CN117660183A - Embryo microfluidic dynamic culture dish and culture method thereof - Google Patents

Abstract

The invention discloses an embryo microfluidic dynamic culture dish and a culture method thereof, which belong to the technical field of biological devices, and comprise at least one microfluidic chip, wherein the microfluidic chip comprises a liquid inlet unit, a culture unit and a liquid outlet unit which are sequentially connected; the culture unit comprises a dynamic culture pond, the dynamic culture pond is provided with at least one embryo culture chamber, the bottom of the embryo culture chamber is provided with embryo culture holes, the embryo culture holes are sequentially provided with an embryo slow moving region, a liquid deformation region and a liquid drainage region along the flowing direction of culture liquid, the liquid inlet unit is connected with the embryo slow moving region, and the liquid outlet unit is connected with the liquid drainage region. Meanwhile, the culture method based on the culture dish is disclosed, and by adopting the embryo microfluidic dynamic culture dish and the culture method thereof, the culture solution is convenient to fill, update and discharge, secretion is cleared completely, meanwhile, the damage of embryos in liquid caused by fluid shear force is reduced, and a proper culture solution environment is provided for the embryos.

Description

Embryo microfluidic dynamic culture dish and culture method thereof

Technical Field

The invention relates to the technical field of biological devices, in particular to an embryo microfluidic dynamic culture dish and a culture method thereof.

Background

Embryo culture is a big spot of modern biotechnology, which means embryo culture under human intervention in laboratory. How to culture a certain amount of embryos, especially blastula, with high quality, excellent development potential and good freezing resistance by using an early embryo in vitro culture technology has become a great subject of embryo culture. The in vitro culture system of early embryo mainly simulates the physiological environment of the parent oviduct as far as possible to construct a culture environment, and although in vitro culture has been developed for decades, the problems of poor embryo quality, low blastula rate and the like still exist. With the improvement of the culture solution formula and the embryo culture system, the static culture system is gradually developed into a dynamic culture system, and the in vitro embryo culture technology is greatly improved.

Conventional culture systems are a culture method in which embryos are cultured alone in a culture medium without adding other cells and their secretion factors, and are classified into a microdroplet method and a plate method. The former is to make 30-100 mu L microdroplet in plastic dish, cover paraffin oil on it, put embryo into it to culture, generally used for embryo culture with small quantity; the latter is the direct culture of embryos in four-well dishes containing 500-800. Mu.L of culture medium, which is typically used for culturing large numbers of embryos. Typically, the embryo may be transplanted after 3 days of culture or transferred again to new culture medium for continued culture until the fifth day of transplantation of the parent. Through continuous improvement of embryo culture solution and gradual improvement of culture conditions, the method is the main method for in-vitro embryo culture at present.

The existing biological culture device is as follows:

the patent publication No. CN102876573A discloses an in vitro culture device for mammalian embryo and a culture method thereof, wherein the in vitro culture device for mammalian embryo drives culture solution and mineral oil in a culture chamber to move through vibration of a membrane with a magnetic sheet arranged in the culture chamber, simulates the embryo to grow and develop under microscopic environmental conditions in the mammal body, stimulates the embryo, and enables the embryo to be in a dynamic, microscopic and three-dimensional growth environment.

The patent with publication number CN202786260U discloses a mammalian embryo swinging incubator, which connects a power transmission shaft of a swinging motor with adjustable angle and speed with an objective table provided with an embryo culture dish, and drives the objective table and the embryo incubator to swing together through the swinging motor, so that embryos are in a dynamic culturing state.

The prior art cannot update and control the culture solution flowing through the embryo cells in time, so that secretion cannot be removed, and a simpler and more efficient culture environment cannot be provided for the embryo cells.

The patent with publication number CN104371919B discloses a micro-fluidic chip, a dynamic culture device and a method for cell culture, which utilize the liquid level difference between a manually controlled nutrition pool and a liquid storage pool to regulate the flowing speed of a driving solution in the micro-fluidic chip, and fix cells through a cell capturing unit, thereby realizing the functions that secretion of embryo cells is easy to diffuse and the cells are fully contacted with nutrient substances and growth factors. However, the liquid exchange operation process is complicated and the operability is poor by manually continuously adjusting the liquid level, and the device does not consider the process of recovering the embryo, so that the embryo is blocked in the cell catcher and cannot be recovered.

