Vitrification freezing treatment device with micro-fluidic chip

Abstract

The utility model provides a vitrification freezing treatment device with a micro-fluidic chip. The micro-fluidic chip comprises a liquid path layer, the liquid path channel is arranged on the liquid path layer, a reagent in the micro-fluidic chip flows through the liquid path channel, and the liquid path channel is provided with a main channel and an auxiliary channel which are crossed; the micro-fluidic chip further comprises an interception structure, the interception structure is used for intercepting reagents and / or cells in the liquid path channels, and the interception structure is arranged at the intersection of the two liquid path channels. The cell vitrification device has the beneficial effects that the cell vitrification device is used for carrying out vitrification on cells around the micro-fluidic chip, and different modules are cooperatively operated, so that the automatic and intelligent vitrification treatment of the cells is realized, and the requirements of users are met.

Description

Technical Field

[0001] The utility model relates to the technical field of cell processing, in particular to a vitrification freezing processing device with a microfluidic chip. Background Art

[0002] Although our company's 2018 patent (application number 201810598384X) has obvious advantages over other vitrification freezing chips in terms of cell capture, precise control of liquid flow rate, and automation of processing procedures, its degree of automation and intelligence still cannot meet user needs and needs to be further improved. Utility Model Content

[0003] The utility model discloses a vitrification freezing treatment device with a microfluidic chip, wherein the microfluidic chip comprises a liquid path layer; a liquid path channel, wherein the liquid path channel is arranged in the liquid path layer, and a reagent in the microfluidic chip flows through the liquid path channel, wherein the liquid path channel has a main channel and an auxiliary channel with intersections; and the microfluidic chip further comprises an interception structure, wherein the interception structure is used for intercepting reagents and / or cells in the liquid path channel, and wherein the interception structure is arranged at the intersection of two liquid path channels.

[0004] As a further improvement of the present invention, the vitrification freezing treatment device further includes a microscope module, and the microscope module is used to observe the cell state.

[0005] As a further improvement of the present invention, the vitrification freezing treatment device also includes a transmission module, which is used to realize the transmission between the microfluidic chip and external substances and energy; the vitrification freezing treatment device also includes a pressure regulating device, which is connected to the transmission module and is used to deliver air pressure to the microfluidic chip.

[0006] As a further improvement of the present invention, the interception structure is a dam body.

[0007] As a further improvement of the present invention, the intersection is in the shape of a square.

[0008] As a further improvement of the present invention, the width of the intersection is wider than the width of the main channel.

[0009] As a further improvement of the present invention, the intersections are symmetrically arranged.

[0010] As a further improvement of the present invention, there is a smooth transition between the intersection and the main channel.

[0011] As a further improvement of the utility model, the dam body is provided with a groove.

[0012] As a further improvement of the present invention, the dam body is roughly in the shape of the letter N.

[0013] As a further improvement of the present utility model, the dam body is arranged at the bottom end of the intersection. The dam body can also be arranged at the upper end or the side wall, etc. However, arranging it at the bottom end is easier to process and facilitates subsequent cooperation with other components, achieving a more efficient and better effect.

[0014] As a further improvement of the present utility model, the height of the upper end of the dam body does not exceed the height of the main channel.

[0015] As a further improvement of the present utility model, the height of the dam body accounts for more than 50% of the height of the main channel. This is convenient for the manufacture of the dam body and the microfluidic chip, and at the same time, the interception effect is more obvious.

[0016] As a further improvement of the present utility model, the height of the dam body accounts for 85 - 95% of the height of the main channel. Since the interception effect of the dam body is negatively correlated with the space size, the higher the dam body, the narrower the space between it and the upper wall of the channel, and the better the interception effect. However, an overly high dam body will greatly reduce the passing efficiency of the liquid. Only when it is 85 - 95% can various performances be balanced, which can not only facilitate the improvement of the passing efficiency, but also facilitate manufacturing, and has an obvious interception effect.

[0017] As a further improvement of the present utility model, the dam body and the liquid path channel of the microfluidic chip are formed into an integral structure by an integral molding method.

[0018] As a further improvement of the present utility model, the dam body and the liquid path channel of the microfluidic chip are arranged at the intersection by bonding, preferably at the bottom end.

[0019] As a further improvement of the present utility model, the dam body and the microfluidic chip are made of the same material. The microfluidic chip in the present utility model can be realized by using various common materials for microfluidic chips.

