CN113789264A - Embryo culture detection device and embryo culture detection method - Google Patents

Embryo culture detection device and embryo culture detection method Download PDF

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Publication number
CN113789264A
CN113789264A CN202110907751.1A CN202110907751A CN113789264A CN 113789264 A CN113789264 A CN 113789264A CN 202110907751 A CN202110907751 A CN 202110907751A CN 113789264 A CN113789264 A CN 113789264A
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culture
embryo
biochemical reaction
chamber
indicator
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朱永刚
史佳明
佟文强
暴钰成
陈华英
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Shenzhen Graduate School Harbin Institute of Technology
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Shenzhen Graduate School Harbin Institute of Technology
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Abstract

The invention provides an embryo culture detection device and an embryo culture detection method. The embryo culture detection device comprises a microfluidic chip, a liquid supply module, a gas-liquid mixing module, a first detection module and a second detection module, wherein the microfluidic chip is provided with a microfluidic channel, a culture chamber and a biochemical reaction chamber, the culture chamber is positioned at the upstream of the biochemical reaction chamber, the liquid supply module supplies reagent for the microfluidic channel, the gas-liquid mixing module supplies culture medium for the microfluidic channel, the first detection module is used for collecting images of embryos, and the second detection module is used for observing reaction results. The embryo culture detection method of the invention is used for collecting the embryo image in the culture chamber and observing the reaction result in the biochemical reaction chamber after culturing the embryo. The embryo culture detection device and the embryo culture detection method provided by the invention can realize the functions of long-term embryo culture, embryo morphology analysis, noninvasive detection of embryo metabolites and the like.

Description

Embryo culture detection device and embryo culture detection method
Technical Field
The invention relates to the technical field of bioengineering, in particular to an embryo culture detection device and an embryo culture detection method.
Background
In recent years, microfluidic chips are widely used for embryo culture, and the chips have micron or nanometer level circuits or liquid channels, which can be used as precise liquid conveying channels, sites for chemical and biological reactions, sites for material synthesis, and the like. However, the existing microfluidic chip has single function, can only realize single culture or observation function, and is not beneficial to research and experiment of embryos.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the embryo culture detection device is based on a microfluidic chip, integrates the functions of long-term controllable culture of embryos, shape analysis of the embryos and detection of physiological indexes, and is beneficial to improving the efficiency of research and experiment on the embryos.
The invention also provides an embryo culture detection method.
An embryo culture detection device according to an embodiment of the first aspect of the invention comprises: the microfluidic chip is provided with a microfluidic channel, a culture chamber and a biochemical reaction chamber, wherein the culture chamber and the biochemical reaction chamber are both communicated with the microfluidic channel, the culture chamber is positioned at the upstream of the biochemical reaction chamber and is used for accommodating embryos, and the microfluidic channel is used for allowing liquid to flow and preventing the embryos from entering; the liquid supply module is connected with the microfluidic chip and can inject an indicator into the microfluidic channel, and the indicator can flow to the biochemical reaction chamber along the microfluidic channel; the gas-liquid mixing module is connected with the microfluidic chip and can inject a culture medium into the microfluidic channel, and the culture medium can sequentially flow through the culture chamber and the biochemical reaction chamber along the microfluidic channel; the first detection module is used for observing the growth condition of the embryo in the culture chamber and collecting an image of the embryo; and the second detection module is used for observing a fluorescent signal generated after the indicator in the biochemical reaction chamber reacts with the metabolite, converting the fluorescent signal into an electric signal and transmitting the electric signal to a computer.
The embryo culture detection device provided by the embodiment of the invention has at least the following beneficial effects: the gas-liquid mixing module can realize the controllable proportion mixing of the culture medium, carbon dioxide and oxygen, the culture medium is injected into the microfluidic chip (the gas-liquid mixing module is used for regulating and controlling the gas environment of the culture medium) through the driving pump, and the culture medium entering the culture chamber provides a proper environment for the culture of embryos; the first observation module can observe the growth condition of the embryo in the culture chamber and take a delayed photograph; after the culture medium is consumed by the embryo, the culture medium is mixed with the indicator in the downstream micro-pipeline and reacts, then the mixture flows to the biochemical reaction chamber for optical detection, the culture medium flowing to the biochemical reaction chamber contains metabolites after the embryo is consumed, the second detection module can observe the reaction result of the indicator and the metabolites to know the content of the metabolites in the culture medium (or know whether the metabolites are contained), and then the second detection module is compared with the data obtained by the channel without the embryo, so that the embryo metabolic data is obtained to further deduce the growth condition of the embryo. Therefore, the embryo culture detection device provided by the invention integrates the functions of long-term controllable embryo culture, embryo morphology analysis and noninvasive embryo metabolite detection, and a user does not need to transfer embryos among different devices or equipment for embryo research or experiment, which is beneficial to improving the embryo research and experiment efficiency.