Patent publication US6193647B1 discloses a microfluidic embryo handling device and method for simulating biological rotation of embryos by designing constriction structures in the micro-channels to locate the egg cells in the channels. However, the egg cells may be damaged by the large fluid shear force, and in addition, this method is not suitable for locating a plurality of egg cells at the same time.

The publication WO 2005/023224 A2 discloses a device for treating cells, embryos or egg cells, a series of micro-pits arranged in sequence under a narrow micro-tunnel, which can be used to sequentially feed egg cells into the micro-pits for positioning and then to achieve subsequent in vitro fertilization. The device has a narrow micro-pipeline width, is difficult to realize a quick liquid change process, and does not consider the problem of crushing and removing redundant cells around cells.

The patent with publication number CN101947124B discloses an integrated microfluidic chip device and a use method thereof, wherein more than one micro-pit for positioning egg cells is arranged, the egg cells are conveyed to the position above one micro-pit of a micro-pit array layer along a micro-pipeline of a micro-pipeline layer by the aid of culture solution, so that the egg cells fall into the micro-pits to realize positioning due to gravity, and the fertilization process, the rapid liquid change process, the embryo culture process and the embryo recovery process are integrally completed. The whole in vitro fertilization process of a single ovum is realized, but due to different sizes of the ovum, the zygote, the blastula and the like, the micro pits with fixed caliber cannot realize the adaptation of the whole process from the ovum to the blastula, and meanwhile, different liquids (buffer solution, mineral oil, liquefied semen and the like) are exchanged, and due to different inertia force and viscosity force, the movement characteristics of the fluid are easily changed in a micro channel, so that the disturbance of microenvironment caused by mixing and doping between fluid layers affects the normal development of the embryo.

The patent with publication number CN211005419U discloses an automatic embryo liquid changing culture dish, each culture unit comprises a liquid injection pool, a culture pool and a waste liquid pool, embryos are placed in micropores in the culture pool, and liquid changing is realized under the action of external air pressure and gravity. The waste liquid flow channel is closer to the embryo, so that the transparent belt on the surface of the blastomere or trophoblast cells in the blastula stage are easily damaged under the negative pressure action of the waste liquid cavity, and bubbles in the culture micropores need to be manually removed in the embryo adding process, so that the complexity and the manual interference are increased.

To sum up, the culture dish in the prior art has the following technical problems:

(1) The culture solution flowing through embryo cells cannot be updated and controlled in time, secretion cannot be discharged, the zona pellucida on the surface of the blastomere or trophoblast cells in the blastocyst stage are damaged under the negative pressure effect of the waste liquid cavity, and the cells are possibly damaged due to the action of larger fluid shearing force.

(2) The liquid exchange operation process is complex and has poor operability, and when different liquids are exchanged, the movement characteristics of the fluids are changed in the micro-channels, so that the disturbance of microenvironment caused by mixing between fluid layers is caused to influence the normal development of embryos.

(3) The process of recovering embryos is not considered, and there is a risk that the embryos are blocked and cannot be recovered, so that a plurality of cells cannot be positioned at the same time.

(4) In the embryo adding process, bubbles in the culture micropores need to be manually removed, and the complexity and the manual interference are increased.

Disclosure of Invention

The invention aims to provide an embryo microfluidic dynamic culture dish and a culture method thereof, which solve the technical problems.

In order to achieve the above purpose, the invention provides an embryo microfluidic dynamic culture dish, which comprises at least one microfluidic chip, wherein the microfluidic chip comprises a liquid inlet unit, a culture unit and a liquid outlet unit which are connected in sequence; the culture unit comprises a dynamic culture pond, the dynamic culture pond is provided with at least one embryo culture chamber, the bottom of the embryo culture chamber is provided with embryo culture holes, the embryo culture holes are sequentially provided with an embryo slow moving region, a liquid deformation region and a liquid drainage region along the flowing direction of culture liquid, the liquid inlet unit is connected with the embryo slow moving region, and the liquid outlet unit is connected with the liquid drainage region.

Preferably, the liquid inlet unit comprises a liquid inlet, the liquid inlet is connected with at least one liquid inlet micro-flow channel, each liquid inlet micro-flow channel is connected with a corresponding embryo culture chamber, and the liquid inlet micro-flow channels adopt a straight line structure or a serpentine structure so that the strokes of all liquid inlet micro-flow channels from the liquid inlet to the corresponding embryo culture chambers are the same.