[0020] As a further improvement of the present utility model, the dam body is made of PDMS material. A PDMS micro - structure is formed on the surface of the microfluidic chip by a molding method. Its mold - turning accuracy is high, reaching the nanometer (nm) level, which can greatly improve the control accuracy of the microfluidic chip in the present invention.

[0021] As a further improvement of the present utility model, one side of the dam body has two supporting feet, and a groove is formed between the two supporting feet. The groove corresponds to the position of the auxiliary channel in the microfluidic chip, so that the reagent 2 in the auxiliary channel can reach the groove.

[0022] As a further improvement of the present utility model, the width between the two supporting feet of the dam body exceeds half of the width of the entire dam body. The width between the supporting feet, that is, the width of the groove, should not be too narrow, as it will generate unnecessary resistance. Therefore, the width of the groove maintains the width of the auxiliary channel. The width of the left supporting foot corresponds to the path length of the reagent passing through the top of the dam body, and the right supporting foot corresponds to the path length of the solution passing through the top of the dam body after confluence. Considering the resistance, the width of the supporting feet should not be too large. Therefore, the width between the two supporting feet of the dam body of the present invention exceeds half of the width of the entire dam body.

[0023] As a further improvement of the present invention, the two supporting feet are respectively the first supporting foot and the second supporting foot. The height of the first supporting foot is lower than that of the second supporting foot. The first supporting foot is located on the side where reagent 1 flows in, and the second supporting foot is located on the side where reagent 1 and reagent 2 flow out after confluence. The setting that the second supporting foot is higher than the first supporting foot will cause the reagent 1 to cross the first supporting foot and confluence with reagent 2, and then due to the higher height of the second supporting foot, it will form a further blockage to the fluid, enabling reagent 1 and reagent 2 to be more fully mixed and further discharging the entrained gas that may be generated due to confluence. In actual use, it is preferably that the height of the second supporting foot is more than 1 / 10 of the height of the first supporting foot, and the effect is significant.

[0024] As a further improvement of the present utility model, an elastic film is also provided on the microfluidic chip, and the elastic film is arranged on the end face of the microfluidic chip on the side where the main channel and the auxiliary channel open.

[0025] As a further improvement of the present utility model, there is a gap between the elastic film and the intercepting structure.

[0026] As a further improvement of the present utility model, the elastic film covers the intersection.

[0027] As a further improvement of the present utility model, the elastic film covers all the main channels and auxiliary channels.

[0028] As a further improvement of the present utility model, the elastic film covers the entire range of the microfluidic chip.

[0029] As a further improvement of the present utility model, the thickness of the elastic film is 50 to 250 microns. Preferably 75 - 150 microns, because at 75 - 150 microns, it is not only convenient for manufacturing but also can have better elasticity and higher response sensitivity in control.

[0030] As a further improvement of the present utility model, the elastic film is made of PDMS material.

[0031] As a further improvement of the present utility model, the microfluidic chip further includes a gas path layer. The gas path layer has a gas path channel connected to an external air pressure source, and the gas path layer and the elastic film cooperate to form a pneumatic microvalve.

[0032] As a further improvement of the present utility model, a concave portion is provided in the area corresponding to the intersection of the gas path layer, and an external air pressure source can be communicated through this concave portion.

[0033] As a further improvement of the present utility model, the area of the concave portion of the gas path layer is not less than the area of the intersection.

[0034] As a further improvement of the present utility model, the shape of the concave portion of the gas path layer is substantially the same as the shape of the intersection, so that the concave portion area can better cover the range of the intersection, and further better realize the application of positive and negative pressures of the air pressure source.

[0035] As a further improvement of the present utility model, the shape of the concave portion of the gas path layer is rectangular or circular.

[0036] As a further improvement of the present utility model, the position of the pneumatic micro-valve corresponds to the position of the intercepting structure.

[0037] As a further improvement of the present utility model, when a negative pressure is applied to the gas path channel in the gas path layer, the elastic film under the gas path channel bends upward and contracts in the direction of the gas path channel space in the gas path layer, so that the passage between the intercepting structure and the elastic film becomes larger.

[0038] As a further improvement of the present utility model, there are two auxiliary channels and two grooves. The two auxiliary channels are respectively located on the left and right sides of the main channel. One groove corresponds to one auxiliary channel, and the other groove corresponds to the other auxiliary channel.