According to some embodiments of the invention, the embryo culture apparatus further comprises a moving platform, the first detection module and the second detection module are both mounted on the moving platform, the chip comprises a plurality of channels, each channel comprises the culture chamber and the biochemical reaction chamber, and the moving platform can move along the arrangement direction of the culture chambers and/or the arrangement direction of the biochemical reaction chambers.
According to some embodiments of the present invention, the microfluidic chip further has an indicator inlet, the microfluidic channel has a main flow section and an indicator inlet section, two ends of the main flow section are respectively communicated with the culture chamber and the biochemical reaction chamber, a start end of the indicator inlet section is communicated with the indicator inlet, and a tail end of the indicator inlet section is communicated with the main flow section.
According to some embodiments of the invention, the microfluidic chip further has a diluent inlet, the microfluidic channel further has a diluent inlet section, the diluent inlet is communicated with a starting end of the diluent inlet section, and a tail end of the diluent inlet section is communicated with the main flow section; the junction of the indicator entry section and the primary flow section is downstream of the junction of the dilution entry section and the primary flow section.
According to some embodiments of the invention, the main flow section comprises a backflow prevention part, a communication position of the dilution inlet section and the main flow section is a first communication position, the backflow prevention part is arranged between the first communication position and the culture chamber, and the backflow prevention part is in a serpentine shape.
According to some embodiments of the invention, the main flow section comprises a mixing portion, the indicator inlet section is in communication with the main flow section at a second communication, the mixing portion is arranged between the second communication and the biochemical reaction chamber, and the mixing portion has a serpentine shape.
According to some embodiments of the invention, the microfluidic chip comprises a substrate and a sealing plug, the microfluidic channel, the culture chamber and the biochemical reaction chamber are all disposed on the substrate, the substrate and the sealing plug are detachably connected, and at least a part of the sealing plug can be inserted into the culture chamber to close the culture chamber.
According to some embodiments of the invention, the gas-liquid mixing module comprises: a peripheral container; the liquid storage container is arranged in the peripheral container, the inner cavity of the liquid storage container is used for storing the culture medium, and the inner cavity of the peripheral container is communicated with the inner cavity of the liquid storage container; a gas supply connected to the peripheral container, the gas supply being configured to controllably introduce gas into the peripheral container; one end of the liquid supply pipeline is connected with the liquid storage container, and the other end of the liquid supply pipeline is connected with the microfluidic chip; the culture medium driving pump is connected with the liquid storage container, or the culture medium driving pump is installed on the liquid supply pipeline and can drive the culture medium to flow to the microfluidic chip along the liquid supply pipeline.
The embryo culture detection method according to the second aspect of the embodiment of the invention comprises the following steps: arranging a culture chamber, a biochemical reaction chamber and a microfluidic channel on the microfluidic chip, wherein the culture chamber and the biochemical reaction chamber are both communicated with the microfluidic channel; delivering culture medium to the culture chamber; placing the embryo into the culture chamber for culture; observing the embryo and collecting an image of the embryo in the culture chamber; inputting an indicator into the microfluidic channel, wherein the indicator and the culture medium enter the biochemical reaction chamber after being mixed; observing the result of the reaction between the indicator and the metabolite in the biochemical reaction chamber.
The embryo culture detection method provided by the embodiment of the invention has at least the following beneficial effects: the method can realize the functions of long-term controllable culture of the embryo, shape analysis of the embryo, noninvasive detection of the embryo metabolite and the like, and is beneficial to improving the efficiency of research and experiment on the embryo.
According to some embodiments of the invention, the culture chamber and the biochemical reaction chamber are each provided in plurality; the step of collecting the images of the embryos is specifically to arrange a first detection module, move the first detection module along the arrangement direction of the culture chambers and sequentially collect the images of the embryos; and arranging a second detection module, moving the second detection module along the arrangement direction of the biochemical reaction chambers after the indicator and the culture medium are mixed and enter the biochemical reaction chambers, sequentially observing the reacted fluorescent signals in the biochemical reaction chambers, converting the optical signals into electric signals and transmitting the electric signals to a computer.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a schematic overall view of an embryo culture apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic view of a gas-liquid mixing module according to some embodiments;
FIG. 3 is a schematic view of a gas-liquid mixing module according to another embodiment;
FIG. 4 is a schematic diagram of a microfluidic chip according to some embodiments;
FIG. 5 is a schematic view of a detection channel;
FIG. 6 is a schematic view of an embryo in a culture chamber;
FIG. 7 is a schematic illustration of an embryo being placed or removed;
FIG. 8 is a schematic diagram of the flow of the embryo culture apparatus.
