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

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

[Technical Field]

[0001] 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. [Background technology]

[0002] In recent years, research and development of three-dimensional cell culture models (organ-on-a-chip devices) that mimic the function or structure of human tissues or organs has been actively pursued for the purpose of drug discovery or elucidating biological phenomena. Behind this active development is the fact that it has become clear that there are many human biological phenomena that cannot be analyzed or elucidated using conventional cell culture in a petri dish or animal experiments. Organ-on-a-chip devices developed to date include the lung-on-a-chip, a pulmonary edema model (Non-Patent Document 1), and the intestine-on-a-chip, a small intestine model (Non-Patent Document 2). A culture model of the outer blood-retinal barrier has also been reported, using a microfluidic chip with a two-layer structure separated by a porous membrane (Patent Document 1).

[0003] However, there are some organs for which organ-on-a-chip devices have not yet been fully developed. The placenta is one such organ. The placenta has different structures and functions between humans and animals. It is also difficult to obtain before birth. Therefore, the development of a human placenta-on-a-chip device is crucial for understanding the molecular and cellular biological phenomena in the human placenta, the mechanisms of placentation, and the mechanisms of pathogenesis.

[0004] Pregnancy-related disorders include hypertension (hypertension) of the uterus (pregnancy), which occurs between the 20th week of pregnancy and 12 weeks after delivery. Pregnancy-related hypertension is estimated to occur in approximately 1 in 20 women and is a leading cause of maternal and perinatal death. The pathogenesis of this disease is complex, but it is thought to result from hypoplasia of the spiral arteries in the uterus, which impairs blood supply to the placenta, resulting in the excessive secretion of factors that inhibit angiogenesis from the placenta, leading to hypertension. In other words, one explanation is that the mother, sensing a lack of blood supply to the placenta, increases her blood pressure in order to provide nutrients to the placenta (fetal side). Under normal conditions, extravillous trophoblast cells (EVT cells), a type of placental cell, invade the mother's spiral arteries and dilate them, a process known as "vascular remodeling." This thickens the spiral arteries and allows a greater volume of blood to be delivered to the placenta. It is known that this vascular remodeling is incomplete in patients with pregnancy-induced hypertension, but the 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. This raises ethical concerns about harming the mother, limiting the number of studies. Pregnancy-induced hypertension is a disease almost exclusively found in humans, and there have been few reports of spontaneous cases in non-human primates. Therefore, information on pregnancy-induced hypertension obtained from animal experiments is limited. Given this background, there is a need for the development of a human placenta-on-a-chip device that can enable detailed investigation of the dynamics of human EVT cells and the factors that control them, in order to elucidate the mechanism behind the development of pregnancy-induced hypertension. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-188723 [Non-patent literature]

[0006] [Non-Patent Document 1] Huh, D., Leslie, DC, Matthews, BD, Fraser, JP, Jurek, S., Hamilton, GA, Ingber, DE (2012). A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci Transl Med, 4(159), 159ra147. [Non-patent document 2] Kasendra, M., Tovaglieri, A., Sontheimer-Phelps, A., Jalili-Firoozinezhad, S., Bein, A., Chalkiadaki, A., Ingber, DE (2018). Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids. Sci Rep, 8(1), 2871. [Non-patent document 3] Khong, TY, De Wolf, F., Robertson, WB, & Brosens, I. (1986). Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants. Br J Obstet Gynaecol, 93(10), 1049-1059. Summary of the Invention [Problem to be solved by the invention]

[0007] Conventionally, cell dynamics have been evaluated by, for example, whether cells on a porous membrane pass through the pores. However, this evaluation system makes it difficult to evaluate cell migration toward and invasion into the three-dimensional vascular network that occurs in vivo. In addition, the pore size of the porous membrane is small, resulting in low cell migration efficiency.

[0008] Therefore, an object of the present invention is to provide a placental cell culture device that enables evaluation of the interaction between placental cells and factors that can act on placental cells, a three-dimensional culture model using the placental cell culture device, a method for preparing the three-dimensional culture model, and a method for evaluating placental cells using the placental cell culture device. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] A placental cell culture device comprising: a placental cell culture section for culturing placental cells; and a factor flow path for presenting factors capable of interacting with the placental cells, wherein the placental cell culture section and the factor flow path are adjacent to each other via a partition member in which shielding sections and communicating sections are arranged alternately, and the communicating sections have 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 having a porosity of 50 to 90%. [3] The placental cell culture device described in [1], wherein the placental cell culture section is filled with a gel containing cell aggregates of the placental cells. [4] A three-dimensional culture model produced using the placental cell culture device described in any one of [1] to [3], wherein the placental cells are cultured in the placental cell culture section, and a factor capable of interacting with the placental cells is present in the factor flow path. [5] A three-dimensional culture model produced using the placental cell culture device described in [2], wherein the placental cells are cultured in the placental cell culture section, and a three-dimensional vascular system is formed in the factor flow path. [6] A three-dimensional culture model produced using the placental cell culture device described in [3], wherein a cell aggregate of the placental cells is cultured in the placental cell culture section, and a vascular wall is formed in the factor flow path. [7] A method for producing a three-dimensional culture model using the placental cell culture device described in [2], comprising the steps of culturing vascular endothelial cells in the factor flow path to form a three-dimensional vasculature, and culturing the three-dimensional vasculature in the factor flow path, and culturing placental cells in the placental cell culture section. [8] A method for producing a three-dimensional culture model using the placental cell culture device described in [3], comprising the steps of culturing vascular endothelial cells in the factor flow path to form a vascular wall, and culturing the vascular wall in the factor flow path and culturing a cell aggregate of the placental cells in the placental cell culture section. [9] 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 described in any one of [1] to [3]. [Effects of the Invention]

[0010] The present invention provides a placental cell culture device that enables evaluation of the interaction between placental cells and factors that can act on placental cells, a three-dimensional culture model using the placental cell culture device, a method for preparing the three-dimensional culture model, and a method for evaluating placental cells using the placental cell culture device. [Brief explanation of the drawings]

[0011] [Figure 1A] FIG. 1 is a perspective view of one embodiment of a placental cell culture device. [Figure 1B] 1B is a cross-sectional view of the placental cell culture device shown in FIG. 1A taken along section line BB. [Figure 1C] 1B is a cross-sectional view of the placental cell culture device shown in FIG. 1A taken along line CC. [Figure 2] FIG. 1B is an exploded view of the placental cell culture device shown in FIG. 1A. [Figure 3] FIG. 1 is a schematic diagram of a mesh sheet used in one embodiment of the placental cell culture device. [Figure 4A] FIG. 1B is a top view of a first substrate used in the placental cell culture device shown in FIG. 1A. [Figure 4B] 4B is an enlarged view of a portion B surrounded by a dashed line in the top view of the first substrate in FIG. 4A. FIG. [Figure 5A] FIG. 1 is a schematic diagram showing the state of formation of a three-dimensional vasculature (E) when vascular endothelial cells are cultured in placental cell culture section 12 filled with medium M3 in placental cell culture device 1. E: Interaction evaluation factor (three-dimensional vasculature). [Figure 5B] FIG. 10 is a schematic diagram showing the state of formation of a three-dimensional vasculature (E) when vascular endothelial cells are cultured in placental cell culture section 12 without filling medium M in placental cell culture device 1. [Figure 6A] Fig. 1 is a schematic diagram showing an example of a three-dimensional culture model including a three-dimensional vasculature. Fig. 2 shows an example of a three-dimensional culture model for evaluating the function of EVT cells. EVT: EVT cells. [Figure 6B] Schematic diagram showing an example of a 3D culture model including a 3D vasculature. An example of a 3D culture model for evaluating chemical permeability across placental cell membranes. ST: ST cells, UD: undifferentiated cells. [Figure 7A] 1 is a schematic diagram showing an example of a three-dimensional culture model including a three-dimensional vasculature. 2 shows an example of a three-dimensional culture model that mimics the process of placental development. [Figure 7B] This is an enlarged view of the area B enclosed by the dashed line in the three-dimensional culture model shown in Figure 7A. FB: fibroblasts. [Figure 8A] FIG. 1 is a perspective view of one embodiment of a placental cell culture device. [Figure 8B] 8B is a cross-sectional view of the placental cell culture device shown in FIG. 8A taken along section line BB. [Figure 9] FIG. 8B is an exploded view of the placental cell culture device shown in FIG. 8A. [Figure 10A] FIG. 8B is a top view of a first substrate used in the placental cell culture device shown in FIG. 8A. [Figure 10B]10B is an enlarged view of a portion B surrounded by a dashed line in the top view of the first substrate in FIG. 10A. [Figure 11A] FIG. 8B is a top view of the placental cell culture device shown in FIG. 8A. [Figure 11B] 11B is an enlarged view of the area B enclosed by the dashed line in the top view of the placental cell culture device shown in Figure 11A. C: Placental cells. [Figure 12A] FIG. 8B is a schematic diagram showing an example of a three-dimensional culture model for evaluating EVT cell migration using the placental cell culture device shown in FIG. 8A. [Figure 12B] FIG. 8B is a schematic diagram showing an example of a three-dimensional culture model for evaluating EVT cell migration using the placental cell culture device shown in FIG. 8A. [Figure 13A] FIG. 2 is a schematic diagram of a second substrate produced in Example 1. [Figure 13B] 1 shows a microscopic image of the mesh sheet used in the placental cell culture device prepared in Example 1. [Figure 14] 1 shows the placental cell culture device prepared in Example 1. [Figure 15A] 1 shows a microscope image of colored water flowing through the central channel of the placental cell culture device equipped with a mesh sheet prepared in Example 1. (+) indicates that the placental cell culture device includes a mesh sheet. [Figure 15B] 1 shows a microscope image of colored water flowing through the central channel of the placental cell culture device without a mesh sheet prepared in Example 1. (-) indicates that the placental cell culture device does not include a mesh sheet. [Figure 16] These are fluorescence microscope images of EVT cells and HUVECs (human umbilical vein endothelial cells) cultured using the placental cell culture device prepared in Example 1. Starting from day 0 after seeding the HUVECs, medium was placed in the central hole of the first substrate and the HUVECs were cultured. (A) is a fluorescence microscope image taken from the side of the placental cell device facing the second substrate. (B) is a fluorescence microscope image taken from the side of the placental cell device. [Figure 17]These are fluorescence microscope images of EVT cells and HUVECs cultured using the placental cell culture device prepared in Example 1. HUVECs were cultured for 0 to 3 days after seeding without placing medium in the central hole. (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. [Figure 18] Schematic diagram of a portion of the placenta in vivo. (A) Schematic diagram of the placenta before CT cells (CT) differentiate into EVT cells (EVT), and (B) Schematic diagram of the placenta after some of the CT cells (CT) differentiate into EVT cells (EVT). V: villi; DM: decidua; ML: myometrium; UA: uterine artery; BL: blood; BF: blood flow; HA: spiral artery; EVT: EVT cells; ST: ST cells; CT: CT cells. [Figure 19A] 1 shows a microscopic image of the EVT cell-containing cell aggregates prepared in Example 2. [Figure 19B] 1 shows a fluorescence microscope image of the EVT cell-containing cell aggregate prepared in Example 2. [Figure 20A] FIG. 10 is a schematic diagram of a first substrate produced in Example 3. [Figure 20B] 1 shows the placenta culture device prepared in Example 3. [Figure 21A] 1 shows a microscopic image of an EVT cell-containing cell aggregate cultured using the placental cultured cell device prepared in Example 3. [Figure 21B] 1 is a graph showing the migration direction of EVT cell-containing cell aggregates cultured using the placental cell culture device prepared in Example 3. The horizontal axis shows the number of days since the EVT cell-containing aggregates were introduced into the placental cell culture device. The vertical axis shows the ratio of the right area (Right) or left area (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. [Figure 22A] 1 shows a microscopic image of EVT cell-containing cell aggregates co-cultured with HUVECs using the placental cultured cell device prepared in Example 3. The EVT cell-containing cell aggregates were co-cultured with HUVEC cells. [Figure 22B]1 is a graph showing the migration direction of EVT cell-containing cell aggregates co-cultured with HUVECs using the placental cell culture device prepared in Example 3. The horizontal axis shows the number of days since the EVT cell-containing aggregates were introduced into the placental cell culture device. The vertical axis shows the ratio of the right area (Right) or left area (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). DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail, with reference to the drawings where necessary. In the drawings, identical or corresponding parts are designated by identical or corresponding reference numerals, and duplicate explanations will be omitted. The dimensional ratios in each drawing may be exaggerated for the purpose of explanation and do not necessarily correspond to the actual dimensional ratios.

