JP2023025739A - Dish for embryo culture, and method for collecting embryo culture solution for chromosome analysis by using the same - Google Patents

Abstract

To enable culture solution to be sucked up for aneuploidy inspection of chromosomes of embryos to be cultured, and dispose wells housing a plurality of embryos adjacent to each other.SOLUTION: A dish for embryo culture comprising a plurality of wells that houses embryos together with culture solution, where the wells are disposed independently and adjacently to each other on the bottom part of the dish for embryo culture, recesses capable of holding the culture solution together with the wells are formed in the bottom part independently of other recesses for each of the wells, a transfer inhibiting part which permits circulation of the culture solution and inhibits transfer of the embryos is provided between the wells and the recess, the volume of the recess is the volume of the culture solution, which is sucked up for aneuploidy inspection of chromosomes of embryos cultured in the wells, or more.SELECTED DRAWING: Figure 5

Description

The present disclosure relates to a dish used for embryo culture and a method for collecting embryo culture medium for chromosome analysis using this dish.

There is an increasing social demand for infertility treatment, and various techniques such as techniques for culturing fertilized eggs and embryos (hereinafter collectively referred to as embryos) and accompanying examinations have been proposed (e.g., patent documents 1). Especially in recent years, the need for chromosomal analysis of embryos in culture has increased. is proposed. Such an examination requires a predetermined amount of culture medium. At present, several microliters to several tens of microliters of culture fluid is sucked up and used for examination.

Japanese Patent Publication No. 2014-507664

However, in recent years, the number of embryos cultured at one time in the embryo culture apparatus has been increased, and the embryos are housed in each of a plurality of, for example, 5 x 5 = 25 microwells provided in one dish and cultured. Such microwells cannot take up the amount of culture medium required for testing. Since human ovum has a diameter of about 150-200 μm, a microwell having a diameter of about 300 μm, for example, can absorb only a small amount of culture solution. Sucking up the required amount of the culture medium in this state is not preferable because the environment for culturing the embryos may change significantly. In addition, simply increasing the capacity of the wells containing the embryos increases the distance from the adjacent wells, which may cause various problems. For example, in an embryo culture apparatus equipped with a time-lapse function for photographing embryos in culture, there is a concern that the number of wells that can be imaged at one time will decrease and the structure of the imaging apparatus will become complicated. In addition, the distance for transferring embryos between wells increases, and the possibility of causing mistakes during transfer also increases.

The present disclosure can be implemented as the following forms or application examples. One aspect of the present disclosure is an embryo culture dish comprising a plurality of wells for accommodating embryos together with a culture medium. In this embryo culture dish, the plurality of wells are arranged independently and adjacently on the bottom of the embryo culture dish, and each well has a recess capable of holding the culture solution together with the well. Independently from the recess, a movement-inhibiting portion is formed in the bottom, and between the well and the recess, permits the flow of the culture solution and inhibits the movement of the embryo, and the volume of the recess is The volume may be equal to or greater than the volume of the culture solution sucked up for chromosomal aneuploidy test of embryos cultured in the wells.

According to such an embryo culture dish, since a plurality of wells are adjacent to each other, when this dish is used in an embryo culture device or the like, the distance between the plurality of wells is short, and handling is easy. The process of aspirating the culture medium for numeracy testing can be performed with less impact on the embryos being cultured. If the distance between multiple wells is short, embryos can be easily moved within the wells, and mistakes during transfer can be reduced. Furthermore, if it is used in a time-lapse embryo culture device, it becomes possible to image a plurality of wells at once. This facilitates non-invasive chromosomal aneuploidy testing of embryos, has almost no effect on the embryos being tested, and increases the success rate of embryo transfer based on the results of aneuploidy testing (pregnancy rate of patients). rate) can also be expected to increase.

The top view of the embryo culture apparatus as 1st Embodiment. FIG. 2 is a plan view illustrating an embryo culture dish arranged in a culture chamber; III-III arrow directional view in FIG. Explanatory drawing which shows the electrical structure of an embryo culture apparatus. The top view which shows an example of arrangement|positioning of the 1st accommodating part 1st accommodating part and the 2nd accommodating part in the dish for embryo cultures. A VI-VI arrow directional view in FIG. 1st accommodating part Explanatory drawing which shows the state with which the 1st accommodating part and the 2nd accommodating part were filled with the culture solution. 1st container part Explanatory drawing which shows the state which sucked out the culture solution of the 1st container part and the 2nd container part. Process drawing which shows the content of collection|extraction and test|inspection processing of the embryo culture medium for a chromosome analysis. Explanatory drawing which shows an example of the display screen of the aneuploidy detector of a chromosome. The top view which shows the shape of the dish for embryo cultures of 2nd Embodiment. The top view which shows the modification of the dish for embryo cultures of 2nd Embodiment. 1st accommodation part Explanatory drawing which shows other embodiment of a 1st accommodation part and a 2nd accommodation part. FIG. 1 is a process diagram showing an overview of embryo culture, preservation, and transplantation. FIG. 4 is an explanatory diagram showing a state in which the embryo culture dish is held by hand.

A. First embodiment:
(A-1) Hardware configuration:
FIG. 1 is a plan view of an embryo culture device (also called an incubator) 10, and FIG. 2 is an explanatory view schematically showing an embryo culture dish arranged in one culture chamber of the embryo culture device 10. This embryo culture apparatus 10 is for culturing eggs that have undergone in vitro fertilization (hereinafter referred to as treated eggs) for a certain period of time in a predetermined culture environment, that is, constant temperature and constant humidity. Since the treated egg is not necessarily a fertilized egg, it is cultured in the embryo culture apparatus 10 for a predetermined period. The treatment of in vitro fertilization may be microinsemination performed under a microscope, or normal in vitro fertilization performed by placing an ovum and sperm together in a predetermined container. The method of fertilization of the treated eggs to be cultured does not matter.