Reference numerals: 101-microfluidic chip, 102-gas-liquid mixing module, 103-liquid supply module, 104-waste liquid collector, 105-mobile platform, 106-first objective lens, 107-camera, 108-first full-reflection mirror, 109-first detection module, 110-second objective lens, 111-optical filter mirror group, 112-laser, 113-second full-reflection mirror, 114-photomultiplier, 115-second detection module, 116-microfluidic channel, 117-embryo, 118-heating plate, 201-peripheral container, 202-liquid storage container, 203-liquid supply pipeline, 204-temperature control unit, 205-culture medium drive pump, 206-gas supply control valve, 207-gas inlet, 208-gas outlet, 401-culture medium inlet, 402-culture chamber, 403-diluent inlet, 404-indicator inlet, 405-backflow prevention part, 406-waste liquid outlet, 407-biochemical reaction chamber, 408-mixing part, 501-substrate, 502-sealing plug, 601-suction head.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In the description of the present invention, it should be understood that the orientation or positional relationship referred to in the description of the orientation, such as the upper, lower, front, rear, left, right, etc., is based on the orientation or positional relationship shown in the drawings, and is only for convenience of description and simplification of description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.
In the description of the present invention, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present numbers, and the above, below, within, etc. are understood as including the present numbers. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present invention, unless otherwise explicitly limited, terms such as arrangement, installation, connection and the like should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific contents of the technical solutions.
In the description of the present invention, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Referring to fig. 1 to 4, the present invention provides an embryo culture detection apparatus, which includes a microfluidic chip 101, a gas-liquid mixing module 102, a liquid supply module 103, a first detection module 109, and a second detection module 115. The microfluidic chip 101 may be made of transparent plastic, glass, or other materials, so as to observe the morphology of the embryo in the microfluidic chip 101 and observe the reaction result after the indicator reacts with the analyte. Referring to fig. 1 and 4, the microfluidic chip 101 has a microfluidic channel 116, a culture chamber 402 and a biochemical reaction chamber 407, the culture chamber 402 and the biochemical reaction chamber 407 are both communicated with the microfluidic channel 116, the culture chamber 402 is located upstream of the biochemical reaction chamber 407, and the culture chamber 402 serves as a space for culturing the embryo 117. The microfluidic channel 116 also has a medium inlet 401 and an indicator inlet 404, both of the medium inlet 401 and the indicator inlet 404 being in communication with the microfluidic channel 116, the medium inlet 401 being located upstream of the culture chamber 402 and the indicator inlet 404 being located upstream of the biochemical reaction chamber 407. The microfluidic channel 116 has a diameter of between one micron and one thousand microns. The microfluidic channel 116 allows fluid to flow, and embryos cannot enter the microfluidic channel 116 due to the cross section of the microfluidic channel 116 and the shape and size differences of the embryos. For example, the size difference may be a diameter difference, and referring to FIGS. 6 and 7, the diameter of the embryo 117 is larger than the diameter of the microfluidic channel 116, so that the embryo 117 does not flow along the microfluidic channel 116 to the biochemical reaction chamber 407.
The liquid supply module 103 is connected to the microfluidic chip 101, and the liquid supply module 103 injects the indicator into the microfluidic channel 116 through the indicator inlet 404, and the indicator flows along the microfluidic channel 116 to the biochemical reaction chamber 407. In addition, the liquid supply module 103 may further inject a diluent (described in detail later) into the micro flow channel 116. The gas-liquid mixing module 102 is connected to the microfluidic chip 101, the gas-liquid mixing module 102 injects a culture medium into the microfluidic channel 116 through the culture medium inlet 401, and the culture medium sequentially flows through the culture chamber 402 and the biochemical reaction chamber 407 along the microfluidic channel 116. The first detection module 109 is configured to observe the growth of the embryo in the culture chamber 402 and collect an image of the embryo 117. The second detection module 115 is used for observing a fluorescence signal after the indicator in the biochemical reaction chamber 407 reacts with the analyte, and after the indicator reacts with the analyte, the generated reactant generates fluorescence under the action of laser, or the generated reactant can change the color of the solution. The type of the indicator is determined by the physiological and biochemical indexes to be measured, and the physiological indexes can be glucose content, dissolved oxygen content, lactic acid content, pH value, active oxygen content and the like in the culture medium.
It should be noted that the terms "upstream" and "downstream" in the present invention are based on the direction of flow of the liquid in the microfluidic channel 116; for example, the culture medium flows from the culture chamber 402 to the biochemical reaction chamber 407 along the microfluidic channel 116, and the culture chamber 402 is located upstream of the biochemical reaction chamber 407, or the biochemical reaction chamber 407 is located downstream of the culture chamber 402.