[0013] [Placenta cell culture device] In one aspect, the present invention provides a placental cell culture device comprising: a placental cell culture section for culturing placental cells; and a factor flow path for presenting a factor capable of interacting with the placental cells, wherein the placental cell culture section and the factor flow path are adjacent to each other via a partition member having alternating arrangements of shielding sections and communicating sections, and the communicating sections have a minimum width of 30 to 500 μm.

[0014] 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 sections and communicating sections are alternately arranged, and the communicating sections have a minimum width of 30 to 500 μm. This allows interaction between placental cells cultured in the placental cell section and factors present in the factor channel. Furthermore, placental cells cultured in the placental cell culture section can pass through the communicating section of the partition member and migrate to the factor channel. The maximum width of the communicating part is, for example, 50 to 500 μm. When the maximum width of the communicating part is within this range, leakage of the medium and the like between the placental cell culture part and the factor flow channel can be appropriately controlled.

[0015] The placental cell culture unit 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.

[0016] The organism from which placental cells are derived is not particularly limited as long as it is an animal having a placenta. Examples of animals from which placental cells are derived include mammals such as primates, rodents, and carnivores. Mammals are preferably primates. Examples of primates include humans, chimpanzees, rhesus monkeys, and marmosets. It is more preferable that the placental cells are human cells.

[0017] Placental cells can be isolated from the placenta or can be derived from undifferentiated cells. TS cells may be derived from blastocysts, may be derived from CT cells, or may be derived from pluripotent stem cells. TS cells can be induced from blastocysts by known methods. For example, cells are isolated from placental tissue by appropriate mechanical and / or enzymatic treatment. The cells are then cultured in a medium that induces TS cells, and TS cells can be established using the expression of TS cell markers (e.g., GATA2-positive, GATA3-positive, TFAP2-positive, ELF5-positive, ZNF750-positive, CDX2-negative) as an indicator. As a method for inducing TS cells from CT cells, for example, the method described in Japanese Patent No. 6400832 can be used. Methods for inducing TS cells from pluripotent stem cells include, for example, methods described in International Publication No. 2020 / 250438.

[0018] CT cells can be isolated from the placenta, for example. CT cells can be isolated from the placenta by, for example, separating cells from placental tissue using appropriate mechanical and / or enzymatic treatment, and isolating the CT cells based on the expression of CT cell markers (CD49f positive, E-cadherin positive, etc.) (Patent Publication No. 6400832; Haider, S., et al., Stem Cell Reports 11, 537-551 (2018)).

[0019] ST cells may be isolated from the placenta or induced from TS cells. ST cells can be isolated from the placenta, for example, by separating cells from placental tissue using appropriate mechanical and / or enzymatic treatments, and then isolating the ST cells using the expression of ST cell markers (e.g., syndecan 1 (SCD1) positivity, human chorionic gonadotropin (hCG) positivity) as an indicator. Methods for inducing ST cells from TS cells include, for example, the methods described in International Publication No. 2020 / 250438.

[0020] EVT cells may be isolated from the placenta or induced from TS cells. EVT cells can be isolated from the placenta, for example, by separating cells from placental tissue using appropriate mechanical and / or enzymatic treatment, and then isolating the EVT cells based on the expression of EVT cell markers (e.g., HLA-G positivity). Methods for inducing EVT cells from TS cells include, for example, the methods described in International Publication No. 2020 / 250438.

[0021] The factor flow path is a flow path for introducing a factor capable of interacting with placental cells. A "factor capable of interacting with placental cells" refers to a factor whose interaction with placental cells is to be evaluated. A factor capable of interacting with placental cells (hereinafter also referred to as an "interaction evaluation factor") may be a factor known to interact with placental cells, or may be a factor whose interaction with placental cells is unknown. The interaction evaluation factor may be a cell, a biological substance, or an exogenous substance. Examples of interaction evaluation factors include vascular endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)), various hormones, various cytokines, various drugs, immune cells, etc.

[0022] First Embodiment 1A to 4B show an example of 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 section and the factor flow path. FIG. 1A is a perspective view of placental cell culture device 1. FIG. 1B is a cross-sectional view of placental cell culture device 1 taken along section line BB. FIG. 1C is a cross-sectional view of placental cell culture device 1 taken along section line CC. FIG. 2 is an exploded view of placental cell culture device 1.

[0023] Placental cell culture device 1 is composed of first substrate 10, second substrate 20, mesh sheet 30, and thin film sheet 40. Placental cell culture device 1 is formed by stacking second substrate 20, thin film sheet 40, mesh sheet 30, and first substrate 10 in this order.

[0024] Placental cell culture device 1 includes a central channel 21, a first side channel 22a, and a second side channel 22b. Placental cell culture device 1 includes 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 culture medium and the like. First port P1 is connected to first side channel 22a via a first port channel p1. Second port P2 is connected to first side channel 22a via a second port channel p2. Third port P3 is connected to second side channel 22b via a third port channel p3. Fourth port P4 is connected to second side channel 22b via a fourth port channel p4. Fifth port P5 and sixth port P6 are connected to central channel 21. Fifth port P5 is provided at one end of central channel 21, and sixth port P6 is provided at the other end of central channel 21.

[0025] 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. 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. 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. 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. 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. 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. The central flow channel 21, the first port flow channel p1, the second port flow channel p2, the third port flow channel p3, and the fourth port flow channel p4 are provided on the second substrate 20.

[0026] In placental cell culture device 1, central channel 21 functions as a channel for factors. Placental cell culture section 12 is provided above central channel 21, which is a channel for factors. Placental cell culture section 12 has side surfaces formed by central hole 11 provided in first substrate 10, and a bottom surface formed by mesh sheet 30 and 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 placental cell culture device 1, mesh sheet 30 functions as a partition member that separates placental cell culture section 12 from the channel for factors (central channel 21).

[0027] (First board) The first substrate 10 is provided with through holes P1a to P6a and a central hole 11. The through holes P1a to P6a are formed at positions that will overlap with the recesses P1b to P6b of the second substrate 20, respectively, when the first substrate 10 is laminated on the second substrate 20.

[0028] Central hole 11 is a through-hole provided in approximately the center of second substrate 20. In placental cell culture device 1, central hole 11, together with mesh sheet 30 and thin film sheet 40, forms placental cell culture section 12. The shape of central hole 11 is not particularly limited. Examples of the planar shape of the central hole include a circle, an ellipse, and a polygon (such as a square, pentagon, or hexagon). The size of central hole 11 is not particularly limited, and examples of the size include a minimum diameter and a maximum diameter of 1 to 20 mm. For example, the size of 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. For example, the size of central hole 11 is preferably 18 mm or less, more preferably 15 mm or less, even more preferably 12 mm or less, and particularly preferably 10 mm or less, for both the minimum diameter and the maximum diameter.

[0029] The thickness of the first substrate 10 is not particularly limited, but may be, for example, 1 to 50 mm. The thickness of the first substrate 10 is, for example, 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. The thickness of the first substrate 10 is, for example, 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.

[0030] (Second board) Fig. 4A is a top view of the second substrate 20. 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.

[0031] The second substrate 20 is formed with recesses P1b to P6b, a first port flow path p1 to a fourth port flow path p4, a central flow path 21, a first side flow path 22a, and a second side flow path 22b. The recesses P1b to P6b are formed at positions that will overlap the through holes P1a to P6a of the first substrate 10, respectively, when the first substrate 10 is laminated.

[0032] Central channel 21 is formed in approximately the center of second substrate 20. In placental cell culture device 1, central channel 21 functions as a factor channel in which an interaction assessment factor is present. Central channel 21 is formed in a position that will at least partially overlap with central hole 11 of first substrate 10 when first substrate 10 is stacked. Recesses P5b and P6b are provided on both ends of central channel 21.

[0033] A first side channel 22a and a second side channel 22b are formed on both sides of central channel 21. A first port channel p1 is connected to one end of first side channel 22a, and a second port channel p2 is connected to the other end of first side channel 22a. A third port channel p3 is connected to one end of second side channel 22b, and a fourth port channel p4 is connected to the other end of second side channel 22b. First side channel 22a and second side channel 22b may be formed on both sides of central channel 21 over the entire length, or may be formed on both sides of a portion of central channel 21. When first side channel 22a and second side channel 22b are formed on both sides of a portion of central channel 21, it is preferable that first side channel 22a and second side channel 22b are formed on both sides of at least the portion of central channel 21 that contacts placental cell culture section 12.

[0034] The width of the central flow channel 21 is not particularly limited, but may be, for example, 500 to 5000 μm. The width of the central flow channel 21 is preferably 700 μm or more, more preferably 800 μm or more, even more preferably 1000 μm or more, and particularly preferably 1200 μm or more. The width of the central flow 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.

[0035] The widths of the first side channel 22a and the second side channel 22b are not particularly limited, but may be, for example, 500 to 3000 μm. The widths of the first side channel 22a and the second side channel 22b are 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 widths of the first side channel 22a and the second side channel 22b are 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 22a and the second side channel 22b may have the same width or different widths.

[0036] The height (depth) of the central channel 21, first side channel 22a, and second side channel 22b is not particularly limited, but may be, for example, 50 to 1000 μm. The height (depth) of the central channel 21, first side channel 22a, and second side channel 22b is preferably 60 μm or more, more preferably 80 μm or more, even more preferably 100 μm or more, and particularly preferably 150 μm or more. The height (depth) of the central channel 21, first side channel 22a, and second side channel 22b is preferably 800 μm or less, more preferably 600 μm or less, even more preferably 500 μm or less, and particularly preferably 300 μm or less. It is preferable that the heights (depths) of the central channel 21, the first side channel 22a, and the second side channel 22b are approximately the same.

[0037] The length of the portion of the central channel 21 where the first side channel 22a and the second side channel 22b are adjacent is not particularly limited, but may be, for example, 500 to 30,000 μm. The length is, for example, 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 length is, for example, preferably 25,000 μm or less, more preferably 200,000 μm or less, even more preferably 15,000 μm or less, and particularly preferably 10,000 μm or less.

[0038] The central channel 21 and the first side channel 22a are separated by a partition member 23a. The central channel 21 and the second side channel 22b are separated by a partition member 23b. The partition members 23a and 23b are composed of a plurality of microposts 24. The plurality of microposts 24 that constitute the partition members 23a and 23b are arranged at predetermined intervals. In the partition members 23a and 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 be polygonal prisms (triangular prisms, quadrangular prisms, etc.) or cylindrical. The microposts 24 are, for example, trapezoidal prisms. 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 greater than the width w1 on the central channel 21 side. This makes it possible to prevent the liquid filled in the central channel 21 from leaking into the first side channel 22a and the second side channel 22b.

[0039] The size of the microposts 24 is not particularly limited, but the width w1 on the central channel 21 side is, for example, 20 to 300 μm. The width w1 is, for example, preferably 25 μm or more, more preferably 30 μm or more, even 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, even more preferably 100 μm or less, and particularly preferably 80 μm or less. The width w2 on the first side channel 22a side or the second side channel 22b side 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, even more preferably 200 μm or less, and particularly preferably 180 μm or less. The length l of the microposts 24 is, for example, 50 to 800 μm. The length l is, for example, preferably 60 μm or more, more preferably 65 μm or more, even more preferably 70 μm or more, and particularly preferably 75 μm or more. The length l is, for example, 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.

[0040] The shortest distance d between two microposts 24 corresponds to the smallest width of the communication portion in the partition member. Examples of the shortest distance d include 30 to 500 μm. The shortest distance d is preferably, for example, 40 μm or more, more preferably 50 μm or more, even more preferably 60 μm or more, and particularly preferably 70 μm or more. The shortest distance d 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.

