CN111793562A

CN111793562A - Culture dish and application thereof

Info

Publication number: CN111793562A
Application number: CN202010579798.5A
Authority: CN
Inventors: 刘洪君
Original assignee: Shenzhen Zhienbo Technology Co ltd
Current assignee: Shenzhen Zhienbo Technology Co ltd
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2020-10-20
Also published as: CN113337401A

Abstract

The invention relates to the field of assisted reproduction, in particular to a culture dish, which comprises a dish main body, wherein a sample adding pool is arranged on the inner bottom surface of the dish main body, a plurality of concave cleaning pools are distributed outside the sample adding pool, and a plurality of concave culture cabins are distributed in the sample adding pool; a cavity contracting from top to bottom is formed in the culture cabin; the cavity comprises an incubation chamber positioned at the bottom and a drainage groove positioned above the incubation chamber; the cultivation chamber and the drainage groove are integrally formed; the invention also discloses application of the culture dish in culturing one or more culture objects, wherein the culture objects comprise at least one of embryos and gametes; the culture dish can not only integrate the advantages of single culture and group culture, thereby being capable of simultaneously culturing one or more gametes or zygotes, but also having various functions, wide application scenes, controllable liquid drop size, convenient operation and easy observation.

Description

Culture dish and application thereof

Technical Field

The invention relates to the technical field of assisted reproduction, in particular to a culture dish and application thereof.

Background

In the past 20 years, the infertility rate of China is increased from 2.5-3% to 12.5-15%, while the number of newborn babies born in China per year is about 1600 thousands, and according to the infertility rate of 12.5-15%, theoretically 200-240 newborn babies can not be born per year. In Vitro Fertilization (ivvitro Fertilization) refers to a technique In which sperm and eggs of a mammal complete the Fertilization process In an environment controlled manually In Vitro, abbreviated as IVF. Because it is inseparable from the embryo transfer technique (ET), also referred to as IVF-ET for short. In Vitro Fertilization (IVF) is used as an assisted fertility measure and can provide pregnant opportunities for infertile couples. Between fertilization (insemination) and transfer, embryos need to be cultured in vitro for 2-6 days. The in vitro culture of embryos is a process for simulating development patterns, environments, nutritional requirements and the like in the embryos and mainly comprises three methods: single culture, group culture and co-culture. Researches prove that the co-culture technology can improve the in vitro culture effect of embryos, overcome in vitro development retardation, increase the number of embryos which develop to the blastocyst stage and improve the quality of the blastocysts; the group culture can effectively improve the development potential of the embryo and improve the planting rate of the embryo; the single culture can correspondingly monitor the development process of the embryo.

The in vitro culture of human embryos is realized by placing a culture dish filled with embryos in a time difference incubator or a conventional carbon dioxide incubator for culture and simultaneously ensuring the temperature, humidity and cleanliness of the culture environment. The devices for in vitro incubation of embryos are mainly culture dishes, which can also be called glass slides or trays, while the culture dishes in the prior art mainly have the following defects:

1. the conventional culture dish has single function, can only realize embryo culture or embryo cleaning, and is inconvenient to operate;

2. the conventional culture dish has a single application scene, namely the same culture dish cannot be suitable for a time difference culture box and a common carbon dioxide culture box;

3. when the number of embryos is large, a large number of culture dishes are needed in the traditional single culture, so that the cost, the operation time and the difficulty are obviously increased;

4. the traditional co-culture and group culture are to culture a plurality of gametes or zygotes in one culture dish, have the problem that the drops of a plurality of groups of culture liquids are adhered because of close distance, and are not easy to operate;

5. the traditional co-culture and group culture can not correspondingly monitor the development process of each embryo one by one, which is not beneficial to embryo selection;

6. the size of the droplets is difficult to control, too large a droplet tends to collapse and spread, and too small a droplet does not provide sufficient nutrition to the embryo.

Chinese patent document CN 201720087281.8, application date 20170123, entitled: the utility model provides an embryo culture dish, discloses an embryo culture dish, and is including ware lid, ware main part and cultivation region, cultivate regional setting in the inboard bottom surface of ware main part, it encloses the fender and encloses the fender in the middle by the outside and separates into a plurality of and cultivates the cell to cultivate the region.

The above patent documents form the culture cells isolated from each other in the same culture dish, and limit the height of the middle barrier, so that a single culture dish is used for culturing multiple groups of embryos, and the multiple groups of embryos are completely isolated from each other, thereby avoiding the effect of mutual confusion. However, complete isolation of multiple sets of embryos presents difficulties in just the realization of co-culture; and the height of the middle enclosure is too high, which also brings obstruction to the control of the size of the liquid drops.

Therefore, a culture dish which can integrate the advantages of single culture and group culture, can simultaneously culture one or more gametes or zygotes, and has the advantages of multiple functions, wide application scenes, controllable droplet size, convenience in operation and easiness in observation is needed.

Disclosure of Invention

It is an object of embodiments described herein to overcome or mitigate the above disadvantages, combine the advantages of single and group cultures, so that the culture of one or more gametes or zygotes can be performed simultaneously, and with multiple functions, a wide range of application scenarios, controllable droplet size, ease of operation, and ease of observation.

In a first aspect of the embodiments described herein, there is provided a culture dish, including a dish main body, a sample adding pool is disposed on an inner bottom surface of the dish main body, a plurality of concave cleaning pools are distributed outside the sample adding pool, and a plurality of concave culture compartments are distributed in the sample adding pool.