Preferably, the liquid outlet unit comprises at least one liquid outlet, the liquid outlet is connected with the embryo culture chamber through at least one liquid outlet micro-flow channel, and the liquid outlet micro-flow channel adopts a straight line structure or a serpentine structure so that the strokes of all liquid outlet micro-flow channels from the liquid outlet to the corresponding embryo culture chamber are the same.

Preferably, the liquid inlet micro-flow channel and the liquid outlet micro-flow channel are arranged on the same plane and horizontally, the width of the liquid inlet micro-flow channel and the liquid outlet micro-flow channel is 100-200 mu m, and the height of the liquid inlet micro-flow channel and the liquid outlet micro-flow channel is 50-100 mu m.

Preferably, the liquid inlet connecting end is provided with a degassing device, the degassing device comprises a degassing pipeline, one end of the degassing pipeline is provided with a degassing membrane and is connected with the liquid inlet connecting end, the degassing pipeline is provided with a degassing vacuum pump for providing negative pressure, and the liquid inlet connecting end is connected with a liquid control device for controlling liquid inlet parameters.

Preferably, each liquid outlet microfluidic channel is provided with a metabolite detection window for sampling, observation and detection;

or the liquid outlet is connected with detection device, and detection device includes metabolite detection chip, and metabolite detection chip's detection inlet is connected through connecting tube with the liquid outlet, is provided with at least one detection microfluidic channel on the metabolite detection chip, and every detection microfluidic channel all is connected with the leakage fluid dram, is provided with metabolite detection window on the detection microfluidic channel.

Preferably, at least one embryo culture chamber is arranged in a space matrix, a culture solution storage layer and an oil cover layer are sequentially arranged above the embryo culture holes, the depth of the embryo culture chamber is 4-8mm, the thicknesses of the culture solution storage layer and the oil cover layer are 2-4mm, the oil cover layer is subjected to hydrophobic treatment relative to the inner wall of the embryo culture chamber, the liquid inlet unit, the embryo culture holes, the inner wall of the culture solution storage layer relative to the embryo culture chamber and the liquid outlet unit are subjected to hydrophilic treatment, a patient information identification area is arranged on the microfluidic chip, and each embryo culture chamber is provided with a digital mark.

Preferably, the width of the bottom of the embryo slow moving zone is 0.25-0.35mm, the height is 0.35-0.4mm, the liquid inlet side of the embryo slow moving zone and the liquid inlet micro-flow channel form an acute angle of 70-85 degrees, and the liquid outlet side of the embryo slow moving zone and the liquid deformation zone form an acute angle of 30-60 degrees;

the liquid deformation area is lower than the liquid inlet micro-flow channel and is 0.1-0.3mm higher than the embryo slow movement area;

the liquid drainage area is of a slope structure, and the gradient of the liquid drainage area is 10-90 degrees.

Preferably, the culture dish cover arranged on the microfluidic chip is further included.

The culture method based on the embryo microfluidic dynamic culture dish comprises the following specific steps:

step S1: preheating an embryo microfluidic dynamic culture dish, and filling a culture solution and a cover oil layer;

step S2: placing embryos one by one in an embryo slow moving region in an embryo culture hole of an embryo culture chamber;

step S3: the liquid inlet connecting end is connected with the liquid control device and the degassing device, is placed in the incubator, and starts the imaging system to collect embryo images;

step S4: filling the culture solution in the cleavage period under the set parameters;

step S5: after culturing for a set liquid exchange time, the liquid outlet is connected with negative pressure equipment, the embryo culture liquid containing the metabolites is discharged to a waste pool bin through a liquid outlet micro-flow channel and the liquid outlet, and waste liquid is automatically returned, when the liquid is discharged, the oil layer is always higher than the liquid level of the culture liquid, and the oil layer stays on the upper layer without mixing;

step S5: when the embryo grows to the D3 stage, filling the blastocyst stage culture solution at a preset flow rate, so that the blastocyst stage culture solution and the blastocyst stage culture solution are gradually diffused;

step S6: and when the embryo develops to the D5 stage, checking the development condition of each embryo, and taking out the qualified embryo.