[0041] The longest distance between two microposts 24 corresponds to the longest width of the communication portion in the partition member. Examples of the longest distance include 50 to 500 μm. The longest distance is preferably, for example, 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 longest distance is preferably, for example, 400 μm or less, more preferably 350 μm or less, even more preferably 300 μm or less, and particularly preferably 250 μm or less.

[0042] The surfaces of the central channel 21, the first side channel 22a, and the second side channel 22b are preferably hydrophobic. If the surfaces of the channels are hydrophobic, when a liquid is filled into one of the channels, surface tension of the liquid is generated between the microposts 24. This prevents the liquid from leaking into an adjacent channel when there is no liquid in the adjacent channel.

[0043] The thickness of the second substrate 20 is not particularly limited, but may be, for example, 0.1 to 50 mm. The thickness of the second substrate 20 is, for example, preferably 0.3 mm or more, more preferably 0.5 mm or more, even more preferably 0.6 mm or more, and particularly preferably 0.8 mm or more. The thickness of the second substrate 20 is, for example, 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.

[0044] The material of the first substrate 10 and the second substrate 20 is not particularly limited, but a material that is highly biocompatible and has high oxygen permeability is preferred. "Oxygen permeability" refers to the property of passing 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 flow channel. The oxygen permeability is, for example, about 100 to 5000 cm 3 / m 2 24h atm. The oxygen permeability is approximately 1100 to 3000 cm 3 / m 2 24h atm, or approximately 1250-2750cm 3 / m 2 24h atm is preferred. 3 / m 2 24h atm is the amount of oxygen that passes through 1m3 of the material in 24 hours under an atmosphere of 1 atmosphere. 2 Capacity per (cm 3 ) Examples of oxygen-permeable materials include oxygen-permeable polymers. Examples of oxygen-permeable polymers include fluororesins and silicones (e.g., polydimethylsiloxane (PDMS)). Among these, PDMS is preferred as the oxygen-permeable polymer.

[0045] The first substrate 10 and the second substrate 20 can be fabricated using known methods such as photolithography, soft lithography, microcontact printing, microfluidic printing, stencil printing, etc. For example, a mold for the first substrate 10 and the second substrate 20 can be fabricated using the photolithography method, and the first substrate 10 and the second substrate 20 can be fabricated using the mold by soft lithography.

[0046] More specifically, a photoresist film is formed by applying a photoresist onto a wafer such as a silicon wafer by spin coating or the like. The photoresist film is then exposed to light through a photomask for the first substrate 10 or the second substrate 20. The unexposed portions of the photoresist film are then removed using a developer to obtain a mold for the first substrate 10 or the second substrate 20. Next, a curable composition is poured into the prepared mold for the first substrate 10 or the mold for the second substrate 20 and cured. The cured product is removed from the mold and processed appropriately to obtain the first substrate 10 or the second substrate 20.

[0047] (mesh sheet) In placental cell culture device 1, placental cell culture section 12 and central channel 21, which is a channel for factors, are at least partially separated by mesh sheet 30. In placental cell culture device 1, mesh sheet 30 functions as a partition member that separates placental cell culture section 12 from central channel 21, which is a channel for factors. The partition member separating placental cell culture section 12 from the factor channel has a configuration in which shielding sections and communicating sections are arranged alternately. In the partition member, the communicating sections have a minimum width of 30 to 500 μm. In the partition member, the communicating sections have a maximum width of 50 to 500 μm, for example. When the minimum and maximum widths of the communicating sections are within the above ranges, placental cells can easily pass through the communicating sections. Furthermore, leakage of the liquid filled in central channel 21 of second substrate 20 into placental cell culture section 12 can be prevented.

[0048] FIG. 3 shows a mesh sheet 30. The mesh sheet 30 is configured by arranging linear members 31 in a mesh pattern. In the mesh sheet 30, the linear members 31 form the shielding portions of the partition member, and the openings 32 form the communicating portions of the partition member. The openings 32 are rectangular. In the mesh sheet 30, the length of the diagonal of the rectangle forming the openings 32 corresponds to the maximum width of the communicating portions of the partition member. The maximum width of the communicating portions is preferably 120 μm or more, more preferably 130 μm or more, even more preferably 140 μm or more, and particularly preferably 150 μm or more. The maximum width of the communicating portions is preferably 400 μm or less, more preferably 300 μm or less, even more preferably 250 μm or less, and particularly preferably 200 μm or less. In mesh sheet 30, the length W1 of the short side of the rectangle forming opening 32 corresponds to the minimum width of the communicating portion in the partition member. The minimum width of the communicating portion is not particularly limited as long as it is in the range of 30 to 500 μm. When the minimum width of the communicating portion is within this range, good efficiency of placental cell migration is maintained. The minimum width of the communicating portion is, for example, preferably 40 μm or more, more preferably 50 μm or more, even 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, even 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 more, more preferably 100 μm or more, even more preferably 120 μm or more, and particularly preferably 150 μm or more. The upper limit of the length W2 of the long side is 500 μm or less. The length W2 of the long side is preferably 400 μm or less, more preferably 300 μm or less, even more preferably 250 μm or less, and particularly preferably 200 μm or less. The shape of the openings of the mesh sheet serving as the partition member is not limited to a rectangular shape like the openings 32. The shape of the openings of the mesh sheet may be, for example, a square, a diamond, a parallelogram, or the like.

[0049] Mesh sheet 30 has a porosity (opening rate) of 50 to 90%. "Porosity (opening rate)" refers to the ratio of the area of the openings to the entire area of mesh sheet 30. For example, the porosity can be calculated using the formula "Porosity (%) = (short side W1 of openings 32 × long side W2 of openings 32 × number of openings 32) / area of mesh sheet 30 × 100." Because mesh sheet 30 has such a high porosity, placental cells can easily pass through openings 32. This increases the efficiency of placental cell migration from the placental cell culture section to central channel 21. The porosity (opening rate) of mesh sheet 30 is preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more.

[0050] The material of the mesh sheet is not particularly limited, but a highly biocompatible material is preferred. Examples of the mesh sheet material 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, fluorine-based resins, and polyamides. Examples of natural fibers include plant fibers such as cotton, hemp, bamboo, and paper; and animal fibers such as wool and silk. The mesh sheet may be a blend of synthetic resin fibers and natural fibers. Examples of ceramics include alumina (Al2O3). Examples of metals include stainless steel, steel, aluminum, gold, and platinum. These materials may also be modified. Examples of such modifications include hydrophilization and coating with extracellular matrices (e.g., collagen, fibronectin, etc.).

[0051] The mesh sheet is not particularly limited as long as it is made of linear members formed into a mesh shape. The mesh sheet may be a woven fabric (either a single raschel or a double raschel), a knitted fabric, a cross-point welding type, an extrusion-molded sheet (such as a trical net or a netron sheet), or a mesh-shaped sheet formed by punching or the like.

[0052] (thin film sheet) In placental cell culture device 1, thin film sheet 40 separates placental cell culture section 12 from first side channel 22a and second side channel 22b. Thin film sheet 40 has thin film opening 41. In placental cell culture device 1, thin film sheet 40 is positioned so that thin film opening 41 overlaps central channel 21. At thin film opening 41, central hole 11 and central channel 21 are adjacent to each other via mesh sheet 30 only, without the thin film sheet 40 in between. Thin film opening 41 can be sized according to central channel 21 in second substrate 20. The width of thin film opening 41 is preferably slightly smaller than the width of central channel 21. The width of thin film opening 41 is, for example, 100 to 3000 μm. The width of thin film opening 41 is preferably 200 μm or more, more preferably 300 μm or more, even 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, even more preferably 1600 μm or less, and particularly preferably 1400 μm or less. The length of the thin film opening 41 is preferably shorter than the length of the central flow path 21. The length of the thin film opening 41 is, for example, 500 to 10,000 μm. The length of the thin film opening 41 is, for example, preferably 1,000 μm or more, more preferably 1,500 μm or more, even more preferably 2,000 μm or more, and particularly preferably 2,500 μm or more. The length of the thin film opening 41 is, for example, preferably 8,000 μm or less, more preferably 7,000 μm or less, even more preferably 6,000 μm or less, and particularly preferably 5,000 μm or less.

[0053] The material of the thin film sheet 40 is not particularly limited, but a highly biocompatible material is preferable. Examples of materials for the thin film sheet 40 include, but are not limited to, polyimide, polyethylene terephthalate (PET), polystyrene, polyethylene, polypropylene, nylon, polytetrafluoroethylene (PTFE), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-hexafluoropropylene copolymer (FEP), tetrafluoroethylene-perfluoroalkylvinylether copolymer (PFA), and polydimethylsiloxane (PDMS). The thin film sheet 40 may be, for example, a resin film formed from the above-mentioned materials.

[0054] Placental cell culture device 1 can be produced by stacking and adhering second substrate 20, thin film sheet 40, mesh sheet 30, and first substrate 10 in this order. When assembling placental cell culture device 1, through-holes P1a-Pa6 of first substrate 10 are aligned with recesses P1b-P6b of second substrate 20, respectively. Furthermore, central channel 21 of second substrate 20 is aligned at least partially with thin film opening 41 of thin film sheet 40. Central hole 11 of first substrate 10 and central channel 21 of second substrate 20 are adjacent at least partially with mesh sheet 30 interposed therebetween.

[0055] When assembling placental cell culture device 1, the surfaces of first substrate 10 and second substrate 20 may be activated to enhance adhesiveness. For example, when first substrate 10 and second substrate 20 are made of PDMS, the surfaces can be activated by oxygen plasma treatment to enhance adhesiveness. In this case, it is preferable to perform a hydrophobic treatment on the flow path surfaces after assembly. When first substrate 10 and second substrate 20 are made of PDMS, the substrate surfaces, including the flow path surfaces, can be hydrophobicized by heat treatment (e.g., at 80°C overnight).

[0056] (3D culture model) The placental cell culture device of this embodiment can be used to prepare 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 an interaction evaluation factor is present in the factor flow path.

[0057] <Interaction evaluation factors> In placental cell culture device 1, central channel 21 functions as a channel for factors. The interaction evaluation factors are not particularly limited, and any factors can be selected depending on the purpose. The interaction evaluation factors present in central channel 21 may be one type or a combination of two or more types.

[0058] The interaction assessment factor can be dissolved or suspended in a buffer solution (PBS, medium, etc.) and introduced into central channel 21 by injecting the solution from fifth port P5 or sixth port P6. A gelling agent may be added to the buffer solution or the like in which the interaction assessment factor is dissolved or suspended, and the buffer solution or the like may be filled into central channel 21 and then gelled. In this way, central channel 21 is filled with a gel containing the interaction assessment factor. In placental cell culture device 1, central channel 21 and placental cell culture section 12 are separated by mesh sheet 30, so that the solution or suspension introduced into central channel 21 does not leak into placental cell culture section 12.

[0059] The gelling agent is not particularly limited, but it is preferable to use one with high biocompatibility. Examples of gelling agents include, but are not limited to, extracellular matrices such as collagen (type I, type II, type III, type V, type XI, etc.), gelatin, elastin, proteoglycans, glycosaminoglycans, fibronectin, vitronectin, laminin, pectin, hyaluronic acid, chitin, and chitosan; polysaccharides such as alginic acid and starch; amino acid polymers such as polylysine and polyarginine; and fibrous proteins such as fibrin. Alternatively, commercially available scaffolds such as Matrigel® (Corning) may be used. When fibrin is used as the gelling agent, it may be prepared as a mixture of fibrinogen and thrombin. In this case, fibrinogen is converted into fibrin by the action of thrombin, resulting in gelation.

