WO2022254481A1

WO2022254481A1 - Placental cell culture device, three-dimensional culture model, method for making three-dimensional culture model, and method for evaluating placental cell

Info

Publication number: WO2022254481A1
Application number: PCT/JP2021/020598
Authority: WO
Inventors: 武志堀; 弘和梶; 大成天野倉; 寛明岡江; 隆博有馬
Original assignee: 国立大学法人東北大学
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2022-12-08
Also published as: JPWO2022254481A1

Abstract

This placental cell culture device comprises a placental cell culture section for culturing placental cells and a channel for a factor in which a factor capable of interacting with the placental cells is located, wherein the placental cell culture section is adjacent to the channel for a factor via a partition member in which a shield part is alternately arranged with a communication part, and the minimum width of the communication part is 30 to 500 μm.

Description

Placental cell culture device, three-dimensional culture model, method for producing three-dimensional culture model, and method for evaluating placental cells

The present invention relates to a placental cell culture device, a three-dimensional culture model, a method for producing a three-dimensional culture model, and a method for evaluating placental cells.

In recent years, research and development of three-dimensional cell culture models (organ-on-a-chip devices) that mimic the functions or structures of human tissues or organs have been actively carried out for drug discovery or elucidation of life phenomena. It is In the background of active development, it has become clear that there are many human life phenomena that cannot be analyzed and elucidated by conventional cell culture in petri dishes or animal experiments. Organ chip devices that have been developed so far include the Lung-on-a-chip for pulmonary edema model (Non-Patent Document 1) and the Intestine-on-a-chip for small intestine model (Non-Patent Document 2). In addition, a culture model of the outer blood-retinal barrier using a microchannel chip with a two-layer structure partitioned by a porous membrane has been reported (Patent Document 1).

However, there are organs for which the development of organ chip devices is insufficient. The placenta is one such organ. The placenta is an organ that differs in structure and function between humans and animals. In addition, it is an organ that is difficult to obtain before childbirth. Therefore, the development of a human placental chip device is extremely important for understanding the molecular and cellular biological phenomena in the human placenta, the mechanism of placenta formation, and the mechanism of pathogenesis.

Diseases associated with the placenta include gestational hypertension. Gestational hypertension is a disease in which hypertension occurs between the 20th week of pregnancy and the 12th week of postpartum. Pregnancy hypertension is said to occur in about 1 out of 20 women, and is a major cause of maternal death and perinatal death. The pathogenesis of this disease is complex, but hypoplasia of the spiral arteries in the uterus occurs and blood supply to the placenta is blocked. thought to occur. In other words, it can be interpreted that the maternal body, sensing insufficient blood supply to the placenta, raises blood pressure in order to supply nutrients to the placenta (fetal side). Under normal conditions, extravillous trophoblast cells (EVT cells), which are a type of placental cell, enter maternal spiral arteries and cause 'vascular remodeling' to enlarge the arteries. becomes thicker and more blood is pumped to the placenta. It is known that this vascular remodeling is incomplete in patients with pregnancy-induced hypertension, but a causal relationship with the onset of pregnancy-induced hypertension has not been clarified (Non-Patent Document 3). When conducting research on vascular remodeling by EVT cells using living tissue, not only placental tissue but also endometrial vascular tissue must be analyzed. Therefore, there is an ethical problem of harming the mother's body, and the number of studies is limited. Gestational hypertension is a disease peculiar to humans, and almost no cases of spontaneous onset have been reported even in non-human primates. Therefore, information on pregnancy-induced hypertension from animal studies is limited.
Against this background, in order to elucidate the onset mechanism of pregnancy-induced hypertension, it is desired to develop a human placenta chip device that enables detailed examination of the dynamics of human EVT cells and factors that control human EVT cells.

JP 2020-188723 A

Conventionally, cell dynamics have been evaluated, for example, by whether cells on porous membranes pass through pores. However, with this evaluation system, it is difficult to evaluate the movement (migration) of cells toward the three-dimensional vascular network and the infiltration into the vascular network (invasion) that occur in vivo. In addition, the pore size of polydural membranes was small, and the efficiency of cell migration was low.

Therefore, the present invention provides a placental cell culture device, a three-dimensional culture model using the placental cell culture device, and a three-dimensional culture model that can evaluate the interaction between placental cells and factors that can act on placental cells. An object of the present invention is to provide a production method and a placental cell evaluation method using the placental cell culture device.

The present invention includes the following aspects.
[1] A placental cell culturing unit for culturing placental cells, and a factor channel in which a factor capable of interacting with the placental cells is present, wherein the placental cell culturing unit and the factor channel are: A placental cell culture device, wherein a shielding portion and a communicating portion are adjacent to each other via alternately arranged partition members, and the communicating portion has a minimum width of 30 to 500 μm.
[2] The placental cell culture device according to [1], wherein the partition member is a mesh sheet with a porosity of 50 to 90%.
[3] The placental cell culture device according to [1], wherein the placental cell culture unit is filled with a gel containing a cell aggregate of the placental cells.
[4] A three-dimensional culture model produced using the placental cell culture device according to any one of [1] to [3], wherein the placental cells are cultured in the placental cell culture unit. , a three-dimensional culture model in which a factor capable of interacting with the placental cells is present in the factor channel;
[5] A three-dimensional culture model produced using the placental cell culture device according to [2], wherein the placental cells are cultured in the placental cell culture unit, and the factor channel is three-dimensionally A three-dimensional culture model in which a vascular system is formed.
[6] A three-dimensional culture model produced using the placental cell culture device according to [3], wherein the cell aggregate of the placental cells is cultured in the placental cell culture unit, and the factor flow A three-dimensional culture model in which vascular walls are formed in tracts.
[7] A method for producing a three-dimensional culture model using the placental cell culture device according to [2], comprising culturing vascular endothelial cells in the factor channel to form a three-dimensional vascular system. and a step of culturing the three-dimensional vascular system in the factor channel and culturing placental cells in the placental cell culture unit.
[8] A method for producing a three-dimensional culture model using the placental cell culture device according to [3], comprising a step of culturing vascular endothelial cells in the factor channel to form a vascular wall; A method for producing a three-dimensional culture model, comprising culturing the vascular wall in the factor channel and culturing a cell aggregate of the placental cells in the placental cell culture unit.
[9] Evaluation of placental cells, including co-culturing placental cells with a factor that can interact with placental cells using the placental cell culture device according to any one of [1] to [3] Method.

According to the present invention, a placental cell culture device, a three-dimensional culture model using the placental cell culture device, and the three-dimensional culture model that can evaluate the interaction between placental cells and factors that can act on placental cells. Methods of making and evaluating placental cells using the placental cell culture device are provided.

1 is a perspective view of an embodiment of a placental cell culture device; FIG. FIG. 1B is a cross-sectional view of the placental cell culture device shown in FIG. 1A taken along line BB. FIG. 1B is a cross-sectional view of the placental cell culture device shown in FIG. 1A taken along line CC. 1B is an exploded view of the placental cell culture device shown in FIG. 1A. FIG. 1 is a schematic diagram of a mesh sheet used for a placental cell culture device of one embodiment. FIG. 1B is a top view of the first substrate used in the placental cell culture device shown in FIG. 1A; FIG. 4B is an enlarged view of a portion B surrounded by a broken line in the top view of the first substrate of FIG. 4A; FIG. FIG. 3 is a schematic diagram showing a formation state of a three-dimensional vascular system (E) when vascular endothelial cells are cultured by filling the placental cell culture portion 12 with medium M3 in the placental cell culture device 1. FIG. E: interaction evaluation factor (three-dimensional vasculature). FIG. 4 is a schematic diagram showing the formation state of a three-dimensional vascular system (E) when vascular endothelial cells are cultured without filling the placental cell culture portion 12 with medium M in the placental cell culture device 1. FIG. 1 is a schematic diagram showing an example of a three-dimensional culture model including a three-dimensional vascular system; FIG. An example of a three-dimensional culture model for functional evaluation of EVT cells is shown. EVT: EVT cells. 1 is a schematic diagram showing an example of a three-dimensional culture model including a three-dimensional vascular system; FIG. An example of a three-dimensional culture model for evaluating the permeability of placental cell membranes to chemical substances is shown. ST: ST cells, UD: undifferentiated cells. 1 is a schematic diagram showing an example of a three-dimensional culture model including a three-dimensional vascular system; FIG. An example of a three-dimensional culture model simulating the process of placental development is shown. FIG. 7B is an enlarged view of portion B surrounded by a dashed line in the three-dimensional culture model shown in FIG. 7A. FB: fibroblast. 1 is a perspective view of an embodiment of a placental cell culture device; FIG. FIG. 8B is a cross-sectional view of the placental cell culture device shown in FIG. 8A taken along line BB. 8B is an exploded view of the placental cell culture device shown in FIG. 8A. FIG. 8B is a top view of the first substrate used in the placental cell culture device shown in FIG. 8A; FIG. 10B is an enlarged view of a portion B surrounded by a broken line in the top view of the first substrate of FIG. 10A; FIG. 8B is a top view of the placental cell culture device shown in FIG. 8A. FIG. FIG. 11B is an enlarged view of portion B surrounded by a dashed line in the top view of the placental cell culture device shown in FIG. 11A. C: Placental cells. FIG. 8B is a schematic diagram showing an example of a three-dimensional culture model for evaluating migration of EVT cells using the placental cell culture device shown in FIG. 8A. FIG. 8B is a schematic diagram showing an example of a three-dimensional culture model for evaluating migration of EVT cells using the placental cell culture device shown in FIG. 8A. 2 is a schematic diagram of a second substrate produced in Example 1. FIG. 1 is a microscope image of a mesh sheet used in a placental cell culture device produced in Example 1. FIG. 1 is a placental cell culture device produced in Example 1. FIG. 4 is a microscopic image when colored water is allowed to flow through the central channel of the placental cell culture device provided with the mesh sheet produced in Example 1. FIG. (+) indicates that the placental cell culture device contains a mesh sheet. 1 is a microscope image when colored water is allowed to flow through the central channel of the placental cell culture device without a mesh sheet produced in Example 1. FIG. (-) indicates that the placental cell culture device does not contain a mesh sheet. 1 is a fluorescence microscope image of EVT cells and HUVEC (Human Umbilical Vein Endothelial Cells) cultured using the placenta-cultured cell device prepared in Example 1. FIG. From the 0th day after HUVEC seeding, HUVEC was cultured by filling the central hole of the first substrate with medium. (A) is a fluorescence microscope image taken from the second substrate side of the placental cell device. (B) is a fluorescence microscope image taken from the side of the placental cell device. 1 is a fluorescence microscope image of EVT cells and HUVEC cultured using the placenta-cultured cell device produced in Example 1. FIG. HUVEC were cultured without medium in the central hole for 0-3 days after seeding. (A) is a fluorescence microscope image taken from the second substrate side of the placental cell device. (B) is a fluorescence microscope image taken from the side of the placental cell device. 1 is a schematic diagram of part of a placenta in vivo. FIG. (A) is a schematic diagram of the placenta before CT cells (CT) differentiate into EVT cells (EVT), and (B) shows a part of the CT cells (CT) after differentiation into EVT cells (EVT). 1 is a schematic diagram of a placenta; FIG. UA: uterine artery; BL: blood; BF: blood flow; HA: spiral artery; EVT: EVT cells; ST: ST cells; 4 is a microscopic image of EVT cell-containing cell aggregates prepared in Example 2. FIG. 4 is a fluorescence microscope image of EVT cell-containing cell aggregates prepared in Example 2. FIG. FIG. 10 is a schematic diagram of a first substrate produced in Example 3; 3 is a placenta culture device produced in Example 3. FIG. 4 is a microscopic image of EVT cell-containing cell aggregates cultured using the placenta-cultured cell device prepared in Example 3. FIG. 10 is a graph showing migration directions of EVT cell-containing cell aggregates cultured using the placenta-cultured cell device prepared in Example 3. FIG. The horizontal axis indicates the number of days elapsed after introduction of the EVT cell-containing aggregates into the placental cell culture device. The vertical axis indicates the ratio of the area on the right side (Right) or the area on the left side (Left) based on the dividing line drawn at the start of culture to the total area of the planar image of the cell aggregate. N=3, mean±SE. 4 is a microscopic image of EVT cell-containing cell aggregates co-cultured with HUVEC using the placenta-cultured cell device prepared in Example 3. FIG. EVT cell-containing cell aggregates were co-cultured with HUVEC cells. 10 is a graph showing migration directions of EVT cell-containing cell aggregates co-cultured with HUVECs using the placenta-cultured cell device prepared in Example 3. FIG. The horizontal axis indicates the number of days elapsed after introduction of the EVT cell-containing aggregates into the placental cell culture device. The vertical axis indicates the ratio of the area on the right side (Right) or the area on the left side (Left) based on the dividing line drawn at the start of culture to the total area of the planar image of the cell aggregate. N=4, mean±SE. *p<0.05 (t-test)

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and overlapping descriptions are omitted. The dimensional ratios in each drawing are exaggerated for explanation and do not necessarily match the actual dimensional ratios.

[Placental cell culture device]
In one aspect, the present invention comprises a placental cell culturing unit for culturing placental cells, and a factor channel in which a factor capable of interacting with the placental cells is present, wherein the placental cell culturing unit and the factor A placental cell culture device is provided in which the flow path and the channel are adjacent to each other via a partition member in which the shielding portions and the communication portions are alternately arranged, and the minimum width of the communication portion is 30 to 500 μm.