Therefore, the embryo microfluidic dynamic culture dish and the culture method thereof have the following beneficial effects:

(1) Through inlet, inlet microfluidic channel, play liquid microfluidic channel and liquid outlet cooperation liquid controlling means in time update and control the culture solution that flows through embryo cell, liquid exchange operation process is simple, only needs the manual work to add the culture solution lid oil and add the embryo after, whole system automatic completion, maneuverability is better. The oil cover layer is always higher than the liquid level of the culture solution during the exchange of the culture solution, only the culture solution is taken away when the culture solution flows, the oil cover layer stays on the upper layer all the time, the mixing is not generated, the microenvironment is stable, and the influence of the disturbance of the culture solution on the embryo development is reduced.

(2) Can thoroughly clear away the embryo secretion through the design of embryo culture hole, and reduce the injury that fluid shear force effect brought, embryo recovery process is simple, and is poor with conventional operation, does not change doctor's operating habit, and embryo culture hole size design has guaranteed that the embryo can not doubly block in the micropore, and does not have the risk of embryo damage. The liquid outlet micro-channel is provided with a liquid deformation area from the embryo slow moving area, and the transparent belt on the surface of the blastomere or trophoblast cells in the blastocyst stage can not be damaged under the negative pressure action of the liquid outlet.

(3) The liquid inlet micro-flow channel, the liquid outlet micro-flow channel and the embryo culture holes are provided with a plurality of digital marks, so that a plurality of cells can be positioned simultaneously.

(4) The degassing device is arranged, so that the air column can be automatically filtered, bubbles in the culture micropores are not required to be manually removed, and the manual interference is reduced.

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 is a schematic diagram of an embryo microfluidic dynamic culture dish according to example 1;

FIG. 2 is a schematic diagram of the structure of an embryo culture well according to the present invention;

FIG. 3 is a cross-sectional view of an embryo culture well according to the present invention;

FIG. 4 is a schematic diagram showing the flow rate of the embryo culture Kong Naliu when the culture solution of the dynamic culture dish flows out;

FIG. 5 is a schematic diagram of the fluid flow rate of a prior art straight wall cell culture well;

FIG. 6 is a schematic diagram of an embryo microfluidic dynamic culture dish according to example 3;

FIG. 7 is a schematic diagram of an embryo microfluidic dynamic culture dish according to example 4;

FIG. 8 is a schematic diagram of an embryo microfluidic dynamic culture dish according to example 6.

Reference numerals

1. A liquid inlet unit; 11. a liquid inlet; 12. a liquid inlet microfluidic channel; 2. a culturing unit; 21. a dynamic culture pond; 22. an embryo culture chamber; 23. embryo culture wells; 231. embryo slow motion region; 232. a liquid deformation zone; 233. a liquid drainage zone; 24. a culture solution storage layer; 25. covering an oil layer; 3. a liquid outlet unit; 31. a liquid outlet; 32. a liquid outlet microfluidic channel; 4. a patient information identification area; 5. a degasser; 51. a degassing conduit; 52. degassing membrane; 53. a degassing vacuum pump; 6. a culture dish cover; 7. a metabolite detection window; 8. a metabolite detection chip; 81. detecting a liquid inlet; 82. a connecting pipe; 83. detecting a microfluidic channel; 84. and a liquid outlet.

Detailed Description

Examples

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.

In the description of the present invention, it should be noted that, directions or positional relationships indicated by terms such as "upper", "lower", "inner", "outer", etc., are directions or positional relationships based on those shown in the drawings, or those that are conventionally put in use, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the apparatus or elements to be referred to must have a specific direction, be constructed and operated in a specific direction, and thus should not be construed as limiting the present invention.

In the description of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.

Some embodiments of the present invention are described in detail below with reference to the accompanying drawings.

Example 1

As shown in fig. 1, an embryo microfluidic dynamic culture dish comprises a microfluidic chip, wherein the microfluidic chip comprises a liquid inlet unit 1, a culture unit 2 and a liquid outlet unit 3 which are sequentially connected. The embryo microfluidic dynamic culture dish of the embodiment can be made of materials such as acrylic (PMMA), dimethyl siloxane (PDMS) or optical glass, has good insulating property and thermal stability, can bear high voltage, has biocompatibility and gas permeability, is suitable for culturing embryo cells, and has excellent optical characteristics and can be applied to various optical detection systems.