[0060] After introducing the interaction assessment factor into the central channel 21, it is preferable to introduce culture medium into the first side channel 22a and the second side channel 22b. The culture medium can be introduced into the first side channel 22a by injecting it through the first port P1 or the second port P2. The culture medium can be introduced into the second side channel 22b by injecting it through the third port P3 or the fourth port P4. By filling the first side channel 22a and the second side channel 22b with culture medium, it is possible to prevent the gel filled in the central channel 21 from drying out. Furthermore, when the interaction assessment factor is a cell, components necessary for the survival of the interaction assessment factor present in the central channel 21, components that control the interaction assessment factor, and the like can be supplied to the interaction assessment factor.

[0061] After introducing the interaction assessment factor into central channel 21, placental cell culture section 12 may be filled with medium. When the interaction assessment factor is a cell, the proliferation of the interaction assessment factor can be controlled by filling placental cell culture section 12 with medium. For example, when the interaction assessment factor is a vascular endothelial cell such as HUVEC, the formation of a three-dimensional vasculature in central channel 21 can be controlled by filling or not filling placental cell culture section 12 with medium.

[0062] 5A is a schematic diagram showing the formation of a three-dimensional vasculature (E) by filling central channel 21 with gel G containing vascular endothelial cells (HUVECs, etc.), followed by filling first side channel 22a, second side channel 22b, and placental cell culture section 12 with media M1 to M3, respectively. When medium M3 is present in placental cell culture section 12, a vasculature is formed not only in the directions of both side channels but also in the direction of placental cell culture section 12. The three-dimensional vasculature functions as an interaction evaluation factor and is indicated by E in the figure.

[0063] 5B is a schematic diagram showing the formation of a three-dimensional vascular system (E) after filling central channel 21 with gel G containing vascular endothelial cells (HUVECs, etc.), filling first side channel 22a and second side channel 22b with media M1 and M2, respectively, and not filling placental cell culture section 12 with media. In the absence of media in placental cell culture section 12, vascular systems are formed in the directions of both side channels, but not in the direction of placental cell culture section 12.

[0064] Figure 18 shows a schematic diagram of EVT cells and maternal blood vessels. EVT cells (EVT) are thought to arise from CT cells (CT) that come into contact with the decidua (DM) and are observed on the surface and inside of the decidua (HA). The DM contains blood vessels (spiral arteries: HA) that transport blood (BL) to the villus (V), and the spiral arteries (HA) have their exits on the surface of the DM (the surface in contact with blood). EVT cells (EVT) infiltrate the inner wall of the vascular system from the exit of the spiral arteries (HA) and remodel the vascular structure. Meanwhile, EVT cells (EVT) that infiltrate into the decidua (DM) are thought to migrate toward the spiral arteries (HA) and infiltrate into the vascular interior from the outside of the spiral arteries (HA). The 3D vascular system shown in Figure 5A can be used to create a 3D culture model in which EVT cells infiltrate into blood vessels from the arterial exit side. The 3D vasculature in Figure 5B can be used to create a 3D culture model in which EVT cells that have invaded the decidua infiltrate into blood vessels.

[0065] A three-dimensional vasculature can be formed by filling central channel 21 with gel G containing vascular endothelial cells (e.g., HUVECs), followed by filling first side channel 22a and second side channel 22b with medium M1 and medium M2, respectively, and culturing the cells. If placental cell culture section 12 is filled with medium M3 during the culturing period, a three-dimensional vasculature as shown in FIG. 5A can be formed. If placental cell culture section 12 is not filled with medium M3 during the culturing period, a three-dimensional vasculature as shown in FIG. 5B can be formed. The culturing period is not particularly limited, and the cells may be cultured until the three-dimensional vasculature reaches the desired state. The culturing period can be, for example, one day or more, two days or more, or three days or more. The upper limit of the culturing period is not particularly limited, but can be, for example, 10 days or less, 8 days or less, or 5 days or less.

[0066] Culture conditions can be those generally used for culturing animal cells, such as a culture temperature of 32 to 40°C (preferably 35 to 38°C) and a CO2 concentration of 2 to 5% (preferably 5%).

[0067] Medium M1 introduced into first side channel 22a, medium M2 introduced into second side channel 22b, and medium M3 introduced into placental cell culture section 12 can be selected appropriately depending on the type of interaction assessment factor. Mediums M1 to M3 may be the same medium or different mediums. When the interaction assessment factor is vascular endothelial cells such as HUVECs, media M1 to M3 can be a medium containing vascular endothelial growth factor (VEGF) or the like, a culture supernatant of fibroblasts (such as the medium for three-dimensional blood vessels used in the Examples described below), or the like.

[0068] ≪Placental cells≫ Placental cells are cultured in placental cell culture section 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 placental cell culture section 12 may be of one type or two or more types. Placental cells may or may not form cell aggregates. Placental cells may be cells attached to the bottom (mesh sheet 30 or thin film sheet 40) of placental cell culture section 12, may be planar cell aggregates (e.g., sheet-like cell aggregates), or may be three-dimensional cell aggregates (e.g., spherical cell aggregates, cell aggregates forming organoids). Cell aggregates may be composed of one type of cell, or may be composed of two or more types of cells.

[0069] Placental cell culture medium The medium used for culturing placental cells can be appropriately selected depending on the purpose. 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.

[0070] Examples of basal media include Doulbecco's modified Eagle's Medium (DMEM), DMEM / F12, IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Ham's F12, RPMI 1640, Fischer's, and mixtures thereof. A preferred basal medium is DMEM / F12.

[0071] The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or serum substitutes, as needed. Examples of serum substitutes include 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, and 3'-thiolglycerol. The basal medium may also contain components, such as lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts, as needed. These components can be used in appropriate combinations.

[0072] The basal medium may be, for example, a medium obtained by adding bovine serum albumin (BSA), ITS-X, L-ascorbic acid, and antibiotics (penicillin, streptomycin, etc.) to the above-mentioned basal medium (e.g., DMEM / F12).Specific examples of the basal medium include TS basal medium and 2D-EVT basal medium used in the Examples described below.

[0073] The growth factor is not particularly limited, but examples thereof include epidermal growth factor (EGF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), neuregulin (NGR), etc. When the medium contains FGF, the medium may also contain heparin. Heparin has the effect of promoting the activity of FGF. Heparin is preferably in the form of a salt.

[0074] Examples of enzyme inhibitors include ROCK inhibitors (Y27632, etc.), GSK3β inhibitors (CHIR99021, etc.), p38 MAPK inhibitors (SB202190, etc.), HDAC inhibitors (valproic acid: VPA, etc.), and ALK inhibitors (A83-01, etc.).

[0075] For example, when the placental cells are TS cells, TS medium can be used, and when TS cells are differentiated into EVT cells, 2D-EVT medium can be used.

[0076] Placental cells can be cultured under conditions generally used for culturing animal cells, such as a culture temperature of 32 to 40°C (preferably 35 to 38°C) and a CO2 concentration of 2 to 5% (preferably 5%).

[0077] In a three-dimensional culture model using placental cell culture device 1, central channel 21 and placental cell culture section 12 are separated by mesh sheet 30. Therefore, placental cells in placental cell culture section 12 can interact with an interaction assessment factor in central channel 21 through openings 32 in mesh sheet 30. Furthermore, placental cells in placental cell culture section 12 can easily migrate to central channel 21 through openings 32 in mesh sheet 30. Therefore, the three-dimensional culture model of this embodiment can be used to evaluate the interaction between placental cells and an interaction assessment factor.

[0078] 6A to 7B show an example of application of the three-dimensional culture model. FIG. 6A shows an example of a three-dimensional culture model for evaluating the function of EVT cells. In the three-dimensional culture model of FIG. 6A, a three-dimensional vasculature (E) is formed in the central channel 21, and EVT cells (EVT) are cultured in the placental cell culture section 12. The EVT cells (EVT) cultured in the placental cell culture section 12 can migrate into the central channel 21 through the openings 32 in the mesh sheet 30. The three-dimensional culture model of FIG. 6A can also be used to evaluate the infiltration of EVT cells into spiral arteries. In the placental cell culture section 12, undifferentiated cells such as TS cells or CT cells may be cultured using an EVT cell differentiation-inducing medium and then induced to differentiate into EVT cells.

[0079] Figure 6B shows an example of a three-dimensional culture model for evaluating the permeability of ST cell membranes to chemicals. In the three-dimensional culture model shown in Figure 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. The placental cell membrane has a structure resembling that of in vivo villi and is composed of ST cells (ST) in the upper layer and undifferentiated cells (UD) (TS cells, CT cells, etc.) in the lower layer. In the three-dimensional culture model shown in Figure 6B, the placental cell membranes are cultured so as to cover the mesh sheet 30 exposed to the placental cell culture section 12. By adding any chemical substance to the medium M3 in the placental cell culture section 12, it is possible to evaluate whether 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 such as a cell matrix and perfusion culturing them using an ST cell induction medium. Examples of ST cell induction media include media prepared by adding a ROCK inhibitor (Y27632, etc.), a GSK3β inhibitor (CHIR99021, etc.), a p38 MAPK inhibitor (SB202190, etc.), and a growth factor (EGF, BMP4, bFGF, etc.) to a basal medium for animal cells (e.g., TS basal medium, etc.).

[0080] FIG. 7A shows an example of a three-dimensional culture model that mimics the placental development process. FIG. 7B is an enlarged view of area B, enclosed by a dashed line, in the three-dimensional culture model shown in FIG. 7A. In the three-dimensional culture model shown in 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. The placental cell aggregates have a structure that mimics the villi in vivo 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 shown in FIG. 7A, fibroblasts (FB) are cultured together with the placental cell aggregates in the placental cell culture section 12. In vivo, as pregnancy progresses and the villi grow, the villi come into contact with the maternal decidua. This contact causes undifferentiated cells, such as CT cells, to emerge from the villi and migrate onto and / or into the decidua, where they come into contact with the decidua. Undifferentiated cells that come into contact with the decidua differentiate into EVT cells and reconstruct maternal blood vessels within the decidua. Therefore, a model of placental development can be constructed by co-culturing placental cell aggregates with fibroblasts in a placental cell culture section. Placental cell aggregates can be produced, for example, by culturing undifferentiated cells (TS cells, CT cells, etc.) in an ST cell induction medium in a well with a non-cell-adhesive inner wall. The ST cell induction medium can be the same as that described above.

[0081] (Method for creating a 3D culture model) The method for producing a three-dimensional culture model of this embodiment includes the steps of introducing an interaction evaluation factor into central channel 21, which is a channel for a factor, and culturing placental cells in placental cell culture section 12 in the presence of the interaction evaluation factor in central channel 21. Methods for introducing the interaction evaluation factor into central channel 21 include the methods described above. Methods for culturing placental cells in placental cell culture section 12 include the methods described above.

[0082] When the interaction evaluation factor present in the factor flow channel is a three-dimensional vasculature, the three-dimensional culture model can be produced by a method including the steps of culturing vascular endothelial cells in central flow channel 21, which is a factor flow channel, to form a three-dimensional vasculature, and culturing the three-dimensional vasculature in central flow channel 21 and culturing placental cells in placental cell culture section 12. Examples of methods for forming a three-dimensional vasculature in central flow channel 21 include the methods described above.

[0083] Culture conditions can be those generally used for culturing animal cells, such as a culture temperature of 32 to 40°C (preferably 35 to 38°C) and a CO2 concentration of 2 to 5% (preferably 5%).

[0084] Second Embodiment 8A to 11B show an example of a placental cell culture device according to the second embodiment. In this embodiment, microposts are used as partition members between the placental cell culture section and the factor flow path. FIG. 8A is a perspective view of placental cell culture device 100. FIG. 8B is a cross-sectional view of placental cell culture device 100 taken along section line BB. FIG. 9 is an exploded view of placental cell culture device 100.

[0085] Placental cell culture device 100 is composed of first substrate 110, second substrate 120, and 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.

[0086] Placental cell culture device 100 includes a central channel 111, a first side channel 112a, and a second side channel 112b. Placental cell culture device 100 includes 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 culture medium and the like. First port P1 is connected to first side channel 112a via a first port channel p1. Second port P2 is connected to first side channel 112a via a second port channel p2. Third port P3 is connected to second side channel 112b via a third port channel p3. Fourth port P4 is connected to second side channel 112b via a fourth port channel p4. Fifth port P5 and sixth port P6 are connected to 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.