As shown in FIG. 1, this embryo culture apparatus 10 has a total of nine culture units for culturing embryos, five culture units 11 to 15 in the lower stage, four culture units 21 to 24 in the upper stage, arrayed. Hereinafter, members related to the culture unit will be referred to as culture units 11 to 24 when collectively referred to. Of course, the number of culture units does not matter. Each of the culture units 11 to 24 includes culture chambers R11 to R24, a lid portion 17 sealing each of the culture chambers R11 to R24, and switches SW11 to SW24. By operating the switches SW11 to SW24, the lids 17 of the corresponding culture units 11 to 24 are opened and closed by drive mechanisms (not shown). Of course, the culture units 11 to 24 may have a structure that is manually opened and closed. Sealing portions made of silicon rubber are provided on the culture chambers R11 to R24 side of the lid portion 17, and when the lid portion 17 is closed, the culture chambers R11 to R24 are kept in an airtight state by the sealing portions. .

Carbon dioxide gas (CO ₂ ) and nitrogen gas (N ₂ ) are supplied to the embryo culture apparatus 10 from an external gas cylinder. Gas ports 18 and 19 through which these gases are supplied are provided on the rear surface of the embryo culture device 10 . Filter ports 28 and 29 to which a filter 27 is attached are also provided on the rear surface of the embryo culture device 10 . This filter 27 is for removing foreign matter such as dust in the outside air taken in by an internal pump (not shown). In the embryo culture apparatus 10, carbon dioxide gas CO ₂ and nitrogen gas N ₂ input from gas ports 18 and 19 and air input through filter 27 are mixed at a predetermined ratio to generate a gas mixture. The ratio of each gas in the air-fuel mixture is measured by a sensor (not shown) and kept at the same ratio at all times.

A display 70 is provided on the surface of the case body 20 . The surface of the display 70 is a touch panel, and various information can be displayed by tapping the displayed buttons. In addition, the case body 20 is provided with general-purpose connectors 26 such as USB-C and Thunderbolt (registered trademark). It is also possible to enter other information. A display example on the display 70 and a case of connecting other devices to the general-purpose connector 26 will be described later.

FIG. 2 shows the culture unit 11 with the cover 17 opened. Culture chambers R11 to R24 are provided in each of the culture units 11 to 24. As shown in FIG. Since the culture chambers R11 to R24 have the same structure, the culture chamber R11 of the culture unit 11 will be described below as an example. A holding frame 180 on which a dish 191 is placed is provided in the culture chamber R11. A supply port 43 and an exhaust port 44 are provided on the bottom surfaces of the culture chambers R11 to R24. The supply port 43 is an opening for supplying the air-fuel mixture to the incubation chamber R11. Also, the exhaust port 44 is an opening for recirculating the air-fuel mixture. The exhaust port 44 is provided at a position covered by the holding frame 180 . As described above, the incubation chamber R11 is made substantially airtight when the lid portion 17 is closed, so that the internal gas environment is kept constant, but the temperature distribution in the incubation chamber R11 is kept uniform. To do so, the air-fuel mixture is supplied from the supply port 43 and exhausted from the exhaust port 44 .

The incubation chamber R11 is provided with panel heaters (not shown) on its side wall and bottom surface. A panel heater keeps the inside of the culture room R11 at a constant temperature. In addition to heaters, temperature sensors, gas concentration sensors, and humidity sensors (not shown) are provided in the incubation chambers R11 to R24, although the description is omitted. Humidity can be detected. By feeding back the signals from these sensors, as a result, the temperature and humidity in the incubation chamber R11 are kept constant, and the gas concentration is also kept constant. Instead of detecting the temperature and the like in the incubation room R11, the temperature, humidity, gas concentration, etc. of the air-fuel mixture supplied from the supply port 43 are kept constant, thereby keeping the environment in the incubation room R11 constant. It is good as

A holding frame 180 in the culture room R11 is arranged with a dish 191 containing an embryo. The dish 191 shown in FIG. 2 has a substantially rectangular shape surrounded by an outer peripheral wall 194, and is further surrounded by a wall 192 at the center of a bottom portion 196, and has five holding portions 200 for holding embryos. 2 rows, 10 in total. A detailed configuration of the housing portion 200 will be described later. A recess 197 is provided at one location on the outer peripheral wall 194 of the dish 191, and the dish 191 can be set on the holding frame 180 by aligning this recess 197 with a projection 182 provided on the holding frame 180. , the dish 191 can be placed in the correct position. Here, the correct position means an appropriate position for capturing an image of a microwell 195 provided in the container 200, which will be described later.

A configuration for imaging the dish 191 from the bottom 196 side will be described with reference to FIG. In the figure, reference H11 indicates a series of side walls and bottom forming an incubation chamber R11. As shown in the figure, a lighting unit 170 provided inside the lid portion 17 is arranged directly above the dish 191 . An opening 198 is provided in the bottom H11 of the incubation chamber R11 directly below the position corresponding to the center of the dish 191, and the camera module 51 and the lens module 52 attached to the case main body 20 are arranged. The dish 191 is illuminated by the illumination unit 170 provided on the lid portion 17 , and the embryo 25 housed in the housing portion 200 is imaged by the camera module 51 . As shown in the figure, the containing section 200 is filled with the culture medium CS, and the embryo 25 is immersed in the culture medium CS. Furthermore, in this state, mineral oil OL is poured into the area surrounded by the wall 192 to prevent evaporation of the culture solution CS, thereby covering the containing section 200 .