In this embodiment, the cleaning tank, the sample adding tank and the culture chamber are arranged simultaneously, so that the embryo or the oocyte can be directly cleaned in the culture dish, and can be directly moved into the culture chamber after being cleaned, so that the culture dish has two functions of cleaning and culturing. The arrangement of a plurality of culture cabins limits the relative positions among a plurality of groups of culture liquid drops and the respective volume sizes of the culture liquid drops, and achieves the effects of controlling the volume of the liquid drops and avoiding the adhesion of the liquid drops. When the culture is carried out, the culture medium is added into the sample adding pool, the culture medium enters each culture cabin under the flowing action, the culture cabins are communicated through the fluid of the culture medium to form a co-culture system, the culture objects of each culture cabin can transmit endocrine factors through the culture medium, and the co-development of a plurality of culture objects is promoted, so that the effects of mutually promoting the growth of embryos in each culture cabin and obviously improving the embryo quality and the blastocyst formation rate are achieved.

As a more preferable embodiment of the present scheme, a cavity which is contracted from top to bottom is formed in the culture cabin; preferably, the cavity comprises an incubation chamber at the bottom, and a drainage slot above the incubation chamber; more preferably, the incubation chamber and the drainage groove are integrally formed.

In a more preferred embodiment of this aspect, the drainage channel is located on the left side of the incubation chamber.

In a more preferred embodiment of the present invention, the cultivation room is in the shape of a hollow truncated cone with a large top and a small bottom, and comprises a bottom surface of the cultivation room and a wall of the cultivation room; preferably, the inclination angle of the culture chamber wall relative to the bottom surface of the culture chamber is larger than that of the drainage groove relative to the bottom surface of the culture chamber; more preferably, the inclination angle of the drainage groove relative to the bottom surface of the culture chamber is 5-15 degrees.

In this embodiment, the drainage groove can play the medium drainage effect, and the drainage groove forms certain angle with the horizontal plane simultaneously, and the culture object gets into the culture chamber along with culture medium through its inclined plane that forms, can cushion the gravity striking effect when culture object gets into, can reduce culture medium and cover medium additional pressure because surface tension causes to the culture object simultaneously.

As a more preferable embodiment of the present disclosure, the plurality of culture compartments are uniformly distributed on the inner side of the sample adding tank and arranged in a circular ring shape, and the plurality of cleaning tanks are uniformly distributed on the outer side of the sample adding tank and arranged in a circular ring shape; preferably, the outer side of each culture cabin is provided with a first mark for positioning the culture cabin; more preferably, the outer side of each cleaning pool is provided with a second mark for positioning the cleaning pool.

In this embodiment, the first and second identifiers are provided for the purpose of positioning the culture objects, so that the cleaning and development effects of the individual culture objects can be monitored in a one-to-one correspondence, and the advantages of single culture and group culture can be retained under the conditions of reduced cost, operation time and difficulty.

As a more preferable embodiment of the present invention, the dish main body includes a dish bottom and a dish wall; preferably, the dish bottom is in a truncated circular shape; more preferably, the bottom end of the dish wall extends downwards to enable the lower surface of the dish bottom to be concave.

In this embodiment, the shape of the dish bottom is defined so that the dish body can form an axial cross section, which can be used as an identification area for the patient, for example, to attach an identification label for the patient, thereby recording and verifying the identification information of the culture object; meanwhile, when the culture box is held by hands or fixed in the culture box, the stability of the culture box can be improved. The lower surface of the dish bottom can form a groove with the dish wall, so that the dish bottom is prevented from scraping due to friction with other contact objects, and embryo observation is influenced.

As a more preferred embodiment of the present disclosure, the sample adding cell includes a bottom surface and a wall, and a top end of the wall is higher than an upper surface of the bottom surface.

As a more preferable embodiment of the scheme, the lower surface of the dish bottom is provided with a plurality of buckles for clamping the dish main body and the incubator; preferably, the incubator comprises a time difference incubator and a carbon dioxide incubator; more preferably, the number of the buckles is 3.

In this embodiment, when the culture dish was placed in the incubator, be located the buckle of dish main part bottom surface can be fixed with the incubator, has reached and has prevented phenomenons such as the dish appears sliding, vibrations to influence embryo culture's effect. When the number of the buckles is 3, a triangular structure can be formed, so that the stability during fixing is improved; meanwhile, the position of the cleaning tank is not overlapped with that of the cleaning tank, so that embryos or gametes in the cleaning tank can be observed under a microscope.

It should be understood that the number and the position of the fasteners in the above embodiments are only one of the embodiments provided for improving the performance of the culture dish, and do not represent that the above limitations are the only possible embodiments.

As a more preferable embodiment of the present invention, the dish further comprises a dish cover which is matched with the dish main body; preferably, a plurality of blocking pieces used for forming a gap between the dish cover and the dish main body are arranged on the lower bottom surface of the dish cover; more preferably, the number of the stoppers is 5.

In this embodiment, the stopper located on the bottom surface of the dish cover can form a certain gap when the dish cover is placed on the dish main body, so as to ensure that gas exchange can be performed between the inside and the outside of the dish main body, thereby balancing the pH and CO of the culture medium in the culture dish₂The effect of concentration; when 5 blocking pieces are arranged, the gap between the dish cover and the dish main body can be ensured to be equal in size.