The gas-liquid mixing module 102 conveys a culture medium to the microfluidic chip 101, and the culture medium entering the culture chamber 402 provides a nutrient environment for culturing the embryo 117; the first detection module 109 can observe the growth of the embryo 117 in the culture chamber 402; the media after the incubation chamber 402 has contacted the embryo 117 (the embryo 117 will consume a portion of the media) will flow to the biochemical reaction chamber 407 and bind to the indicator; the medium flowing to the biochemical reaction chamber 407 contains metabolites of the embryo 117 (some metabolites may be used as the analyte), and the second detection module 115 can observe the result of the reaction between the indicator and the analyte to obtain the content of the analyte in the medium (or obtain whether the analyte is contained), so as to further deduce the metabolic condition and growth condition of the embryo. Therefore, the embryo culture detection device provided by the invention integrates the functions of long-term controllable embryo culture, embryo morphology analysis and noninvasive embryo metabolite detection, and a user does not need to transfer embryos among different devices or equipment for embryo research or experiment, which is beneficial to improving the embryo research and experiment efficiency.
The specific structure of the microfluidic chip 101 will be described first. Referring to fig. 4, the microfluidic channel 116 includes a medium inlet section, a main flow section, a waste liquid discharge section, a dilution inlet section, and an indicator inlet section, and the microfluidic chip 101 has a medium inlet 401, a diluent inlet 403, a culture chamber 402, a biochemical reaction chamber 407, an indicator inlet 404, and a waste liquid discharge port 406. Referring to fig. 4, the medium inlet section corresponds to the flow section from point n to point p, i.e., the medium inlet section corresponds to the n-p section; the main flow section corresponds to the section a-b-f-g-q; the waste liquid discharge section corresponds to the d-e section; diluting and entering a section corresponding to the h-f section; the indicator entering section corresponds to the m-g section or the r-c section.
Referring to fig. 4, both ends of the medium inlet section are respectively communicated with a medium inlet 401 and a culture chamber 402, and the medium flows from the medium inlet 401 to the culture chamber 402 along the medium inlet section. The two ends of the main flow section are respectively communicated with the culture chamber 402 and the biochemical reaction chamber 407, and the culture medium flows from the culture chamber 402 to the biochemical reaction chamber 407 along the main flow section. The two ends of the waste liquid discharge section are respectively communicated with the biochemical reaction chamber 407 and the waste liquid discharge port 406, and the waste liquid in the biochemical reaction chamber 407 flows to the waste liquid discharge port 406 along the waste liquid discharge section; referring to FIG. 1, the embryo culture apparatus further comprises a waste collector 104, the waste collector 104 is connected to a waste outlet 406 via a pipe, and waste flows into the waste collector 104.
For the detection of some biochemical indicators, the analyte in the culture medium needs to be diluted to a certain concentration before being combined with the indicator, for example, when glucose in the culture medium is detected, the culture medium needs to be diluted first, so that the microfluidic channel 116 has a dilution inlet section. The two ends of the dilution inlet section are respectively communicated with the diluent inlet 403 and the main flow section, and the diluent can flow into the dilution inlet section from the diluent inlet 403 and finally join the solution in the main flow section. The communication of the indicator inlet section with the main flow section (which may correspond to point c or point g in fig. 4, where point g is taken as an example) is downstream of the communication of the diluent inlet section with the main flow section (which may correspond to point f in fig. 4). In the process that the culture medium flows in the main flow section, the culture medium is mixed with the diluent and then mixed with the indicator, and the mixed solution finally enters the biochemical reaction chamber 407. In some embodiments, the main flow section includes a backflow prevention portion 405, a communication position between the dilution inlet section and the main flow section is a first communication position (which may correspond to a point f in fig. 4), a backflow prevention portion 405 is disposed between the first communication position and the culture chamber 402, the backflow prevention portion 405 is in a serpentine shape, and a zigzag shape of the backflow prevention portion 405 increases resistance of liquid flowing in the backflow prevention portion 405, so as to prevent diluent or indicator from flowing backwards into the culture chamber 402 to contaminate the embryo 117.
In some embodiments, the main flow section further comprises a mixing portion 408, the shape of the mixing portion 408 is similar to the shape of the backflow prevention portion 405, the mixing portion 408 is also serpentine, and the zigzag shape of the mixing portion 408 increases the resistance of the liquid flowing in the mixing portion 408, so that the time for the liquid to flow in the mixing portion 408 is increased, which is beneficial to the sufficient mixing between the liquids. The communication between the indicator inlet section and the main flow section is a second communication (which may correspond to point c or point g in fig. 4), and a mixing part 408 is disposed between the second communication and the biochemical reaction chamber 407, and the mixing part 408 can allow the culture medium (or the diluted culture medium) to be sufficiently mixed with the indicator. A mixing part 408 is provided between the connection between the dilution inlet section and the main flow section (which may correspond to point f in FIG. 4) and the connection between the indicator inlet section and the main flow section (which may correspond to point g in FIG. 4), and the mixing part 408 can sufficiently mix the culture medium and the diluent.