[0039] As a further improvement of the present utility model, there are two auxiliary channels. The two auxiliary channels are located on the same side of the main channel, and the groove corresponds to the two auxiliary channels.

[0040] As a further improvement of the present utility model, the length of the groove is greater than 0% of the length of the dam body and less than 100% of the length of the dam body.

[0041] As a further improvement of the present utility model, the length of the groove accounts for 20 - 80% of the length of the dam body.

[0042] As a further improvement of the present utility model, the intercepting structure is a single-channel dam body. There are two single-channel dam bodies, namely the first single-channel dam body and the second single-channel dam body. The first single-channel dam body is arranged on the auxiliary channel, and the second single-channel dam body is arranged on the main channel.

[0043] As a further improvement of the present utility model, the single-channel dam body is generally in a long strip shape.

[0044] As a further improvement of the present utility model, the upper end height of the first single-channel dam body does not exceed the height of the auxiliary channel, and the upper end height of the second single-channel dam body does not exceed the height of the main channel.

[0045] As a further improvement of the present utility model, the upper end height of the first single-channel dam body accounts for more than 50% of the height of the auxiliary channel, and the upper end height of the second single-channel dam body accounts for more than 50% of the height of the main channel.

[0046] As a further improvement of the present utility model, the upper end height of the first single-channel dam body accounts for 85 - 95% of the height of the auxiliary channel, and the upper end height of the second single-channel dam body accounts for 85 - 95% of the height of the main channel.

[0047] As a further improvement of the present utility model, the single-channel dam body is made of the same material as the microfluidic chip.

[0048] As a further improvement of the present utility model, the single-channel dam body is made of PDMS material.

[0049] As a further improvement of the present utility model, the microfluidic chip further includes a gas path layer. The gas path layer has a gas path channel connected to an external air pressure source. The gas path layer and the elastic film cooperate to form a pneumatic microvalve. There are two pneumatic microvalves, one of which corresponds to the position of the first single-channel dam body, and the other corresponds to the position of the second single-channel dam body.

[0050] As a further improvement of the present utility model, the microfluidic chip includes a liquid path layer and an elastic film;

[0051] The liquid path layer is provided with a cell loading channel, and a single-channel dam body is provided in the cell loading channel. The single-channel dam body is used to intercept cells in the cell loading channel;

[0052] The elastic film is arranged above the cell loading channel. The elastic film covers the single-channel dam body. There is a gap between the elastic film and the single-channel dam body. The elastic film maintains a planar state. Cells in the cell loading channel will be blocked by the single-channel dam body, and reagents in the cell loading channel can pass through the single-channel dam body; when the elastic film bends upward, the passage between the single-channel dam body and the elastic film becomes larger, and cells can flow over the single-channel dam body along with the reagent.

[0053] As a further improvement of the present utility model, the liquid path layer further includes a cell processing channel, and the cell processing channel is communicated with the cell loading channel. When the elastic film bends upward, the passage between the single-channel dam body and the elastic film becomes larger, and cells can flow over the single-channel dam body along with the solution and enter the cell processing channel.

[0054] As a further improvement of the present utility model, a cell processing area is provided in the cell processing channel for capturing cells.

[0055] As a further improvement of the present invention, the single-channel dam body is roughly in the shape of a long strip. The dam body can also be in other shapes or forms in actual use, as long as it can intercept the reagent. The roughly long strip shape is convenient for production and processing and the precision is convenient for accurate control, which can greatly improve the control accuracy of the microfluidic chip. Of course, other forms can also be used.

[0056] As a further improvement of the present invention, a single-channel dam body is arranged at the bottom end of the liquid channel. The dam body is arranged at the bottom end, which can conveniently position and place the single-channel dam body, is easy to produce and process, and the precision is easy to accurately control, which can greatly improve the control precision of the microfluidic chip. Of course, other forms of position settings can also be used, such as setting the single-channel dam body on the side wall of the liquid channel, etc., as long as the interception effect can be achieved.

[0057] As a further improvement of the present invention, the microfluidic chip also includes an air path layer, the air path layer has an air path channel connected to an external air pressure source, the air path layer and the elastic film cooperate to form a pneumatic microvalve, and the position of the pneumatic microvalve corresponds to the position of the single-channel dam body; negative pressure is applied to the air path channel in the air path layer, and the elastic film under the air path channel bends upward, so that the passage between the single-channel dam body and the elastic film becomes larger, thereby allowing the cells to cross the single-channel dam body along with the reagent flow; positive pressure is applied to the air path channel in the air path layer, and the elastic film under the air path channel bends downward, so that the elastic film contacts the single-channel dam body, thereby achieving isolation of the cell loading channel.