It should be understood that the focus of this embodiment is on enabling the capsule to form a void with the capsule body, and therefore all ways of achieving this effect are within the scope of the invention, not limited to the provision of the stop. Meanwhile, the stopper may be cylindrical, square, rectangular or irregular in shape, etc., regardless of the shape, for the purpose of achieving the above-mentioned corresponding functions; that is, the shape of the buckle is not limited, and the skilled person can select the buckle according to the needs.

As a more preferable embodiment of the scheme, the capsule is made of polystyrene, and the transparency is 88% -100%; the dish body is made of polycarbonate, and the transparency is 88% -100%; the surface of the dish body is subjected to hydrophilic processing, and the bottom of the dish body is subjected to polishing processing.

It should be understood that the materials of the dish body and the dish cover are for the convenience of observation under a microscope and photographing and imaging by a camera, and for the convenience of forming droplets by adhering liquid, and the materials of the dish cover and the dish body are not limited, that is, other materials capable of achieving the effects can also be used for the culture dish in the scheme.

In another aspect of the embodiments described herein, there is provided a use of the culture dish for culturing one or more culture objects, the culture objects including at least one of embryos and gametes.

As a more preferred embodiment of this solution, the application comprises the following steps:

s1, adding a cleaning medium into the cleaning pool;

s2, adding a culture medium into the sample adding pool, enabling the culture medium to enter a culture cabin and enter an incubation chamber through the drainage groove;

s3, adding a covering medium into the dish main body to cover the cleaning pool and the sample adding pool;

s4, balancing the culture dish obtained in the step S3 in an incubator overnight or standing for more than 12 hours;

s5, taking out the culture dish obtained in the step S4, placing the culture object in the cleaning pool for cleaning, and then moving the culture object into the culture cabin;

s6, placing the culture dish obtained in the step S5 in an incubator, and culturing the culture object.

As a more preferred embodiment of this aspect, the culture medium comprises at least one of a one-step broth, a cleavage broth, and a blastocyst broth;

the covering medium comprises at least one of oil for in vitro assisted reproductive culture, mineral oil, paraffin oil and oil for embryo culture.

The invention has the beneficial effects that:

1. according to the culture dish, the culture cabins are arranged in the sample adding pool, so that the volume of liquid drops can be controlled, the liquid drops can be prevented from being adhered, and the embryo quality and the blastocyst formation rate can be remarkably improved; and the periphery of each culture chamber is provided with a digital identifier, so that embryos can be positioned, the development of single embryos can be correspondingly monitored one by one, and the effects of keeping the advantages of single culture and group culture are finally achieved under the conditions of reducing cost, operation time and difficulty.

2. According to the culture dish, the side wall is obliquely arranged, so that a culture medium can conveniently enter the culture cabin, the gravity impact of embryos and the culture cabin due to vertical entering can be avoided, and the effects of preventing the embryos from being damaged and improving the liquid feeding smoothness are achieved.

3. The culture dish is not only suitable for the culture box with the time difference, but also suitable for the common culture box.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a schematic structural view of the capsule of the present invention;

FIG. 2 is a bottom view of the capsule of the present invention;

FIG. 3 is a schematic view of the construction of the capsule body according to the present invention;

FIG. 4 is a bottom view of the capsule body of the present invention;

FIG. 5 is a top plan view of the culture chamber of the present invention;

FIG. 6 schematically shows a perspective view of a culture dish according to the invention;

FIG. 7 is a top perspective view of the culture compartment of the present invention;

FIG. 8 is an inverted top view of the capsule body of the present invention;

FIG. 9 is a cross-sectional perspective view of the culture dish of the present invention;

FIG. 10 is a partial top view of the capsule body of the present invention;

FIG. 11 is an enlarged view of portion A of FIG. 9;

in the figure, 1, a dish body; 101. a dish bottom; 1011. buckling; 102. a dish wall; 2. a sample adding pool; 201. the bottom surface of the sample adding pool; 202. the sample adding pool wall; 3. a cleaning tank; 301. cleaning the bottom surface of the pool; 302. cleaning the pool wall; 303. a second identifier; 4. a culture compartment; 401. an incubation chamber; 4011. the bottom surface of the cultivation room; 4012. the walls of the cultivation chamber; 402. a drainage groove; 403. a first identifier; 404. a cavity; 5. a dish cover; 501. a stopper; 6. a culture dish.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described with reference to the accompanying drawings.

In the description of the present invention, it is to be noted that, unless otherwise explicitly specified and defined, terms used herein should be construed according to meanings that are generally understood by those of ordinary skill in the art to which the present disclosure pertains.

As used herein, the terms "upper," "lower," "front," "rear," "left," "right," "top," "bottom," "inner," "outer," and the like are used in the orientation or positional relationship indicated in the drawings for convenience in describing the invention and for simplicity in description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the invention.

By way of example, the "top" of the culture dish 6 is the surface of the culture dish 6 that faces upward when the culture dish 6 is in normal use (e.g., when containing embryos or gametes, culture medium, and overlay medium); the "bottom" of the culture dish 6 is the surface of the culture dish 6 that faces downwards when the culture dish 6 is in normal use; the edge surface of the dish body 1 of the culture dish 6 substantially orthogonal to its axis of extension may be referred to as the end of the culture dish 6; the edge surface of the dish body 1 of the culture dish 6 substantially parallel to its axis of extension may be referred to as the side of the culture dish 6.

As used herein, the terms "mounted," "disposed," "sleeved/connected," "connected," and the like are to be construed broadly, e.g., "connected," which may be a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements.