A placental cell culture device is a device for culturing placental cells. The placental cell culture device includes a placental cell culture section and a factor channel. The placental cell culture section and the factor channel are adjacent to each other via a partition member. The partition member has a structure in which shielding portions and communication portions are alternately arranged, and the minimum width of the communication portion is 30 to 500 μm. Therefore, the placental cells cultured in the placental cell part can interact with the factor present in the factor channel. In addition, the placental cells cultured in the placental cell culture section can pass through the communication section of the partition member and move to the channel for the factor.
The maximum width of the communicating portion is 50 to 500 μm. When the maximum width of the communication portion is within the above range, leakage of medium or the like between the placental cell culture portion and the factor channel can be appropriately controlled.

The placental cell culture department is a culture vessel for culturing placental cells. "Placental cells" refer to cells that constitute the placenta and their progenitor cells. Examples of placental cells include trophoblast stem cells (TS cells), cytotrophoblast cells (CT cells), syncytiotrophoblast cells (ST cells), and EVT cells. is mentioned.

The organism from which placental cells are derived is not particularly limited as long as it is an animal with a placenta. Animals from which placental cells are derived include, for example, mammals such as primates, rodents, and carnivores. Mammals are preferably primates. Primates include, for example, humans, chimpanzees, rhesus monkeys, marmosets, and the like. More preferably, the placental cells are human cells.

Placental cells may be those isolated from the placenta or may be derived from undifferentiated cells.
TS cells may be derived from blastocysts, CT cells, or pluripotent stem cells.
Induction of TS cells from blastocysts can be performed by known methods. For example, mechanical and/or enzymatic treatments are optionally used on placental tissue to separate cells. Then, after culturing the cells in a medium that induces TS cells, TS cells are established using the expression of TS cell markers (GATA2-positive, GATA3-positive, TFAP2-positive, ELF5-positive, ZNF750-positive, CDX2-negative, etc.) as indicators. can be done.
As a method for inducing TS cells from CT cells, for example, the method described in Japanese Patent No. 6400832 can be used.
As a method for inducing TS cells from pluripotent stem cells, for example, the method described in International Publication No. 2020/250438 and the like can be used.

For example, CT cells isolated from the placenta can be used. Isolation of CT cells from the placenta can be achieved, for example, by appropriately using mechanical and/or enzymatic treatments on the placental tissue to separate the cells and determine the expression of CT cell markers (CD49f-positive, E-cadherin-positive, etc.). As an indicator, CT cells can be isolated (Patent No. 6400832, Haider, S., et al., Stem Cell Reports 11, 537-551 (2018).)

ST cells may be isolated from the placenta or derived from TS cells. Isolation of ST cells from the placenta, for example, is performed by appropriately using mechanical and/or enzymatic treatments on the placental tissue to separate the cells and detect ST cell markers (Syndecan 1 (SCD1) positive, human chorionic gonadotropin (hCG ) positive, etc.) can be used as an index to isolate ST cells.
Methods for inducing ST cells from TS cells include, for example, the methods described in International Publication No. 2020/250438 and the like.

EVT cells may be isolated from placenta or may be derived from TS cells. Isolation of EVT cells from the placenta can be achieved, for example, by appropriately using mechanical and/or enzymatic treatment on the placental tissue to separate the cells, and using the expression of an EVT cell marker (such as HLA-G positive) as an index, EVT cells can be isolated.
Methods for inducing EVT cells from TS cells include, for example, the methods described in International Publication No. 2020/250438.

The factor channel is a channel for the presence of factors that can interact with placental cells. A "factor capable of interacting with placental cells" refers to a factor whose interaction with placental cells is evaluated. Factors that can interact with placental cells (hereinafter also referred to as "interaction evaluation factors") may be factors that are known to interact with placental cells, or factors that are unknown to interact with placental cells. It can be a factor. The interaction evaluation factor may be a cell, a biological substance, or an ex vivo substance. Examples of interaction evaluation factors include vascular endothelial cells (eg, human umbilical vein endothelial cells (HUVEC)), various hormones, various cytokines, various drugs, immune cells, and the like.

<First Embodiment>
1A to 4B show an example of the placental cell culture device 1 of the first embodiment. In this embodiment, a mesh sheet with a porosity of 50 to 90% is used as a partition member between the placental cell culture portion and the factor channel. FIG. 1A is a perspective view of placental cell culture device 1. FIG. FIG. 1B is a cross-sectional view of the placental cell culture device 1 taken along line BB. FIG. 1C is a cross-sectional view of the placental cell culture device 1 taken along line CC. FIG. 2 is an exploded view of the placental cell culture device 1. FIG.

The placental cell culture device 1 is composed of a first substrate 10, a second substrate 20, a mesh sheet 30, and a thin film sheet 40. Placental cell culture device 1 is formed by laminating second substrate 20, thin film sheet 40, mesh sheet 30, and first substrate 10 in this order.

The placental cell culture device 1 includes a central channel 21, a first side channel 22a, and a second side channel 22b. The placental cell culture device 1 is provided with a first port P1, a second port P2, a third port P3, a fourth port P4, a fifth port P5, and a sixth port P6 as introduction/exhaust ports for culture medium or the like. there is The first port P1 is connected to the first side flow path 22a through the first port flow path p1. The second port P2 is connected to the first side flow path 22a through a second port flow path p2. The third port P3 is connected to the second side flow path 22b through a third port flow path p3. The fourth port P4 is connected to the second side flow path 22b through a fourth port flow path p4. The fifth port P5 and sixth port P6 are connected to the central channel 21 . The fifth port P5 is provided at one end of the central flow passage 21, and the sixth port P6 is provided at the other end of the central flow passage 21. As shown in FIG.

The first port P1 is composed of a through hole P1a provided in the first substrate 10 and a recess P1b provided in the second substrate 20. As shown in FIG. The second port P2 is composed of a through hole P2a provided in the first substrate 10 and a recess P2b provided in the second substrate 20. As shown in FIG. The third port P3 is composed of a through hole P3a provided in the first substrate 10 and a recess P3b provided in the second substrate 20. As shown in FIG. The fourth port P4 is composed of a through hole P4a provided in the first substrate 10 and a recess P4b provided in the second substrate 20. As shown in FIG. The fifth port P5 is composed of a through hole P5a provided in the first substrate 10 and a recess P5b provided in the second substrate 20. As shown in FIG. The sixth port P6 is composed of a through hole P6a provided in the first substrate 10 and a recess P6b provided in the second substrate 20. As shown in FIG.
The central channel 21 , the first port channel p<b>1 , the second port channel p<b>2 , the third port channel p<b>3 , and the fourth port channel p<b>4 are provided on the second substrate 20 .

In the placental cell culture device 1, the central channel 21 functions as a factor channel. Placental cell culture section 12 is provided above central channel 21, which is a channel for factors. The placental cell culture portion 12 has a side portion formed by the central hole 11 provided in the first substrate 10 , and a bottom portion formed by the mesh sheet 30 and the thin film sheet 40 . Placental cell culture section 12 and central channel 21 are adjacent to each other with mesh sheet 30 interposed therebetween. In the placental cell culture device 1, the mesh sheet 30 functions as a partition member that separates the placental cell culture section 12 and the channel for factor (central channel 21).

(First substrate)
The first substrate 10 is provided with through holes P1a to P6a and a central hole 11. As shown in FIG. The through holes P1a to P6a are formed at positions that overlap with the recessed portions P1b to P6b of the second substrate 20, respectively, when stacked on the second substrate 20. As shown in FIG.

The central hole 11 is a through hole provided substantially in the center of the second substrate 20 . In placental cell culture device 1 , central hole 11 forms placental cell culture part 12 together with mesh sheet 30 and thin film sheet 40 . The shape of the central hole 11 is not particularly limited. The planar shape of the central hole includes, for example, a circular shape, an elliptical shape, a polygonal shape (square shape, pentagonal shape, hexagonal shape, etc.), and the like. Although the size of the central hole 11 is not particularly limited, for example, both the minimum diameter and the maximum diameter are 1 to 20 mm. The size of the central hole 11 is preferably 2 mm or more, more preferably 3 mm or more, even more preferably 4 mm or more, and particularly preferably 5 mm or more, for both the minimum diameter and the maximum diameter. The size of the central hole 11 is preferably 18 mm or less, more preferably 15 mm or less, still more preferably 12 mm or less, and particularly preferably 10 mm or less, for each of the minimum diameter, b, and maximum diameter, for example.

Although the thickness of the first substrate 10 is not particularly limited, it may be 1 to 50 mm, for example. For example, the thickness of the first substrate 10 is preferably 2 mm or more, more preferably 3 mm or more, still more preferably 4 mm or more, and particularly preferably 5 mm or more. For example, the thickness of the first substrate 10 is preferably 40 mm or less, more preferably 30 mm or less, even more preferably 20 mm or less, and particularly preferably 15 mm or less.

(Second substrate)
4A is a top view of the second substrate 20. FIG. FIG. 4B is an enlarged view of a portion B surrounded by a dashed line in the top view of the second substrate 20 shown in FIG. 4A.

The second substrate 20 is formed with recesses P1b to P6b, a first port channel p1 to a fourth port channel p4, a central channel 21, a first side channel 22a, and a second side channel 22b. .
The recesses P1b to P6b are formed at positions overlapping the through holes P1a to P6a of the first substrate 10 when the first substrates 10 are stacked.

The central channel 21 is formed substantially in the center of the second substrate 20 . In the placental cell culture device 1 , the central channel 21 functions as a factor channel for allowing interaction evaluation factors to exist. The central flow path 21 is formed at a position at least partially overlapping with the central hole 11 of the first substrate 10 when the first substrates 10 are laminated. Both ends of the central flow path 21 are provided with recesses P5b and P6b.

A first side channel 22a and a second side channel 22b are formed on both sides of the central channel 21, respectively. A first port flow path p1 is connected to one end of the first side flow path 22a, and a second port flow path p2 is connected to the other end of the first side flow path 22a. A third port flow path p3 is connected to one end of the second side flow path 22b, and a fourth port flow path p4 is connected to the other end of the second side flow path 22b. The first side flow channel 22a and the second side flow channel 22b may be formed on both sides over the entire length of the central flow channel 21, or may be formed on both sides of a portion of the central flow channel 21. . When the first side flow channel 22a and the second side flow channel 22b are formed on both sides of a part of the central flow channel 21, at least on both sides of the part where the central flow channel 21 contacts the placental cell culture section 12, It is preferable that the first side flow path 22a and the second side flow path 22b are formed respectively.

Although the width of the central channel 21 is not particularly limited, it may be, for example, 500 to 5000 μm. The width of the central channel 21 is preferably 700 μm or more, more preferably 800 μm or more, still more preferably 1000 μm or more, and particularly preferably 1200 μm or more. The width of the central channel 21 is preferably 4000 μm or less, more preferably 3000 μm or less, even more preferably 2000 μm or less, and particularly preferably 1800 μm or less.

The widths of the first side flow channel 22a and the second side flow channel 22b are not particularly limited, but may be 500 to 3000 μm, for example. The width of the first side flow channel 22a and the second side flow channel 22b is preferably 600 μm or more, more preferably 700 μm or more, even more preferably 800 μm or more, and particularly preferably 900 μm or more. The width of the first side flow channel 22a and the second side flow channel 22b is preferably 2500 μm or less, more preferably 2000 μm or less, still more preferably 1500 μm or less, and particularly preferably 1200 μm or less. The first side flow channel 22a and the second side flow channel 22b may have the same width or different widths.

The height (depth) of the central flow channel 21, the first side flow channel 22a, and the second side flow channel 22b is not particularly limited, but is, for example, 50 to 1000 μm. The height (depth) of the central flow channel 21, the first side flow channel 22a, and the second side flow channel 22b is preferably 60 μm or more, more preferably 80 μm or more, even more preferably 100 μm or more, and 150 μm or more. is particularly preferred. The height (depth) of the central flow channel 21, the first side flow channel 22a, and the second side flow channel 22b is preferably 800 µm or less, more preferably 600 µm or less, even more preferably 500 µm or less, and 300 µm or less. is particularly preferred.
The height (depth) of the central flow channel 21, the first side flow channel 22a, and the second side flow channel 22b are preferably substantially the same.

In the central flow path 21, the length of the portion where the first side flow path 22a and the second side flow path 22b are adjacent is not particularly limited, but for example, it is 500 to 30000 μm. For example, the length is preferably 600 μm or more, more preferably 700 μm or more, still more preferably 800 μm or more, and particularly preferably 900 μm or more. For example, the length is preferably 25000 μm or less, more preferably 200000 μm or less, even more preferably 15000 μm or less, and particularly preferably 10000 μm or less.

The central channel 21 and the first side channel 22a are partitioned by a partition member 23a. The central channel 21 and the second side channel 22b are partitioned by a partition member 23b. The partitioning member 23a and the partitioning member 23b are composed of a plurality of microposts 24. As shown in FIG. A plurality of microposts 24 constituting the partition member 23a and the partition member 23b are arranged at predetermined intervals. In the partitioning member 23a and the partitioning member 23b, the microposts 24 constitute shielding portions, and the gaps between the microposts 24 constitute communicating portions.
The shape of the microposts 24 is not particularly limited. The microposts 24 may have a polygonal prism shape (triangular prism, square prism, etc.) or a cylindrical shape. The micropost 24 has, for example, a trapezoidal prism shape. It is preferable that the width w2 of the microposts 24 on the first side channel 22a side or the second side channel 22b side is larger than the width w1 on the central channel 21 side. As a result, the liquid filled in the central flow path 21 can be prevented from leaking into the first side flow path 22a and the second side flow path 22b.