[0058] As a further improvement of the present invention, the microfluidic chip includes a liquid circuit layer, the liquid circuit layer is provided with a cell recovery channel, the cell recovery channel is provided with a valve at the inlet end facing the inside of the microfluidic chip, and the valve enables cells to pass through, thereby enabling cells to enter the cell recovery channel from the inlet end.

[0059] As a further improvement of the present invention, the valve has a single-channel dam body, which is used to intercept cells and reagents and allows gas to freely enter the cell recovery channel.

[0060] As a further improvement of the present invention, the valve also includes an elastic film, which is arranged above the single-channel dam body. When the elastic film bends upward, the passage between the single-channel dam body and the elastic film becomes larger, and the recoil reagent entrains the cells through the single-channel dam body and the cell recovery channel to complete cell recovery.

[0061] As a further improvement of the present utility model, the microfluidic chip further includes a gas path layer. The gas path layer has a gas path channel connected to an external air pressure source. The gas path layer and the elastic film cooperate to form a pneumatic valve, and the position of the pneumatic valve corresponds to the position of the single-channel dam. Applying a negative pressure to the gas path channel in the gas path layer causes the elastic film under the gas path channel to bend upward, increasing the passage between the single-channel dam and the elastic film, so that cells can flow over the single-channel dam with the solution and enter the cell recovery channel. Applying a positive pressure to the gas path channel in the gas path layer causes the elastic film under the gas path channel to bend downward, bringing the elastic film into contact with the single-channel dam, thereby closing the cell recovery channel and preventing the reagent from entering the cell recovery channel.

[0062] As a further improvement of the present utility model, the microfluidic chip includes a liquid path layer. The liquid path layer has liquid path channels, and dams are provided at the intersections of two liquid path channels.

[0063] The microfluidic chip further includes a gas path layer and an elastic film sandwiched between the gas path layer and the liquid path layer. The gas path layer and the elastic film cooperate to form a pneumatic micro-valve, which is used to control the opening and closing of each liquid path channel in the liquid path layer.

[0064] The liquid path channels include a cell loading channel, a cell processing channel, a cell recovery channel, a first channel, a second channel, a mixing channel, a third channel, and a waste liquid channel. The pneumatic micro-valves include a first pneumatic micro-valve, a second pneumatic micro-valve, a third pneumatic micro-valve, a fourth pneumatic micro-valve, a fifth pneumatic micro-valve, a sixth pneumatic micro-valve, and a seventh pneumatic micro-valve. The intersections include a first intersection, a second intersection, and a third intersection. Dams are provided at the first intersection, the second intersection, and the third intersection. A cell processing area is provided in the cell processing channel.

[0065] The first channel is provided with a first pneumatic micro-valve, and the second channel is provided with a second pneumatic micro-valve. The intersection position of the first channel and the second channel forms a first intersection.

[0066] One end of the mixing channel is connected to the first intersection, and the other end of the mixing channel is connected to the cell processing channel. The third channel is connected to the mixing channel. The intersection position of the third channel and the mixing channel forms a second intersection. The third channel is provided with a third pneumatic micro-valve.

[0067] The waste liquid channel, the cell processing channel, and the cell recovery channel are connected in sequence. The waste liquid channel is provided with a seventh pneumatic micro-valve, and the cell recovery channel is provided with a fifth pneumatic micro-valve.

[0068] The backflush channel is connected to the waste liquid channel. The intersection position of the backflush channel and the waste liquid channel forms a third intersection. The backflush channel is provided with a sixth pneumatic micro-valve.

[0069] The cell loading channel is connected to the cell processing channel. The cell loading channel is provided with a fourth pneumatic micro-valve.

[0070] As a further improvement of the present utility model, an air passage corresponding to the position of the intersection is provided in the air passage layer. When a negative pressure is applied to the air passage, the elastic film arches up.

[0071] As a further improvement of the present utility model, air passages corresponding to the positions of the first intersection, the second intersection and the third intersection are respectively provided in the air passage layer. When negative pressures are applied to the three air passages, the elastic films above the first intersection, the second intersection and the third intersection arch up.