Referring to fig. 6 and 7, in some embodiments, the microfluidic chip 101 includes a substrate 501 and a sealing plug 502, and the substrate 501 and the sealing plug 502 are detachably connected. The sealing plug 502 may be made of plastic. The microfluidic channel 116, the medium inlet 401, the diluent inlet 403, the culture chamber 402, the biochemical reaction chamber 407, the indicator inlet 404, and the waste liquid discharge port 406 are all opened on the substrate 501, and at least a part of the sealing plug 502 can be inserted into the culture chamber 402 to seal the culture chamber 402. In the present application, sealing growth chamber 402 specifically prevents growth chamber 402 from being in direct communication with the external environment. When it is desired to remove an embryo 117 from the culture chamber 402, the user can remove the sealing plug 502 and then insert the tip 601 into the culture chamber 402 and aspirate the embryo 117 in the culture chamber 402 using the tip 601; when it is desired to add an embryo 117 to the culture chamber 402, the user can remove the sealing plug 502 and then insert the tip 601 into the culture chamber 402 and inject the embryo that would have been captured by the tip 601 into the culture chamber 402.
Other modules of the embryo culture apparatus will be described.
The liquid supply module 103 may specifically include a plurality of syringe pumps, different liquid (such as indicator, diluent, etc.) are stored in different syringe pumps, and output ends of the syringe pumps are connected to the diluent inlet 403 and the indicator inlet 404 through pipes.
Referring to fig. 2 and 3, in some embodiments, the gas-liquid mixing module 102 includes a peripheral container 201, a reservoir container 202, a gas supply, a supply line 203, and a medium-driven pump 205. The inner part of the peripheral container 201 and the inner part of the liquid storage container 202 are both provided with cavities, the liquid storage container 202 is arranged inside the peripheral container 201, the inner cavity of the liquid storage container 202 is used for storing culture media, and the inner cavity of the peripheral container 201 is communicated with the inner cavity of the liquid storage container 202. The gas supplier (not shown) may be a gas tank or a gas bottle storing gas, and the gas supplier is connected to the peripheral container 201 through a pipeline, and the gas supplier is used for inputting gas into the peripheral container 201, and since the inner cavity of the peripheral container 201 is communicated with the inner cavity of the liquid storage container 202, part of the gas enters the liquid storage container 202 to contact with the culture medium and dissolve into the culture medium.
The gas may be oxygen, nitrogen, carbon dioxide, etc., and may provide essential components for the growth of the embryo or components for regulating the culture medium. A gas supply control valve 206 is installed on the pipeline, the flow rate of the gas and the relative concentration between different gases can be adjusted by adjusting the opening degree of the plurality of gas supply control valves 206, so as to adjust the parameters of the culture medium, for example, the amount of carbon dioxide dissolved into the culture medium can be adjusted by changing the flow rate of the carbon dioxide, so as to adjust the pH value of the culture medium; the dissolved oxygen concentration of the culture medium can be adjusted by changing the flow rate of oxygen.
One end of the liquid supply pipeline 203 is connected with the liquid storage container 202, and the other end of the liquid supply pipeline 203 is connected with the microfluidic chip 101. The culture medium mixed with the gas flows to the microfluidic chip 101 along the liquid supply line 203 and enters the microfluidic channel 116 from the culture medium inlet 401. The flow of the medium is driven by the medium-driving pump 205, and the medium-driving pump 205 may be connected to the reservoir 202 or mounted on the liquid supply line 203 depending on the kind of the medium-driving pump 205.
Referring to fig. 2, if the medium-driving pump 205 is a pressure pump, the medium-driving pump 205 is connected to the liquid storage container 202; the liquid storage container 202 has an air inlet 207 and an air outlet 208, the culture medium driving pump 205 is connected with the air inlet 207 through a hose, when the culture medium is conveyed to the microfluidic chip 101, the air outlet 208 needs to be closed, the pressure pump conveys air into the liquid storage container 202 (the air conveyed by the pressure pump can be air mixed by oxygen and carbon dioxide according to a specific ratio) to increase the air pressure in the liquid storage container 202, and the culture medium is discharged out of the liquid storage container 202 after the air pressure is increased.
Referring to fig. 3, if the medium-driving pump 205 is a peristaltic pump, the medium-driving pump 205 is installed on the liquid supply line 203, and both the gas inlet 207 and the gas outlet 208 are kept open when the peristaltic pump is operated. The specific structure and operation principle of the pressure pump and the peristaltic pump belong to the well-known technology, and are not described in detail here. The micro fluidic chip 101 may be fed with a Flow-stop (Flow-stop) medium injection method, for example, flowing for 2min after stopping for 2h, so that the embryo can sufficiently consume the nutrients in the medium.
Referring to fig. 2, in some embodiments, the gas-liquid mixing module 102 further includes a temperature control unit 204, and the temperature control unit 204 may be a cooling/heating unit, and an internal unit of the cooling/heating unit is installed inside the peripheral container 201. The temperature control module is used for adjusting the ambient temperature in the peripheral container 201, the temperature control unit can be used for refrigerating or heating by changing the operation mode of the temperature control unit 204, and the refrigerating power or heating power of the refrigerating module can be changed by changing the operation parameters of the temperature control module, so that the temperature of the culture medium is in a proper temperature range.