Next, processing performed in the embryo culture apparatus 10 will be described. FIG. 4 is an explanatory diagram showing the electrical configuration of the embryo culture device 10. As shown in FIG. As illustrated, the embryo culture apparatus 10 is provided with a control section 60 . The control unit 60 includes a well-known CPU 61, ROM 62, and RAM 63, as well as a memory interface 64 for exchanging data with a memory card 65, a general-purpose I/O interface 66 for exchanging signals with external equipment, and an incubation room R11. ~ Incubation room interface 67 for exchanging signals for controlling the environment in R 24, camera interface 68 for performing imaging instructions and acquisition of captured images with camera module 51, video for displaying images on display 70 It includes an interface (abbreviated as video I/F) 69 and the like.

The CPU 61 is a high-speed processor having a function as a DSP for high-speed processing of fertilization determination based on images, which will be described later, and a vector operation function for performing determination processing using a neural network or the like. The ROM 62 stores a program for realizing processing in the embryo culture apparatus 10 including time-lapse control processing, which will be described later. The CPU 61 implements necessary processing and control by appropriately reading such programs from the ROM 62, developing them in the RAM 63, and executing them. In the memory card 65, in addition to the image captured by the camera module 51, information regarding the culture environment such as the temperature of each of the culture chambers R11 to R24 is recorded. Instead of the memory card 65, a magnetic storage medium such as a hard disk or a semiconductor storage medium such as an SSD may be used. Alternatively, the data may be stored in a so-called cloud connected via a network.

A touch panel is provided on the surface of the display 70 , and by touching the display 70 , buttons and the like displayed on the display 70 can be selected. Although the display 70 is provided integrally with the embryo culture device 10 in this embodiment, it may be provided separately from the embryo culture device 10 and connected by wire or wirelessly. Alternatively, the embryo culture apparatus 10 and a computer may be connected via a network or the like, and the display of the computer may be used as the display 70 . Alternatively, a highly portable terminal such as a mobile phone or tablet may be used as the display 70 .

The general-purpose I/O interface 66 includes switches SW11 to SW24 provided in the embryo culture apparatus 10, a driving device 71 for opening and closing the lid portion 17, a warning device 72 for generating a warning sound, and a mixture adjustment for adjusting the mixture. It is connected to the device 73 and the like. The air-fuel mixture adjustment device 73 includes a pressure adjustment valve that adjusts the pressure of carbon dioxide gas, nitrogen gas, etc. supplied to the gas ports 18 and 19, a pump for taking in the atmosphere through the filter 27, and a culture chamber R11- It includes a pump that feeds the air-fuel mixture to R24. The culture chamber interface 67 is connected to the panel heaters provided in each of the culture chambers R11 to R24, the control valves V11 to V24 for controlling the supply amount of the air-fuel mixture, and the like. The control valves V11 to V24 are provided in pipes extending from the gas mixture supply pipe 84 for supplying the gas mixture to the culture chambers R11 to R24.

A computer (hereinafter abbreviated as PC) 90 is connected to the general-purpose I/O interface 66 via the general-purpose connector 26 . The PC 90 includes a rotary control device 95 in addition to a display 91 and a keyboard (not shown). The control device 95 has a small-diameter dial 96 mounted on a substantially circular base portion 97. By rotating the dial 96 left and right, the multiple time-lapse images displayed on the display 91 are moved back and forth in chronological order. It is possible to use such as moving and displaying. Such devices are also called rotary selectors. The dial 96 can not only be rotated left and right, but also can be pushed in, and when one of the currently displayed images is selected, it realizes a "determine" operation similar to the operation with a mouse button. Needless to say, a dedicated button may be provided on the base portion 97 to assign the determination and selection operations. For example, if the time-lapse images are taken every 10 minutes for 24 hours, there are 6×24=144 images for each embryo. Such a rotary pointing device is useful when quickly referring to such a large number of images and confirming the determination of fertilization.

(A-2) Dish details:
5 is an enlarged view of a portion of the dish 191 surrounded by the wall 192, and FIG. 6 is a sectional view taken along line VI-VI in FIG. As shown in the figure, in a substantially rectangular area surrounded by the wall 192, 5 x 2 rows of storage units 200 are arranged vertically symmetrically in the figure. A plurality of drop placing portions 186 for placing the cleaning culture solution CS are provided outside the ten storage portions 200 near the wall 192 . The drop placement section 186 may simply place a drop of the culture medium CS on the bottom 196, but the bottom 196 may be provided with a recess or a hydrophilic region to facilitate placement of the culture medium CS. good too.

As illustrated, each housing portion 200 is formed in a shape in which a substantially circular first housing portion 210 and a trapezoidal second housing portion 220 elongated in the height direction are connected. The first accommodating portion 210 corresponds to the well, and the second accommodating portion 220 corresponds to the recess. A microwell 195 having a diameter larger than that of the embryo 25 is further formed in the center of the first housing portion 210 . Therefore, the ten microwells 195 are arranged close to each other, and are contained in one imaging area PA of the camera module 51 arranged below the dish 191 . In other words, it is possible to image the existing regions of all the microwells 195 in one imaging operation, and if the embryos 25 are housed in the microwells 195, the state of the embryos 25 can be imaged at once. The photographed images are divided into images of the respective embryos 25 and stored in the memory card 65 or the like.

The first accommodation portion 210 and the second accommodation portion 220 are connected to each other through a connecting portion, as shown in FIG. This portion is the passage portion. A partition portion 230 slightly higher than the bottom surface of the second housing portion 220 is formed in the passage portion as a movement inhibiting portion. The partitions 230 are provided so as to protrude from both sides of the connecting portion in the width direction toward the central portion, and the partitioning portions 230 protruding from both sides are located at the center of the connecting portion, that is, the position where the VI-VI cutting line passes. So, they are facing each other with a small gap left. This gap is smaller than the smallest possible diameter of the embryo 25, which in this embodiment is about 50 μm. Therefore, the embryos 25 housed in the microwells 195 do not move toward the second housing section 220 due to the movement of the culture medium CS even if the housing sections 200 are filled with the culture medium CS.