[0072] As a further improvement of the present utility model, a single-channel dam body is provided in the liquid passage. The number of single-channel dam bodies is the same as the number of pneumatic microvalves, and one single-channel dam body is correspondingly arranged at the position of each pneumatic microvalve.

[0073] As a further improvement of the present utility model, the transmission module includes a device main body, a liquid storage container, a first chip interface, a cell loading container, a first interface, and a cell recovery device. The liquid storage container is installed on the device main body. One end of the first chip interface is communicated with the liquid storage container, and the other end of the first chip interface is used for communicating with the liquid passage of the microfluidic chip;

[0074] The cell loading container is installed on the device main body. One end of the first interface is communicated with the cell loading container, and the other end of the first interface is used for communicating with the cell loading passage of the microfluidic chip;

[0075] The cell recovery device is installed on the device main body. The cell recovery device is detachably connected to the device main body, and the cell recovery device is used for communicating with the cell recovery passage of the microfluidic chip.

[0076] As a further improvement of the present utility model, the transmission module further includes a waste liquid hole, and the waste liquid hole is arranged on the device main body and is used for communicating with the waste liquid passage of the microfluidic chip.

[0077] As a further improvement of the present utility model, the transmission module further includes a second chip interface. One end of the second chip interface is communicated with the waste liquid hole, and the other end of the second chip interface is used for communicating with the waste liquid passage of the microfluidic chip.

[0078] As a further improvement of the present utility model, the transmission module further includes a first sealing ring, and the first sealing ring is installed at the connection between the other end of the first chip interface and the liquid passage, so as to ensure the airtightness of the connection between the other end of the first chip interface and the liquid passage.

[0079] As a further improvement of the present utility model, the transmission module further includes a second sealing ring and a third sealing ring. The second sealing ring is installed at the connection between the other end of the second chip interface and the waste liquid channel, thereby ensuring the airtightness of the connection between the other end of the second chip interface and the waste liquid channel; the third sealing ring is installed at the connection between the other end of the first interface and the cell loading channel, thereby ensuring the airtightness of the connection between the other end of the first interface and the cell loading channel.

[0080] As a further improvement of the present utility model, the solution transmission device further includes a connection mechanism for connecting the device main body to the microfluidic chip.

[0081] As a further improvement of the present utility model, the device main body is a clamp.

[0082] As a further improvement of the present utility model, the clamp includes an upper clamp body and a lower clamp body.

[0083] As a further improvement of the present utility model, the liquid storage container, the cell loading container and the cell recovery device are installed on the upper clamp body.

[0084] As a further improvement of the present utility model, the microfluidic chip is arranged on the lower clamp body.

[0085] As a further improvement of the present utility model, a slope is provided inside the cell loading container.

[0086] As a further improvement of the present utility model, the cell loading container is in the shape of a funnel with a wider upper part and a narrower lower part.

[0087] As a further improvement of the present utility model, the separation mechanism includes a solenoid valve switch and a cantilever, and the cantilever is connected to the valve core of the solenoid valve switch.

[0088] As a further improvement of the present utility model, the transmission module further includes a recovery hole, and the recovery hole is arranged on the device main body and is recessed into the device main body.

[0089] As a further improvement of the present utility model, the transmission module further includes a second interface. One end of the second interface is communicated with the cell recovery device, and the other end of the second interface is used for communicating with the cell recovery channel of the microfluidic chip.

[0090] As a further improvement of the present utility model, the transmission module further includes a third interface. One end of the third interface is communicated with the recovery hole, and the other end of the second chip interface is used for communicating with the cell recovery channel of the microfluidic chip.

[0091] As a further improvement of the present utility model, the transmission module further includes a fourth sealing ring, and the fourth sealing ring is installed at the connection between the other end of the second interface and the cell recovery channel, so as to ensure the airtightness of the connection between the other end of the second interface and the cell recovery channel;

[0092] The transmission module further includes a fifth sealing ring, and the fifth sealing ring is installed at the connection between the other end of the third interface and the cell recovery channel, so as to ensure the airtightness of the connection between the other end of the third interface and the cell recovery channel.

[0093] As a further improvement of the present utility model, an air circuit interface is provided on the device main body.

[0094] As a further improvement of the present utility model, an observation window is provided on the device main body.