Referring to fig. 1, in some embodiments, the embryo culture apparatus further includes a heating plate 118, the heating plate 118 is attached to the bottom of the substrate 501 of the microfluidic chip 101, a heating area of the heating plate 118 is a flow section (which may correspond to a medium inlet section) before the medium enters the culture chamber 402, and the heating plate 118 is also used for adjusting the temperature of the medium entering the culture chamber 402.
Referring to fig. 1, in some embodiments, the first detection module 109 includes a first objective lens 106, a first full-reflection mirror 108 and a camera 107, the first objective lens 106 faces the culture chamber 402, light from the culture chamber 402 passes through the first objective lens 106 and is reflected by the first full-reflection mirror 108, the light is received by the camera 107, and the camera 107 thus obtains an image of the embryo in the culture chamber 402. The first objective lens 106 is used for magnifying the image to make up for the deficiency of the capability of the common camera 107 to capture small-sized objects.
In some embodiments, the second detection module 115 includes a second objective lens 110, a laser 112, a filter mirror group 111, a second full-reflective mirror 113, and a photomultiplier 114. The second objective 110 faces the biochemical reaction chamber 407, the laser emitted by the laser 112 is reflected to the biochemical reaction chamber 407 by the optical filter mirror group 111, if the solution in the biochemical reaction chamber 407 contains the substance to be detected, the reactant generated after the reaction of the indicator and the substance to be detected generates fluorescence under the action of the laser, the fluorescence passes through the second objective 110 and the optical filter mirror group 111 and is reflected to the photomultiplier 114 by the second full-reflection mirror 113, and the photomultiplier 114 converts the optical signal into an electrical signal and transmits the signal to the computer, thereby realizing the observation of the embryo physiological index detection result.
In order to increase the number of culturable embryos per microfluidic chip 101, the culture chamber 402 is provided in plurality, and correspondingly, the biochemical reaction chamber 407 is provided in plurality. In order to observe the morphology and the physiological index detection results of a plurality of embryos, the embryo culture device is provided with a moving platform 105, the first detection module 109 and the second detection module 115 are both installed on the moving platform 105, and the moving platform 105 can move along the arrangement direction of the plurality of culture chambers 402 and the arrangement direction of the plurality of culture chambers 402. The first detection module 109 moves with the moving platform 105 to observe embryos in different culture chambers 402, and the second detection module 115 moves with the moving platform 105 to observe reactants in different biochemical reaction chambers 407. The movable platform 105 may be driven by a motor, a lead screw slider mechanism, or the like. It should be noted that, the first detection module 109 and the second detection module 115 can be integrally packaged and then installed on the movable platform 105, and fig. 1 is only a simple drawing for showing that the first detection module 109 and the second detection module 115 are installed on the movable platform 105, and does not mean that only the camera 107 and the photomultiplier 114 are installed on the movable platform 105.
The invention also provides an embryo culture detection method, which can realize the functions of long-term controllable culture, embryo morphology analysis, noninvasive detection of embryo metabolites and the like on embryos and is beneficial to improving the efficiency of embryo research and experiments. The method comprises the following steps:
delivering culture medium to culture chamber 402;
placing embryo 117 into culture chamber 402;
observing the embryo 117 and collecting an image of the embryo 117;
the indicator is input into the microfluidic channel 116, and the indicator and the culture medium are mixed and then enter the biochemical reaction chamber 407;
the result of the reaction between the indicator and the analyte in the biochemical reaction chamber 407 is observed.
Referring to fig. 4, in some embodiments, the culture chamber 402 and the biochemical reaction chamber 407 are each provided in plurality, and in this case, the first detection module 109 moves in the arrangement direction of the plurality of culture chambers 402 and sequentially collects images of the plurality of embryos 117, and similarly, after the indicator is mixed with the culture medium and enters the biochemical reaction chamber 407, the second detection module 115 moves in the arrangement direction of the plurality of biochemical reaction chambers 407 and sequentially observes the reaction results in the plurality of biochemical reaction chambers 407. Referring to fig. 1, 4 and 7, the method will now be described in more detail in general order of operation:
(1) one incubation chamber 402 corresponds to one main channel, the number of main channels to be observed at this time is set in a controller (the controller is not specifically shown), the number of main channels to be observed is recorded as i, and an array A [ i ] is set]＝{A1，A2，A3…… Ai}，AiIs the distance between each main channel and a first reference point.
(2) One biochemical reaction chamber 407 corresponds to one detection channel,setting the number of the detection channels needing to be observed in the controller, recording the number of the detection channels needing to be observed as j, and setting an array B [ j ]]＝{B1，B2，B3……Bj}， BjIs the distance between each detection channel and the second reference point. The second reference point may refer to one of the detection channels, for example, the plurality of biochemical reaction chambers 407 are arranged in the front-back direction, the plurality of detection channels are also arranged in the front-back direction, and the front one of the detection channels may be selected as the second reference point. In some cases, the first reference point and the second reference point may also be set to one and the same point or position. Meanwhile, the biochemical indexes corresponding to each detection channel need to be set, and the moving sequence of the plurality of detection channels is determined according to the biochemical indexes corresponding to each detection channel.