The size of the microposts 24 is not particularly limited, but the width w1 on the side of the central channel 21 is, for example, 20 to 300 μm. For example, the width w1 is preferably 25 μm or more, more preferably 30 μm or more, still more preferably 35 μm or more, and particularly preferably 40 μm or more. The width w1 is, for example, preferably 200 μm or less, more preferably 150 μm or less, still more preferably 100 μm or less, and particularly preferably 80 μm or less.
The width w2 on the side of the first side flow path 22a or the side of the second side flow path 22b is, for example, 50 to 500 μm. The width w2 is, for example, preferably 60 μm or more, more preferably 70 μm or more, even more preferably 80 μm or more, and particularly preferably 90 μm or more. The width w2 is, for example, preferably 400 μm or less, more preferably 300 μm or less, still more preferably 200 μm or less, and particularly preferably 180 μm or less.
The length l of the microposts 24 may be 50-800 μm. For example, the length l is preferably 60 μm or more, more preferably 65 μm or more, still more preferably 70 μm or more, and particularly preferably 75 μm or more. For example, the length l is preferably 600 μm or less, more preferably 500 μm or less, even more preferably 400 μm or less, and particularly preferably 300 μm or less.
The height of the microposts 24 is preferably the same as the height (depth) of the central channel 21 .

The shortest distance d between two microposts 24 corresponds to the minimum width of the communicating portion of the partition member. The shortest distance d is, for example, 30 to 500 μm. The shortest distance d is, for example, preferably 40 μm or longer, more preferably 50 μm or longer, still more preferably 60 μm or longer, and particularly preferably 70 μm or longer. The shortest distance d is, for example, preferably 400 μm or less, more preferably 350 μm or less, still more preferably 300 μm or less, and particularly preferably 250 μm or less.

The longest distance between two microposts 24 corresponds to the maximum width of the communicating portion of the partition member. Examples of the longest distance include 50 to 500 μm. The longest distance is, for example, preferably 60 μm or longer, more preferably 70 μm or longer, still more preferably 80 μm or longer, and particularly preferably 90 μm or longer. The longest distance is, for example, preferably 400 μm or less, more preferably 350 μm or less, even more preferably 300 μm or less, and particularly preferably 250 μm or less.

The channel surfaces of the central channel 21, the first side channel 22a, and the second side channel 22b are preferably hydrophobic. If the surface of the channel is hydrophobic, surface tension of the liquid is generated between the microposts 24 when any one of the channels is filled with liquid. This prevents liquid from leaking into the adjacent channel when there is no liquid in the adjacent channel.

Although the thickness of the second substrate 20 is not particularly limited, it may be 0.1 to 50 mm, for example. For example, the thickness of the second substrate 20 is preferably 0.3 mm or more, more preferably 0.5 mm or more, still more preferably 0.6 mm or more, and particularly preferably 0.8 mm or more. For example, the thickness of the second substrate 20 is preferably 40 mm or less, more preferably 20 mm or less, even more preferably 10 mm or less, and particularly preferably 5 mm or less.

Materials for the first substrate 10 and the second substrate 20 are not particularly limited, but materials having high biocompatibility and high oxygen permeability are preferable. "Oxygen permeability" means the property of being permeable to molecular oxygen. By using an oxygen-permeable material for the first substrate 10 and the second substrate 20, oxygen can reach the inside of each channel. The oxygen permeability is, for example, about 100 to 5000 cm ³ /m ² ·24 h·atm. The oxygen permeability is preferably about 1100-3000 cm ³ /m ² ·24 h·atm, or about 1250-2750 cm ³ /m ² ·24 h·atm. "cm ³ /m ² ·24 h·atm" represents the capacity (cm ³ ) of oxygen per 1 m ² that permeates in 24 hours under an environment of 1 atmospheric pressure.
Oxygen permeable materials include oxygen permeable polymers. Examples of oxygen-permeable polymers include fluororesins and silicones (eg, polydimethylsiloxane (PDMS), etc.). Among them, the oxygen-permeable polymer is preferably PDMS.

The first substrate 10 and the second substrate 20 can be produced using known methods such as photolithography, soft lithography, microcontact printing, microfluidic, and stencil methods. For example, a mold for the first substrate 10 and the second substrate 20 can be produced using a photolithography method, and the first substrate 10 and the second substrate 20 can be produced using the mold using a soft lithography method.

More specifically, a photoresist film is formed by coating a wafer such as a silicon wafer with a photoresist by spin coating or the like. Next, the photoresist film is exposed through a photomask for the first substrate 10 or the second substrate 20 . Next, the unexposed portion of the photoresist film is removed with a developer to obtain the mold for the first substrate 10 or the mold for the second substrate 20 .
Next, the curable composition is poured into the prepared mold for the first substrate 10 or the mold for the second substrate 20 and cured. The first substrate 10 or the second substrate 20 can be obtained by removing the cured product from the mold and processing it appropriately.

(mesh sheet)
In the placental cell culture device 1 , the placental cell culture portion 12 and the central channel 21 as a channel for factors are at least partially separated by the mesh sheet 30 . In the placental cell culture device 1, the mesh sheet 30 functions as a partition member that separates the placental cell culture portion 12 and the central channel 21, which is the channel for factors.
The partition member that separates the placental cell culture section 12 and the factor channel has a structure in which shielding sections and communicating sections are alternately arranged. In the partition member, the minimum width of the communicating portion is 30 to 500 μm. In the partition member, the maximum width of the communicating portion is, for example, 50 to 500 μm. When the minimum width and the maximum width of the communicating portion are within the above range, placental cells can easily pass through the communicating portion. In addition, it is possible to prevent the liquid filled in the central channel 21 of the second substrate 20 from leaking into the placental cell culture section 12 .

FIG. 3 shows a mesh sheet 30. FIG. The mesh sheet 30 is configured by arranging linear members 31 in a mesh pattern. In the mesh sheet 30, the linear member 31 constitutes the shielding portion of the partition member, and the opening 32 constitutes the communicating portion of the partition member. The opening 32 has a rectangular shape. In the mesh sheet 30, the length of the diagonals of the rectangles forming the openings 32 corresponds to the maximum width of the communicating portion of the partition member. The maximum width of the communicating portion is preferably 120 μm or more, more preferably 130 μm or more, still more preferably 140 μm or more, and particularly preferably 150 μm or more. The maximum width of the communicating portion is preferably 400 μm or less, more preferably 300 μm or less, still more preferably 250 μm or less, and particularly preferably 200 μm or less.
In the mesh sheet 30, the length W1 of the short sides of the rectangles forming the openings 32 corresponds to the minimum width of the communicating portion of the partition member. The minimum width of the communicating portion is not particularly limited as long as it is in the range of 30-500 μm. When the minimum width of the communicating portion is within the above range, the placental cell migration efficiency is favorably maintained. The minimum width of the communicating portion is, for example, preferably 40 μm or more, more preferably 50 μm or more, still more preferably 60 μm or more, and particularly preferably 70 μm or more. The upper limit of the minimum width of the communicating portion is 500 μm or less. The minimum width of the communicating portion is preferably 400 μm or less, more preferably 300 μm or less, still more preferably 200 μm or less, and particularly preferably 150 μm or less.
The length W2 of the long side of the rectangle forming the opening 32 is, for example, preferably 80 μm or longer, more preferably 100 μm or longer, even more preferably 120 μm or longer, and particularly preferably 150 μm or longer. The upper limit of the length W2 of the long side is 500 μm or less. The long side W2 is preferably 400 μm or less, more preferably 300 μm or less, still more preferably 250 μm or less, and particularly preferably 200 μm or less.
The shape of the opening of the mesh sheet, which is the partition member, is not limited to a rectangular shape like the opening 32 . The shape of the openings of the mesh sheet may be, for example, a square shape, a rhombus shape, a parallelogram shape, or the like.

The mesh sheet 30 has a porosity (opening ratio) of 50 to 90%. “Porosity (opening ratio)” refers to the ratio of the area of the openings to the area of the entire mesh sheet 30 . For example, the porosity (%) is expressed by the formula: "short side W1 of opening 32 x long side W2 of opening 32 x number of openings 32)/area of mesh sheet 30 x 100". can ask. Since the mesh sheet 30 has a high porosity as described above, placental cells can easily pass through the openings 32 . Therefore, the transfer efficiency of placental cells from the placental cell culture section to the central channel 21 is high. The porosity (opening ratio) of the mesh sheet 30 is preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more.

The material of the mesh sheet is not particularly limited, but a material with high biocompatibility is preferable. Examples of materials for the mesh sheet include synthetic resins, natural fibers, glass fibers, carbon fibers, ceramics, and metals. Examples of synthetic resins include water-insoluble resins such as polyethylene, polypropylene, polyester, fluororesin, and polyamide. Examples of natural fibers include vegetable fibers such as cotton, hemp, bamboo and paper; animal fibers such as wool and silk. The mesh sheet may be a blend of synthetic resin fibers and natural fibers. Ceramics include alumina (Al ₂ O ₃ ) and the like. Metals include stainless steel, steel, aluminum, gold, platinum and the like. Also, these materials may be modified. Examples of such modifications include hydrophilic treatment, coating with extracellular matrix (eg, collagen, fibronectin, etc.), and the like.

The mesh sheet is not particularly limited as long as the linear members are formed in a mesh shape. The mesh sheet is formed into a mesh shape by woven fabric (single raschel or double raschel), knitting, cross-point welding type, extruded sheet (trical net, netron sheet, etc.), punching processing, or the like. good.

(thin film sheet)
In the placental cell culture device 1, the thin film sheet 40 separates the placental cell culture section 12 from the first side channel 22a and the second side channel 22b. The thin film sheet 40 has thin film openings 41 . In placental cell culture device 1 , thin film sheet 40 is arranged such that thin film opening 41 overlaps central channel 21 . In the thin film opening 41 , the central hole 11 and the central channel 21 are adjacent to each other with only the mesh sheet 30 interposed therebetween without the thin film sheet 40 interposed therebetween. The membrane opening 41 can be sized according to the central channel 21 in the second substrate 20 . The width of the membrane opening 41 is preferably slightly smaller than the width of the central channel 21 . The width of the thin film opening 41 is, for example, 100 to 3000 μm. The width of the thin film opening 41 is preferably 200 μm or more, more preferably 300 μm or more, still more preferably 4000 μm or more, and particularly preferably 450 μm or more. The width of the thin film opening 41 is preferably 2000 μm or less, more preferably 1800 μm or less, still more preferably 1600 μm or less, and particularly preferably 1400 μm or less.
The length of membrane opening 41 is preferably shorter than the length of central channel 21 . The length of the thin film opening 41 is, for example, 500 to 10000 μm. The length of the thin film opening 41 is, for example, preferably 1000 μm or longer, more preferably 1500 μm or longer, even more preferably 2000 μm or longer, and particularly preferably 2500 μm or longer. For example, the length of the thin film opening 41 is preferably 8000 μm or less, more preferably 7000 μm or less, even more preferably 6000 μm or less, and particularly preferably 5000 μm or less.

The material of the thin film sheet 40 is not particularly limited, but a material with high biocompatibility is preferable. Examples of materials for the thin film sheet 40 include polyimide, polyethylene terephthalate (PET), polystyrene, polyethylene, polypropylene, nylon, polytetrafluoroethylene (PTFE), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene- Hexafluoropropylene copolymer (FEP), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), polydimethylsiloxane (PDMS), and the like, but are not limited to these. For the thin film sheet 40, for example, a resin film made of the above materials can be used.

The placental cell culture device 1 can be produced by laminating and bonding the second substrate 20, the thin film sheet 40, the mesh sheet 30, and the first substrate 10 in this order. When assembling the placental cell culture device 1, the through holes P1a to Pa6 of the first substrate 10 and the recesses P1b to P6b of the second substrate 20 are aligned with each other. Also, the central channel 21 of the second substrate 20 and the thin film opening 41 of the thin film sheet 40 are at least partially aligned. At least part of the central hole 11 of the first substrate 10 and the central channel 21 of the second substrate 20 are arranged to be adjacent to each other with the mesh sheet 30 interposed therebetween.

When assembling the placental cell culture device 1, the surfaces of the first substrate 10 and the second substrate 20 may be activated to enhance adhesiveness. For example, if the first substrate 10 and the second substrate 20 are made of PDMS, the surfaces can be activated by oxygen plasma treatment to enhance adhesion. In this case, it is preferable to perform a hydrophobizing treatment on the surface of the flow path after assembly. When the first substrate 10 and the second substrate 20 are made of PDMS, the substrate surface including the channel surface can be hydrophobized by heat treatment (for example, 80° C. overnight).

(Three-dimensional culture model)
The placental cell culture device of this embodiment can be used to produce a three-dimensional culture model containing placental cells. In the three-dimensional culture model, placental cells are cultured in the placental cell culture section, and interaction evaluation factors are present in the factor channel.

≪Interaction evaluation factor≫
In the placental cell culture device 1, the central channel 21 functions as a factor channel. The interaction evaluation factor is not particularly limited, and any factor can be selected according to the purpose. The interaction evaluation factors present in the central channel 21 may be of one type or a combination of two or more types.

The interaction evaluation factor can be dissolved or suspended in a buffer solution (PBS, medium, etc.) and introduced into the central flow channel 21 by injecting it from the fifth port P5 or the sixth port P6. A gelling agent may be added to a buffer solution or the like for dissolving or suspending the interaction evaluation factor, and gelation may be performed after filling the central channel 21 . As a result, the central channel 21 is filled with the gel containing the interaction evaluation factor. In the placental cell culture device 1, the central channel 21 and the placental cell culture section 12 are separated by the mesh sheet 30, so that the solution or suspension introduced into the central channel 21 is transferred to the placental cell culture section 12. No leakage.