[0095] As a further improvement of the present utility model, the pressure regulating device includes a pneumatic generating module, a pneumatic distribution module, and a pneumatic output interface;

[0096] The pneumatic distribution module includes a regulating valve. The input end of the regulating valve is communicated with the pneumatic generating module through a pipeline, and the output end of the regulating valve is communicated with the pneumatic output interface through a pipeline;

[0097] The pneumatic output interface is used to connect to the transmission module.

[0098] As a further improvement of the present utility model, the pneumatic generating module includes a constant positive pressure module, a constant negative pressure module, a positive pressure source module, a negative pressure source module, a fine-tuning positive pressure module, and a fine-tuning negative pressure module. The constant positive pressure module is used to generate a constant positive air pressure, the constant negative pressure module is used to generate a constant negative air pressure, the positive pressure source module is used to generate a positive air pressure, the positive pressure source module is connected to the fine-tuning positive pressure module, the fine-tuning positive pressure module is used to adjust the positive air pressure output by the positive pressure source module, the negative pressure source module is used to generate a negative air pressure, the negative pressure source module is connected to the fine-tuning negative pressure module, and the fine-tuning negative pressure module is used to adjust the negative air pressure output by the negative pressure source module.

[0099] As a further improvement of the present utility model, the regulating valve includes a first regulating valve, a second regulating valve, a third regulating valve, a fourth regulating valve, and a fifth regulating valve, and the pneumatic output interface includes a first pneumatic output interface, a second pneumatic output interface, a third pneumatic output interface, a fourth pneumatic output interface, and a fifth pneumatic output interface;

[0100] For the first regulating valve, the first interface of the first regulating valve is communicated with the constant positive pressure module, the second interface of the first regulating valve is communicated with the constant negative pressure module, the third interface of the first regulating valve is communicated with the first pneumatic output interface, and the first regulating valve is used to realize the switching mode between constant positive pressure and constant negative pressure;

[0101] The first interface of the second regulating valve communicates with the fine-tuning positive pressure module, the second interface of the second regulating valve communicates with the external atmospheric pressure, the third interface of the second regulating valve communicates with the second air pressure output interface, and the second regulating valve is used to realize the switching mode of fine-tuning positive pressure and atmospheric pressure;

[0102] The first interface of the third regulating valve communicates with the fine-tuning negative pressure module, the second interface of the third regulating valve communicates with the external atmospheric pressure, the third interface of the third regulating valve communicates with the third air pressure output interface, and the third regulating valve is used to realize the switching mode of fine-tuning negative pressure and atmospheric pressure;

[0103] The first interface of the fourth regulating valve communicates with the constant positive pressure module, the second interface of the fourth regulating valve communicates with the constant negative pressure module, the third interface of the fourth regulating valve communicates with the external atmospheric pressure, the fourth interface of the first regulating valve communicates with the fourth air pressure output interface, and the fourth regulating valve is used to realize the switching mode of constant positive pressure, constant negative pressure, and atmospheric pressure;

[0104] The first interface of the fifth regulating valve communicates with the fine-tuning positive pressure module, the second interface of the fifth regulating valve communicates with the fine-tuning negative pressure module, the third interface of the fifth regulating valve communicates with the external atmospheric pressure, the fourth interface of the fifth regulating valve communicates with the fifth air pressure output interface, and the fifth regulating valve is used to realize the switching mode of fine-tuning positive pressure, fine-tuning negative pressure, and atmospheric pressure.

[0105] As a further improvement of the present invention, the transmission module is connected to the microfluidic chip, the microfluidic chip includes a gas path layer, an elastic film sandwiched between the gas path layer and the liquid path layer, the liquid path layer is provided with a plurality of liquid path channels, and a pneumatic microvalve is formed by the cooperation of the gas path layer and the elastic film, and the pneumatic microvalve is used to control the opening and closing of each liquid path channel in the liquid path layer;

[0106] The liquid path channels include a cell loading channel and a cell recovery channel, the pneumatic microvalves include a fourth pneumatic microvalve and a fifth pneumatic microvalve, the fourth pneumatic microvalve is provided in the cell loading channel, and the fifth pneumatic microvalve is provided in the cell recovery channel;

[0107] The liquid path channels further include a cell processing channel, a first channel, a second channel, a mixing channel, a third channel, a backflush channel, and a waste liquid channel, the pneumatic microvalves include a first pneumatic microvalve, a second pneumatic microvalve, a third pneumatic microvalve, a sixth pneumatic microvalve, and a seventh pneumatic microvalve, and a cell processing area is provided in the cell processing channel;