The culture unit 2 comprises a dynamic culture pond 21, wherein the dynamic culture pond 21 is provided with at least one embryo culture chamber 22, and generally comprises 12-18 embryo culture chambers 22 to meet the requirement of culture quantity, the embodiment is provided with 16 embryo culture chambers 22, the 16 embryo culture chambers 22 are arranged in a space matrix, and the 16 embryo culture chambers 22 are arranged in two longitudinal columns to reduce the volume of a culture dish as much as possible.

Embryo culture room 22 bottom is provided with embryo culture hole 23, and embryo culture hole 23 has set gradually embryo slow motion district 231, liquid deformation district 232 and liquid drainage district 233 along the culture solution flow direction, and feed liquor unit 1 is connected with embryo slow motion district 231, and liquid outlet unit 3 is connected with liquid drainage district 233 as shown in fig. 2. The width of the bottom of the embryo slow moving area 231 is 0.25-0.35mm, the height is 0.35-0.4mm, the liquid inlet side of the embryo slow moving area 231 forms an acute angle of 70-85 degrees with the liquid inlet micro-flow channel 12, and the liquid outlet side of the embryo slow moving area 231 forms an acute angle of 30-60 degrees with the liquid deformation area 232; embryo slow motion zone 231 can keep embryos in the zone following the fluid flow rate of the sudden drop of liquid in the process of feeding the culture solution to generate micro motion due to the angle design and the bottom size, so that embryos are not rolled or even separated from embryo culture holes 23 under the condition of being impacted by the fluid, and the embryos are lost.

The liquid deformation area 232 is lower than the liquid inlet micro-flow channel 12 and higher than the embryo slow motion area 231, and the height difference is 0.1-0.3mm; the liquid deformation area 232 is provided with a liquid buffer area and a liquid drainage area 233 which are slightly lower than the liquid inlet micro-flow channel 12 and slightly higher than the bottom surface of the embryo culture hole 23, the liquid deformation area 232 increases the liquid path pressure due to the height difference of abrupt structural rise, the liquid generates rheology, the liquid flow velocity is increased, and secretion generated in the embryo culture process is turned over.

The liquid drainage area 233 is of a slope structure, the gradient of the liquid drainage area 233 is 10-90 degrees, the liquid drainage area 233 is a slope extending upwards from the liquid deformation area 232 to the liquid micro-flow channel 32, and the continuously-lifted height difference further enables liquid to be boosted, so that embryo secretion is thoroughly taken away. The embryo culture well 23 is sized to ensure that embryos are not locked and that embryos are not subject to possible damage from fluid shear forces in the liquid, and to clean the embryo secretions. And the zona pellucida on the surface of the blastomere or trophoblast cells in blastocyst stage are not damaged under the negative pressure of the liquid outlet 31.

As shown in FIG. 3, a culture solution storage layer 24 and an oil cover layer 25 are sequentially arranged above the embryo culture holes 23, the depth of the embryo culture chamber 22 is 4-8mm, so that only a single embryo is supported and accommodated, the thicknesses of the culture solution storage layer 24 and the oil cover layer 25 are 2-4mm, the oil cover layer 25 is subjected to hydrophobic treatment relative to the inner wall of the embryo culture chamber 22, and the liquid inlet unit 1, the embryo culture holes 23 and the culture solution storage layer 24 are subjected to hydrophilic treatment relative to the inner wall of the embryo culture chamber 22 and the liquid outlet unit 3, so that bubbles are not generated in liquid flowing into the embryo culture micropores in the culture process. The inner and outer surfaces should be kept clean and free of insoluble particulates and bacteria. Before addition of embryos, it is necessary to manually add pre-equilibrated broth to the broth storage layer 24 and then rapidly cover the oil layer 25 to prevent evaporation of the broth from affecting its osmotic pressure. The culture solution storage layer 24 is always higher than the liquid level of the culture solution, only the culture solution is taken away in the process of changing the culture solution, and mineral oil stays on the upper layer and is not mixed and doped to cause disturbance of microenvironment so as to influence normal development of embryos. The microfluidic chip is provided with a patient information identification area 4, and the patient information identification area 4 is attached with a two-dimensional code, a bar code or an electronic tag so as to avoid embryo loss or embryo misplacement. Each embryo culture chamber 22 is provided with a digital identifier, which marks the embryo.