(3) Setting the number of biochemical indexes to be detected in a controller, and recording the number as n; setting an array C [ n ]]＝{C1，C2， C3，……，CnAnd the biochemical indexes can be glucose, dissolved oxygen, lactic acid, pH value, active oxygen and the like.
(4) Setting two time constants T1And T2，T1Indicates the time T that the first detection module 109 needs to stay after moving to a position of a main channel2Indicating the time required for the second detection module 115 to stay after moving to a position of a certain detection channel.
(5) The heating plate 118 located in the microfluidic chip 101 is turned on to preheat, and then the gas-liquid mixing module 102 is started to adjust parameters such as the pH value and the dissolved oxygen concentration of the culture medium to a proper range, and then the culture medium is delivered to the culture chamber 402 of the microfluidic chip 101. At this time, the pumping device of the diluent can be activated to inject the diluent into the microfluidic chip 101, and the diluent will be mixed with the culture medium flowing out of the culture chamber 402 and enter the biochemical reaction chamber.
(6) The sealing plug 502 is pulled out, and the embryo is placed in the microfluidic chip 101 for culturing.
(7) The mobile platform 105 is reset, and the first detection module 109 starts from the initial position (the first reference point) along multiple linesThe culture chambers 402 are moved in the arrangement direction by the distance A1，A2，A3……AiAnd when the first detecting module 109 moves to the position of the cultivation room 402, it stays for a period of time, the length of the stay time is T1(ii) a During the dwell period, the first detection module 109 captures the growth of the embryo. The first detection module 109 finishes shooting the last embryo and then finishes shooting one round, and then the first detection module 109 can reset to the initial position and move again after resetting, and then shooting the next round of embryos is carried out.
(8) After the embryo shooting is completed (specifically, after a certain round of shooting is completed), the liquid supply module 103 is started, and the liquid supply module 103 injects liquid (indicator) into the microfluidic chip 101.
(9) The movable platform 105 is reset, and the second detection module 115 moves from the initial position (the second reference point) along the arrangement direction of the plurality of biochemical reaction chambers 407 to the corresponding positions of the plurality of biochemical reaction chambers 407 by the movement distance B1， B2，B3，……Bj(not necessarily moving in sequence from 1 to j) and staying at each position for a period of time T2. During the period, the controller is used to collect the signal of the photomultiplier light-increasing output and store the signal into a file dedicated to the current object to be detected (for example, in the first round of detection, the signal record is stored in the corresponding index C1In the file(s). The respective laser 112 and liquid supply module are turned off after the detection is finished.
It is necessary to explain the detecting sequence of the second detecting module 115, and the second detecting module 115 does not move B in sequence1， B2，B3，……BjIn particular, the distance (c) is shifted according to the biochemical marker to be detected. Taking fig. 5 as an example, four detection channels are sequentially arranged from front to back, and for convenience of description, a distance between a certain detection channel and a reference point is directly used as a reference for the detection channel; that is, the detection channels are respectively denoted as B1，B2，B3，B4. While FIG. 5 has two indicator inlets 404, the two indicator inlets 404 being used to inject different indicatorsAgent, two indicator inlets 404 are distinguished in fig. 5 by different fill lines. For convenience of explanation, it is assumed here that the indicator inlet 404 on the left side is an inlet for a glucose indicator, and the indicator inlet 404 on the right side is an inlet for a dissolved oxygen indicator.
In order to facilitate subsequent data screening and sorting, the second detection module 115 detects one biochemical indicator after detecting another biochemical indicator. For example, the detection channel related to dissolved oxygen is detected first, and the second detection module moves to B1 and then to B4; then, the second detection module 115 is reset to the initial position; then, the second detection module moves to B2 and then to B3 for detecting the glucose-related detection channel.
(10) And judging whether the detected physiological and biochemical index number k is greater than or equal to n. If k is less than n, indicating that the physiological index substance to be detected exists, assigning value to k (k is k +1), and returning the program to the process (7) to execute again; if k is larger than or equal to n, the physiological index substance to be detected does not exist, and the detection process is ended.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims (10)

1. Embryo culture detection device, its characterized in that includes:
the microfluidic chip is provided with a microfluidic channel, a culture chamber and a biochemical reaction chamber, wherein the culture chamber and the biochemical reaction chamber are both communicated with the microfluidic channel, the culture chamber is positioned at the upstream of the biochemical reaction chamber and is used for accommodating embryos, and the microfluidic channel is used for allowing liquid to flow and preventing the embryos from entering;
the liquid supply module is connected with the microfluidic chip and can inject an indicator into the microfluidic channel, and the indicator can flow to the biochemical reaction chamber along the microfluidic channel;
the gas-liquid mixing module is connected with the microfluidic chip and can inject a culture medium into the microfluidic channel, and the culture medium can sequentially flow through the culture chamber and the biochemical reaction chamber along the microfluidic channel;
the first detection module is used for observing the growth condition of the embryo in the culture chamber and collecting an image of the embryo;
and the second detection module is used for observing a fluorescent signal generated after the indicator in the biochemical reaction chamber reacts with the metabolite, and converting the fluorescent signal into an electric signal to be output.