[0108] The first channel is provided with a first pneumatic microvalve, the second channel is provided with a second pneumatic microvalve, and the second channel communicates with the first channel;

[0109] One end of the mixing channel is communicated with the first channel, the other end of the mixing channel is communicated with the cell processing channel, the third channel is communicated with the mixing channel, and a third pneumatic micro-valve is provided in the third channel;

[0110] The waste liquid channel, the cell processing channel and the cell recovery channel are communicated in sequence, and a seventh pneumatic micro-valve is provided in the waste liquid channel;

[0111] The backflush channel is communicated with the waste liquid channel, a sixth pneumatic micro-valve is provided in the backflush channel, and the cell loading channel is communicated with the cell processing channel;

[0112] There are five first air pressure output interfaces. One ends of the five first air pressure output interfaces are connected in parallel with the third interface of the first regulating valve. The other ends of the five first air pressure output interfaces are respectively communicated with the air passage channels of the air passage layers of the first pneumatic micro-valve, the second pneumatic micro-valve, the third pneumatic micro-valve, the sixth pneumatic micro-valve and the seventh pneumatic micro-valve, so as to realize the switching of constant positive pressure and constant negative pressure of the first pneumatic micro-valve, the second pneumatic micro-valve, the third pneumatic micro-valve, the sixth pneumatic micro-valve and the seventh pneumatic micro-valve;

[0113] There are three second air pressure output interfaces. One ends of the three second air pressure output interfaces are connected in parallel with the third interface of the second regulating valve. The other ends of the three second air pressure output interfaces are respectively communicated with the first channel, the second channel and the backflush channel;

[0114] The cell loading channel is communicated with the second air pressure output interface, or the waste liquid channel is communicated with the third air pressure output interface;

[0115] There are two fourth air pressure output interfaces. One ends of the two fourth air pressure output interfaces are connected in parallel with the fourth interface of the fourth regulating valve. The other ends of the two fourth air pressure output interfaces are respectively communicated with the air passage channels of the air passage layers of the fourth pneumatic micro-valve and the fifth pneumatic micro-valve, so as to realize the switching of constant positive pressure, constant negative pressure and atmospheric pressure of the fourth pneumatic micro-valve and the fifth pneumatic micro-valve;

[0116] The fifth air pressure output interface is communicated with the third channel.

[0117] As a further improvement of the present utility model, the vitrification freezing treatment device includes an automatic operation device. The automatic operation device includes a robotic arm, a camera and a controller. A pick-and-place mechanism is provided at the end of the robotic arm. The pick-and-place mechanism is used for picking up and putting down items. The camera is used for collecting images of the items, judging what kind of items through the images, manipulating the robotic arm to move through the controller, and controlling the pick-and-place mechanism to complete the picking or putting-down action through the controller.

[0118] As a further improvement of the present utility model, the pick-and-place mechanism is a mechanical clamp.

[0119] As a further improvement of the present utility model, the camera is mounted on the robotic arm.

[0120] As a further improvement of the present utility model, the microscopic module includes a mechanical positioning device, a lens, and a first camera located above the lens. The mechanical positioning device is used to move the microscopic module and / or the workbench.

[0121] As a further improvement of the present utility model, the mechanical positioning device is a robotic arm or an electric guide rail.

[0122] As a further improvement of the present utility model, the lens is a lens with adjustable magnification.

[0123] As a further improvement of the present utility model, the microscopic module further includes a first light source, and the first light source, the lens, and the first camera are integrated.

[0124] As a further improvement of the present utility model, the microscopic module further includes a fixed microscopic observation system. The fixed microscopic observation system includes an objective lens, a piezoelectric objective lens positioner, an inverted DIC microscopic imaging system, a second light source, and a second camera.

[0125] As a further improvement of the present utility model, the vitrification cryopreservation device further includes an AI module. The AI module includes an item recognition algorithm, a cell recognition algorithm, and a cell processing parameter algorithm.

[0126] The item recognition algorithm is based on the image data collected by the camera in the automated operation device and assists the robotic arm in grasping objects.

[0127] The cell recognition algorithm gives a judgment on the cell state based on the image data collected by the microscopic module and assists in completing automated cell manipulation.