The liquid inlet unit 1 of this embodiment includes a liquid inlet 11, the liquid inlet 11 is connected with a plurality of liquid inlet micro-flow channels 12, each liquid inlet micro-flow channel 12 is connected with a corresponding embryo culture chamber 22, in order to ensure that the strokes from the liquid inlet 11 to the corresponding embryo culture chamber 22 are the same, the liquid inlet micro-flow channel 12 with a shorter linear distance from the liquid inlet 11 to the corresponding embryo culture chamber 22 adopts a linear structure, and the liquid inlet micro-flow channel 12 with a longer linear distance from the liquid inlet 11 to the corresponding embryo culture chamber 22 adopts a serpentine structure, so as to ensure that the culture liquid reaches the culture pond with the same liquid flow rate. The liquid outlet unit 3 of this embodiment includes a liquid outlet 31 to realize simpler liquid path interface control, the liquid outlet 31 is connected with a plurality of embryo culture chambers 22 through a plurality of liquid outlet micro-flow channels 32, and the adoption of the straight line structure or the serpentine structure of the liquid outlet micro-flow channels 32 makes the strokes of all liquid outlet micro-flow channels 32 from the liquid outlet 31 to the corresponding embryo culture chambers 22 the same. To ensure that the cell waste liquid reaches the liquid outlet 31 at the same liquid flow rate, the principle is the same as that of the liquid inlet unit 1. The liquid inlet micro-flow channel 12 and the liquid outlet micro-flow channel 32 are arranged on the same plane and horizontally, the width of the liquid inlet micro-flow channel 12 and the liquid outlet micro-flow channel 32 is 100-200 mu m, and the height is 50-100 mu m. The liquid inlet 11 and the liquid outlet 31 can be locked with a liquid control system to realize filling, updating and discharging of the nutrient solution. The liquid exchange operation process is simple, and the manual intervention is less.

As shown in fig. 4-5, the flow field in the embryo culture well 23 at the time of draining can be found to have a certain flow rate of the liquid near the cells in the embryo culture well 23, which is superior to the conventional straight wall cell culture well, which indicates that the cell culture well of the present embodiment is more suitable for the draining of cell metabolites.

Example 2

The difference between this embodiment and the embodiment is that: the liquid outlet 31 is provided with a plurality of, and every liquid outlet microfluidic channel 32 is connected with a corresponding liquid outlet 31, and secretion of every embryo can be collected to every liquid outlet 31, is convenient for the detection to single embryo secretion.

Example 3

This embodiment differs from embodiment 1 in that: the embodiment is further provided with a degassing device 5, as shown in fig. 6, for separating the gas in the liquid inlet micro-channel and the gas in the liquid inlet pipeline, so as to avoid generating bubbles in the use process and influencing embryo observation and culture effects. The degasser 5 is arranged at the connecting end of the liquid inlet 11, the degasser 5 comprises a degasser pipeline 51, one end of the degasser pipeline 51 is provided with a degasser film 52 and is connected with the connecting end of the liquid inlet 11, the degasser film 52 can permeate gas but not permeate liquid, namely, the waterproof breathable film is a novel high polymer waterproof material, and the waterproof breathable film adopts a PE high polymer breathable film. The degassing pipeline 51 is provided with a degassing vacuum pump 53 for providing negative pressure, and the degassing device 5 generates a vacuum environment outside the degassing pipeline 51 through the degassing vacuum pump 53 when in use, so that the mixed gas in the liquid is pumped out through vacuum, and the liquid continues to flow along the pipeline, thereby realizing gas-liquid separation.

Example 4

As shown in fig. 7, this embodiment differs from embodiment 1 in that: a culture dish cover 6 is arranged on the microfluidic chip to reduce the probability of pollution and reduce the loss of liquid temperature.

Example 5

This embodiment differs from embodiment 1 in that: each of the outlet microfluidic channels 32 is provided with a metabolite detection window 7 for sampling, observation and detection. Different apparent morphologies of a large amount of secretions on the surface of the microcarrier, such as filiform winding of collagen fibers, a large amount of cell gap structures of the shed cells and the like, can be observed through a light mirror imaging system. The metabolite detection window 7 can be directly connected with a sample inlet of detection equipment, and the morphological structure of secretion cells can be detected through sampling, the protein content can be measured, and the cell count and the proportion of various types can be calculated. Meanwhile, the metabolite detection window 7 can be used as a sampling port, and an operator can manually sample embryo secretion and can be used for detection of cell markers, cell ploidy analysis, single cell sequencing and the like so as to obtain more information about embryo development.