As used herein, the term "gamete" refers to a mature cell produced by the reproductive system when an organism is sexually reproduced, abbreviated as a germ cell. Gametes are classified into male gametes (male gametes) and female gametes (female gametes). Animal female gametes are commonly referred to as ova (ova or egg) and animal male gametes are referred to as sperm (sperm). In the field of assisted reproduction, the term "gamete" generally refers to primary oocytes, secondary oocytes, and mature oocytes and sperm. Primary oocytes are oocytes that undergo meiosis following mitotic proliferation of the oogonium. The primary oocyte of a human is present at birth as a tetraploid cell. And (3) completing the first maturation division of the primary oocyte 36-48 hours before ovulation to form a secondary oocyte. And performing second meiosis on the secondary oocyte to form a mature oocyte, wherein the mature oocyte is spherical, an oocyte nucleus is arranged in an egg, an oocyte membrane is arranged outside the egg nucleus, and a layer of colloidal membrane is wrapped on the surface of the mature oocyte and is called a zona pellucida.

As used herein, the term "embryo" is approximately spherical, and consists of one or more cells (blastomeres) surrounded by a gelatinous shell (an acellular matrix called zona pellucida).

In certain aspects, the term "embryo" may sometimes be used for the fertilized gamete (ovum) that becomes a fetus at this stage after implantation into the uterus until 8 weeks after fertilization. According to this term, a fertilized gamete may sometimes be referred to as a pre-embryo or fertilized egg before implantation occurs. However, for convenience, the term "embryo" may also sometimes be used to encompass the fertilized egg stage prior to implantation, and the method will generally be followed herein.

That is, the term "embryo" is used in a broad sense to cover all stages of development from fertilization of gametes to morula, blastocyst stage, hatching and implantation, which means that it may be used herein to refer to the following stages: fertilization gametes, 2-cells, 4-cells, 8-cells, 16-cells, densification, morula, blastocyst, expansion blastocyst, and hatching blastocyst, and all stages in between (e.g., 3-cells or 5-cells). Also, the terms "embryo" and "fertilized egg" are used interchangeably herein.

Furthermore, embryos incubated using the culture dish 6 according to the present invention may be frozen beforehand. For example, embryos are cryopreserved immediately after fertilization (e.g., at the 1-cell stage) and then thawed; alternatively, they may be freshly prepared, for example embryos freshly prepared from gametes by IVF or ICSI techniques.

As mentioned above, embryos/gametes are sometimes stored/maintained in the culture dish 6, for example during In Vitro Fertilization (IVF) processes or immature oocyte culture (IVM). As mentioned above, the culture dish 6 for assisted reproduction will comprise a plurality of culture compartments 4 for accommodating embryos or gametes to be cultured. The embryos or gametes in the respective culture compartments 4 are immersed in the culture medium covered by the oil layer (covering medium).

The possibility of using the culture dish 6 in an experiment for handling multiple embryos from the same patient or from multiple patients means that the ease of use and the rationality of the design of the culture dish 6 are important. The culture dish 6 is designed for this purpose in order to help the operator to better achieve the overall goals of the experiment, such as reducing the risk of handling errors (e.g. embryo or gamete confusion, no identification), minimizing the use of consumables (e.g. culture oil) and facilitating embryo imaging analysis. In order to solve the above problems, the present invention is designed to have the dimensions and height of the main body 1 of the culture dish 6 and the positioning marks of the culture compartment 4 and the washing tank 3, respectively.

FIGS. 1-10 schematically illustrate embodiments of the culture dish 6 of the present invention in some cases.

The culture dish 6 comprises a dish body 1, which dish body 1 can be manufactured according to conventional techniques, such as injection moulding of a suitable plastic material. In some cases, culture dish 6 further includes a removable dish lid 5 that is separate from dish body 1. It is not the specific technique used to manufacture the culture dish 6 that is important for the culture dish 6 of the present invention, but the shape and configuration of the culture dish 6. In this regard, the manufacturing techniques and materials for the capsule body 1 and capsule 5 are substantially the same. In particular, the culture dish 6 may be formed by injection molding of substantially transparent polymers (e.g., PS and PC).

As can be seen from FIG. 6, in the example, the end of the culture dish 6 is formed in an arc shape, and the dish main body 1 is square in axial section.

The outer angle between the side of the culture dish 6 and the axial section is arc-shaped. The axial cross-section of the culture dish 6 in the specific example has a length L of 42 + -0.05 mm, a characteristic width W (at the widest point) of around 43.8mm and a height H of about 10.6 mm. However, it should be understood that other sizes and shapes of the culture dish 6 may be selected according to the current implementation to match the geometry of the incubator for the particular moveout used for incubation and equilibration of the culture dish 6. For example, according to various embodiments of the present invention, and according to an implementation, the culture dish 6 may have a characteristic length L selected from: 10-70 mm, 20-60 mm, 30-50 mm, 40-45 mm; and/or a characteristic width W selected from the following ranges: 10-80 mm, 25-65 mm, 33-55 mm, 40-47 mm; and/or a characteristic height H selected from the following ranges or less: 8mm, 6mm, 5.5mm, 4.5mm, etc. However, it should be understood that the overall dimensions of the culture dish 6 are not essential and may be chosen adaptively according to the current implementation and the intended use.

Referring to FIGS. 1-10, a culture dish 6 is used to culture one or more gametes/zygotes, such as embryos or gametes.