Filling and suctioning of the culture solution CS into the container 200 will be described. FIG. 7 shows how the container 200 is filled with the culture solution CS using the pipette 240 . The culture solution CS is filled up to near the upper limit of the storage section 200 . The volume of the container 200 is predetermined, and in the present embodiment, the volume of the culture solution to be aspirated, which will be described later, is 30 μl. After sucking the culture medium CS, it can be transferred to the storage section 200 . Of course, if the volume of the culture solution to be sucked is further reduced, the volume of the container 200 may be set accordingly. Thereafter, after covering the culture medium CS with mineral oil OL, a glass capillary is placed above the microwell 195 and the embryo 25 is placed inside the microwell 195 .

In this embodiment, after culturing the embryo 25 is continued, the culture solution CS in the container 200 is aspirated, and chromosome aneuploidy is detected. In that case, as shown in FIG. In FIG. 8, the culture solution CS still remains at the bottom of the second container 220, but in reality, the first container 210 and the second container 220 communicate with each other through the gap of the partition 230. Therefore, when all the culture medium CS in the second container 220 is sucked up, the water level of the culture medium CS in the first container 210 matches the bottom of the second container 220 . Even in this state, the embryo 25 is still submerged in the culture solution CS. In addition, even if the culture medium CS in the first container 210 moves to the second container 220 as the pipette 240 sucks it up, the gap between the partitions 230 is sufficient for the diameter of the embryo 25. Since it is narrow, the embryo 25 does not flow out from the microwell 195 to the second container 220 due to the flow of the culture solution CS.

(A-3) Culture and examination of embryos:
Next, examination of embryos cultured using this dish 191 will be described. FIG. 9 is a flow chart showing a sampling/examination processing routine of the embryo culture medium for chromosome analysis. In the present embodiment, treated eggs that have undergone fertilization treatment are cultured by the embryo culture apparatus 10. In the course of culture, chromosome aneuploidy tests are performed on the embryos being cultured. This test is called the pre-implantation chromosome aneuploidy test (PGT-A). Preimplantation chromosomal aneuploidy testing is often performed by biopsying an embryo such as a blastocyst in culture, that is, by collecting about 6 to 10 cells from the trophectoderm of the blastocyst. In embodiments, the medium in which the embryos were immersed is used. The culture solution contains DNA released from embryos in culture, and by subjecting this to a gene sequencer, chromosomal aneuploidy can be tested.

When this process is started, first, 40 μl of the culture solution is filled into each of the ten storage portions 200 provided in the dish 191 (step S300). As the culture solution CS, a single medium (one-step type culture solution) added with phenol red was used in this embodiment. Since the storage part 200 has a volume of 50 μl, the storage part 200 does not overflow with the culture solution CS even when 40 μl of the culture solution CS is filled. After filling the culture solution CS into all the ten containers 200, the inside of the wall 192 is filled with the mineral oil OL, and each container 200 filled with the culture solution CS is covered with the mineral oil OL (step S310).

In this state, next, the embryo 25 is housed in the microwell 195 of each housing section 200 (step S320). At this time, if necessary, measures are taken to remove air bubbles present in the culture solution CS. One embryo 25 is accommodated in one microwell 195, and this is repeated until all microwells 195 are completed (step S325).

After the embryos 25 are accommodated in all the microwells 195, the dish 191 is set in one of the culture chambers R11-R24 of the culture units 11-24 of the embryo culture apparatus 10 (step S330). Specifically, the lid 17 of one of the culture chambers is opened, and the dish 191 is placed in alignment with the holding frame 180 . After that, the lid part 17 is closed to start culturing.

As already described, the embryo culture apparatus 10 maintains the environment in the culture room R11 in a state suitable for culture, and the camera module 51 that photographs the dish 191 from below captures each container of the dish 191 at predetermined intervals. While imaging the embryos 25 housed in the 200 microwells 195, a time-lapse culture process (step S340) is performed to culture the embryos. This time-lapse culturing process continues until 72 hours have passed since the start of culturing, which is defined as a predetermined time (step S345).

When 72 hours have passed since the start of culture, the lid 17 is opened, the dish 191 is taken out from the culture chamber R11 (step S350), and the culture solution CS is sucked up from each container 200 (step S360). Specifically, as shown in FIG. 8, the tip of the pipette 240 is inserted into the second container 220 filled with each container 200 to suck up the culture solution CS. At this time, all the culture medium CS in the second container 220 is sucked up. When all the culture medium CS in the second container 220 is sucked up, the culture medium CS in the first container 210 communicating with it is also sucked out. Since it is provided at a position deeper than the bottom of the portion 220, the culture medium CS in the microwell 195 is not sucked out. Therefore, even if the pipette 240 is inserted into the second container 220 and all the culture medium CS in the second container 220 is sucked out, the embryo 25 is still immersed in the culture medium CS. When the culture medium CS is sucked out by the pipette 240, the culture medium CS may be sucked out while measuring so that the volume of the culture medium CS becomes 30 μl. The aspirated culture medium CS is individually placed in an analysis sample case of a gene sequencer, which will be described later.

The embryos 25 remaining in the microwells 195 may be filled with a new culture medium CS from the second container 220 and continue to be cultured, or once removed from the microwells 195 and cryopreserved for transplantation. You may In the above example, the culture medium CS was sucked up 72 hours after the start of the culture, but taking advantage of the fact that the embryo culture apparatus 10 is a time-lapse embryo culture apparatus, the observation of the embryo is continued, and the completion of the blastocyst is confirmed. Alternatively, the culture medium CS may be sucked up. In this case, it often takes about 120 hours from the start of culture. Completion of the blastocyst can be determined by AI that has performed supervised machine learning in advance on the photographed image.