2. The embryo culture detection device according to claim 1, further comprising a moving platform, wherein the first detection module and the second detection module are both mounted on the moving platform, the culture chambers and the biochemical reaction chambers are both provided in plurality, and the moving platform can move along the arrangement direction of the culture chambers and/or the arrangement direction of the biochemical reaction chambers.
3. The embryo culture detection device according to claim 1, wherein the microfluidic chip further comprises an indicator inlet, the microfluidic channel comprises a main flow section and an indicator inlet section, two ends of the main flow section are respectively communicated with the culture chamber and the biochemical reaction chamber, a starting end of the indicator inlet section is communicated with the indicator inlet, and a tail end of the indicator inlet section is communicated with the main flow section.
4. The embryo culture detection device according to claim 3, wherein the microfluidic chip further has a diluent inlet, the microfluidic channel further has a diluent inlet section, the diluent inlet is communicated with the beginning end of the diluent inlet section, and the end of the diluent inlet section is communicated with the main flow section; the junction of the indicator entry section and the primary flow section is downstream of the junction of the dilution entry section and the primary flow section.
5. The embryo culture detection device of claim 4, wherein the main flow section comprises a backflow prevention portion, the communication between the dilution inlet section and the main flow section is a first communication, the backflow prevention portion is arranged between the first communication and the culture chamber, and the backflow prevention portion is serpentine.
6. The embryo culture detection device according to claim 1, wherein the second detection module comprises a laser, a second objective lens, a second full-reflection mirror, a filter lens group and a photomultiplier tube; the second objective faces the biochemical reaction chamber, the optical filter mirror group can reflect laser generated by the laser to the biochemical reaction chamber, the laser generated by the laser is used for exciting reactants in the biochemical reaction chamber to generate the fluorescent signal, the fluorescent signal is transmitted to the photomultiplier tube sequentially through the second objective, the optical filter mirror group and the second full-reflection mirror, and the photomultiplier tube is used for converting the fluorescent signal into an electric signal.
7. The embryo culture detection device according to claim 1, wherein the microfluidic chip comprises a substrate and a sealing plug, the microfluidic channel, the culture chamber and the biochemical reaction chamber are all disposed on the substrate, the substrate and the sealing plug are detachably connected, and at least a part of the sealing plug can be inserted into the culture chamber to close the culture chamber.
8. The embryo culture detection device of claim 1, wherein the gas-liquid mixing module comprises:
a peripheral container;
the liquid storage container is arranged in the peripheral container, the inner cavity of the liquid storage container is used for storing the culture medium, and the inner cavity of the peripheral container is communicated with the inner cavity of the liquid storage container;
a refrigeration device connected to the interior of the peripheral container, the refrigeration device for regulating the temperature of the interior of the peripheral container;
a gas supply connected to the peripheral container for supplying CO2、O2And N2Inputting into the peripheral container;
one end of the liquid supply pipeline is connected with the liquid storage container, and the other end of the liquid supply pipeline is connected with the microfluidic chip;
the culture medium driving pump is connected with the liquid storage container, or the culture medium driving pump is installed on the liquid supply pipeline and can drive the culture medium to flow to the microfluidic chip along the liquid supply pipeline.
9. The embryo culture detection method is characterized by comprising the following steps:
arranging a culture chamber, a biochemical reaction chamber and a microfluidic channel on the microfluidic chip, wherein the culture chamber and the biochemical reaction chamber are both communicated with the microfluidic channel;
delivering culture medium to the culture chamber;
placing the embryo into the culture chamber for culture;
observing the embryo and collecting an image of the embryo in the culture chamber;
inputting an indicator into the microfluidic channel, wherein the indicator and the culture medium enter the biochemical reaction chamber after being mixed;
observing the result of the reaction between the indicator and the metabolite in the biochemical reaction chamber.
10. The embryo culture detection method according to claim 9, wherein a plurality of culture chambers and biochemical reaction chambers are provided;
the step of collecting the images of the embryos is specifically to arrange a first detection module, move the first detection module along the arrangement direction of the culture chambers and sequentially collect the images of the embryos;
and arranging a second detection module, moving the second detection module along the arrangement direction of the plurality of biochemical reaction chambers and observing and recording the reaction results in the plurality of biochemical reaction chambers in sequence after the indicator is mixed with the culture medium and enters the biochemical reaction chambers.
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