Example 6

As shown in fig. 8, this embodiment differs from embodiment 2 in that: the liquid outlet 31 is connected with detection device and has culture dish lid 6, and detection device includes metabolite detection chip 8, and the detection inlet 81 of metabolite detection chip 8 is connected through connecting tube 82 with liquid outlet 31, is provided with a plurality of detection microfluidic channel 83 on the metabolite detection chip 8, and every detection microfluidic channel 83 all is connected with fluid-discharge outlet 84, is provided with metabolite detection window 7 on the detection microfluidic channel 83.

Example 7

The culture method based on the embryo microfluidic dynamic culture dish comprises the following specific steps:

step S1: the embryo microfluidic dynamic culture dish was preheated and filled with culture fluid and oil cap layer 25.

Firstly, taking out an embryo microfluidic dynamic culture dish on a clean workbench, manually filling pre-balanced culture solution into an embryo culture hole 23 and a culture solution storage layer 24 by using a Pasteur tube under a body view mirror, and quickly filling by using a mineral oil material cover oil layer 25 after filling, wherein mineral oil cannot overflow an embryo culture chamber 22 in the filling process.

Step S2: embryos are placed one by one in the embryo transfer zone 231 in the embryo culture well 23 of the embryo culture chamber 22. After the injection, the patient information is checked, embryos are manually placed one by one under a stereoscopic vision in the embryo transfer area 231 of the embryo culture well 23, and the number is checked and recorded.

Step S3: the liquid inlet 11 is connected with the liquid control device and the degasser 5, and is placed in the incubator, and the imaging system is started to collect embryo images, which is used for recording the whole culturing process, so that the embryo state can be analyzed later. The liquid control device can control the proper temperature, humidity, gas concentration and the like of the feed liquid.

Step S4: filling the culture solution in the cleavage stage under the set parameters.

Step S5: after culturing for a set liquid exchange time, the liquid outlet 31 is connected to negative pressure equipment, the embryo culture liquid containing the metabolites is discharged to a waste pool bin through the liquid outlet micro-flow channel 32 and the liquid outlet 31, and the liquid waste is automatically returned, and the oil layer 25 is always higher than the liquid level of the culture liquid during liquid discharge, so that the oil layer 25 stays on the upper layer and is not mixed.

Step S5: when the embryo grows to the D3 stage, the blastocyst stage culture solution is filled at a preset flow rate, so that the blastogenesis stage culture solution and the blastocyst stage culture solution are gradually diffused, the process of slowly changing the concentration of the liquid ensures that the osmotic pressure of the embryo does not change suddenly, and the embryo is always in a culture environment with little change, so that the interference is less.

Step S6: and when the embryo develops to the D5 stage, checking the development condition of each embryo, and taking out qualified high-quality embryos.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.

Claims (10)

1. An embryo microfluidic dynamic culture dish, which is characterized in that: the device comprises at least one micro-fluidic chip, wherein the micro-fluidic chip comprises a liquid inlet unit, a culture unit and a liquid outlet unit which are sequentially connected; the culture unit comprises a dynamic culture pond, the dynamic culture pond is provided with at least one embryo culture chamber, the bottom of the embryo culture chamber is provided with embryo culture holes, the embryo culture holes are sequentially provided with an embryo slow moving region, a liquid deformation region and a liquid drainage region along the flowing direction of culture liquid, the liquid inlet unit is connected with the embryo slow moving region, and the liquid outlet unit is connected with the liquid drainage region.

2. An embryo microfluidic dynamic culture dish as claimed in claim 1, wherein: the liquid inlet unit comprises a liquid inlet, the liquid inlet is connected with at least one liquid inlet micro-flow channel, each liquid inlet micro-flow channel is connected with the corresponding embryo culture chamber, and the liquid inlet micro-flow channels adopt a straight line structure or a snake-shaped structure to enable the strokes of all liquid inlet micro-flow channels from the liquid inlet to the corresponding embryo culture chambers to be the same.

3. An embryo microfluidic dynamic culture dish as claimed in claim 2, wherein: the liquid outlet unit comprises at least one liquid outlet, the liquid outlet is connected with the embryo culture chamber through at least one liquid outlet micro-flow channel, and the liquid outlet micro-flow channel adopts a straight line structure or a serpentine structure to enable the strokes of all liquid outlet micro-flow channels from the liquid outlet to the corresponding embryo culture chamber to be the same.