Although the gelling agent is not particularly limited, it is preferable to use one with high biocompatibility. Examples of gelling agents include collagen (type I, type II, type III, type V, type XI, etc.), gelatin, elastin, proteoglycan, glycosaminoglycan, fibronectin, vitronectin, laminin, pectin, hyaluronic acid, chitin. , and extracellular matrices such as chitosan; polysaccharides such as alginic acid and starch; and amino acid polymers such as polylysine and polyarginine; fibrous proteins such as fibrin; A commercially available scaffold such as Matrigel® (CORNING) may also be used. When fibrin is used as a gelling agent, it may be prepared as a mixture of fibrinogen and thrombin. In this case, thrombin converts fibrinogen into fibrin and gels.

After introducing the interaction evaluation factor into the central channel 21, it is preferable to introduce the culture medium into the first side channel 22a and the second side channel 22b. The medium can be introduced into the first side channel 22a by injecting the medium from the first port P1 or the second port P2. The medium can be introduced into the second side channel 22b by injecting the medium from the third port P3 or the fourth port P4. By filling the medium in the first side channel 22a and the second side channel 22b, the gel filled in the central channel 21 can be prevented from drying out. Further, when the interaction evaluation factor is a cell, a component necessary for the survival of the interaction evaluation factor present in the central channel 21, a component controlling the interaction evaluation factor, etc. are supplied to the interaction evaluation factor. can be done.

After introducing the interaction evaluation factor into the central flow path 21, the placental cell culture section 12 may be filled with medium. When the interaction-evaluating factor is a cell, the growth of the interaction-evaluating factor can be controlled by filling the placental cell culture section 12 with medium. For example, when the interaction evaluation factor is vascular endothelial cells such as HUVEC, the formation of a three-dimensional vascular system in the central channel 21 can be controlled by filling or not filling the placental cell culture section 12 with medium. .

FIG. 5A shows that after filling gel G containing vascular endothelial cells (HUVEC, etc.) in central channel 21, media M1 to M3 are added to first side channel 22a, second side channel 22b, and placental cell culture section 12. are respectively filled to form a three-dimensional vascular system (E). When the medium M3 is present in the placental cell culture section 12, a vascular system is formed not only in the direction of both side channels but also in the direction of the placental cell culture section 12. The three-dimensional vasculature functions as an interaction evaluation factor and is indicated by E in the figure.

FIG. 5B shows that after the central channel 21 is filled with a gel G containing vascular endothelial cells (HUVEC, etc.), the first side channel 22a and the second side channel 22b are filled with media M1 and M2, respectively, and placenta FIG. 10 is a schematic diagram when a three-dimensional vascular system (E) is formed without filling the cell culture section 12 with a culture medium. If no culture medium exists in the placental cell culture section 12 , a vascular system is formed in the direction of both side channels, but no vascular system is formed in the direction of the placental cell culture section 12 .

Fig. 18 is a schematic diagram of EVT cells and maternal blood vessels. EVT cells (EVT) are thought to arise from CT cells (CT) in contact with the decidua (DM) and are found on and within the decidua (HA). In the decidua (DM), there are blood vessels (spiral arteries: HA) for sending blood (BL) to the villus (V) side, and spiral arteries ( HA) exit. EVT cells (EVT) infiltrate the inner wall of blood vessels from the outlet of spiral arteries (HA) and reconstruct thick blood vessels. On the other hand, EVT cells (EVT) that have infiltrated the decidua (DM) are thought to migrate toward the spiral artery (HA) and infiltrate from the outside of the spiral artery (HA) into the inside of the blood vessel. The three-dimensional vascular system in FIG. 5A can be used to create a three-dimensional culture model in which EVT cells invade blood vessels from the arterial exit side. The three-dimensional vasculature of FIG. 5B can be used to create a three-dimensional culture model in which EVT cells that have invaded the decidua invade blood vessels.

The three-dimensional vascular system is formed by filling gel G containing vascular endothelial cells (HUVEC, etc.) in central channel 21, and then adding medium M1 and medium M2 to first side channel 22a and second side channel 22b. It can be performed by filling each and culturing. When the placental cell culture section 12 is filled with medium M3 during the culture period, a three-dimensional vascular system as shown in FIG. 5A can be formed. If the placental cell culture section 12 is not filled with medium M3 during the culture period, a three-dimensional vascular system as shown in FIG. 5B can be formed. The culture period is not particularly limited, and the culture may be continued until the three-dimensional vascular system reaches a desired state. The culture period can be, for example, 1 day or longer, 2 days or longer, or 3 days or longer. The upper limit of the culture period is not particularly limited, but can be, for example, 10 days or less, 8 days or less, or 5 days or less.

The culture conditions can be those generally used for culturing animal cells. For example, the culture temperature can be 32-40° C. (preferably 35-38° C.) and the CO ₂ concentration can be 2-5% (preferably 5%).

The medium M1 introduced into the first side flow channel 22a, the medium M2 introduced into the second side flow channel 22b, and the medium M3 introduced into the placental cell culture unit 12 are appropriately selected according to the type of interaction evaluation factor. can be done. The media M1 to M3 may be the same media or different media. When the interaction evaluation factor is a vascular endothelial cell such as HUVEC, the media M1 to M3 include a medium containing a vascular endothelial growth factor (VEGF) and the like, a culture supernatant of fibroblasts (described later). The three-dimensional blood vessel culture medium used in the example of 1) and the like can be used.

≪Placental cells≫
Placental cells are cultured in the placental cell culture unit 12 . The type of placental cells to be cultured is not particularly limited, and any placental cells can be used depending on the purpose. Placental cells cultured in the placental cell culture unit 12 may be of one type, or may be of two or more types. Placental cells may or may not form cell aggregates. The placental cells may be cells attached to the bottom of the placental cell culture unit 12 (mesh sheet 30 or thin film sheet 40), or planar cell aggregates (eg, sheet-like cell aggregates). It may well be a three-dimensional cell aggregate (eg, spherical cell aggregate, cell aggregate forming an organoid). A cell aggregate may be composed of one type of cell, or may be composed of two or more types of cells.

• Culture medium for placental cells The medium used for culturing placental cells can be appropriately selected depending on the purpose. As the medium, for example, a basal medium generally used for culturing animal cells can be used. The medium used for culturing placental cells may be a basal medium supplemented with growth factors, various enzyme inhibitors, and the like.

Examples of basal media include Doulbecco's modified Eagle's Medium (DMEM) medium, DMEM/F12 medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Ham's F12 medium, Examples include RPMI1640 medium, Fischer's medium, and mixed medium thereof. Preferred basal media include, for example, DMEM/F12.

The basal medium may contain serum (such as fetal bovine serum (FBS)) or a serum substitute, if necessary. Serum replacements include, for example, albumin, transferrin, sodium selenite, ITS-X (Invitrogen), knockout serum replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, Insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, etc. The basal medium optionally contains lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth It may contain components such as factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. These can be used in combination as appropriate.

As a basal medium, for example, bovine serum albumin (BSA), ITS-X, L-ascorbic acid, and antibiotics (penicillin, streptomycin, etc.) are added to the above basal medium (eg, DMEM/F12). media. Specific examples of the basal medium include the TS basal medium and 2D-EVT basal medium used in Examples described later.

The growth factor is not particularly limited, but examples include epidermal growth factor (EGF), fibroblast growth factor (FGF), bone morphogenic protein (BMP), neuregulin ( Neuregulin: NGR) and the like. When the medium contains FGF, the medium may contain heparin. Heparin has the effect of promoting the activity of FGF. Heparin is preferably in the form of a salt.

Examples of enzyme inhibitors include ROCK inhibitors (Y27632, etc.), GSK3β inhibitors (CHIR99021, etc.), p38 MAPK inhibitors (SB202190, etc.), HDAC inhibitors (valproic acid: VPA, etc.), ALK inhibitors (A83- 01 etc.).

For example, when the placental cells are TS cells, TS medium can be used. When TS cells are differentiated into EVT cells, 2D-EVT medium can be used.

Culture conditions for placental cells can be those generally used for culturing animal cells. For example, the culture temperature can be 32-40° C. (preferably 35-38° C.) and the CO ₂ concentration can be 2-5% (preferably 5%).

In the three-dimensional culture model using the placental cell culture device 1, the central channel 21 and the placental cell culture section 12 are separated by the mesh sheet 30. Therefore, the placental cells of the placental cell culture section 12 and the interaction evaluation factor of the central channel 21 can interact via the openings 32 of the mesh sheet 30 . Also, the placental cells in the placental cell culture unit 12 can easily move to the central channel 21 through the openings 32 of the mesh sheet 30 . Therefore, the three-dimensional culture model of this embodiment can be used to evaluate interactions between placental cells and interaction evaluation factors.

Figures 6A-7B show an application example of the three-dimensional culture model.
FIG. 6A is an example of a three-dimensional culture model for functional evaluation of EVT cells. In the three-dimensional culture model of FIG. 6A, a three-dimensional vascular system (E) is formed in the central channel 21 and EVT cells (EVT) are cultured in the placental cell culture section 12 . EVT cells (EVT) cultured in the placental cell culture unit 12 can move to the central channel 21 through the openings 32 of the mesh sheet 30 . In the three-dimensional culture model of FIG. 6A, the infiltration of EVT cells into spiral arteries can be evaluated. In the placental cell culture unit 12, undifferentiated cells such as TS cells or CT cells may be cultured using a differentiation-inducing medium for EVT cells to induce differentiation into EVT cells.

FIG. 6B shows an example of a three-dimensional culture model for evaluating chemical substance permeability to ST cell membranes. In the three-dimensional culture model of FIG. 6B, a three-dimensional vascular system (E) is formed in the central channel 21 and placental cell membranes are cultured in the placental cell culture section 12 . Placental cell membranes have a structure that mimics in vivo villi, and are composed of upper layer ST cells (ST) and lower layer undifferentiated cells (UD) (TS cells, CT cells, etc.). In the three-dimensional culture model of FIG. 6B, placental cell membranes are cultured so as to cover the mesh sheet 30 exposed to the placental cell culture section 12 . By adding an arbitrary chemical substance to the medium M3 of the placental cell culture unit 12, it is possible to evaluate whether or not the chemical substance permeates the placental cell membrane. Placental cell membranes can be prepared, for example, by attaching undifferentiated cells (TS cells, CT cells, etc.) to a scaffold material such as a cell matrix, and perfusion culturing them using an ST cell induction medium. Examples of ST cell induction media include basal media for animal cells (e.g., TS basal medium, etc.), ROCK inhibitors (Y27632, etc.), GSK3β inhibitors (CHIR99021, etc.), p38 MAPK inhibitors (SB202190, etc.), and media supplemented with growth factors (EGF, BMP4, bFGF, etc.) and the like.

Fig. 7A shows an example of a three-dimensional culture model simulating the process of placental development. FIG. 7B is an enlarged view of portion B surrounded by a dashed line in the three-dimensional culture model shown in FIG. 7A. In the three-dimensional culture model of FIG. 7A, a three-dimensional vascular system (E) is formed in the central channel 21 and placental cell aggregates are cultured in the placental cell culture section 12 . Placental cell aggregates have a structure that mimics in vivo villi, and are composed of outer layer ST cells (ST) and inner undifferentiated cells (UD) (TS cells, CT cells, etc.). In the three-dimensional culture model of FIG. 7A, fibroblasts (FB) are cultured together with placental cell aggregates in the placental cell culture section 12 . In vivo, as the pregnancy progresses and the villi grow, a phenomenon occurs in which the villi come into contact with the maternal decidua. This contact causes undifferentiated cells such as CT cells to emerge from within the villi and migrate onto and/or into the decidua to contact the decidua. Undifferentiated cells that come into contact with the decidua differentiate into EVT cells and remodel maternal blood vessels within the decidua. Therefore, by co-cultivating placental cell aggregates and fibroblasts in the placental cell culture section, a placental development process model can be constructed. Placental cell aggregates can be prepared, for example, by culturing undifferentiated cells (TS cells, CT cells, etc.) using an ST cell-inducing medium in wells having cell-non-adhesive inner walls. As the ST cell induction medium, the same medium as described above can be used.

(Method for producing three-dimensional culture model)
The method for producing a three-dimensional culture model of the present embodiment comprises a step of introducing an interaction evaluation factor into the central channel 21, which is a factor channel, and allowing the interaction evaluation factor to exist in the central channel 21, placental cells and culturing the placental cells in the culture unit 12 . Methods for introducing interaction evaluation factors into the central channel 21 include the methods described above. Methods for culturing placental cells in the placental cell culture unit 12 include the methods described above.

When the interaction evaluation factor present in the factor channel is a three-dimensional vascular system, the three-dimensional culture model cultures vascular endothelial cells in the central channel 21, which is the factor channel, to form a three-dimensional vascular system. and culturing the three-dimensional vascular system in the central channel 21 and culturing the placental cells in the placental cell culturing section 12 . Methods for forming a three-dimensional vascular system in the central channel 21 include the methods described above.

The culture conditions can be those generally used for culturing animal cells. For example, a culture temperature of 32-40° C. (preferably 35-38° C.) and a CO ₂ concentration of 2-5% (preferably 5%) can be used.

<Second embodiment>
8A-11B show an example of the placental cell culture device of the second embodiment. In this embodiment, a micropost is used as a partition member between the placental cell culture section and the factor channel. 8A is a perspective view of placental cell culture device 100. FIG. FIG. 8B is a cross-sectional view of the placental cell culture device 100 taken along line BB. FIG. 9 is an exploded view of the placental cell culture device 100. FIG.

The placental cell culture device 100 is composed of a first substrate 110, a second substrate 120, and a ring member 130. Placental cell culture device 100 is formed by stacking second substrate 120, first substrate 110, and ring member 130 in this order.