[0087] In placental cell culture device 100, central channel 111 functions as a placental cell culture section. Communication hole 115 is provided above central channel 111, which serves as a placental cell culture section. Ring member 130 is laminated on first substrate 110 so that communication hole 115 is located within the ring hole. In placental cell culture device 100, first side channel 112a and / or second side channel 112b function as a channel for factors.

[0088] (First board) Fig. 10A is a top view of first substrate 110. Fig. 10B is an enlarged view of part B surrounded by a dashed line in the top view of first substrate 110 shown in Fig. 10A. The first substrate 110 is provided with a central channel 111, a first side channel 112a, a second side channel 112b, a first port P1 to a sixth port P6, and a first port channel p1 to a fourth port channel p4. The central channel 111, the first side channel 112a, the second side channel 112b, and the first port channel p1 to the fourth port channel p4 are closed on a first surface side of the first substrate 110, and are open on a second surface side of the first substrate 110. The first substrate 110 is stacked on the second substrate 120 so that the second surface faces the second substrate 120. As a result, the second substrate 120 forms the bottom surface of each channel of the first substrate 110.

[0089] Central channel 111 is formed in the approximate center of first substrate 110. In placental cell culture device 100, central channel 111 functions as a placental cell culture section.

[0090] A first side channel 112a and a second side channel 112b are formed on both sides of the central channel 111. A first port channel p1 is connected to one end of the first side channel 112a, and a second port channel p2 is connected to the other end of the first side channel 112a. A third port channel p3 is connected to one end of the second side channel 112b, and a fourth port channel p4 is connected to the other end of the second side channel 112b. The first side channel 112a and the second side channel 112b may be formed on both sides of the entire length of the central channel 111, or may be formed on both sides of a portion of the central channel 111. When the first side channel 112a and the second side channel 112b are formed on both sides of a portion of the central channel 111, it is preferable that the first side channel 112a and the second side channel 112b are formed at least on both sides of a position where the communication hole 115 is formed.

[0091] A communication hole 115 is provided above the central channel 111. The communication hole 115 is a through-hole that penetrates from the first surface side of the first substrate 10 to the central channel 111. The central channel 111 communicates with an external region on the first surface side of the first substrate 110 via the communication hole 115. The size of the communication hole 115 is not particularly limited, but is preferably smaller than the width of the central channel 111. The communication hole 115 has a diameter, for example, of 0.05 to 20 mm. The size of the communication hole 115 is preferably, for example, 0.1 mm or more, more preferably 0.2 mm or more, even more preferably 0.3 mm or more, and particularly preferably 0.4 mm or more. The size of the communication hole 115 is, for example, preferably 15 mm or less, more preferably 10 mm or less, even more preferably 5 mm or less, and particularly preferably 1 mm or less.

[0092] The width of the central channel 111 is not particularly limited, but may be, for example, 500 to 5000 μm. The width of the central channel 111 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 central channel 111 is preferably 5000 μm or less, more preferably 4000 μm or less, even more preferably 3000 μm or less, and particularly preferably 2500 μm or less.

[0093] The widths of the first side channel 112a and the second side channel 112b are not particularly limited, but may be, for example, 500 to 3000 μm. The widths of the first side channel 112a and the second side channel 112b are 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 widths of the first side channel 112a and the second side channel 112b are 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.

[0094] The height (depth) of the central channel 111, first side channel 112a, and second side channel 112b is not particularly limited, but may be, for example, 50 to 1000 μm. The height (depth) of the central channel 111, first side channel 112a, and second side channel 112b is preferably 20 μm or more, more preferably 40 μm or more, even more preferably 50 μm or more, and particularly preferably 80 μm or more. The height (depth) of the central channel 111, first side channel 112a, and second side channel 112b is preferably 700 μm or less, more preferably 500 μm or less, even more preferably 300 μm or less, and particularly preferably 200 μm or less. It is preferable that the heights (depths) of the central channel 111, the first side channel 112a, and the second side channel 112b are approximately the same.

[0095] The length of the portion of the central channel 111 where the first side channel 112a and the second side channel 112b are adjacent is not particularly limited, but may be, for example, 500 to 30,000 μm. The length is, for example, 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 length is, for example, preferably 25,000 μm or less, more preferably 20,000 μm or less, even more preferably 15,000 μm or less, and particularly preferably 10,000 μm or less.

[0096] Central channel 111 and first side channel 112a are separated by partition member 113a. Central channel 111 and second side channel 112b are separated by partition member 113b. Partition members 113a and 113b are composed of a plurality of microposts 114. The plurality of microposts 114 constituting partition members 113a and 113b are arranged at predetermined intervals. In partition members 113a and 113b, microposts 114 form shielding portions, and gaps between microposts 114 form communicating portions. In placental cell culture device 100, central channel 111 functions as a placental cell culture portion, and first side channel 112a or second side channel 112b functions as a channel for factors. Partition member 113a or partition member 113b is a partition member that separates central channel 111, which is a placental cell culture section, from first side channel 112a or second side channel 112b, which is a channel for factors.

[0097] The shape of the microposts 114 is not particularly limited. The microposts 114 may be polygonal prisms (triangular prisms, quadrangular prisms, etc.) or cylindrical. The microposts 114 are, for example, trapezoidal prisms. 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 greater than the width w1 on the central channel 21 side. This makes it possible to prevent the liquid filled in the central channel 111 from leaking into the first side channel 112a and the second side channel 112b.

[0098] 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, even 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, even more preferably 100 μm or less, and particularly preferably 80 μm or less. The width w2 on the first side channel 112a side or the second side channel 112b side 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, even more preferably 200 μm or less, and particularly preferably 150 μm or less. The length l of the microposts 114 is, for example, 40 to 500 μm. The length l 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 length l is, for example, preferably 400 μm or less, more preferably 300 μm or less, even 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 .

[0099] The shortest distance d between two microposts 114 corresponds to the minimum width of the communication part in the partition member. The shortest distance d is 30 to 500 μm. When the minimum width of the communication part is within this range, the migration efficiency of placental cells is maintained at a good level. The shortest distance d is, for example, preferably 40 μm or more, more preferably 50 μm or more, even more preferably 60 μm or more, and particularly preferably 70 μm or more. The shortest distance d 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.

[0100] The longest distance between two microposts 114 corresponds to the longest width of the communication portion in the partition member. Examples of the longest distance include 50 to 500 μm. The longest distance is preferably, for example, 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 longest distance is preferably, for example, 400 μm or less, more preferably 350 μm or less, even more preferably 300 μm or less, and particularly preferably 250 μm or less.

[0101] The surfaces of the central channel 111, the first side channel 112a, and the second side channel 112b are preferably hydrophobic. If the surfaces of the channels are hydrophobic, when one of the channels is filled with liquid, surface tension of the liquid is generated between the microposts 114. This prevents the liquid from leaking into an adjacent channel when there is no liquid in the adjacent channel.

[0102] The thickness of the first substrate 110 is not particularly limited, but may be, for example, 0.1 to 50 mm. The thickness of the first substrate 110 is, for example, preferably 0.3 mm or more, more preferably 0.5 mm or more, even more preferably 0.6 mm or more, and particularly preferably 0.8 mm or more. The thickness of the second substrate 20 is, for example, 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.

[0103] The material of the first substrate 110 is not particularly limited, but is preferably a material that is highly biocompatible and highly oxygen permeable. Examples of oxygen permeable materials include oxygen permeable polymers. Examples of oxygen permeable polymers include fluororesin and silicone (e.g., PDMS). Of these, PDMS is a preferred oxygen permeable polymer.

[0104] The first substrate 110 can be fabricated using known methods such as photolithography, soft lithography, microcontact printing, microfluidic printing, and stencil printing. For example, a mold for the first substrate 110 can be fabricated using photolithography, and the mold can be used to fabricate the first substrate 110 by soft lithography. Except for the shape of the mold, the first substrate 110 can be fabricated in the same manner as the first substrate 10 and second substrate 20 of the first embodiment.

[0105] (Second board) The second substrate 120 can be a flat plate of approximately the same size as the first substrate 110. There are no particular limitations on the material of the second substrate 120, but a material with high biocompatibility is preferable. Examples of materials for the second substrate 120 include glass, various synthetic resins, and metal.

[0106] (Ring member) The ring member 130 is stacked on the first surface of the first substrate 110 so that the communication hole 115 of the first substrate 110 is positioned within the ring hole. The ring member 130, together with the first substrate 110, forms an upper well 131. The side surface of the upper well 131 is formed by the inner ring wall of the ring member 130, and the bottom surface is formed by the first surface of the first substrate 110. The upper well 131 is in communication with the central channel 111 via the communication hole 115.

[0107] The ring member 130 has a ring hole that is 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, more preferably 3 mm or more, even more preferably 4 mm or more, and particularly preferably 5 mm or more. The size of the ring hole is preferably 15 mm or less, more preferably 12 mm or less, even more preferably 10 mm or less, and particularly preferably 8 mm or less. The outer periphery 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 outer periphery of the ring member 130 is, for example, 3 to 30 mm in diameter. The outer periphery is preferably 4 mm or more, more preferably 5 mm or more, even more preferably 6 mm or more, and particularly preferably 7 mm or more. The outer periphery size 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.

[0108] The material of the ring member 130 is not particularly limited, but a highly biocompatible material is preferable. Examples of materials for the ring member 130 include, but are not limited to, polyimide, polyethylene terephthalate (PET), polystyrene, polyethylene, polypropylene, nylon, polytetrafluoroethylene (PTFE), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-hexafluoropropylene copolymer (FEP), tetrafluoroethylene-perfluoroalkylvinylether copolymer (PFA), and polydimethylsiloxane (PDMS). The ring member 130 can be formed of, for example, PDMS.

[0109] Placental cell culture device 100 can be produced by stacking and adhering second substrate 120, first substrate 110, and ring member 130 in that order. When assembling placental cell culture device 1, the second surface of first substrate 110 faces second substrate 120. Communication hole 115 is positioned within the ring hole of ring member 130.

[0110] When assembling placental cell culture device 100, the surfaces of first substrate 110 and / or second substrate 120 may be activated to enhance adhesiveness. For example, if first substrate 110 and / or second substrate 120 are made of PDMS, the surfaces can be activated by oxygen plasma treatment to enhance adhesiveness. In this case, it is preferable to perform a hydrophobic treatment on the flow path surfaces after assembly. If first substrate 110 and / or second substrate 120 are made of PDMS, the substrate surfaces, including the flow path surfaces, can be hydrophobized by heat treatment (e.g., at 80°C overnight).

[0111] In the placental cell culture device of this embodiment, central channel 111, which is the placental cell culture section, may be filled with a gel containing cell aggregates of placental cells. Cell aggregates C of placental cells may be composed of one type of placental cell, or may be composed of two or more types of placental cells. Cell aggregates C of placental cells preferably contain EVT cells. Cell aggregates containing EVT cells can be prepared, for example, by culturing TS cells in an EVT cell induction medium in the wells of an agarose microwell plate.

[0112] Examples of EVT cell induction media include media prepared by adding a ROCK inhibitor (Y27632, etc.), a p38 MAPK inhibitor (SB202190, etc.), and growth factors (EGF, NGR1, etc.) to a basal medium for animal cells (e.g., TS basal medium, etc.). Extracellular matrices such as Matrigel (registered trademark) (CORNING) may also be added to the EVT cell induction medium. Specific examples of EVT cell induction media include the 3D-EVT medium and 3D-EVT-Dox medium used in the Examples.

[0113] Culture conditions can be those generally used for culturing animal cells, such as a culture temperature of 32 to 40°C (preferably 35 to 38°C) and a CO2 concentration of 2 to 5% (preferably 5%).