The dish body 1 includes 16 culture compartments 4 for culturing embryos or gametes. During normal use, a culture medium (e.g. a one-step broth) is filled in culture compartments 4 containing embryos or gametes, and in use a single culture compartment 4 contains only one embryo or gamete. For example, if a given culture dish 6 is used to culture 12 embryos or gametes from a patient, the embryos or gametes should be placed in 12 culture compartments 4 in the culture dish 6, respectively, so that all 12 culture compartments 4 have embryos or gametes and the remaining 4 compartments have no embryos or gametes. In other words, it is not uncommon to cultivate embryos or gametes of different patients simultaneously in the same culture dish 6 or slide, or to receive embryos of different patients in the remaining culture compartments 4 for cultivation during human assisted reproduction, although there may be empty culture compartments 4 in the same culture dish 6.

In addition, although a single culture compartment 4 can only hold 1 embryo or gamete during normal use, multiple embryos or gametes can actually be held in a single culture compartment 4. While it may be desirable in some experimental studies to place multiple embryos or gametes in the same culture compartment 4 to study the interaction of growth factors secreted by multiple subjects to population-cultured embryos or gametes, an embryo or gamete placement scenario based on the above-described study objectives is unnecessary. The invention overcomes the defects of the prior culture dish 6, and simultaneously, based on the research consideration of the prior art, the culture cabins 4 are arranged in a circular ring shape, the middle part forms a sample adding pool 2, the sample adding pool 2 is mutually communicated with each culture cabin 4 through culture media added in the sample adding pool 2, embryos or gametes are placed in the independent culture cabins 4, one cabin position is ensured to only contain 1 embryo or gamete, nutrient metabolites (such as growth factors) secreted by the embryos or gametes in the culture cabins 4 are transmitted/diffused through the culture media in the sample adding pool 2, the nutrient secretions are effectively shared and exchanged, the aim of culturing a technical personnel group is realized, and meanwhile, the embryos and the gametes are provided with sufficient nutrition and a wide growth and development space. The operation of microscopic imaging or camera-down photographing of embryos or gametes by technicians or embryologists is also facilitated, the imaging effect is clearer, embryo overlap or embryo ghosting is avoided, and at the same time, the harmful effects on healthy embryos due to necrosis of one or more embryos or gametes which may occur in group culture are effectively avoided by removing necrotic embryos or gametes in the independent culture compartments 4.

In this embodiment, 16 culture compartments 4 are uniformly distributed around the sample addition pool 2 and arranged in a circular ring shape, and the direct distances between the walls of adjacent culture compartments 4 are equal, but the distances between different culture compartments 4 and the end and the side of the culture dish 6 are different. The culture compartments 4 in the same culture dish 6 have the same shape and size.

At present, technicians have the problem that identification and positioning difficulty of each embryo and gamete is high through the position and the shape of the culture cabin 4 in embryo or gamete transfer operation, the existing culture dish 6 is usually designed to carry out memory identification through taking the relative position of the culture cabin 4 and the end part or the side part of the dish main body 1 as a reference standard, but operators face a large number of embryos or gametes, the memory burden is undoubtedly increased for the operators, and meanwhile accurate positioning of the embryos or gametes is difficult to guarantee. The existing other design solution of the culture dish 6 is that the digital identification is carried out at the bottom of the culture cabin 4 to identify and position the embryo or gamete, so that the problem of embryo identification and identification is solved, but the digital identification carried out at the bottom of the culture cabin 4 can influence the effect of light transmission through the bottom surface and the surface flatness of the bottom surface, and can also shield the observation under a microscope, thereby influencing the imaging effect under a germ cell microscope.

The invention aims at the defects of the prior art and the use requirements of operators in the field of assisted reproduction, and creatively designs the culture dish 6. The positioning of the culture compartments 4 is not dependent on the position of each culture compartment 4 from the end and the side for identification, and meanwhile, in order to avoid the marks from affecting embryo observation and surface flatness of the hole, first marks 403 (arabic numerals) corresponding to each other are arranged on the peripheral edge of each culture compartment 4 and are arranged clockwise, so that each culture compartment 4 can be clearly positioned and identified by the peripheral arabic numerals, and the effect can be seen in fig. 10.