The placental cell culture device 100 includes a central channel 111, a first side channel 112a, and a second side channel 112b. The placental cell culture device 100 has a first port P1, a second port P2, a third port P3, a fourth port P4, a fifth port P5, and a sixth port P6 as inlet/outlet ports for the culture solution or the like. there is The first port P1 is connected to the first side flow path 112a through the first port flow path p1. The second port P2 is connected to the first side flow path 112a by a second port flow path p2. The third port P3 is connected to the second side flow path 112b by a third port flow path p3. The fourth port P4 is connected to the second side flow path 112b through a fourth port flow path p4. The fifth port P5 and sixth port P6 are connected to the central channel 111 . The fifth port P5 is provided at one end of the central flow passage 111, and the sixth port P6 is provided at the other end of the central flow passage 111. As shown in FIG.

In the placental cell culture device 100, the central channel 111 functions as a placental cell culture section. A communication hole 115 is provided in the upper part of the central channel 111, which is the placental cell culture part. The ring member 130 is stacked on the first substrate 110 such that the communication hole 115 is positioned within the ring hole. In the placental cell culture device 100, the first side channel 112a and/or the second side channel 112b function as channel for factors.

(First substrate)
10A is a top view of the first substrate 110. FIG. FIG. 10B is an enlarged view of a portion B surrounded by a dashed line in the top view of the first substrate 110 shown in FIG. 10A.
The first substrate 110 includes a central flow channel 111, a first side flow channel 112a, a second side flow channel 112b, a first port P1 to a sixth port P6, and a first port flow channel p1 to a fourth port flow channel p4. is provided. The central flow channel 111, the first side flow channel 112a, the second side flow channel 112b, and the first port flow channel p1 to the fourth port flow channel p4 are closed on the first surface side of the first substrate 110, The second surface side of the first substrate 110 is open. The first substrate 110 is stacked on the second substrate 120 such that the second surface faces the second substrate 120 . Thus, the second substrate 120 forms the bottom surface of each channel of the first substrate 110 .

The central channel 111 is formed substantially in the center of the first substrate 110 . In the placental cell culture device 100, the central channel 111 functions as a placental cell culture section.

A first side channel 112a and a second side channel 112b are formed on both sides of the central channel 111, respectively. A first port flow path p1 is connected to one end of the first side flow path 112a, and a second port flow path p2 is connected to the other end of the first side flow path 112a. A third port flow path p3 is connected to one end of the second side flow path 112b, and a fourth port flow path p4 is connected to the other end of the second side flow path 112b. The first side flow channel 112a and the second side flow channel 112b may be formed on both sides over the entire length of the central flow channel 111, or may be formed on both sides of a portion of the central flow channel 111. . When the first side flow channel 112a and the second side flow channel 112b are formed on both sides of a part of the central flow channel 111, at least the first side flow channel is formed on both sides of the position where the communication hole 115 is formed. 112a and the second side channel 112b are preferably formed respectively.

A communication hole 115 is provided in the upper part of the central flow path 111 . The communication hole 115 is a through hole penetrating from the first surface side of the first substrate 10 to the central flow path 111 . The central channel 111 communicates with the external region on the first surface side of the first substrate 110 through a communication hole 115 . Although the size of the communication hole 115 is not particularly limited, it is preferably smaller than the width of the central channel 111 . The communication hole 115 has a diameter of 0.05 to 20 mm, for example. The size of the communication hole 115 is preferably, for example, a hole diameter of 0.1 mm or more, more preferably a hole diameter of 0.2 mm or more, even more preferably a hole diameter of 0.3 mm or more, and particularly preferably a hole diameter of 0.4 mm or more. The size of the communication hole 115 is preferably, for example, a hole diameter of 15 mm or less, more preferably a hole diameter of 10 mm or less, even more preferably a hole diameter of 5 mm or less, and particularly preferably a hole diameter of 1 mm or less.

Although the width of the central channel 111 is not particularly limited, it may be 500 to 5000 μm, for example. The width of the central channel 111 is preferably 600 μm or more, more preferably 700 μm or more, still more preferably 800 μm or more, and particularly preferably 900 μm or more. The width of the central channel 111 is preferably 5000 μm or less, more preferably 4000 μm or less, still more preferably 3000 μm or less, and particularly preferably 2500 μm or less.

The widths of the first side flow channel 112a and the second side flow channel 112b are not particularly limited, but may be 500 to 3000 μm, for example. The width of the first side flow channel 112a and the second side flow channel 112b is preferably 600 μm or more, more preferably 700 μm or more, even more preferably 800 μm or more, and particularly preferably 900 μm or more. The width of the first side channel 112a and the second side channel 112b is preferably 2500 μm or less, more preferably 2000 μm or less, even more preferably 1500 μm or less, and particularly preferably 1200 μm or less. The first side channel 112a and the second side channel 112b may have the same width or different widths.

The height (depth) of the central flow channel 111, the first side flow channel 112a, and the second side flow channel 112b is not particularly limited, but is, for example, 50 to 1000 μm. The height (depth) of the central flow channel 111, the first side flow channel 112a, and the second side flow channel 112b is preferably 20 μm or more, more preferably 40 μm or more, even more preferably 50 μm or more, and 80 μm or more. is particularly preferred. The height (depth) of the central flow channel 111, the first side flow channel 112a, and the second side flow channel 112b is preferably 700 μm or less, more preferably 500 μm or less, even more preferably 300 μm or less, and 200 μm or less. is particularly preferred.
It is preferable that the heights (depths) of the central flow channel 111, the first side flow channel 112a, and the second side flow channel 112b are substantially the same.

In the central channel 111, the length of the portion where the first side channel 112a and the second side channel 112b are adjacent is not particularly limited, but may be 500 to 30000 μm, for example. For example, the length is preferably 600 μm or more, more preferably 700 μm or more, still more preferably 800 μm or more, and particularly preferably 900 μm or more. For example, the length is preferably 25000 μm or less, more preferably 20000 μm or less, even more preferably 15000 μm or less, and particularly preferably 10000 μm or less.

The central channel 111 and the first side channel 112a are separated by a partition member 113a. The central channel 111 and the second side channel 112b are partitioned by a partition member 113b. The partitioning member 113a and the partitioning member 113b are composed of a plurality of microposts 114. As shown in FIG. A plurality of microposts 114 constituting the partition member 113a and the partition member 113b are arranged at predetermined intervals. In the partitioning member 113a and the partitioning member 113b, the microposts 114 constitute shielding portions, and the gaps between the microposts 114 constitute communicating portions. In the placental cell culture device 100, the central channel 111 functions as a placental cell culture part, and the first side channel 112a or the second side channel 112b functions as a factor channel. The partition member 113a or the partition member 113b is a partition member that separates the central channel 111, which is the placental cell culture part, from the first side channel 112a or the second side channel 112b, which is the factor channel.

The shape of the microposts 114 is not particularly limited. The microposts 114 may have a polygonal prism shape (triangular prism, square prism, etc.) or a cylindrical shape. The micropost 114 has, for example, a trapezoidal prism shape. It is preferable that the width w2 of the microposts 114 on the first side channel 112a side or the second side channel 112b side is larger than the width w1 on the central channel 21 side. As a result, the liquid filled in the central flow path 111 can be prevented from leaking into the first side flow path 112a and the second side flow path 112b.

The size of the microposts 114 is not particularly limited, but the width w1 on the central channel 111 side is, for example, 10 to 300 μm. The width w1 is, for example, preferably 20 μm or more, more preferably 30 μm or more, still more preferably 40 μm or more, and particularly preferably 45 μm or more. The width w1 is, for example, preferably 200 μm or less, more preferably 150 μm or less, still more preferably 100 μm or less, and particularly preferably 80 μm or less.
The width w2 on the side of the first side flow path 112a or the side of the second side flow path 112b is, for example, 30 to 500 μm. The width w2 is, for example, preferably 50 μm or more, more preferably 60 μm or more, even more preferably 70 μm or more, and particularly preferably 80 μm or more. The width w2 is, for example, preferably 400 μm or less, more preferably 300 μm or less, still more preferably 200 μm or less, and particularly preferably 150 μm or less.
The length l of the microposts 114 may be 40-500 μm. For example, the length l is preferably 50 μm or more, more preferably 60 μm or more, still more preferably 70 μm or more, and particularly preferably 80 μm or more. The length l is, for example, preferably 400 μm or less, more preferably 300 μm or less, still more preferably 200 μm or less, and particularly preferably 100 μm or less.
The height of the microposts 114 is preferably the same as the height (depth) of the central channel 111 .

The shortest distance d between two microposts 114 corresponds to the minimum width of the communicating portion of the partition member. The shortest distance d is 30-500 μm. When the minimum width of the communicating portion is within the above range, the placental cell migration efficiency is favorably maintained. The shortest distance d is, for example, preferably 40 μm or longer, more preferably 50 μm or longer, still more preferably 60 μm or longer, and particularly preferably 70 μm or longer. The shortest distance d is, for example, preferably 400 μm or less, more preferably 350 μm or less, still more preferably 300 μm or less, and particularly preferably 250 μm or less.

The longest distance between two microposts 114 corresponds to the maximum width of the communicating portion of the partition member. Examples of the longest distance include 50 to 500 μm. The longest distance is, for example, preferably 60 μm or longer, more preferably 70 μm or longer, still more preferably 80 μm or longer, and particularly preferably 90 μm or longer. The longest distance is, for example, preferably 400 μm or less, more preferably 350 μm or less, even more preferably 300 μm or less, and particularly preferably 250 μm or less.

The channel surfaces of the central channel 111, the first side channel 112a, and the second side channel 112b are preferably hydrophobic. If the channel surface is hydrophobic, surface tension of the liquid is generated between the microposts 114 when any one of the channels is filled with liquid. This prevents liquid from leaking into the adjacent channel when there is no liquid in the adjacent channel.

Although the thickness of the first substrate 110 is not particularly limited, it may be 0.1 to 50 mm, for example. For example, the thickness of the first substrate 10 is preferably 0.3 mm or more, more preferably 0.5 mm or more, still more preferably 0.6 mm or more, and particularly preferably 0.8 mm or more. For example, the thickness of the second substrate 20 is preferably 40 mm or less, more preferably 20 mm or less, even more preferably 10 mm or less, and particularly preferably 5 mm or less.

The material of the first substrate 110 is not particularly limited, but a material with high biocompatibility and high oxygen permeability is preferable. Oxygen permeable materials include oxygen permeable polymers. Examples of oxygen-permeable polymers include fluororesins, silicones (eg, PDMS, etc.), and the like. Among them, the oxygen-permeable polymer is preferably PDMS.

The first substrate 110 can be manufactured using known methods such as photolithography, soft lithography, microcontact printing, microfluidic, and stencil. For example, a mold for the first substrate 110 can be manufactured using a photolithography method, and the first substrate 110 can be manufactured using the mold using a soft lithography method. It can be manufactured in the same manner as the first substrate 10 and the second substrate 20 of the first embodiment, except that the shape of the mold is different.

(Second substrate)
A flat plate having substantially the same size as the first substrate 110 can be used for the second substrate 120 . The material of the second substrate 120 is not particularly limited, but a material with high biocompatibility is preferable. Examples of materials for the second substrate 120 include glass, various synthetic resins, and metals.

(Ring member)
The ring member 130 is stacked on the first surface of the first substrate 110 such that the communication hole 115 of the first substrate 110 is positioned within the ring hole. The ring member 130 forms an upper well 131 together with the first substrate 110 . The upper layer well 131 has a side surface formed by the ring inner wall of the ring member 130 and a bottom surface formed by the first surface of the first substrate 110 . The upper layer well 131 communicates with the central channel 111 through a communication hole 115 .

The ring member 130 has a ring hole larger than the size of the communication hole 115 . The size of the ring hole of the ring member 130 is, for example, 1 to 20 mm in diameter. The size of the ring hole is preferably 2 mm or more in diameter, more preferably 3 mm or more in diameter, still more preferably 4 mm or more in diameter, and particularly preferably 5 mm or more in diameter. The size of the ring hole is preferably 15 mm or less in diameter, more preferably 12 mm or less in diameter, even more preferably 10 mm or less in diameter, and particularly preferably 8 mm or less in diameter. The size of the outer circumference of the ring member 130 is not particularly limited as long as it does not block the first port P1 to the sixth port P6 formed on the first substrate 110 when stacked on the first substrate 110. The size of the outer circumference of the ring member 130 is, for example, 3 to 30 mm in diameter. The size of the outer circumference is preferably 4 mm or more in diameter, more preferably 5 mm or more in diameter, still more preferably 6 mm or more in diameter, and particularly preferably 7 mm or more in diameter. The size of the outer circumference is preferably 20 mm or less in diameter, more preferably 15 mm or less in diameter, even more preferably 12 mm or less in diameter, and particularly preferably 10 mm or less in diameter.

The material of the ring member 130 is not particularly limited, but a material with high biocompatibility is preferable. Examples of materials for the ring member 130 include polyimide, polyethylene terephthalate (PET), polystyrene, polyethylene, polypropylene, nylon, polytetrafluoroethylene (PTFE), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene- Hexafluoropropylene copolymer (FEP), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), polydimethylsiloxane (PDMS), and the like, but are not limited to these. The ring member 130 can be made of PDMS, for example.

The placental cell culture device 100 can be produced by laminating and bonding the second substrate 120, the first substrate 110, and the ring member 130 in this order. When assembling the placental cell culture device 1 , the second surface of the first substrate 110 faces the second substrate 120 . The communication hole 115 is positioned inside the ring hole of the ring member 130 .