[0114] Cell aggregates of placental cells can be introduced into central channel 111 by suspending them in a gelling solution containing a gelling agent and injecting the suspended cell aggregates through communicating hole 115, or through fifth port P5 or sixth port P6. The number of placental cell aggregates introduced into central channel 111 may be one, or two or more. For example, one cell aggregate can be introduced into central channel 111 through communicating hole 115. In this case, the cell aggregate introduced into central channel 111 can be present below communicating hole 115. Central channel 111 is separated from first side channel 112a and second side channel 112b by partition member 113a and partition member 113b, respectively. Therefore, the gelling solution introduced into central channel 111 does not leak into first side channel 112a and second side channel 112b. After central channel 111 is filled with the gelling solution containing cell aggregates of placental cells, the gelling solution is allowed to gel. This allows for the production of placental cell culture device 100, in which central channel 111, the placental cell culture section, is filled with a gel containing cell aggregates of placental cells.

[0115] The gelling agent used in the gelling solution is not particularly limited, and the same as those described above can be used. Specific examples of the gelling solution include a mixed solution of Matrigel (registered trademark) (CORNING) and collagen I.

[0116] When central channel 111 is filled with a gel containing placental cells, first side channel 112a, second side channel 112b, and upper well 131 may be filled with a culture medium to prevent the gel from drying out. The culture medium can be selected appropriately depending on the type of placental cells introduced into central channel 111. Specific examples of culture media include TS basal medium used in the examples.

[0117] (3D culture model) The placental cell culture device of this embodiment can be used to create a three-dimensional culture model containing placental cells. In the three-dimensional culture model, placental cells are cultured in a placental cell culture section, and an interaction evaluation factor is present in a factor channel. FIGS. 11A to 12B show an example of the three-dimensional culture model of this embodiment. In the three-dimensional culture model of FIGS. 11A to 12B, a gel G containing cell aggregates C of placental cells is filled in central channel 111. Medium M1 containing an interaction evaluation factor E is introduced into first side channel 112a. Medium M2 is introduced into second side channel 112b. Medium M3 is introduced into upper well 131. Medium M1 to M3 may be the same medium or different mediums.

[0118] ≪Placental cells≫ In placental cell culture device 100, central channel 111 functions as a placental cell culture section. The placental cells cultured in central channel 111 are preferably cell aggregates. Cell aggregates of placental cells can be introduced into central channel 111 as described above.

[0119] By introducing a gelling solution containing cell aggregates C of placental cells into central channel 111 through communication hole 115, cell aggregates C can be present in central channel 111 at the position where communication hole 115 is provided. By using cell aggregates of placental cells, placental cell migration can be accurately evaluated. For example, if the shape of cell aggregates C extends toward the factor channel side where interaction evaluation factor E is present, it can be evaluated that interaction evaluation factor E induces positive migration of placental cells (e.g., Figure 12B). Conversely, if the distribution area of cell aggregates C on the factor channel side where interaction evaluation factor E is present decreases, it can be evaluated that interaction evaluation factor E induces negative migration of placental cells.

[0120] <Interaction evaluation factors> In placental cell culture device 100, first side channel 112a and / or second side channel 112b function as a channel for a factor. The interaction evaluation factor is not particularly limited, and any factor can be selected depending on the purpose. The interaction evaluation factor present in central channel 21 may be one type, or a combination of two or more types.

[0121] 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 through 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 through the third port P3 or the fourth port P4.

[0122] The medium used for dissolving or suspending the interaction assessment factor can be appropriately selected depending on the types of placental cells and interaction assessment factor cultured in central channel 111. Specific examples of the medium include the assay medium used in the Examples.

[0123] When the interaction evaluation factor is vascular endothelial cells such as HUVECs, the vascular endothelial cells can be suspended in a medium, filled into first side channel 112a or second side channel 112b, and cultured to form a vascular wall along partition member 113a or partition member 113b (e.g., FIG. 11B). The vascular wall as shown in FIG. 11B can be formed by introducing a medium containing vascular endothelial cells into first side channel 112a, leaving placental cell culture device 100 with first side channel 112a facing upward, and culturing the cells for a desired period of time (e.g., about 10 to 60 minutes).

[0124] The interaction evaluation factor may be introduced into either the first side channel 112a or the second side channel 112b, or into both. When evaluating placental cell migration using a three-dimensional culture model, the interaction evaluation factor is preferably introduced into either the first side channel 112a or the second side channel 112b. In this case, only the medium may be introduced into the side channel into which no interaction evaluation factor has been introduced. Alternatively, different interaction evaluation factors may be introduced into the first side channel 112a and the second side channel 112b, respectively.

[0125] In the three-dimensional culture model of the present embodiment, it is preferable to fill upper well 131 with culture medium. By filling upper well 131 with culture medium, placental cells introduced into central channel 111 can be maintained well.

[0126] (Method for creating a 3D culture model) The method for producing a three-dimensional culture model of this embodiment includes the steps of filling central channel 111, which is a placental cell culture section, with a gel containing cell aggregates of placental cells, and causing an interaction assessment factor to be present in first side channel 112a and / or second side channel 112b, which are factor channels, and culturing the cell aggregates of placental cells in central channel 111. Methods for filling central channel 111 with a gel containing cell aggregates of placental cells include the methods described above. Methods for causing an interaction assessment factor to be present in first side channel 112a and / or second side channel 112b include the methods described above.

[0127] When the interaction evaluation factor present in the factor channel is a vascular wall, the three-dimensional culture model can be produced by a method including the steps of filling central channel 111, which is a placental cell culture section, with a gel containing cell aggregates of placental cells, culturing vascular endothelial cells in first side channel 112a and / or second side channel 112b, which are factor channels, to form a vascular wall, and culturing the vascular wall in first side channel 112a and / or second side channel 112b, and culturing cell aggregates of placental cells in central channel 111. Examples of methods for forming a vascular wall in first side channel 112a and / or second side channel 112b include the methods described above.

[0128] Culture conditions can be those generally used for culturing animal cells, such as a culture temperature of 32 to 40°C (preferably 35 to 38°C) and a CO2 concentration of 2 to 5% (preferably 5%).

[0129] [Method for evaluating placental cells] In one aspect, the invention provides 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 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.

[0130] The placental cell culture device of the first embodiment can be used to evaluate various functions of placental cells. For example, by culturing placental cell structures (placental organoids) that mimic in vivo villus structures in the placental cell culture section, the responses of placental cells to various interaction evaluation factors can be evaluated.

[0131] The placental cell device of the second embodiment is suitable for evaluating placental cell migration. In the placental cell device of the second embodiment, a gel containing cell aggregates of placental cells is filled into the central channel, and the side channels are used as flow channels for factors. This allows for accurate evaluation of the effects of various interaction evaluation factors on placental cell migration by observing changes in the shape of the cell aggregates of placental cells. [Example]

[0132] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0133] [Example 1] In Example 1, a three-dimensional culture model containing a HUVEC vascular network and EVT cells was prepared using the placental cell culture device of the first embodiment described above (see FIGS. 1A to 4B).

[0134] <Preparation of placental cell culture device> (Photolithographic mold fabrication) Photolithography was used to fabricate molds for the first and second substrates of the placental cell culture device. First, a high-purity silicon wafer (AS ONE) was spin-coated with a permanent epoxy negative photoresist, SU-8 2100 (KAYAKU Advanced Materials), at 500 rpm for 10 seconds, followed by 1500 rpm for 30 seconds. The silicon wafer with the spin-coated photoresist was heated on a hot plate at 65°C for 5 minutes, then at 95°C for 45 minutes. This resulted in the fabrication of a 200 μm-thick SU-8 film. Two SU-8 films were fabricated, one for the first substrate and one for the second substrate. Photomasks for each substrate were fabricated using a DWL200 laser lithography system (Heidelberg Instruments). The 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). The molds were then heated on a hot plate at 65°C for 5 minutes and then on a hot plate at 95°C for 15 minutes. They were then cooled at room temperature for 5 minutes. After cooling, each mold was placed in a stirred SU-8 developer (KAYAKU Advanced Materials) and reacted for 1.5 to 2 hours to remove uncured SU-8. Each mold was then washed with 86% ethanol-IP (denatured) (FUJIFILM Wako). Each mold was then heat-treated at 65°C for 2 to 3 minutes, then at 150°C for 10 minutes.

[0135] (Fabrication of the first and second substrates by soft lithography) The first and second substrates were fabricated by soft lithography using a mold fabricated by photolithography. A PDMS solution was prepared by mixing the base resin and curing agent in a 10:1 ratio and poured into the mold. After degassing, the PDMS solution was cured by heating at 80°C overnight. The PDMS solution was poured into the mold to a thickness of approximately 1 mm for the second substrate and approximately 1 cm for the first substrate. Each substrate component was cut out from the mold using a disposable scalpel (Kai Industries) and tweezers. Using the grooves patterned on the PDMS component for the first substrate as a reference, holes for introducing and discharging liquids into and from each channel formed on the second substrate were drilled using a biopsy trephine (Kai Industries). The central hole was 6 mm in diameter. The two ports connected to the first side channel on the second substrate were 6 mm in diameter, and the two ports connected to the second side channel on the second substrate were 4 mm in diameter. The two ports connected to the central channel on the second substrate were 1.5 mm in diameter. FIG. 13A shows a schematic diagram of the second substrate thus fabricated.

[0136] (Assembly of placental cell culture device) A placental cell culture device was assembled using the first and second substrates prepared as described above (see Figure 2).

[0137] A micromesh sheet (8 mm × 8 mm, opening: long side 160 to 170 μm, short side 110 to 115 μm) (Mizuta Manufacturing Co., Ltd.) was prepared as a mesh sheet. Figure 13B shows a photograph of the mesh sheet used.

[0138] A thin PET sheet (25 μm thick, AS ONE) was processed to a 10 mm x 5 mm size. A 5% 3-aminopropyltriethoxysilane solution (Sigma) was prepared in purified water. The processed polyethylene terephthalate (PET) sheet was silanized by placing it in the 3-aminopropyltriethoxysilane solution and treating it at 80°C for 30 minutes. The silanization was performed to enhance adhesion to the PDMS component. A 0.5 mm x 4.5 mm opening was created in the center of the silanized PET sheet using a biopsy trephine with a modified tip. A thin layer of PDMS solution was applied around the opening, and a mesh sheet was attached to cover it. Hereafter, the PET sheet with the attached mesh sheet is referred to as the "mesh sheet."

[0139] The surface of each substrate was activated by oxygen plasma treatment for 20 seconds using a small plasma device (PM100) (Yamato Scientific Co., Ltd.). The surface activation treatment was performed to improve adhesion between the first and second substrates and between each substrate and the mesh sheet. The mesh sheet was sandwiched and bonded between the surface-activated first and second substrates. Bonding was achieved by heating on a hot plate at 150°C for 10 minutes. During assembly, the opening in the PET sheet was aligned with the center of the central flow path on the second substrate. The central hole in the first substrate was aligned with the opening in the PET sheet.

[0140] The oxygen plasma treatment rendered the surface of the channel formed on the second substrate highly hydrophilic. In this state, it was not possible to introduce solutions by taking advantage of the hydrophobicity of PDMS, i.e., to selectively introduce solutions into a specific channel (the central channel). Therefore, to make the hydrophilic PDMS surface hydrophobic, the assembled device was treated overnight at 80°C. To prepare the device for cell culture, it was sterilized by UV treatment for 30 minutes. Figure 14 shows a photograph of the fabricated placental cell culture device.

[0141] <Culture medium> (3D blood vessel culture medium) Human umbilical vein endothelial cells (RFP-HUVEC) (Angio Proteome) were maintained and managed using EGM-2 Endothelial Cell Growth Medium-2 BulletKit (LONZA). To efficiently create a 3D vascular network composed of RFP-HUVECs in fibrin gel, we used conditioned medium derived from human lung fibroblasts (NHLFs) (LONZA). NHLFs were maintained in DMEM medium (Thermo Fisher Scientific) containing 10% FBS. When the NHLFs reached 70-100% of the dish area, the medium was replaced with EGM-2 medium. After culturing for 3 days, the supernatant was used as the conditioned medium. This conditioned medium was used as the medium for 3D blood vessels.