In this embodiment, the cultivation vessel 4 is symmetrically arranged along the center line of the long end, and the whole cultivation vessel 4 is horseshoe-shaped, and the detailed structure is shown in fig. 7. The bottom surface of the culture cabin 4 is a classical round surface with the radius of 0.15 mm; the vertical distance from the bottom surface to the opening end of the culture cabin 4 is 1mm, namely the depth of the culture cabin 4 is 1 mm; the maximum width of the open end of the culture compartment 4 is 1mm, and the length of the culture compartment 4 is 2.5 mm. The bottom surface of the culture cabin 4 is subjected to surface polishing treatment, so that the phenomenon that an incident light source is refracted or scattered due to too low surface flatness is avoided, and imaging observation on a culture object is influenced. In fig. 11, a cavity 404 which is contracted from top to bottom is formed in the culture compartment 4, the cavity 404 comprises an incubation chamber 401 positioned at the bottom and a drainage groove 402 positioned above the incubation chamber 401, and the incubation chamber 401 and the drainage groove 402 are integrally formed; the cultivation room 401 is in a hollow round table shape with a large top and a small bottom, and comprises a cultivation room bottom 4011 and a cultivation room wall 4012; the bottom end of the drainage channel 402 is connected to the top end of the culture chamber wall 4012. The incubation chamber 401 is where the culture object (e.g., an embryo or gamete) is placed during incubation. The characteristic width/diameter of the bottom 4011 of the incubation chamber is set to be in the order of mm, for example, about 0.3 mm. The width of the bottom plate of the culture chamber 4 is limited in consideration of the size of the culture object (such as an embryo or a gamete), the size of the embryo or the gamete is usually between several tens of micrometers and 200 μm, the size of the space of the culture chamber 401 can be reasonably limited according to the size of the moving space of the culture object, the relative movement of the culture object should be avoided as much as possible, and the culture medium in the space of the culture chamber 401 can be ensured to fully meet the growth requirement of the culture object. The drainage groove 402 is located on the left side of the cultivation room 401, the bottom end of the drainage groove is a flat inclined plane, and a certain included angle is formed between the drainage groove and the cultivation room wall 4012, and the included angle can be selected from the following characteristic angles: 120 °, 130 °; 150 degrees. In fig. 7, the width of the drainage groove 402 gradually narrows from the left end of the culture compartment 4 to the culture chamber 401, and the drainage groove 402 forms a certain range of characteristic included angle with the bottom surface 4011 of the culture chamber: the inclination of the included angle is 5-15 degrees, 7-14 degrees and 9-12 degrees, so that the drainage groove 402 forms a certain gradient, the culture medium (such as one-step culture solution) in the sample adding pool 2 is promoted to reach the culture chamber 401 through the drainage groove 402, and meanwhile, the gradient of the drainage groove 402 enables an operator to transfer or place embryos or gametes more conveniently, namely, the embryos or gametes can slowly enter the culture chamber 401 through the drainage groove 402, so that the embryos or gametes are prevented from being impacted when directly and vertically entering the culture chamber 401 under the action of gravity, and the structural function of the embryos or gametes is prevented from being damaged.

The culture dish 6 comprises a main body 1 and a culture dish wall 102, wherein the main body 1 comprises a dish bottom 101 and a dish wall 102 which vertically surrounds the edge of the dish bottom 101 and is connected with the dish bottom 101; the upper surface of the dish bottom 101 is provided with 1

sample adding pool

2 and 8 concave cleaning pools 3.8 wash pond 3 and be the ring shape evenly distributed in the outside of application of sample pond 2 to about the roughly circular symmetry of vertical axis, the degree of depth of every washing pond 3 is about 0.25 ~ 0.5mm, and the diameter is about 9mm, and all can hold 15 ~ 31mm³Clear ofThe distance between the ends of adjacent washing tanks 3 is about 2 mm. The embryo or the gamete is placed in the cleaning pool 3 to be sufficiently cleaned and remove harmful substances in the embryo or the gamete, and early preparation is made for later culture in the culture cabin 4.

The sample adding pool 2 comprises a sample adding pool bottom surface 201 and a sample adding pool wall 202 which is arranged at the edge of the sample adding pool bottom surface 201 in a surrounding manner and is connected with the sample adding

pool bottom surface

201, and 16 culture compartments 4 which are concave and are uniformly distributed in a ring shape are arranged on the upper surface of the sample adding pool bottom surface 201 at intervals.

The culture dish 6 is used when the dish body 1 also needs to hold a certain amount of covering medium over the embryo or gamete and the culture medium, i.e. micro-droplet culture technique. Therefore, a medium (e.g., embryo culture oil) is covered over the sample addition well 2 containing the embryo or gamete and the culture medium to form a covering layer; the cover medium is less dense relative to the culture medium and is a hydrophobic substance that does not mix with the culture medium. The covering medium is positioned between the culture medium and the environment around the culture dish 6, provides a protective barrier for the growth and development of embryos or gametes, and is beneficial to maintaining the stability of the pH value, osmotic pressure and humidity of the culture medium, preventing the environment of the culture medium from being polluted, preventing the water of the culture medium from evaporating and reducing the using amount of the culture medium. Wherein the coating medium (e.g., embryo culture oil) is generally transparent.

As shown in fig. 1, the internal geometry of the capsule 5 may be selectively matched to the shape and size of the cross section of the upper end surface of the capsule body 1 in the horizontal cross section, so that the capsule 5 is fitted to the capsule body 1. The height of the dish cover 5 is about 5mm, and the center of the dish cover 5 is provided with an annular area, namely an embryo or gamete culture observation area.

Another important aspect of the capsule 5, as shown in figure 6, is that it needs to be wider than the capsule body 1; that is, when the dish lid 5 is coupled to the dish main body 1, the dish lid 5 can completely cover the opening portion at the upper end of the dish main body 1. Meanwhile, the dish body 1 and the dish cover 5 are not cylindrical, but are truncated cylindrical, that is, the dish bottom 101 is truncated circular; and the axial section of the dish cover 5 and the dish main body 1 is in arc surface transition with a certain radian with the columnar ring surface, so that the two surfaces are prevented from being in direct contact to form a sharp angle. This means that when the operator holds the culture dish 6 by its side, the dish 6 can be held around by the index finger and the thumb, the presence of the axial section enhances the stability of holding the dish 6, reducing the risk of falling off; the existence of the cambered surface avoids the risk that the formed acute angle causes scratch or conical pain to the finger.