When assembling the placental cell culture device 100, the surfaces of the first substrate 110 and/or the second substrate 120 may be activated to enhance adhesiveness. For example, if the first substrate 110 and/or the second substrate 120 are made of PDMS, an oxygen plasma treatment can activate the surface to improve adhesion. In this case, it is preferable to perform a hydrophobizing treatment on the surface of the flow path after assembly. When the first substrate 110 and/or the second substrate 120 are made of PDMS, heat treatment (eg, 80° C. overnight) can hydrophobize the substrate surface including the channel surface.

In the placental cell culture device of the present embodiment, the central channel 111, which is the placental cell culture part, may be filled with gel containing cell aggregates of placental cells. The cell aggregate C of placental cells may be composed of one type of placental cells, or may be composed of two or more types of placental cells. The cell aggregate C of placental cells preferably contains EVT cells. A cell aggregate containing EVT cells can be prepared, for example, by culturing TS cells in wells of an agarose microwell plate using an EVT cell induction medium.

EVT cell induction media include, for example, animal cell basal media (e.g., TS basal medium, etc.), ROCK inhibitors (Y27632, etc.), p38 MAPK inhibitors (SB202190, etc.), and growth factors (EGF, NGR1, etc.). ) and the like. The EVT cell induction medium may be supplemented with an extracellular matrix such as Matrigel® (CORNING). Specific examples of the EVT cell induction medium include 3D-EVT medium and 3D-EVT-Dox medium used in Examples.

The placental cell aggregates are suspended in a gelling solution containing a gelling agent and injected from the communication hole 115, the fifth port P5 or the sixth port P6, thereby forming the placental cell aggregates in the central channel 111. body can be introduced. The number of placental cell aggregates to be introduced into the central channel 111 may be one, or two or more. For example, one cell aggregate can be introduced into the central channel 111 through the communication hole 115 . In this case, the cell aggregate introduced into the central channel 111 can exist below the communicating hole 115 .
The central flow path 111 and the first side flow path 112a and the second side flow path 112b are separated by

partition members

113a and 113b, respectively. Therefore, the gelling solution introduced into the central channel 111 does not leak into the first side channel 112a and the second side channel 112b. After the central channel 111 is filled with a gelling solution containing cell aggregates of placental cells, the gelling solution is gelled. As a result, the placental cell culture device 100 can be obtained in which the central channel 111, which is the placental cell culture portion, is filled with the gel containing the cell aggregates of placental cells.

The gelling agent used in the gelling solution is not particularly limited, and the same gelling agent as described above can be used. A specific example of the gelling solution is a mixed solution of Matrigel (registered trademark) (CORNING) and collagen I.

When the center channel 111 is filled with a gel containing placental cells, the first side channel 112a, the second side channel 112b, and the upper well 131 may be filled with medium to prevent the gel from drying. good. The medium can be appropriately selected according to the type of placental cells introduced into central channel 111 . Specific examples of the medium include the TS basal medium used in Examples.

(Three-dimensional culture model)
The placental cell culture device of this embodiment can be used to produce a three-dimensional culture model containing placental cells. In the three-dimensional culture model, placental cells are cultured in the placental cell culture section, and interaction evaluation factors are present in the factor channel. 11A-12B show an example of the three-dimensional culture model of this embodiment. In the three-dimensional culture models of FIGS. 11A to 12B, the central channel 111 is filled with gel G containing cell aggregates C of placental cells. Medium M1 containing interaction evaluation factor E is introduced into first side channel 112a. A culture medium M2 is introduced into the second side channel 112b. Medium M3 is introduced into the upper layer well 131 . The media M1 to M3 may be the same media or different media.

≪Placental cells≫
In the placental cell culture device 100, the central channel 111 functions as a placental cell culture section. The placental cells cultured in the central channel 111 are preferably cell aggregates. Cell aggregates of placental cells can be introduced into the central channel 111 as described above.

By introducing the gelling solution containing the cell aggregates C of placental cells into the central channel 111 through the communicating holes 115, the cell aggregates C are allowed to exist at the positions where the communicating holes 115 are provided in the central channel 111. be able to. Migration of placental cells can be accurately evaluated by using cell aggregates of placental cells. For example, when the shape of the cell aggregate C extends to the side of the factor channel where the interaction evaluation factor E is present, it can be evaluated that the interaction evaluation factor E causes positive migration of placental cells ( For example, FIG. 12B). Conversely, in the cell aggregate C, when the distribution area on the side of the factor channel where the interaction evaluation factor E is present is reduced, it can be evaluated that the interaction evaluation factor E causes negative migration of placental cells. can

≪Interaction evaluation factor≫
In the placental cell culture device 100, the first side channel 112a and/or the second side channel 112b function as channel for factors. The interaction evaluation factor is not particularly limited, and any factor can be selected according to the purpose. The interaction evaluation factors present in the central channel 21 may be of one type or a combination of two or more types.

The interaction evaluation factor can be introduced into the first side channel 112a by dissolving or suspending it in a medium or the like and injecting it from the first port P1 or the second port P2. Also, the solution or suspension of the interaction evaluation factor can be introduced into the second side channel 112b by injecting it from the third port P3 or the fourth port P4.

The medium used for dissolving or suspending the interaction-evaluating factors can be appropriately selected according to the types of placental cells cultured in the central channel 111 and the interaction-evaluating factors. Specific examples of the medium include the assay medium used in Examples.

When the interaction evaluation factor is a vascular endothelial cell such as HUVEC, the vascular endothelial cell is suspended in a medium, filled in the first side channel 112a or the second side channel 112b and cultured to obtain partition member 113a or A vessel wall can be formed along the partition member 113b (eg, FIG. 11B). After introducing a culture medium containing vascular endothelial cells into the first side channel 112a, placental cell culture device 100 was left standing with first side channel 112a facing up. , can be formed by culturing for an arbitrary time (for example, about 10 to 60 minutes).

The interaction evaluation factor may be introduced into either the first side channel 112a or the second side channel 112b, or may be introduced into both. When evaluating placental cell migration using a three-dimensional culture model, the interaction evaluation factor is preferably introduced into either one of the first side channel 112a and the second side channel 112b. In this case, only the culture medium may be introduced into the side channel into which the interaction evaluation factor has not been introduced. Alternatively, different interaction evaluation factors may be introduced into the first side channel 112a and the second side channel 112b.

In the three-dimensional culture model of this embodiment, it is preferable to fill the upper well 131 with medium. By filling the upper layer well 131 with medium, the placental cells introduced into the central channel 111 can be well maintained.

(Method for producing three-dimensional culture model)
The method for producing a three-dimensional culture model according to the present embodiment includes a step of filling a gel containing a cell aggregate of placental cells in the central channel 111, which is a placental cell culture part, and a first side channel, which is a channel for factors. culturing cell aggregates of placental cells in central channel 111 with an interaction evaluator present in 112a and/or second side channel 112b. Examples of the method for filling the central channel 111 with the gel containing the cell aggregates of placental cells include the methods described above. Methods for causing the interaction evaluation factor to exist in the first side channel 112a and/or the second side channel 112b include the methods described above.

When the interaction evaluation factor present in the factor channel is a vascular wall, the three-dimensional culture model includes a step of filling gel containing placental cell aggregates in the central channel 111, which is the placental cell culture part; a step of culturing vascular endothelial cells in the first side channel 112a and/or the second side channel 112b, which are channels for factors, to form a vascular wall; culturing the vessel wall in channel 112 b and culturing a cell aggregate of placental cells in central channel 111 . Methods for forming a blood vessel wall in the first side channel 112a and/or the second side channel 112b include the above-described methods.

[Method for evaluating placental cells]
In one aspect, the present invention provides a placental cell evaluation method comprising co-culturing placental cells with a factor capable of interacting with placental cells using the placental cell culture device described above.
The placental cell culture device may be the placental cell culture device of the first embodiment or the placental cell culture device of the second embodiment.

The placental cell culture device of the first embodiment can be used to evaluate various functions of placental cells. For example, by culturing cell structures (placental organoids) of placental cells that mimic in vivo villous structures and the like in a placental cell culture unit, responses of placental cells to various interaction evaluation factors can be evaluated. .

The placental cell device of the second embodiment is suitable for evaluating migration of placental cells. In the placental cell device of the second embodiment, the central channel is filled with a gel containing placental cell aggregates, and the side channels are used as factor channels. Accordingly, by observing changes in the shape of cell aggregates of placental cells, it is possible to accurately evaluate the effects of various interaction evaluation factors on the migration of placental cells.

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
In Example 1, a three-dimensional culture model containing HUVEC vascular networks and EVT cells was produced using the placental cell culture device of the first embodiment (see FIGS. 1A to 4B).

<Production of placental cell culture device>
(Preparation of template by photolithography)
Photolithography was used to create templates for the first and second substrates of the placental cell culture device. First, a high-purity silicon wafer (AS ONE) was spin-coated with permanent film epoxy negative photoresist SU-8 2100 (KAYAKU Advanced Materials) (500 rpm for 10 seconds, then 1500 rpm for 30 seconds). The photoresist spin-coated silicon wafer was heated on a hot plate at 65° C. for 5 minutes and then on a hot plate at 95° C. for 45 minutes. Thus, an SU-8 membrane with a thickness of 200 μm was produced. Two SU-8 films were prepared, one for the first substrate and the other for the second substrate. A photomask for each substrate was produced using a laser lithography apparatus DWL200 (Heidelberg Instruments). A photomask for the first substrate was placed on the SU-8 film for the first substrate mold, and UV irradiation was performed (27 seconds). A photomask for the second substrate was placed on the SU-8 film for the second substrate mold, and UV irradiation was performed (27 seconds). After that, it was heated on a 65° C. hot plate for 5 minutes and on a 95° C. hot plate for 15 minutes. It was then cooled at room temperature for 5 minutes. Each cooled mold was placed in agitated SU-8 developer (KAYAKU Advanced Materials) and allowed to react for 1.5 to 2 hours to remove uncured SU-8. Each template was then washed with 86% ethanol-IP (denatured) (FUJIFILM Wako). Each mold was then heat treated at 65° C. for 2-3 minutes and then at 150° C. for 10 minutes.

(Preparation of first substrate and second substrate by soft lithography)
A first substrate and a second substrate were produced by soft lithography using a template produced by photolithography. A PDMS solution was prepared by mixing the main agent and the curing agent at a ratio of 10:1, and poured into the prepared mold. After degassing, the PDMS solution was cured by overnight treatment at 80°C. The PDMS solution was poured so that the second substrate had a thickness of about 1 mm and the first substrate had a thickness of about 1 cm. Using a disposable scalpel (Kai Industries) and tweezers, each board component was cut out from the mold. With reference to the grooves patterned in the PDMS component for the first substrate, holes for liquid introduction/exhaust to each channel formed in the second substrate were made using a biopsy trepan (Kai Industries). The central hole was Φ6 mm. Two ports connected to the first side flow path of the second substrate were set to Φ6 mm, and two ports connected to the second side flow path of the second substrate were set to Φ4 mm. Two ports connected to the central channel of the second substrate were φ1.5 mm. FIG. 13A shows a schematic diagram of the manufactured second substrate.

(Assembly of placental cell culture device)
A placental cell culture device was assembled using the first and second substrates produced as described above (see FIG. 2).

A micromesh sheet (8 mm x 8 mm, opening: long side 160-170 μm, short side 110-115 μm) (Mizuta Seisakusho) was prepared as a mesh sheet. FIG. 13B shows a photograph of the mesh sheet used.

A PET thin film sheet (thickness 25 μm, AS ONE) was processed to 10 mm x 5 mm. A 5% 3-aminopropyltriethoxysilane solution (Sigma) was prepared in purified water. The processed polyethylene terephthalate (PET) sheet was silanized by placing it in a 3-aminopropyltriethoxysilane solution and treating at 80° C. for 30 minutes. The silanization treatment was performed to enhance adhesion to the PDMS member. A 0.5 mm x 4.5 mm opening was made in the center of the silanized PET sheet using a biopsy trepan with a modified tip. A PDMS solution was applied thinly around the opening, and a mesh sheet was attached so as to cover the opening. Hereinafter, the PET sheet to which the mesh sheet is adhered is referred to as "meshed sheet".

The surface of each substrate was treated with oxygen plasma for 20 seconds using a small plasma device (PM100) (Yamato Scientific Co., Ltd.) to activate the surface of each substrate. The surface activation treatment was performed to improve the adhesiveness between the first substrate and the second substrate and the adhesiveness between each substrate and the sheet with mesh. A sheet with a mesh was sandwiched and adhered between the first and second substrates whose surfaces were activated. Adhesion was performed by heat treatment on a hot plate at 150° C. for 10 minutes. During assembly, the opening provided in the PET sheet was made to overlap with the vicinity of the center of the central channel of the second substrate. Also, the central hole of the first substrate was made to overlap the opening provided in the PET sheet.

Due to the oxygen plasma treatment, the surface of the channel formed on the second substrate became highly hydrophilic. In this state, introduction of a solution using the hydrophobicity of PDMS, that is, selective introduction of a solution into a specific channel (central channel) cannot be performed. Therefore, the assembled device was treated overnight at 80° C. to render the hydrophilized PDMS surface hydrophobic. In order to use the device for cell culture, it was sterilized by UV treatment for 30 minutes. FIG. 14 shows a photograph of the fabricated placental cell culture device.

<Culture medium>
(Three-dimensional blood vessel culture medium)
Human umbilical vein endothelial cells (RFP-HUVEC) (Angio Proteomie) were maintained using EGM-2 Endothelial Cell Growth Medium-2 Bullet Kit (LONZA).
A conditioned medium derived from human lung fibroblasts (NHLF) (LONZA) was used to efficiently fabricate three-dimensional vascular networks composed of RFP-HUVECs in fibrin gels. NHLF was maintained and managed in 10% FBS-containing DMEM medium (Thermo Fisher). When NHLF reached 70-100% of the dish area, the medium was replaced with EGM-2 medium, and the supernatant obtained after culturing for 3 days was used as a conditioned medium. This conditioned medium was used as a three-dimensional vascular medium.