This process is repeated for all storage units 200 of one dish 191, and the culture medium CS is sucked out from all storage units 200 and transferred to analysis sample cases (step S365). It is set in a sequencer, and an aneuploidy test is performed by the gene sequencer (step S370). In this embodiment, "MiSeq" manufactured by Illumina, Inc. was used as a gene sequencer. After that, the process exits to "END" and terminates this processing routine.

The culture medium CS placed in the analysis sample case contains DNA fragments released from the embryo during culture. A piece of DNA was originally separated from the 23 pairs of genes and contains the information of the original gene. For this reason, when the culture solution contained in the analysis sample case is analyzed by a sequencer (so-called next-generation sequencer) that can use the NGS method in which a template prepared using a sample of normal human genes is set on a semiconductor chip, Excess and deficiency can be easily explained for 23 pairs of genes. As such a next-generation sequencer, for example, "NextSeq550" manufactured by Iiiumina, Inc. and "Ion GneStudio GX5" manufactured by Ion Torrent can be used.

FIG. 10 shows an example of analysis results. As shown in the figure, the next-generation sequencer 300 as an analyzer includes a storage unit 310 that stores a sample case for analysis, an operation unit 320 that instructs work such as template preparation and the start of analysis, and displays analysis results. A display unit 330 and the like are provided. When the sample cases for analysis into which the culture medium CS in which the embryos 25 have been cultured are respectively transferred are set in the container 310 of the next-generation sequencer 300 that has been prepared for template preparation, etc., and the start of analysis is instructed. After several hours to ten and several hours, the number of genes contained in the culture medium CS is displayed, as illustrated in FIG.

In the illustrated example, 3 instead of 2 gene pairs were found for the 10th gene, indicating that the 10th chromosome is redundant, that is, the embryos analyzed have chromosomal aneuploidy. I understand. Utilizing this analysis result, among the embryos being cultured in the embryo culture apparatus 10, embryos for which aneuploidy was not found are used for transplantation to patients, thereby increasing infertility treatment, that is, increasing the pregnancy rate. be able to. This is because it is known that embryos with aneuploidy in chromosomes are highly likely to fail to implant even if transplanted, or to miscarry in early pregnancy even if implanted. Moreover, in this method, the embryo 25 can be examined non-invasively. Compared to the method of extracting several trophectderm (TE) cells from the blastocyst and testing for chromosomal aneuploidy, the effect on the embryo was small, and the embryo 25 was immersed in the culture medium CS. Since aneuploidy analysis can be performed in this state, the effect on the embryo can be minimized.

As described above, according to the dish 191 of the present embodiment, when performing an aneuploidy test using the culture medium CS in which the embryos 25 are cultured, the embryos 25 are accommodated even if the culture medium CS is sucked up. Since the culture medium CS in the microwell 195 remains without fail, the influence of the uptake of the culture medium CS can be suppressed. In addition, since the dish 191 of this embodiment has 5×2 rows of microwells 195 arranged close to each other, the embryos 25 can be easily moved in the microwells 195, and mistakes during transfer can be reduced. The dish 191 of this embodiment can image the state of the embryo 25 in each microwell 195 at once when used in the time-lapse embryo culture apparatus. Therefore, it is possible to provide a plurality of microwells 195 in one dish 191 without moving the camera module 51, thereby reducing the number of imaging times in time-lapse embryo culture.

In the present embodiment, the bottom portion of the second containing portion 220 is rounded toward the center similarly to the first containing portion 210. Therefore, when the culture solution CS is sucked up, the culture solution CS in the second containing portion 220 is can be efficiently sucked up without waste. Moreover, even if all of the culture medium CS in the second storage section 220 is sucked up by the pipette 240, the bottom of the partition section 230 is raised, so that the liquid level of the culture medium CS in the first storage section 210 is higher than the bottom. never go down. Therefore, the culture medium CS in the microwell 195 in which the embryo 25 is present will not be accidentally pumped out.

B. Second embodiment:
(B-1) Shape of dish:
Next, a second embodiment of the dish will be described. In the second embodiment, as shown in FIG. 11, the shape of the dish 191A is different, but other than this, that is, the sampling and examination processing routine of the embryo culture medium for chromosome analysis, etc. are the same as in the first embodiment. A dish 191A of the second embodiment uses a container 200 having the same shape as that of the first embodiment. The number and the shape of the first accommodating portion 210 and the second accommodating portion 220 are also the same. However, in the second embodiment, the first accommodating portions 210 of the ten accommodating portions 200 are arranged closer than in the first embodiment. In order to arrange the first accommodation portions 210 close to each other, each accommodation portion 200 is arranged to open further outward toward the outer side. Therefore, the space L1 in the column direction between the first housing portions 210 in each row is narrower than the space L2 in the column direction between the second housing portions 220 provided corresponding to the first housing portions 210 . The interval L2 is the distance between the centers of the areas of the second accommodating portions 220 . As a result, in the dish 191A of the second embodiment, the imaging range PA2 for imaging the ten first housing portions 210 at once can be made narrower than the imaging range PA1 of the first embodiment. Therefore, it is also possible to increase the resolution of the embryo 25 in each microwell 195 imaged by the camera module 51 . Note that the size of the imaging range PA2 may be the same size as in the first embodiment, and the number of storage units 200 may be increased to, for example, 6×2 rows.

According to the dish 191A of the second embodiment, the imaging range of each of the plurality of microwells 195 can be narrowed, and the size of the container 200 for storing the culture medium CS for chromosomal aneuploidy test can be sufficiently increased. can be secured. Other effects are the same as those of the first embodiment.

