combinations.

[0088] Example 5 In Example 4, the cultured embryos were removed with a glass pipette and immersed in a fibronectin solution prepared at 15 μg / mL in phosphate buffer (Thermo Fisher Scientific, 14190144), followed by incubation at 37°C under 5% CO for 10 minutes to apply fibronectin to the embryo surface. Other procedures were the same as in Example 4.

[0089] Example 6: Embryo with enhanced implantation ability for transplantation No. 3 In Example 4, the embryos after culture were removed with a glass pipette and immersed in a 0.1% fibronectin solution (Sigma-Aldrich, F0895-2MG), and then incubated at 37°C under 5% CO for 1 minute to apply fibronectin to the embryo surface. Other procedures were the same as in Example 4.

[0090] Example 7: Embryo with enhanced implantation ability for transplantation No. 4 In Example 4, the embryos after culture were removed with a glass pipette and immersed in a 0.1% fibronectin solution (Sigma-Aldrich, F0895-2MG), and then incubated at 37°C under 5% CO for 60 minutes to apply fibronectin to the embryo surface. Other procedures were the same as in Example 4.

[0091] (result) The results are shown in Table 1.

[0092] [Table 1]

[0093] As shown in Table 1, when the fibronectin concentration was low, such as 15 μg / mL, contact for 10 minutes did not enhance embryonic cell adhesion. Similarly, even when the fibronectin concentration was high, such as 1 mg / mL, contact for a short period of 1 minute did not enhance embryonic cell adhesion. On the other hand, at 1 mg / mL, the desired enhancing effect was confirmed with a contact time of 10 minutes or more.

[0094] From these results, it can be inferred that the embryos of Examples 4 and 7 achieved a particularly significant enhancement in implantation ability. [Prior art documents] [Patent documents]

[0095] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-94045 [Patent Document 2] International Publication No. 2020 / 170727 [Non-patent literature]

[0096] [Non-Patent Document 1] Japan Society of Obstetrics and Gynecology, 2020 Clinical Results of In Vitro Fertilization, Embryo Transfer, etc. [Non-patent document 2] Ministry of Health, Labour and Welfare, Overview of the Annual Total (Rounded Figures) of the Monthly Vital Statistics Report for 2020 (Reiwa 2), Table 4 [Non-patent document 3] Green CJ et al., 2015, Hum. Reprod., 30(2): 284-298 [Non-patent document 4] Larson RC et al., 1992, J. Reprod. Fertil., 96: 289-297 [Non-Patent Document 5] Takeuchi, K. et al., 1998, Journal of Mammalian Egg Research, 5(2): 105-112

Claims

1. A cell-containing container, The container recesses, and a capillary fixing portion for fixing a capillary on or inside the recess; Equipped with The recessed portion is A cell-accommodating chamber formed by the recess, the tip end of the capillary tube, and the capillary tube fixing portion when the capillary tube is fixed Equipped with The cell-containing chamber A communication part that communicates with the outside of the container Equipped with a portion of the inner surface of the cell-containing chamber that comes into contact with the cells is made of a low-adhesion material; When the capillary tube is fixed, the tip of the capillary tube does not come into contact with the cell, the communicating portion equalizes the pressure between the cell-containing chamber and the outside of the container when the capillary is fixed. The container.

2. The container according to claim 1 , wherein the horizontal cross-sectional area of ​​the cell-containing chamber gradually decreases toward the bottom of the cell-containing chamber.

3. 2. The container according to claim 1, wherein the opening area of ​​the upper surface of the recess has an equivalent circle diameter of 0.1 mm to 20 mm.

4. The container of claim 1 , wherein the cell is a single cell.

5. 5. The container of claim 4, wherein the single cell is a placental embryo.

6. The container according to claim 5, wherein the placental embryo is an embryo with enhanced implantation ability.

7. A cell collection kit comprising the container according to any one of claims 1 to 6 and a capillary tube.

8. A method for recovering embryos with enhanced implantation ability, comprising: a step of contacting the surface of a placental embryo with a solution of a polypeptide containing an RGD motif in vitro in the container according to any one of claims 1 to 6 to produce an embryo with enhanced implantation ability; Fixing a capillary to the container; and a step of applying suction pressure to the inside of the tubule to recover the implantation ability enhanced embryos. A method comprising:

9. The method of claim 8, wherein the inner diameter of the tubule is greater than the diameter of the embryo with enhanced implantation competence.

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