(TS basal medium)
A TS basal medium was prepared by adding the following components to DMEM/F12 medium (FUJIFILM Wako). The concentrations given below are the final concentrations of each component in TS basal medium.
Bovine serum albumin (BSA) (FUJIFILM Wako) 0.15%
Penicillin 5,000 units/mL
Streptomycin (Thermo Fisher Scientific) 5,000 μg/mL
ITS-X (FUJI FILM Wako) 1%
KSR (Thermo Fisher Scientific) 1%
L-ascorbic acid (FUJIFILM Wako) 0.2mM

(TS medium)
A TS medium was prepared by adding the following components to a TS basal medium. The concentrations shown below are the final concentrations of each component in TS medium.
Y27632 (FUJIFILM Wako) 2.5 μM
EGF (FUJIFILM Wako) 25ng/mL
VPA (FUJI FILM Wako) 0.8 mM
A83-01 (FUJIFILM Wako) 5 μM
CHIR99021 (FUJIFILM Wako) 2 μM

(2D-EVT basal medium)
A 2D-EVT basal medium was prepared by adding the following components to DMEM/F12 medium (FUJIFILM Wako). The concentrations shown below are the final concentrations of each component in 2D-EVT basal medium.
Bovine serum albumin (BSA) (FUJIFILM Wako) 0.3%
ITS-X (FUJI FILM Wako) 1%
Penicillin 5,000 units/mL
Streptomycin (Thermo Fisher Scientific) 5,000 μg/mL

(2D-EVT medium)
A 2D-EVT medium was prepared by adding the following components to a 2D-EVT basal medium. The concentrations shown below are the final concentrations of each component in the 2D-EVT medium.
Y27632 (FUJI FILM Wako) 2.5 μM
KSR (Thermo Fisher Scientific) 4%
A83-01 (FUJIFILM Wako) 7.5 μM
NRG1 (Cell Signaling) 50ng/mL

<Production of EVT cells>
(Preparation of EGFP-TS cells)
Genetically modified TS cells (EGFP-TS cells) were used. EGFT-TS cells express EGFP by doxycycline hyclate (Sigma-Aldrich) treatment. EGFP-TS cells were prepared as follows.
According to the method previously reported (Takahashi et al., PNAS, 2019, 116 (52) 26606-26613), the EGFP gene was inserted into the pCS-3G vector to prepare a lentivirus. After infecting TS cells with the prepared lentivirus, single-cell cloning was performed to select EGFP-TS cells that highly express EGFP in a doxycycline-dependent manner.
EGFP-TS cells were maintained and managed with TS medium.

(Induction of differentiation from EGFP-TS cells to EVT cells)
A 2D-EVT medium supplemented with 0.2 mg/mL of Matrigel (Corning) was used to induce differentiation from EGFP-TS cells to EVT cells.

<Confirmation of effectiveness of mesh sheet>
The placental cell culture device of Example 1 must have a structure that allows cells to move across the boundary between the first substrate and the second substrate. On the other hand, it is necessary that the liquid filled in the central channel of the second substrate does not leak to the first substrate side. In the placental cell culture device of Example 1, a mesh sheet (openings: long side 160-170 μm, short side 110-115 μm; porosity 50% or more; FIG. 13B) was used as the partition member having the above performance.

In order to confirm the effectiveness of the mesh sheet, a leakage test of the color liquid filled in the central channel was conducted. For the leak test, a placental cell culture device using a sheet with mesh and a placental cell culture device using a PET sheet (without mesh sheet attached) were used.
The results are shown in Figures 15A and 15B. FIG. 15A shows the results of a placental cell culture device using a sheet with mesh. FIG. 15B shows the results of a placental cell culture device using a PET sheet (without mesh sheet attached). In the placental cell culture device using the sheet with mesh, leakage of the color liquid from the central channel of the second substrate to the central hole of the first substrate was not confirmed (Fig. 15A). On the other hand, in the placental cell culture device using the PET sheet (without mesh sheet attached), the color liquid leaked from the central channel of the second substrate to the central hole of the first substrate (Fig. 15B). From this result, it was confirmed that by installing the mesh sheet between the central flow path of the second substrate and the central hole of the first substrate, it was possible to prevent liquid from leaking from the central flow path to the central hole.

In the cell culture device described in JP-A-2020-188723, a porous membrane is provided at the boundary between the lower layer and the upper layer. In this case, the presence of the porous membrane prevents the liquid introduced into the lower layer from leaking to the upper layer. Also, cells in the upper layer can migrate to the lower layer through the porous membrane. However, there are problems associated with the use of porous membranes. For example, since the porous polycarbonate membrane has poor light transmission, it is difficult to observe the morphology of cells in the upper layer with a microscope from the bottom of the device. Even when a PET porous membrane with relatively high light transmittance is used, it is difficult to clearly observe the contours of cells. Furthermore, the pore size of commercially available porous membranes is generally about 0.4 to 8 μm, and the ratio of the total pore area to the membrane area (porosity) is about 15% at maximum. Therefore, although cells can pass through, for example, 8 μm pores, their migration efficiency is poor.

The mesh sheet used in Example 1 has large openings and a high porosity, so cells can easily pass through the mesh sheet. In addition, since the light transmittance is high, the cells present in the central hole of the first substrate can be microscopically observed from the second substrate side through the mesh sheet. Furthermore, leakage of liquid from the second substrate to the first substrate can be prevented. Therefore, the mesh sheet is effective as a partition member between the first substrate and the second substrate.

<Co-culture of HUVEC vascular network and EVT cells using placental cell culture device (1)>
A HUVEC suspension of 1.0×10 ⁷ cells/mL was prepared. Next, fibrinogen from bovine plasma (SIGMA) was dissolved in PBS(−) and filtered to prepare a 10 mg/mL fibrinogen solution. HUVEC suspension and fibrinogen solution were mixed at a ratio of 1:3. Thrombin (Sigma) (50 U/mL) was added to this mixture to a final concentration of 1 U/mL and quickly introduced into the central channel of the second substrate. Fibrinogen turns into fibrin by the action of thrombin and gels. Placed in a CO ₂ incubator at 37° C. for 5 minutes to gel the fibrin gel in the central channel. The first and second side channels (approximately 250 μL/channel) and the central hole (approximately 200 μL/hole) were filled with three-dimensional vascular medium. The next day, the media in the first side channel, the second side channel and the central hole were replaced with three-dimensional vascular media.

Three days after HUVEC seeding, TS cells were seeded in the central hole of the first substrate according to the following procedure. In order to coat the inside of the central hole with Matrigel (Corning), about 200 μL of Matrigel diluted 40-fold with PBS(−) was placed in the central hole and allowed to stand in an incubator at 37° C. for 1 hour. EGFP-TS cells (5.0×10 ⁵ cells/mL) suspended in EVT basal medium were prepared. 10 μL of cell suspension, 200 μL of 2D-EVT medium and 5 μL of about 10 mg/mL Matrigel were mixed and added to the central hole of the first substrate (5×10 ³ cells/hole). Six days after HUVEC seeding, a media change in the central hall was performed. The medium used for medium replacement was 2D-EVT medium containing 400-fold diluted Matrigel (final concentration: about 0.025 mg/mL) and 1 μg/mL doxycycline hyclate. Cultures were terminated 9 days after HUVEC seeding. Regarding the first side channel and the second side channel, the medium was replaced with a three-dimensional vascular medium (conditioned medium) 3 days, 5 days, and 7 days after HUVEC seeding.

(Fluorescence microscope observation)
A 4% paraformaldehyde (PFA) (FUJIFILM Wako) solution was introduced into the placental cell culture device and left at room temperature for 10 minutes to fix the cells. After that, the cells were washed with a 2% FBS/PBS(-) solution.
Cells were observed using an all-in-one fluorescence microscope BZ-X810 (KEYENCE).

(result)
FIG. 16 shows fluorescence microscope images 9 days after HUVEC seeding (6 days after EGFP-TS cell seeding). From the fluorescence microscope image (B) taken from the side of the placental cell device, it was confirmed that EGFP-TS cells were present at the height of the mesh sheet of the placental cell culture device. It was also confirmed that HUVEC existed in the area under the mesh sheet. From the fluorescence microscope image (A) taken from the second substrate side of the placental cell device, it was confirmed that HUVEC had a three-dimensional vascular network structure. EGFP-TS cells in the placental cell culture device were thought to be differentiated into EVT cells because a medium that differentiates into EVT cells was used. Some EGFP-TS cells (EVT cells) descended to the position where the HUVEC vascular network exists, suggesting the possibility of interacting with the HUVEC cells.

<Co-culture of HUVEC vascular network and EVT cells using placental cell culture device (2)>
From day 0 to day 3 after HUVEC seeding, except that no three-dimensional vascular medium was added to the central hole, <Co-culture of HUVEC vascular network and EVT cells using a placental cell culture device (1 )> was cultured in the same manner.

(result)
FIG. 17 shows fluorescence microscope images 9 days after HUVEC seeding (6 days after EGFP-TS cell seeding). The HUVEC vascular network was formed more planar than the fluorescence microscope image shown in FIG. That is, the HUVEC vascular network had a structure in which the vessels did not extend to the central hole. These results suggested that the period of vascular network formation up to 3 days after HUVEC seeding may be important in determining the orientation of the HUVEC vascular network. When medium was present in the central hole, the seeded HUVEC would extend the vascular network towards the central hole (see Figure 5A). On the other hand, in the absence of medium in the central hole, the seeded HUVECs would not extend the vascular network towards the central hole (see FIG. 5B).

Fig. 18 is a schematic diagram of EVT cells and maternal blood vessels in vivo. EVT cells (EVT) are thought to arise from CT cells (CT) that contact the decidua (DM) and are found on and within the decidua (DM). In the decidua (DM), there are blood vessels (spiral arteries: HA) for sending blood (BL) to the villus (V) side, and these blood vessels are located on the surface of the decidua (DM) (surface in contact with blood). There is an exit. EVT cells (EVT) can infiltrate the inner wall of the blood vessel from the outlet of the blood vessel and reconstruct the blood vessel thicker. On the other hand, EVT cells (EVT) that have infiltrated the decidua (DM) are thought to migrate toward the spiral artery (HA) and infiltrate from the outside of this blood vessel into the inside of the blood vessel. It is believed that EVT cells that have infiltrated blood vessels through two routes in this way reconstruct thicker blood vessels. Vascular invasion of EVT cells via these two pathways can be modeled by using the placental cell culture device of Example 1. The placental cell culture device of Example 1 can control the formation of HUVEC blood vessels in the central hole side of the first substrate only by inserting or not inserting the medium in the central hole of the first substrate at the initial stage of culture. The control of extending HUVEC blood vessels toward the central hall can be used to create a model in which EVT cells invade blood vessels from the artery exit side. Controlling HUVEC vessels not to extend toward the central hole can be used to create a model in which EVT cells that have entered the decidua invade blood vessels.

[Example 2]
In Example 2, a production test of an EVT cell-containing cell structure to be cultured in the placental cell culture device of the second embodiment (see FIGS. 8A to 10B) was conducted.

<Culture medium>
The same TS basal medium and TS medium as above were used.

(3D-EVT medium)
A 3D-EVT medium was prepared by adding the following components to a TS basal medium. The concentrations shown below are the final concentrations of each component in the 3D-EVT medium.
Y27632 (FUJIFILM Wako) 2.5 μM
EGF (FUJIFILM Wako) 25ng/mL
NRG1 (cell signaling) 100 ng/mL
SB202190 (FUJIFILM Wako) 2 μM
Matrigel (Corning) 0.2 mg/mL

(3D-EVT-Dox medium)
A 3D-EVT medium was prepared by adding the following components to a TS basal medium. The concentrations shown below are the final concentrations of each component in the 3D-EVT medium.
Y27632 (FUJIFILM Wako) 2.5 μM
EGF (FUJIFILM Wako) 25ng/mL
NRG1 (cell signaling) 100 ng/mL
SB202190 (FUJIFILM Wako) 2 μM
Matrigel (Corning) 0.2 mg/mL
Doxycycline hyclate (Sigma-Aldrich) 2 μg/mL

(assay medium)
An assay medium was prepared by adding the following components to TS basal medium. The concentrations given below are the final concentrations in the assay medium.
Doxycycline hyclate (Sigma-Aldrich) 2 μg/mL

<Preparation of EVT cell-containing cell structure>
EGFP-TS cells were maintained and managed with TS medium. EGFP-TS cells were collected from the dish for maintenance culture and suspended in TS basal medium to prepare a cell suspension. 10 μL of cell suspension (0.5×10 ⁵ cells/mL) was placed in each well of PrimeSurface 96U plate (Sumitomo Bakelite) containing 90 μL of 3D-EVT medium. On day 3 of culture, 50 μL of 3D-EVT-Dox medium was added. On day 4 of culture, the produced cell aggregates were moved into a 3.5 cm dish together with the medium. On the other hand, 30 μL of Matrigel-growth factor reduced (about 10 mg/mL, Corning) and 20 μL of collagen I (5 mg/mL, AteloCell IAC-50, Koken) were mixed on ice to prepare a mixed gel solution. The cell aggregates in the dish were sucked up with a pipetman tip, and only the cell aggregates were transferred into the mixed gel solution. At this time, care was taken not to allow the culture medium to enter the mixed gel solution as much as possible. Next, using a Pipetman, the cell aggregates were sucked up along with about 7 μL of the mixed gel solution and transferred into wells of a 4-well plate. The 4-well plate was placed in a 37° C. 5% CO ₂ incubator for about 15 minutes to gel the mixed gel solution. 200 μL of assay medium was then added. Cultivation of cell aggregates was started under an environment of 37° C. and 5% CO ₂ . One day after starting the culture in the gel (5th day after cell seeding), the medium was replaced with 200 μL of the assay medium.