[0142] (TS basal medium) TS 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 the 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 (FUJIFILM Wako) 1% KSR(Thermo Fisher Scientific) 1% L-ascorbic acid (FUJIFILM Wako) 0.2mM

[0143] (TS medium) TS medium was prepared by adding the following components to 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 (FUJIFILM Wako) 0.8mM A83-01 (FUJIFILM Wako) 5 μM CHIR99021 (FUJIFILM Wako) 2 μM

[0144] (2D-EVT basal medium) To the DMEM / F12 medium (FUJIFILM Wako), the following components were added to prepare the 2D-EVT basal medium. The concentrations shown below are the final concentrations of each component in the 2D-EVT basal medium. Bovine serum albumin (BSA) (FUJIFILM Wako) 0.3% ITS-X (FUJIFILM Wako) 1% Penicillin 5,000 units / mL Streptomycin (Thermo Fisher Scientific) 5,000 μg / mL

[0145] (2D-EVT medium) To the 2D-EVT basal medium, the following components were added to prepare the 2D-EVT medium. The concentrations shown below are the final concentrations of each component in the 2D-EVT medium. Y27632 (FUJIFILM Wako) 2.5 μM KSR (Thermo Fisher Scientific) 4% A83-01 (FUJIFILM Wako) 7.5 μM NRG1 (Cell Signaling) 50 ng / mL

[0146] <Preparation of EVT cells> (Preparation of EGFP-TS cells) Genetically modified TS cells (EGFP-TS cells) were used. EGFP-TS cells express EGFP upon treatment with doxycycline hyclate (Sigma-Aldrich). The method for preparing EGFP-TS cells was carried out as follows. Following a previously reported method (Takahashi et al., PNAS, 2019, 116 (52) 26606-26613), lentivirus was produced by inserting the EGFP gene into the pCS-3G vector. TS cells were infected with the lentivirus, and single-cell cloning was performed to select EGFP-TS cells that overexpressed EGFP in a doxycycline-dependent manner. EGFP-TS cells were maintained in TS medium.

[0147] (Induction of differentiation from EGFP-TS cells to EVT cells) To induce differentiation of EGFP-TS cells into EVT cells, 2D-EVT medium containing 0.2 mg / mL of Matrigel (Corning) was used.

[0148] <Confirming the effectiveness of the mesh sheet> The placental cell culture device of Example 1 must have a structure that allows cells to migrate across the boundary between the first and second substrates. At the same time, it is necessary that the liquid filled in the central channel of the second substrate does not leak toward the first substrate. In the placental cell culture device of Example 1, a mesh sheet (opening: long side 160-170 μm, short side 110-115 μm; porosity 50% or more; Figure 13B) was used as a partition member that meets the above-mentioned performance.

[0149] To confirm the effectiveness of the mesh sheet, a leakage test of the colored solution filled in the central channel was conducted using a placenta cell culture device with a mesh sheet and a placenta cell culture device with a PET sheet (without a mesh sheet). The results are shown in Figures 15A and 15B. Figure 15A shows the results for a placental cell culture device using a mesh sheet. Figure 15B shows the results for a placental cell culture device using a PET sheet (no mesh sheet attached). In the placental cell culture device using a mesh sheet, no leakage of colored liquid from the central channel of the second substrate to the central hole of the first substrate was observed (Figure 15A). On the other hand, in the placental cell culture device using a PET sheet (no mesh sheet attached), colored liquid leaked from the central channel of the second substrate to the central hole of the first substrate (Figure 15B). These results confirmed that by placing a mesh sheet between the central channel of the second substrate and the central hole of the first substrate, leakage of liquid from the central channel to the central hole can be prevented.

[0150] The cell culture device described in JP 2020-188723 A has a porous membrane at the boundary between the lower and upper layers. The porous membrane prevents liquid introduced into the lower layer from leaking into the upper layer. Furthermore, cells in the upper layer can migrate through the porous membrane to the lower layer. However, there are problems with using porous membranes. For example, polycarbonate porous membranes have poor optical transparency, making it difficult to observe the morphology of cells in the upper layer from the bottom of the device using a microscope. Even when using a PET porous membrane, which has relatively good optical transparency, it is difficult to clearly observe the cell outlines. Furthermore, the hole size of commercially available porous membranes is generally around 0.4 to 8 μm, and the ratio of total pore area to membrane area (porosity) is a maximum of around 15%. Therefore, although cells can pass through 8 μm pores, for example, migration efficiency is poor.

[0151] The mesh sheet used in Example 1 has large openings and a high porosity, allowing cells to easily pass through the mesh sheet. Furthermore, because of its high light transmittance, cells present in the central hole of the first substrate can be observed under a microscope from the second substrate side through the mesh sheet. Furthermore, it can prevent liquid from leaking from the second substrate to the first substrate. Therefore, the mesh sheet is effective as a partition member between the first and second substrates.

[0152] <Co-culture of HUVEC vascular networks and EVT cells using a placenta cell culture device (1)> 1.0×10 7 A HUVEC suspension was prepared at 100 cells / mL. Next, fibrinogen from bovine plasma (Sigma) was dissolved in PBS(-) and filtered to prepare a 10 mg / mL fibrinogen solution. The HUVEC suspension and fibrinogen solution were mixed at a 1:3 ratio. Thrombin (Sigma) (50 U / mL) was added to this mixture to a final concentration of 1 U / mL, and the mixture was quickly introduced into the central channel of the second substrate. The action of thrombin converted fibrinogen to fibrin, forming a gel. The substrate was then placed in a CO2 incubator at 37°C for 5 minutes to allow the fibrin gel in the central channel to gel. The first and second side channels (approximately 250 μL / channel) and the central hole (approximately 200 μL / hole) were filled with 3D vessel medium. The next day, the medium in the first and second side channels and the central hole was replaced with 3D vessel medium.

[0153] Three days after seeding the HUVECs, TS cells were seeded in the central hole of the first substrate according to the following procedure. To coat the central hole with Matrigel (Corning), approximately 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 cells) suspended in EVT basal medium were then seeded. 5 10 μL of the cell suspension, 200 μL of 2D-EVT medium, and 5 μL of approximately 10 mg / mL Matrigel were mixed and added to the central hole of the first substrate (5 × 10 3Six days after seeding HUVECs, the medium in the central hole was replaced. The medium used for the replacement was 2D-EVT medium containing 400-fold diluted Matrigel (final concentration: approximately 0.025 mg / mL) and 1 μg / mL doxycycline hyclate. The culture was terminated 9 days after seeding HUVECs. For the first and second side channels, the medium was replaced with 3D vessel medium (conditioned medium) 3, 5, and 7 days after seeding HUVECs.

[0154] (Fluorescence microscope observation) A 4% paraformaldehyde (PFA) solution (FUJIFILM Wako) was introduced into the placental cell culture device and left at room temperature for 10 minutes to fix the cells, after which the cells were washed with a 2% FBS / PBS(-) solution. The cells were observed using an all-in-one fluorescence microscope BZ-X810 (KEYENCE).

[0155] (result) Figure 16 shows fluorescence microscopy images taken 9 days after seeding HUVECs (6 days after seeding EGFP-TS cells). A fluorescence microscopy image (B) taken from the side of the placental cell device confirmed that EGFP-TS cells were present at the height of the mesh sheet of the placental cell culture device. HUVECs were also confirmed to be present in the area below the mesh sheet. A fluorescence microscopy image (A) taken from the second substrate side of the placental cell device confirmed that the HUVECs had formed a three-dimensional vascular network structure. Because a medium for differentiating into EVT cells was used, it was believed that the EGFP-TS cells in the placental cell culture device had differentiated into EVT cells. Some EGFP-TS cells (EVT cells) had descended to the level of the HUVEC vascular network, suggesting the possibility that they were interacting with HUVEC cells.

[0156] <Co-culture of HUVEC vascular networks and EVT cells using a placenta cell culture device (2)> The culture was performed in the same manner as in <Co-culture of HUVEC vascular networks and EVT cells using a placenta cell culture device (1)>, except that no 3D blood vessel medium was added to the central hole from day 0 after HUVEC seeding to day 3 after seeding.

[0157] (result) Figure 17 shows a fluorescence microscopy image taken 9 days after seeding HUVECs (6 days after seeding EGFP-TS cells). The HUVEC vascular network was flatter than the fluorescence microscopy image shown in Figure 16. In other words, the HUVEC vascular network did not extend toward the central hole. These results suggest that the period of vascular network formation up to 3 days after seeding HUVECs may be important in determining the orientation of the HUVEC vascular network. When medium is present in the central hole, seeded HUVECs are expected to extend their vascular network toward the central hole (see Figure 5A). On the other hand, when medium is not present in the central hole, seeded HUVECs are expected not to extend their vascular network toward the central hole (see Figure 5B).

[0158] Figure 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 come into contact with the decidua (DM) and are observed on the surface and inside of the decidua (DM). Within the decidua (DM), there are blood vessels (spiral arteries: HA) that transport blood (BL) to the villi (V) side, and these blood vessels have their outlets on the surface of the decidua (DM) (the surface in contact with blood). EVT cells (EVT) infiltrate the inner wall of the blood vessel from the vascular outlet and remodel the blood vessel into a thicker one. Meanwhile, EVT cells (EVT) that infiltrate into the decidua (DM) are thought to migrate toward the spiral arteries (HA) and infiltrate from the outside of these blood vessels into the inside of the blood vessel. It is thought that EVT cells that infiltrate into the blood vessel via these two routes remodel the blood vessel into a thicker one. The vascular infiltration of EVT cells via these two routes can be modeled 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 toward the central hole simply by placing or not placing culture medium in the central hole of the first substrate at the early stage of culture. Controlling the extension of HUVEC blood vessels toward the central hole can be used to create a model in which EVT cells infiltrate into blood vessels from the arterial outlet side. Controlling the extension of HUVEC blood vessels toward the central hole can be used to create a model in which EVT cells that have invaded the decidua infiltrate into blood vessels.

[0159] [Example 2] In Example 2, a production test was carried out for an EVT cell-containing cell construct to be cultured in the placental cell culture device of the second embodiment described above (see FIGS. 8A to 10B).

[0160] <Culture medium> The TS basal medium and TS medium used were the same as those described above.

[0161] (3D-EVT medium) 3D-EVT medium was prepared by adding the following components to 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) 25 ng / mL NRG1 (Cell signaling) 100 ng / mL SB202190 (FUJIFILM Wako) 2 μM Matrigel (Corning) 0.2 mg / mL

[0162] (3D-EVT-Dox medium) The following components were added to the TS basal medium to prepare the 3D-EVT 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) 25 ng / 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

[0163] (assay medium) The following components were added to the TS basal medium to prepare the assay medium. The concentrations shown below are the final concentrations in the assay medium. Doxycycline hyclate (Sigma-Aldrich) 2 μg / mL

[0164] <Preparation of cell constructs containing EVT cells> EGFP-TS cells were maintained and managed with the TS medium. The EGFP-TS cells were collected from the dish for maintenance culture, suspended in the TS basal medium, and a cell suspension was prepared. 10 μL of the cell suspension (0.5×10 5Cells (1000 cells / mL) were placed into each well of a 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 resulting cell aggregates were transferred to a 3.5 cm dish along with the medium. Meanwhile, 30 μL of Matrigel-growth factor reduced (approximately 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 then aspirated with a Pipetman tip, and only the cell aggregates were transferred into the mixed gel solution. Care was taken to minimize the amount of medium entering the mixed gel solution. Next, the cell aggregates were aspirated with approximately 7 μL of mixed gel solution using a Pipetman and transferred into the wells of a 4-well plate. The 4-well plate was placed in a 37°C, 5% CO2 incubator for approximately 15 minutes to allow the mixed gel solution to gel. Then, 200 μL of assay medium was added. Cell aggregate culture was initiated in a 37°C, 5% CO2 environment. One day after the start of culture in the gel (day 5 after cell seeding), the medium was replaced with 200 μL of assay medium.