4. A dynamic culture dish for micro-fluidic embryos according to claim 3, wherein: the liquid inlet micro-flow channel and the liquid outlet micro-flow channel are arranged on the same plane and horizontally, the width of the liquid inlet micro-flow channel and the liquid outlet micro-flow channel is 100-200 mu m, and the height of the liquid inlet micro-flow channel and the liquid outlet micro-flow channel is 50-100 mu m.

5. An embryo microfluidic dynamic culture dish as claimed in claim 2, wherein: the liquid inlet connecting end is provided with a degassing device, the degassing device comprises a degassing pipeline, one end of the degassing pipeline is provided with a degassing membrane and is connected with the liquid inlet connecting end, the degassing pipeline is provided with a degassing vacuum pump for providing negative pressure, and the liquid inlet connecting end is connected with a liquid control device for controlling liquid inlet parameters.

6. A dynamic culture dish for micro-fluidic embryos according to claim 3, wherein: each liquid outlet microfluidic channel is provided with a metabolite detection window for sampling, observation and detection;

or the liquid outlet is connected with detection device, and detection device includes metabolite detection chip, and metabolite detection chip's detection inlet is connected through connecting tube with the liquid outlet, is provided with at least one detection microfluidic channel on the metabolite detection chip, and every detection microfluidic channel all is connected with the leakage fluid dram, is provided with metabolite detection window on the detection microfluidic channel.

7. An embryo microfluidic dynamic culture dish as claimed in claim 1, wherein: at least one embryo culture chamber is space matrix arrangement, embryo culture hole top has set gradually culture solution reservoir and cover oil reservoir, embryo culture chamber degree of depth is 4-8mm, the thickness of culture solution reservoir and cover oil reservoir is 2-4mm, cover oil reservoir carries out hydrophobic treatment relative to embryo culture chamber's inner wall, feed liquor unit, embryo culture hole, culture solution reservoir carries out hydrophilic treatment relative to embryo culture chamber's inner wall and play liquid unit, be provided with patient information identification area on the microfluidic chip, every embryo culture chamber all is provided with the digital identification.

8. An embryo microfluidic dynamic culture dish as claimed in claim 1, wherein: the width of the bottom of the embryo slow moving zone is 0.25-0.35mm, the height is 0.35-0.4mm, the liquid inlet side of the embryo slow moving zone and the liquid inlet micro-flow channel form an acute angle of 70-85 degrees, and the liquid outlet side of the embryo slow moving zone and the liquid deformation zone form an acute angle of 30-60 degrees;

the liquid deformation area is lower than the liquid inlet micro-flow channel and is 0.1-0.3mm higher than the embryo slow movement area;

the liquid drainage area is of a slope structure, and the gradient of the liquid drainage area is 10-90 degrees.

9. An embryo microfluidic dynamic culture dish as claimed in claim 1, wherein: the culture dish cover is arranged on the microfluidic chip.

10. A culture method based on an embryo microfluidic dynamic culture dish as claimed in any one of claims 1-9, characterized by the specific steps of:

step S1: preheating an embryo microfluidic dynamic culture dish, and filling a culture solution and a cover oil layer;

step S2: placing embryos one by one in an embryo slow moving region in an embryo culture hole of an embryo culture chamber;

step S3: the liquid inlet connecting end is connected with the liquid control device and the degassing device, is placed in the incubator, and starts the imaging system to collect embryo images;

step S4: filling the culture solution in the cleavage period under the set parameters;

step S5: after culturing for a set liquid exchange time, the liquid outlet is connected with negative pressure equipment, the embryo culture liquid containing the metabolites is discharged to a waste pool bin through a liquid outlet micro-flow channel and the liquid outlet, waste liquid is automatically recovered, the oil layer is always higher than the liquid level of the culture liquid during liquid discharge, and the oil layer stays on the upper layer without mixing;

step S5: when the embryo grows to the D3 stage, filling the blastocyst stage culture solution at a preset flow rate, so that the blastocyst stage culture solution and the blastocyst stage culture solution are gradually diffused;

step S6: and when the embryo develops to the D5 stage, checking the development condition of each embryo, and taking out the qualified embryo.