(B-2) Modification 1:
FIG. 12 shows a dish 191B that is a modification of the dish 191A of the second embodiment. In this example, replenisher storage in which equilibration drops 251 and 252 for additional injection of the culture solution CS are placed in spaces on both sides of the arrangement direction of the first storage portions 210 generated by arranging the first storage portions 210 close to each other. department. This drop is a supplemental culture medium, and is prepared for additionally injecting the culture medium CS into the containing section 200 in order to continue the culture when the culture medium CS is sucked up from the containing section 200 . Since it is placed on the dish 191B while being covered with the mineral oil OL during the culture, it does not evaporate and is placed in the same environment (temperature, humidity, etc.) as the culture solution CS in the container 200. Therefore, even if the additional culture medium CS is added in place of the sucked-up culture medium CS, the added culture medium CS is the same as the culture medium with which the embryo 25 has been in contact so far, and is suitable.

In the above-described first and second embodiments, the storage unit 200 has a volume of 50 μl, is filled with 40 μl of the culture medium, and sucks out 30 μl of the culture medium for inspection. Capacity is just one example. If the volume of the culture solution required for the chromosome aneuploidy test becomes small and the volume of the culture solution CS to be sucked out becomes, for example, about 10 μl, the volume of the container 200 is set to about 20 μl, and the volume of the culture solution CS to be filled is set to 15 μl. It doesn't matter how much. In general, if the volume of the container 200 is V200, the volume of the culture medium CS to be filled is Vcs, and the volume of the culture medium CS to be sucked out for testing is Vout, then:
Vout < Vcs ≤ V200
satisfy the relationship with Also, assuming that the volume of the microwell 195 is Vmw,
Vout + Vmw ≤ Vcs
Then, when the culture medium CS is sucked out, the culture medium remains in the microwell 195, which is preferable.

(B-3) Modification 2:
Another example of the arrangement of the housing portion 200 is shown in FIG. In this example, a large number of the first accommodating portions 210 of the accommodating portion 200 are arranged close to each other. In this example, the housing portion 200 is arranged on the circumference, and the first housing portions 210 having respective microwells 195 are provided in 8 pieces on the inner peripheral side and 8 pieces on the outer peripheral side, for a total of 16 pieces. Even in this case, the same effects as in the second embodiment can be obtained, and the imaging range PA3 required for imaging many microwells 195 can be reduced.

C. Embryo culture, storage, and transfer procedures:
In the above-described embodiment, the handling of the embryo after sucking up the culture medium CS was not particularly described, but an example of handling the embryo for transplantation will be described. FIG. 14 is a process diagram for explaining procedures for culturing, preserving, and transplanting embryos. After the end of the processing routine shown in FIG. 9, first, the processing of additionally injecting the culture medium CS into each container 200 is performed (step S400). This is performed until additional orders are completed for all the storage units 200 (step S405).

When additional pouring to all of the storage units 200 is completed, the dish 191 is returned to the culture room R11 of the embryo culture apparatus 10 (step S410). When the cover 17 of the culture chamber R11 is closed, culturing is resumed and the embryos are continued to be cultured (step S420). Cultivation is restarted and the cells are waited until about 40 hours have passed (step S425). When 40 hours have passed, the results of the chromosomal aneuploidy test are read from the results of the next-generation sequencer (step S430). Based on the results, normal embryos are selected from the plurality of embryos tested for chromosomal aneuploidy (step S440).

If there is an embryo confirmed to have chromosomal aneuploidy among the examined embryos, it is regarded as not a normal embryo, and the embryos other than the embryo judged to be abnormal (hereinafter referred to as normal embryos) are cryopreserved and sent to the patient. When the embryo becomes suitable for transplantation, the frozen-preserved embryo is thawed and transplanted into the patient (step S450). This completes the embryo culture, preservation, and transplantation procedures.

When the dish 191 is taken out of the incubation room R11 (FIG. 9, steps S330 and S350) and returned to the incubation room R11 (FIG. 14, step S410), the user directly touches the dish 191 with his finger HD. Grasping and manipulating. This state is illustrated in FIG. The dish 191 used in each of the above-described embodiments has a substantially rectangular shape, and is sandwiched and held between the thumb and the opposite finger such as the middle finger. An outer peripheral wall 194 of the dish 191 is provided with a depression 400 as a gripping assist structure. Therefore, when the dish 191 is gripped with the fingers HD, the fingers fit into the depressions 400 of the outer peripheral wall 194 and the dish 191 can be held firmly. Note that the recess 400 is provided on at least one of the four sides of the substantially rectangular dish 191 . Dimples 400 may be provided at two locations where the thumb and the opposite finger are in contact. Instead of the depression 400, the outer surface of the outer peripheral wall 194 may be provided with fine irregularities. In this case, unevenness may be provided only at the recess 400, but the outside of the outer peripheral wall 194 may be widely uneven. The unevenness may be formed by roughening the surface of the molding die when the dish 191 is formed, or may be formed later by roughening the surface by a technique such as sandblasting. Note that illustration of the recess 197 is omitted in the figure.

D. Other forms:
(1) The embryo culture dish of the present disclosure can be implemented as an embryo culture dish having a plurality of wells for accommodating embryos together with a culture solution. In this embryo culture dish, the plurality of wells are arranged independently and adjacently on the bottom of the embryo culture dish, and each well has a recess capable of holding the culture solution together with the well. Independently from the recess, a movement-inhibiting portion is formed in the bottom, and between the well and the recess, permits the flow of the culture solution and inhibits the movement of the embryo, and the volume of the recess is The volume may be equal to or greater than the volume of the culture solution sucked up for chromosomal aneuploidy test of embryos cultured in the wells.