[0165] (immunocyte staining) The assay medium in the wells was removed, and 4% paraformaldehyde (PFA) (FUJIFILM Wako) was added. The cell aggregates were fixed in paraformaldehyde for 40 minutes. The paraformaldehyde solution in the wells was then replaced with phosphate buffered saline (PBS) (FUJIFILM Wako). To permeabilize the cells, the PBS (-) in the wells was replaced with 500 μL of 0.3% Triton X-100 (FUJIFILM Wako) diluted in PBS (-), and the cell aggregates were treated at room temperature for 1 hour. The cell aggregates were then washed once with 500 μL of PBS (-).

[0166] The primary antibody solution was prepared by adding PE-conjugated anti-HLA-G antibody [MEM-G / 9] (ab24384) (1x200 dilution) to antibody diluent (PBS[-] containing 0.1% Tween 20 and 2% FBS). 500 μL of the primary antibody solution was added to each well and incubated overnight at 4°C. The next day, the primary antibody solution was removed from the well, and the cell aggregates were washed with PBS(-) (500 μL three times). The cell aggregates were analyzed using an all-in-one fluorescence microscope BZ-X800 (Keyence).

[0167] (result) Figure 19A shows a microscopic image of cell aggregates after migration into the mixed gel. EVT cells can adopt a spindle-shaped, elongated morphology within the gel, but few spindle-shaped cells were observed around the cell aggregates immediately after migration (Day 0). On Day 1 and Day 2 after migration into the mixed gel, spindle-shaped cells were observed around the cell aggregates.

[0168] Figure 19B shows the results of immunocytostaining of cell aggregates on day 2. The cell aggregates expressed HLA-G, a marker for EVT cells. The center of the cell aggregates was not stained with HLA-G. Possible reasons for this include (1) the antibody being unable to reach the center of the cell aggregates, and (2) the presence of many undifferentiated cells near the center of the cell aggregates that do not express HLA-G. Doxycycline treatment confirmed EGFP expression in the cells of the cell aggregates. These results confirmed that the above method can be used to create cell aggregates surrounded by EVT cells.

[0169] [Example 3] In Example 3, a three-dimensional culture model containing an EVT cell-containing cell construct was produced using the placental cell culture device of the second embodiment described above (see FIGS. 8A to 10B). <Preparation of placental cell culture device> (Photolithographic mold fabrication) A mold for the first substrate of the placental cell culture device was fabricated using photolithography. First, a high-purity silicon wafer (AS ONE) was spin-coated with a permanent epoxy negative photoresist SU-8 2100 (KAYAKU Advanced Materials) at 500 rpm for 10 seconds, followed by 3000 rpm for 30 seconds. The photoresist-coated silicon wafer was then heated on a hot plate at 65°C for 5 minutes and then at 95°C for 20 minutes. This resulted in the formation of a 100 μm-thick SU-8 film. A photomask for the first substrate was then fabricated using a laser lithography system DWL200 (Heidelberg Instruments). The photomask for the first substrate was placed on the SU-8 film for the first substrate mold and exposed to UV light for 20 seconds. The wafer was then heated on a hot plate at 65°C for 5 minutes and then at 95°C for 10 minutes. It was then cooled at room temperature for 5 minutes. The cooled mold was placed in a stirred SU-8 developer (KAYAKU Advanced Materials) and reacted for 30 minutes to remove uncured SU-8. The mold was 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.

[0170] (Fabrication of the first substrate by soft lithography) The first substrate was fabricated by soft lithography using a mold for the first substrate fabricated by photolithography. A PDMS solution was prepared by mixing the base resin and curing agent in a 10:1 ratio and poured into the mold. After degassing, the PDMS solution was cured by heating at 65°C overnight. The first substrate components were removed from the mold using a disposable scalpel or other tool. Holes for introducing and discharging liquid into and from the flow channel were opened using a biopsy trephine (2 mm and 6 mm diameter). Figure 20A shows a schematic diagram of the fabricated first substrate.

[0171] The PDMS ring to be attached to the first substrate was fabricated by punching a PDMS sheet with two types of biopsy trephines (Φ8 mm and Φ6 mm).

[0172] (Assembly of the placental cell culture device) Using the first substrate and the second substrate prepared as described above, a placental cell culture device was assembled (see Fig. 9).

[0173] As the second substrate, cover glass (Matsunami Glass) was used.

[0174] The surface of the flow channel 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 substrate and the second substrate were adhered and treated at 80 °C overnight. In order to use the device for cell culture, sterilization treatment was performed by performing UV treatment for 30 minutes. Fig. 20B shows a photograph of the prepared placental cell culture device.

[0175] <Preparation of EVT cell-containing cell constructs and migration of EVT cells (1)> Similar to Example 2, cell aggregates were prepared from EGFP-TS cells. First, EGFP-TS cells were collected from the dish for maintenance culture, suspended in TS basal medium, and a cell suspension was prepared. 10 μL of the cell suspension (0.8×10 5 cells / mL) was placed in each well of a PrimeSurface 96U plate containing 90 μL of 3D-EVT medium. On the 3rd day of culture, 50 μL of 3D-EVT-Dox medium was added. On the 4th day of culture, the prepared cell aggregates were transferred 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 aspirated with a pipette tip, and only the cell aggregates were transferred into the mixed gel solution. At this time, care was taken to ensure that as little medium as possible entered the mixed gel solution.

[0176] Next, using a pipette, the cell aggregates were sucked up along with approximately 7 μL of the mixed gel solution and introduced through a 0.5 mm diameter communication hole in the placental cell culture device. The placental cell culture device was then placed in a 6 cm dish. Carefully preventing the gel inside the device from drying out using gauze moistened with ultrapure water, the device / dish was placed in a 37°C, 5% CO2 incubator. After leaving the device for approximately 15 minutes to allow the mixed gel solution to gel, a PDMS ring was placed on the first substrate to form an upper well. 60 μL of assay medium was added to the upper well. The central channel of the placental cell culture device contained the mixed gel and one cell aggregate. 40 μL of assay medium was added to each of the first and second side channels. Cell aggregate culture within the device was initiated using placental cell culture in a 37°C, 5% CO2 environment. On day 1 (day 5 of cell seeding) and day 2 (day 6 of cell seeding) after initiating culture in the placental cell culture device, the medium was replaced with 50 μL of assay medium (upper well, first side channel, second side channel).

[0177] (result) Figure 21A shows fluorescence microscopy images of cell aggregates introduced into the placental cell culture device on days 1 (day 5 from the start of culture), 2 (day 6 from the start of culture), and 3 (day 7 from the start of culture). Doxycycline confirmed that the cells in the cell aggregates expressed EGFP. On day 2 (day 6 from the start of culture), some cells had a spindle shape similar to EVT cells. Figure 21A confirms that EVT cells migrated to the left or right to a similar extent.

[0178] On day 1 after the cell aggregates were introduced into the placental cell culture device, a dividing line was drawn on a planar image of the cell aggregates observed from the second substrate side, dividing the area of the planar image into approximately equal halves. Using this dividing line as a reference, the areas on the left and right sides of the dividing line were analyzed for the cell aggregates on days 5 (day 1 after introduction), 6 (day 2 after introduction), and 7 (day 3 after introduction) from the start of culture 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 (Left) or right (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. From the results in Fig. 21B, it was also confirmed that EVT cells spread evenly to the left and right.

[0179] <Production of EVT cell-containing cell structure and migration of EVT cells (2)> A cell aggregate was prepared from EGFP-TS cells in the same manner as described above. First, EGFP-TS cells were collected from the dish for maintenance culture, suspended in TS basal medium, and a cell suspension was prepared. 10 μL of the cell suspension (0.65×10 5 cells / mL) was placed in each well of a PrimeSurface 96U plate containing 90 μL of 3D-EVT medium. On the 4th day of culture, the prepared cell aggregate was transferred into a 3.5 cm dish together with the medium. In the same manner as described above, the cell aggregate was introduced into the placental cell culture device, and the first side flow path and the second side flow path were filled with the medium. Culturing of the cell aggregate in the placental cell culture device was started under an environment of 37 °C and 5% CO2.

[0180] One to two hours after introducing the cell aggregate into the placental cell device, HUVEC cells were introduced into the first side flow path of the device. As a procedure, first, a suspension of RFP-HUVEC (5×10 6A concentration of 100 cells / mL was prepared in EGM-2 medium. Using a cut-off Pipetman tip (for 2-20 μL), 7 μL of RFP-HUVEC suspension was added to the second port (P2) of the placental cell culture device (see Figure 8A). As a control, 7 μL of EGM-2 was added to the fourth port (P4). The placental cell culture device was placed vertically with the first side channel containing HUVECs facing up, secured with a large clip, and placed in a 5% CO2 incubator at 37°C. After 25 minutes, the placental cell culture device was horizontally repositioned and culture was initiated. On days 1 (day 5 after cell seeding) and 2 (day 6 after cell seeding) after initiating cell aggregate culture in the placental cell culture device, the medium was replaced with 50 μL of assay medium (upper well, first side channel, and second side channel).

[0181] (result) Figure 22A shows fluorescence microscopy images taken on days 1 (day 5 from the start of culture), 2 (day 6 from the start of culture), and 3 (day 7 from the start of culture) after introducing cell aggregates into the placental cell device. The results in Figure 22A confirmed that EVT cells migrated toward the first side channel where HUVECs were present.

[0182] As described above, a dividing line was drawn on a planar image of the cell aggregates observed from the second substrate side of the placental cell culture device, and the areas of the cell aggregates on the left (HUVEC(-)) and right (HUVEC(+)) of the dividing line were measured. The results are shown in Figure 22B. Two days after co-culture of EVT cells and HUVECs (sixth day from the start of culture), it was confirmed that EVT cells had significantly migrated toward HUVECs.

[0183] These results demonstrated that the placental cell culture device of Example 3 could be used to evaluate the migration of EVT cells.

[0184] While preferred embodiments of the present invention have been described and illustrated, it should be understood that these are exemplary of the present invention and should not be considered limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is not to be deemed limited by the foregoing description, but is limited only by the scope of the appended claims. [Industrial Applicability]

[0185] The present invention provides a placental cell culture device that enables evaluation of the interaction between placental cells and factors that can act on placental cells, a three-dimensional culture model using the placental cell culture device, a method for preparing the three-dimensional culture model, and a method for evaluating placental cells using the placental cell culture device. [Explanation of symbols]

[0186] 1,100 placental cell culture devices 10,110 First board 11 Central Hall 12 Placental Cell Culture Department 20,120 Second board 21,111 Central Channel 22a, 112a First side flow path 22b, 112b Second side channel 23a, 23b, 113a, 113b Partition members 24,114 microposts 30 mesh sheet 31 Linear members 32 Opening 40 Thin Film Sheet 41 Thin Film Opening 115 Communication hole 130 Ring member 131 Upper Well P1~P6 1st port to 6th port p1~p4 1st port flow passage to 4th port flow passage

Claims

1. a placental cell culture unit for culturing placental cells; a factor flow path for providing a factor capable of interacting with the placental cells; The placental cell culture section and the factor flow path are adjacent to each other via a partition member in which shielding sections and communication sections are alternately arranged, and the minimum width of the communication sections is 30 to 500 μm. Placental cell culture device.

2. The placental cell culture device according to claim 1 , wherein the partition member is a mesh sheet having a porosity of 50 to 90%.

3. The placental cell culture device according to claim 1 , wherein the placental cell culture section is filled with a gel containing cell aggregates of the placental cells.

4. 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 section; a factor capable of interacting with the placental cells is present in the factor channel; 3D culture model.

5. 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 section; A three-dimensional vascular system is formed in the factor channel. 3D culture model.

6. 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 section, A blood vessel wall is formed in the factor channel. 3D culture model.

7. A method for producing a three-dimensional culture model using the placental cell culture device according to claim 2, comprising: Cultivating 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 section; A method for producing a three-dimensional culture model, comprising:

8. A method for producing a three-dimensional culture model using the placental cell culture device according to claim 3, comprising: a step of culturing vascular endothelial cells in the factor channel to form a vascular wall; culturing the vascular wall cells in the factor channel and culturing cell aggregates of the placental cells in the placental cell culture section; A method for producing a three-dimensional culture model, comprising:

9. 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 of any one of claims 1 to 3.

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