The dish lid 5 prevents contaminants (e.g., dust or particles) outside the culture dish 6 from entering the dish body 1. A clearance of about 1.1mm exists between the dish cover 5 and the dish main body 1, and the clearance is generated by the contact of a plurality of stoppers 501 arranged on the lower surface of the dish cover 5 and the top end of the dish main body 1. Gas (e.g. CO) in the incubator₂Or oxygen) can freely enter the interior of the dish body 1 through the gap for gas exchange, since the culture medium for in vitro culture of embryos contains bicarbonate components when CO is present₂When entering the culture medium through the gap, the pH value of the culture medium can be quickly balanced by forming a carbonate buffer pair with bicarbonate.

In fig. 8, the dish body 1 includes a dish bottom 101 and a dish wall 102, and the dish bottom 101 and the dish wall 102 together define the volume of the culture dish 6. The bottom end of the dish wall 102 extends downwards, so that the lower surface of the dish bottom 101 is concave, and the depth of the concave part is 1.1 mm; when culture dish 6 was placed in the incubator, ware main part 1 was as the strong point with the bottom of ware wall 102, made the ware end 101 be in unsettled state, avoided the ware end 101 in placing or removing of culture dish 6 in-process by scraping flower or wearing and tearing, can reduce simultaneously and be infected with fingerprint or other stained scattering and/or the shadow that causes to influence the formation of image observation of cultivateing the object. A plurality of cultivation cabins 4 are ring shape evenly distributed in the inboard of application of sample pond 2, make its lower surface at the dish bottom 101 form a ring shape anchor ring, and this anchor ring is through polishing processing, and the roughness is homogeneous, when light passes through the anchor ring, can not change the original route of light, does benefit to the focus imaging. The lower surface of the dish bottom 101 is provided with 3 buckles 1011, and the connecting lines of the buckles form a triangular shape. When the culture dish 6 was arranged in the incubator, buckle 1011 can be with the draw-in groove looks gomphosis that corresponds in the incubator, make culture dish 6 fix in the incubator to can avoid 6 vibrations of culture dish or remove in the incubator, influence embryo or gamete growth and development.

In fig. 10, the culture chamber 4 is disposed in the middle of the dish bottom 101 and is uniformly distributed on the upper surface of the bottom surface 201 of the sample adding pool in a circular ring shape. The material of the dish body 1 is PC. In fig. 9, the sample addition cell 2 includes a bottom surface 201 and a wall 202, and the top end of the wall 202 is higher than the upper surface of the cell bottom 101; the diameter of the sample adding pool 2 is about 10mm, and the depth of the sample adding pool 2 is 2.4 mm. It will be appreciated that the overall size and geometry of the sample application reservoir 2 is not essential and may be selected according to the current implementation, for example being adapted according to the number and arrangement of the culture compartments 4 housed within the sample application reservoir 2. The plurality of culture compartments 4 are all arranged on the upper surface of the bottom surface 201 of the sample adding pool, and during the culture period, the sample adding pool 2 is filled or partially filled with culture media, so that the culture media can circulate among different culture compartments 4. In other words, the culture medium can be added into the sample adding pool 2, so that not only can the connection between different culture compartments 4 be established, but also a plurality of culture compartments 4 can be filled with the culture medium at the same time, the operation time for adding the culture medium into a single culture compartment 4 is shortened, and the pollution risk is reduced. Wherein the surface of the culture medium filled in the culture compartment 4 is at the same level as the top of the culture compartment 4, however, the surface of the culture medium may be lower or higher than the top surface of the culture compartment 4 in an actual process.

In fig. 9, a plurality of cleaning tanks 3 are each in the shape of a circular basin and are arranged in the upper surface of a dish bottom 101; the cleaning tank 3 may be a member additionally attached to the dish bottom 101, or may be integrally formed with the dish bottom 101. The maximum diameter of the washing tank 3 is 7.8mm and the depth is 1 mm. The cleaning pool 3 comprises a cleaning pool bottom surface 301 and a cleaning pool wall 302, the cleaning pool 3 shrinks from top to bottom from outside to inside, and the inclination angle of the cleaning pool wall 302 relative to the cleaning pool bottom surface 301 is 15 degrees, 30 degrees or 45 degrees, the inclination angle is helpful for an operator to identify the horizontal page of the cleaning medium (such as phosphate buffer solution or gamete buffer solution), and simultaneously, the addition amount of the required cleaning medium can be reduced. In fig. 10, a second mark 303 (english letter) is provided on the periphery of each cleaning pool 3, and each letter mark corresponds to each cleaning pool 3 one-to-one, so that the culture object can be conveniently positioned and identified in the cleaning process.

A plurality of washing ponds 3 are the outside of ring shape evenly distributed at application of sample pond 2, a plurality of cultivation cabins 4 are the inboard of ring shape evenly distributed at application of sample pond 2, make the operator can move embryo or gamete to wash in washing pond 3 from each angle in 360 degrees of annular, and cultivate in placing cultivation cabin 4 nearby, need not to cultivate because operator's operating habits is different (for example some user custom left hand operation embryo, some user custom right hand operation embryo), and adjust the operating position of washing and two steps of cultivateing.