(Immune cell staining)
The assay medium in the wells was removed, 4% paraformaldehyde (PFA) (FUJIFILM Wako) was added, and the cell aggregates were fixed by soaking in paraformaldehyde for 40 minutes. After that, the paraformaldehyde solution in the wells was replaced with phosphate buffered saline (PBS) (-) (FUJIFILM Wako). For permeabilization, the PBS (-) in the wells was replaced with 0.3% Triton X-100 (FUJIFILM Wako) diluted with 500 μL of PBS (-), and the cell aggregates were allowed to stand at room temperature for 1 hour. processed. The cell aggregates were washed once with 500 μL of PBS(-).

Anti-HLA-G antibody with PE [MEM-G/9] (ab24384) (x1/200 dilution) was added to the antibody diluent (PBS [-] containing 0.1

% Tween

20 and 2% FBS) to prepare the primary antibody solution. 500 μL of the primary antibody solution was added to the wells and allowed to stand at 4° C. overnight. The next day, the primary antibody solution was removed from the wells, and the cell aggregates were washed with PBS(-) (3 times with 500 μL). Cell aggregates were analyzed using an all-in-one fluorescence microscope BZ-X800 (Keyence).

(result)
FIG. 19A shows a microscopic image of cell aggregates after migration into the mixed gel. Although EVT cells can assume a spindle-shaped elongated form in the gel, almost no spindle-shaped cells were observed around the cell aggregates immediately after migration (Day 0). One day (Day 1) and two days (Day 2) after migration into the mixed gel, spindle-shaped cells were observed around the cell aggregates.

FIG. 19B shows the results of immunocytostaining of Day 2 cell aggregates. Cell aggregates expressed HLA-G, a marker for EVT cells. There was no HLA-G staining near the center of the cell aggregate. The reasons for this are (1) the possibility that the antibody could not reach the vicinity of the center of the cell aggregate, and (2) the cells near the center of the cell aggregate were undifferentiated cells that do not express HLA-G. Many possibilities were considered. By doxycycline treatment, EGFP expression in the cells of the cell aggregate could be confirmed. From these results, it was confirmed that the above method can produce cell aggregates surrounded by EVT cells.

[Example 3]
In Example 3, a three-dimensional culture model containing an EVT cell-containing cell structure was produced using the placental cell culture device of the second embodiment (see FIGS. 8A to 10B).
<Production of placental cell culture device>
(Preparation of template by photolithography)
A template for the first substrate of the placental cell culture device was made using a photolithography method. First, a high-purity silicon wafer (AS ONE) was spin-coated with permanent epoxy negative photoresist SU-8 2100 (KAYAKU Advanced Materials) (500 rpm for 10 seconds, then 3000 rpm for 30 seconds). The photoresist spin-coated silicon wafer was heated on a 65° C. hotplate for 5 minutes and then on a 95° C. hotplate for 20 minutes. Thus, an SU-8 membrane with a thickness of 100 μm was produced. A photomask was produced for the first substrate using a laser lithography apparatus DWL200 (Heidelberg Instruments). A photomask for the first substrate was placed on the SU-8 film for the first substrate mold, and UV irradiation was performed (20 seconds). After that, it was heated on a 65° C. hot plate for 5 minutes and on a 95° C. hot plate for 10 minutes. It was then cooled at room temperature for 5 minutes. The cooled mold was placed in agitated SU-8 developer (KAYAKU Advanced Materials) and allowed to react for 30 minutes to remove uncured SU-8. The templates were then washed with 86% ethanol-IP (denatured) (FUJIFILM Wako). The mold was then treated at 65°C for 3 minutes and 150°C for 6 minutes.

(Preparation of first substrate by soft lithography)
A first substrate was produced by soft lithography using a first substrate mold produced by photolithography. A PDMS solution was prepared by mixing a main agent and a curing agent at a ratio of 10:1, and poured into the prepared mold. After degassing, the PDMS solution was cured by overnight treatment at 65°C. Using a disposable scalpel or the like, the first board component was removed from the mold. Holes for fluid introduction/exhaust into the channel were made with biopsy trepans (Φ2 mm and Φ6 mm). FIG. 20A shows a schematic diagram of the manufactured first substrate.

A PDMS ring attached to the first substrate was produced by punching out a PDMS sheet with two types of biopsy trepans (Φ8 mm and Φ6 mm).

(Assembly of placental cell culture device)
A placental cell culture device was assembled using the first and second substrates produced as described above (see FIG. 9).

A cover glass (Matsunami Glass) was used as the second substrate.

The flow path surface side of the first substrate and the surface of the second substrate were treated with oxygen plasma for 2 minutes using a small plasma device (PM100) (Yamato Scientific Co., Ltd.). The first and second substrates were bonded together and treated at 80° C. overnight. In order to use the device for cell culture, it was sterilized by UV treatment for 30 minutes. FIG. 20B shows a photograph of the fabricated placental cell culture device.

<Preparation of EVT cell-containing cell structure and migration of EVT cells (1)>
Cell aggregates were prepared from EGFP-TS cells in the same manner as in Example 2. First, EGFP-TS cells were collected from a dish for maintenance culture and suspended in TS basal medium to prepare a cell suspension. 10 μL of cell suspension (0.8×10 ⁵ cells/mL) was placed in each well of PrimeSurface 96U plate containing 90 μL of 3D-EVT medium. On day 3 of culture, 50 μL of 3D-EVT-Dox medium was added. On day 4 of culture, the produced cell aggregates were moved into a 3.5 cm dish together with the medium. On the other hand, 30 μL of GF-reduced Matrigel and 20 μL of collagen I were mixed on ice to prepare a mixed gel solution. The cell aggregates in the dish were sucked up with a pipetman tip, and only the cell aggregates were transferred into the mixed gel solution. At this time, care was taken not to allow the culture medium to enter the mixed gel solution as much as possible.

Next, using Pipetman, the cell aggregate was sucked up together with about 7 μL of the mixed gel solution, and the cell aggregate was introduced through a communicating hole of φ0.5 mm provided in the placental cell culture device. Placental cell culture devices were then placed in 6 cm dishes. The device/dish was placed in a 37° C., 5% CO ₂ incubator, taking care not to dry out the gel inside the device using gauze or the like moistened with ultrapure water. After standing still for about 15 minutes to gel the mixed gel solution, a PDMS ring was placed on the first substrate to form an upper layer well. 60 μL of assay medium was placed in the upper wells. The central channel of the placental cell culture device contained a mixed gel and one cell aggregate. 40 μL of assay medium was introduced into each of the first side channel and the second side channel. Cultivation of cell aggregates in the device was started with placental cell culture under an environment of 37° C. and 5% CO ₂ . On the 1st day (5th day of cell seeding) and 2nd day (6th day of cell seeding) after starting the culture in the placental cell culture device, the medium was replaced with 50 μL of the assay medium (upper layer well, first side channel, second side channel).

(result)
In FIG. 21A, the cell aggregates were introduced into the placental cell culture device on day 1 (day 5 from the start of culture), day 2 (day 6 from the start of culture), and day 3 (day 7 from the start of culture). Fluorescence microscopy images of eyes) are shown. Doxycycline confirmed that the cells in the cell aggregate expressed EGFP. Some of the cells had an EVT cell-like spindle shape on day 2 (day 6 from the start of culture). Figure 21A confirmed that EVT cells migrated to the left or right to similar extents.

On the first day after introducing the cell aggregates into the placental cell culture device, the dividing line dividing the area of the planar view image of the cell aggregates observed from the second substrate side substantially evenly to the left and right. pulled. Using this dividing line as a reference, for the cell aggregates on the 5th day (1st day after introduction), 6th day (2nd day after introduction), and 7th day (3rd day after introduction) from the start of culture, Areas to the left and right of the dividing line were analyzed using ImageJ 1.52a (National Institutes of Health).
The results are shown in FIG. 21B. Each plot is the area of the cell aggregate on the left or right side of the dividing line when the total area of the image of the cell aggregate observed from the second substrate side is set to 1. The result of FIG. 21B also confirmed that the EVT cells spread evenly to the left and right.

<Preparation of EVT cell-containing cell structure and migration of EVT cells (2)>
Cell aggregates were prepared from EGFP-TS cells in the same manner as above. First, EGFP-TS cells were collected from a dish for maintenance culture and suspended in TS basal medium to prepare a cell suspension. 10 μL of cell suspension (0.65×10 ⁵ cells/mL) was placed in each well of PrimeSurface 96U plate containing 90 μL of 3D-EVT medium. On day 4 of culture, the produced cell aggregates were moved into a 3.5 cm dish together with the medium. Similar to the above, the cell aggregates were introduced into the placental cell culture device and the first side channel and the second side channel were filled with medium. Cultivation of the cell aggregates in the placental cell culture device was started under an environment of 37° C. and 5% CO ₂ .

One to two hours after introducing the cell aggregates into the placental cell device, HUVEC cells were introduced into the first side channel of the device. As a procedure, first, a suspension of RFP-HUVEC (5×10 ⁶ cells/mL) was prepared in EGM-2 medium. Using a truncated Pipetman tip (for 2-20 μL), 7 μL of RFP-HUVEC suspension was introduced through the second port P2 of the placental cell culture device (see FIG. 8A). As a control, 7 μL of EGM-2 was added through the fourth port P4. The placental cell culture device was set upright so that the first side channel into which HUVECs were introduced faced up, fixed with a large clip, and placed in a 5% CO ₂ incubator at 37°C in this vertical state. After 25 minutes, the placental cell culture device was leveled and culture started. On the 1st day (5th day of cell seeding) and 2nd day (6th day of cell seeding) after starting the culture of the cell aggregates in the placental cell culture device, 50 μL of the assay medium was used. exchanged (upper well, first side channel, second side channel).

(result)
In FIG. 22A, the cell aggregates were introduced into the placental cell device on day 1 (day 5 from the start of culture), day 2 (day 6 from the start of culture), and day 3 (day 7 from the start of culture). ) shows a fluorescence microscope image. The results in FIG. 22A confirm that EVT cells migrate toward the first side channel where HUVEC reside.

In the same manner as described above, a dividing line is drawn on the planar view image of the cell aggregate observed from the second substrate side of the placental cell culture device, and the left side (HUVEC (−)) and the right side (HUVEC (+)) of the dividing line are drawn. The area of cell aggregates was measured.
The results are shown in FIG. 22B. Two days after co-culturing EVT cells and HUVECs (day 6 from the start of culture), it was confirmed that EVT cells migrated significantly toward HUVECs.

These results show that the placental cell culture device of Example 3 can be used to evaluate migration of EVT cells.

While the preferred embodiments of the invention have been described and illustrated above, it should be understood that they are intended to be illustrative of the invention and should not be taken as limiting. Additions, omissions, substitutions, and other changes can be made without departing from the spirit or scope of the invention. Accordingly, the present invention should not be viewed as limited by the foregoing description, but only by the scope of the appended claims.

1,100 Placental cell culture device 10,110 First substrate 11 Central hole 12 Placental cell culture part 20,120 Second substrate 21,111

Central channel

22a, 112a

First side channel

22b, 112b

Second side channel

23a , 23b, 113a,

113b partition member

24, 114 micropost 30 mesh sheet 31 linear member 32 opening 40 thin film sheet 41 thin film opening 115 communicating hole 130 ring member 131 upper layer wells P1 to P6 first to sixth ports p1 ~ p4 1st port flow path ~ 4th port flow path

Claims

a placental cell culture unit for culturing placental cells;
a factor channel in which a factor capable of interacting with the placental cells is present;
The placental cell culture part and the factor channel are adjacent to each other via a partition member in which shielding parts and communicating parts are alternately arranged, and the minimum width of the communicating part is 30 to 500 μm Placenta cell culture device.
The placental cell culture device according to claim 1, wherein the partition member is a mesh sheet with a porosity of 50 to 90%.
The placental cell culture device according to claim 1, wherein the placental cell culture unit is filled with a gel containing the cell aggregates of the placental cells.
A three-dimensional culture model produced using the placental cell culture device according to any one of claims 1 to 3,
The placental cells are cultured in the placental cell culture unit,
a factor capable of interacting with the placental cells is present in the factor channel;
3D culture model.
A three-dimensional culture model produced using the placental cell culture device according to claim 2,
The placental cells are cultured in the placental cell culture unit,
A three-dimensional vascular system is formed in the factor channel,
3D culture model.
A three-dimensional culture model produced using the placental cell culture device according to claim 3,
A cell aggregate of the placental cells is cultured in the placental cell culture unit,
a blood vessel wall is formed in the factor channel;
3D culture model.
A method for producing a three-dimensional culture model using the placental cell culture device according to claim 2,
culturing vascular endothelial cells in the factor channel to form a three-dimensional vascular system;
culturing the three-dimensional vascular system in the factor channel and culturing placental cells in the placental cell culture unit;
A method for producing a three-dimensional culture model, comprising:
A method for producing a three-dimensional culture model using the placental cell culture device according to claim 3,
culturing vascular endothelial cells in the factor channel to form a vascular wall;
a step of culturing the vascular wall in the factor channel and culturing a cell aggregate of the placental cells in the placental cell culture unit;
A method for producing a three-dimensional culture model, comprising:
A method for evaluating placental cells, comprising co-culturing placental cells with a factor capable of interacting with placental cells using the placental cell culture device according to any one of claims 1 to 3.