According to such an embryo culture dish, since a plurality of wells are adjacent to each other, when this dish is used in an embryo culture device or the like, the distance between the plurality of wells is short, and handling is easy. The process of aspirating the culture medium for numeracy testing can be performed with less impact on the embryos being cultured. Moreover, since the movement inhibiting portion is provided between the well and the recess, the culture medium around the embryo can be sucked up while the embryo cultured in the well remains in the well. In addition, if the distance between multiple wells is short, embryos can be easily moved within the wells, and mistakes during transfer can be reduced. Furthermore, if it is used in a time-lapse embryo culture device, it becomes possible to image a plurality of wells at once. This facilitates non-invasive chromosomal aneuploidy testing of embryos, has almost no effect on the embryos being tested, and increases the success rate of embryo transfer based on the results of aneuploidy testing (pregnancy rate of patients). rate) can also be expected to increase.

The number of wells per dish for embryo culture may be plural, and may be at least two, and may be any number as long as the wells are adjacent to each other and the accommodating portions can be arranged. The well may have a hemispherical shape, an elliptical shape in plan view, or a parabolic shape in vertical cross section, depending on the shape of the embryo to be accommodated. Moreover, the plurality of wells does not necessarily have to have the same shape, and may have different shapes. By doing so, it becomes possible to visually recognize in which well the embryo is to be manipulated in the microscope field of view.

(2) In such a configuration, at least some of the plurality of wells are arranged in two rows, and each of the recesses provided corresponding to the wells arranged in one row corresponds to the wells in the other row. The recess provided corresponding to the well may be provided on the opposite side across the well and have a shape extending outward from the corresponding well over a predetermined length. By doing so, it becomes easy to arrange a plurality of subsequent wells adjacent to each other and to arrange a storage portion of sufficient size. The predetermined length may be adjusted according to the size of the container, particularly the volume of the culture solution to be contained. The accommodating portion may be rectangular, or may be trapezoidal in which the lateral length increases with increasing distance from the well. Of course, it may be elliptical or oval in plan view. In addition, in the depth direction, the depth may be uniform throughout the accommodating portion, or may be curved such that the depth increases toward the center, similar to the well. In the latter case, when the culture solution is sucked up, the culture solution gathers in the deepest part, so the culture solution in the container can be efficiently sucked up.

(3) In such a configuration, the spacing in the row direction between the wells in each row may be narrower than the spacing in the row direction between the recesses provided corresponding to the wells. By doing so, the wells can be arranged more efficiently, and the number of wells provided in the embryo culture dish can be increased. Therefore, if this embryo culture dish is used in a time-lapse embryo culture apparatus or the like, it is possible to increase the number of wells that can be imaged at one time.

(4) In such a configuration, all the wells in the embryo culture dish are moved relative to the embryo culture dish by a camera provided in a time-lapse embryo culture apparatus in which the embryo culture dish is housed. It may be arranged in a range in which an image can be captured without In this way, the camera provided in the time-lapse embryo culture apparatus can image all the wells at once, and there is no need to move the camera with respect to the embryo culture dish. Of course, it is also possible to provide a plurality of sets of wells and recesses in one embryo culture dish, and to move the camera to sequentially take images.

(5) In such a configuration, the movement-inhibiting portion may be a passage portion connecting the well and the concave portion, the passage portion being shallower than the depth of the well. In this way, even if all the culture solution in the recess is sucked up with a pipette or the like, the depth of the movement-inhibiting portion is shallower than the depth of the well, so the liquid level of the culture solution in the well does not fall below the passage. Therefore, the culture medium in the wells containing embryos to be cultured is not accidentally pumped out. Needless to say, the movement-inhibiting portion only needs to inhibit migration of the embryo from the well, and the depth of the movement-inhibiting portion may be set to the same extent as the depth of the well. In this case, it is possible to suck up all the culture medium in the well, but if it is necessary to leave the culture medium around the embryo, the amount of sucking up can be adjusted. For example, an adapter may be provided at the tip of the pipette used when sucking up the culture fluid, which is spaced apart from the bottom of the recess by a predetermined distance. In this way, when the culture solution is sucked up with a pipette, a predetermined height of the culture solution can be left from the bottom of the recess.

(6) In such a configuration, the movement inhibiting portion includes a passage portion provided at a connection portion between the well and the recess and having a gap through which the culture medium flows, and the gap of the passage portion It may be narrower than the minimum diameter of the embryo contained in the well. In this way, migration of embryos within the well can be easily inhibited. A passageway may simply be formed as a gap narrower than the size of the embryo. In this case, the width of the passage itself may be narrowed, or a wide passage may be provided with a fence or mesh to inhibit migration of the embryos.

(7) In such a configuration, the well may have a microwell at the bottom of the well that is smaller than the well and larger than the embryo to be accommodated. This allows the position of the embryo to be within the microwell and limits the position of the embryo to be imaged. It is also desirable for culturing embryos, as it can limit movement of embryos during culture. Of course, the microwell may be omitted. It is also possible to limit the movement of the embryos in the well by providing a ring-shaped protuberance with a diameter slightly smaller than the diameter of the embryo at the bottom of the well.

(8) In such a configuration, the bottom surface of the embryo culturing dish may be provided with a replenishing liquid storage section for storing a replenishing liquid for replenishing the culture liquid in the recess. In this way, the equilibrated medium that has been acclimatized to the environment during cultivation can be easily replenished.

(9) In such a configuration, an outer peripheral wall that defines the outer periphery is provided, and the outer surface of the outer peripheral wall is provided with a gripping assist structure that assists the user's finger gripping when gripping the embryo culture dish. good. This facilitates handling of the embryo culture dish.