In addition to the above-described features of the culture dish 6, there are various distinctive features on the exterior of the culture dish 6 that facilitate its use. For example, the culture dish 6 has a substantially cylindrical or basin shape, and can be stably placed in a time-lapse incubator for culture or in a general carbon dioxide incubator for culture. The versatility of the culture dish 6 contributes to widening the use scenario of the culture dish 6 (for example, some laboratories are not equipped with a matching equation of time incubator, and the culture dish 6 of the present invention can be used as well). The situation that the existing culture dish 6 can only be used in a single scene (for example, the culture dish can only be used in a time difference incubator or can only be used in a carbon dioxide incubator) is overcome. Meanwhile, the front surface of the culture dish 6 is an axial section and can be used as a patient information identification area or a patient information identification system pasting area. The flat part of the dish main body 1 can be written (pasted) with a patient information identification label or a patient one-to-one checking system chip, so that an operator can conveniently identify the identity information of an embryo or gamete in the operation process, the phenomenon that the operator mistakenly pairs the transplanted embryo and a parent in the in vitro fertilization embryo transplantation process is avoided, and the ethical problem is avoided.

As with the features of the culture dish 6 described above, other embodiments of the invention may modify the various exemplary dimensions and geometric configurations described above. For example, the overall shape and size of the culture dish 6 may be selected according to the culture box device to be used. In addition, the number of the culture compartments 4 arranged on the upper surface of the dish bottom 101 in the above example is 16, but the number and the spatial arrangement of the culture compartments 4 may be different for different requirements. It will also be appreciated that in the above embodiments the culture dish 6 is used centrally for the culture of embryos or gametes, but that according to other embodiments of the invention the culture dish 6 may be used for the culture of other objects. It will also be appreciated that culture dishes 6 according to other embodiments of the present invention may comprise some or all of the features of the culture dish 6 described above, in other words, the various features of the embodiments of the present invention are independent and beneficial. For example, according to some embodiments of the present invention, the culture dish 6 can be configured as a culture compartment 4 having a design of the above-mentioned type, but the culture dish 6 may not include the sample addition reservoir 2, i.e., the culture compartment 4 can be used independently to achieve the culture effect. In summary, it will be appreciated that embodiments of the invention may comprise any suitable combination of the features described above, and that functionally-independent features may be combined in some embodiments, partially or wholly.

Claims

1. The utility model provides a culture dish, its characterized in that, includes ware main part (1), the interior bottom surface of ware main part (1) is equipped with application of sample pond (2), application of sample pond (2) distribute outward have a plurality of recessed washing ponds (3), it has a plurality of recessed cultivation cabins (4) to distribute in application of sample pond (2).

2. A culture dish according to claim 1, wherein the culture compartment (4) is internally provided with a cavity (404) which is contracted from top to bottom; preferably, the cavity (404) comprises an incubation chamber (401) at the bottom, and a drainage channel (402) above the incubation chamber (401); more preferably, the incubation chamber (401) and the drainage groove (402) are integrally formed.

3. A culture dish according to claim 2, wherein the culture chamber (401) is in the shape of a hollow truncated cone with a large top and a small bottom, and comprises a culture chamber bottom surface (4011) and a culture chamber wall (4012); preferably, the angle of inclination of said incubation chamber wall (4012) relative to the incubation chamber floor (4011) is greater than the angle of inclination of said drainage slots (402) relative to the incubation chamber floor (4011); more preferably, the inclination angle of the drainage groove (402) relative to the bottom surface (4011) of the culture chamber is 5-15 degrees.

4. A culture dish according to claim 2, wherein a plurality of said culture compartments (4) are uniformly distributed in a circular ring shape inside said sample adding well (2), and a plurality of said cleaning wells (3) are uniformly distributed in a circular ring shape outside said sample adding well (2); preferably, the outer side of each culture compartment (4) is provided with a first mark (403) for positioning the culture compartment (4); more preferably, the outer side of each cleaning pool (3) is provided with a second mark (303) for positioning the cleaning pool (3).

5. A culture dish according to claim 2, wherein the dish body (1) comprises a dish bottom (101) and a dish wall (102); preferably, the dish bottom (101) is in a truncated circular shape; more preferably, the bottom end of the dish wall (102) extends downwards to enable the lower surface of the dish bottom (101) to be concave.

6. A culture dish according to claim 5, wherein the sample application reservoir (2) comprises a reservoir bottom surface (201) and a reservoir wall (202), and the top end of the reservoir wall (202) is higher than the upper surface of the dish bottom (101).

7. A culture dish according to claim 5, wherein the lower surface of the dish bottom (101) is provided with a plurality of buckles (1011) for clamping the dish body (1) with the culture box; preferably, the incubator comprises a time difference incubator and a carbon dioxide incubator.

8. A culture dish according to claim 5, further comprising a dish lid (5) arranged in cooperation with the dish body (1); preferably, a plurality of stoppers (501) for forming a gap between the dish cover (5) and the dish main body (1) are arranged on the lower bottom surface of the dish cover (5).

9. Use of a culture dish according to any of claims 2-8 for cultivating one or more culture objects, wherein the culture objects comprise at least one of embryos and gametes.

10. Use according to claim 9, characterized in that it comprises the following steps:

s1, adding a cleaning medium into the cleaning tank (3);

s2, adding a culture medium into the sample adding pool (2), enabling the culture medium to enter a culture cabin (4) and enter an incubation chamber (401) through the drainage groove (402);

s3, adding a covering medium into the dish main body (1) to cover the cleaning pool (3) and the sample adding pool (2);

s4, balancing the culture dish (6) obtained in the step S3 in an incubator overnight or standing for more than 12 hours;

s5, taking out the culture dish (6) obtained in the step S4, putting the culture object into the cleaning pool (3) for cleaning, and then moving the culture object into the culture cabin (4);

s6, placing the culture dish (6) obtained in the step S5 into an incubator, and culturing the culture object.