(10) In such a configuration, the well and the recess of the embryo culture dish are filled with the culture medium, and the well filled with the culture medium contains the embryo to be cultured, and the culture medium is filled. The well and the recess are covered with an oil that suppresses the evaporation of the culture solution, and after a predetermined time from the placement of the embryo in the well, a predetermined volume of the culture solution is sucked up from the recess, The sucked culture medium may be subjected to analysis by an analyzer capable of analyzing chromosomal aneuploidy using DNA contained in the culture medium. In this way, chromosomal aneuploidy testing on embryos can be non-invasive and has little effect on the embryos being tested. Therefore, it can be expected to increase the success rate of embryo transfer (patient pregnancy rate) based on the results of the aneuploidy test.

The present disclosure is not limited to the embodiments described above, and can be implemented in various configurations without departing from the scope of the present disclosure. For example, the technical features in the embodiments corresponding to the technical features in the respective modes described in the Summary of the Invention column may be used to solve some or all of the above problems, or Substitutions and combinations may be made as appropriate to achieve part or all. Also, if the technical features are not described as essential in this specification, they can be deleted as appropriate.

The present disclosure is not limited to the embodiments described above, and can be implemented in various configurations without departing from the scope of the present disclosure. For example, the technical features of the embodiments corresponding to the technical features in each form described in the outline of the invention are used to solve some or all of the above problems, or Alternatively, replacements and combinations can be made as appropriate to achieve all. Also, if the technical features are not described as essential in this specification, they can be deleted as appropriate. For example, part of the configuration implemented by hardware in the above embodiments can be implemented by software.

DESCRIPTION OF SYMBOLS 10... Embryo culture apparatus, 11-24... Culture unit, 17... Lid part, 18... Gas port, 20... Case main body, 25... Embryo, 26... General-purpose connector, 27... Filter, 28... Filter port, 43... Supply port , 44... exhaust port, 51... camera module, 52... lens module, 60... control unit, 61... CPU, 62... ROM, 63... RAM, 64... memory interface, 65... memory card, 66... general-purpose I/O interface , 67... Incubation chamber interface, 68... Camera interface, 70... Display, 71... Drive device, 72... Warning device, 73... Mixture adjustment device, 84... Mixture supply pipe, 91... Display, 95... Control device, 96 Dial 97 Base portion 170 Lighting unit 180 Holding frame 182 Convex portion 186 Drop arrangement portion 191, 191A, 191B Dish 192 Wall 194 Peripheral wall 195 Microwell , 196...Bottom 197...Recess 198...Opening 200...Accommodating part 210...First accommodating part 220...Second accommodating part 230...Partition part 240...Pipette 251...Equalizing drop for additional injection , 300... Next-generation sequencer, 310... Storage unit, 320... Operation unit, 330... Display unit, HD... Fingers

Claims (10)

An embryo culture dish comprising a plurality of wells for accommodating embryos together with a culture medium,
placing the plurality of wells independently and adjacently on the bottom of the embryo culture dish;
Forming a depression capable of holding the culture solution together with the well in the bottom independently of other depressions for each of the wells,
a movement-inhibiting portion that permits the flow of the culture medium and inhibits movement of the embryo is provided between the well and the recess;
A dish for embryo culture, wherein the volume of the recess is equal to or greater than the volume of the culture solution sucked up for aneuploidy test of the chromosome of the embryo cultured in the well. At least part of the plurality of wells are arranged in two rows, and each of the recesses provided corresponding to the wells arranged in one row corresponds to the wells in the other row. 2. The embryo culture dish according to claim 1, wherein the recess is provided on the opposite side of the well and has a shape extending outward from the corresponding well over a predetermined length. 3. The embryo culture dish according to claim 2, wherein the row-wise spacing of the wells in each row is narrower than the row-wise spacing of the recesses provided corresponding to the wells. All of the wells in the embryo culture dish are within a range that can be imaged by a camera provided in a time-lapse embryo culture apparatus in which the embryo culture dish is accommodated without moving relative to the embryo culture dish. 4. The embryo culture dish according to any one of claims 1 to 3, which is arranged in a . The embryo culture according to any one of claims 1 to 4, wherein the movement-inhibiting portion is a passage portion connecting the well and the recess, the passage portion being shallower than the depth of the well. dish. The movement-inhibiting portion is provided at a connection portion between the well and the recess, and includes a passage portion having a gap through which the culture medium flows,
the gap in the passage portion is narrower than the minimum diameter of the embryo accommodated in the well;
The embryo culture dish according to any one of claims 1 to 5. The embryo culture dish according to any one of claims 1 to 6, wherein the well has a microwell at the bottom of the well that is smaller than the outer shape of the well and larger than the outer shape of the embryos to be accommodated. 8. The embryo culture dish according to any one of claims 1 to 7, wherein the bottom surface of the embryo culture dish is provided with a replenisher containing portion for containing a replenishment culture solution for replenishing the culture solution in the recess. Embryo culture dish. The embryo culture dish according to any one of claims 1 to 8,
Equipped with an outer peripheral wall that divides the outer periphery,
An embryo culture dish, wherein the outer surface of the outer peripheral wall is provided with a grip assisting structure that assists a user's fingers to grip the embryo culture dish when gripping the embryo culture dish. filling the well and the recess of the embryo culture dish according to any one of claims 1 to 9 with the culture solution,
An embryo to be cultured is housed in the well filled with the culture solution,
covering the well filled with the culture medium and the recess with oil that suppresses evaporation of the culture medium;
a predetermined amount of the culture medium is sucked up from the recess after a predetermined time from the placement of the embryo in the well;
A method for collecting embryo culture medium for chromosome analysis, wherein the sucked culture medium is subjected to analysis by an analyzer capable of chromosomal aneuploidy analysis using DNA contained in the culture medium.

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