[0128] The cell processing parameter algorithm is based on the image data collected by the microscopic module during the cell processing process. According to the change characteristics of the cell shape during the processing process, it gives cell processing parameters as a reference.

[0129] The beneficial effects of the present utility model are as follows: The present utility model focuses on the process of vitrification cryopreservation of embryonic cells (such as oocytes, etc.) on a microfluidic chip. Different modules cooperate to achieve automated and intelligent vitrification cryopreservation of embryonic cells, meeting the user's needs. Description of the Drawings

[0130] Figure 1 It is a schematic structural diagram of a liquid path layer provided with a dam body;

[0131] Figure 2 It is a schematic structural diagram of an embodiment of a liquid path layer provided with a dam body;

[0132] Figure 3 It is a schematic structural diagram of another embodiment of the liquid path layer provided with a dam body;

[0133] Figure 4 It is a schematic structural diagram of an elastic film;

[0134] Figure 5 It is a schematic diagram of the principle of a microfluidic chip;

[0135] Figure 6 It is a schematic structural diagram of the liquid path layer provided with a single-channel dam body;

[0136] Figure 7 It is a schematic diagram of a microfluidic chip with a cell loading function;

[0137] Figure 8 It is a schematic diagram of cell loading;

[0138] Figure 9 It is a schematic diagram of a microfluidic chip with a cell recovery function;

[0139] Figure 10 It is a schematic structural diagram of the liquid path layer;

[0140] Figure 11 It is a schematic structural diagram of the transmission module;

[0141] Figure 12 It is a schematic structural diagram of the upper clamp body;

[0142] Figure 13 It is a schematic diagram of the principle of the pressure regulating device;

[0143] Figure 14 It is a schematic structural diagram of the automatic operation device;

[0144] Figure 15 It is a schematic diagram of the principle of the microscopic module.

[0145] Figure 16 It is a schematic structural diagram of an embodiment of the present invention.

[0146] Figure 17 It is a schematic structural diagram of the cell recovery device, wherein the cryopreservation tube does not extend into the recovery hole.

[0147] Figure 18 It is a schematic structural diagram of the cell recovery device, wherein the cryopreservation tube has extended into the recovery hole. Detailed implementation manners

[0148] The present utility model discloses a vitrification freezing treatment method, which includes the following steps:

[0149] Step (1): Pre-fill the reagent in the liquid path channel of the microfluidic chip;

[0150] Step (2): Inject the cells into the liquid path channels of the microfluidic chip;

[0151] Step (3): Treat the cells in the microfluidic chip with reagents;

[0152] Step (4): Recover after cell treatment.

[0153] The vitrification freezing treatment method includes step (1), and the said step (1) includes:

[0154] The reagents introduced into the microfluidic chip include reagent 1 flowing through the main channel 4 and reagent 2 flowing through the auxiliary channel 5;

[0155] After reagent 1 is input via the corresponding input port, it flows in the main channel 4 of the microfluidic chip; after reagent 2 is input via the corresponding input port, it flows in the auxiliary channel 5 of the microfluidic chip;

[0156] There is an intersection between the main channel 4 and the auxiliary channel 5, so that the reagents in the main channel 4 and the auxiliary channel 5 can converge at the intersection;

[0157] This step (1) also includes a confluence step. In the said confluence step, the reagents are intercepted by an interception structure, so as to achieve that before confluence, any one reagent has a certain cross-overlap with the channel where the other reagent is located without blocking it.

[0158] As an embodiment of this step (1), the interception structure is the dam 2. In the confluence step, by controlling the flow of the reagents in the main channel 4 and the auxiliary channel 5, the reagent 2 input from the auxiliary channel 5 reaches the intersection first. There is a dam 2 at the intersection. The reagent 2 is intercepted first by using the dam 2, so that the reagent 2 cannot cross the dam 2;

[0159] Subsequently, the reagent 1 input from the main channel 4 also reaches the intersection;

[0160] After the reagent 1 crosses the dam 2 at the intersection, it contacts and converges with the reagent 2 to form a combined flow, and the combined flow continues to flow downstream.

[0161] The dam 2 can be a physical structure that can intercept liquids (such as various reagents) or solids (such as cells to be treated) in the channels of the microfluidic chip. Since the dam 2 is set in the channel, it causes a certain resistance or block to the passage of liquids or solids. The dam 2 can be of various shapes, or other components or mechanisms in other relevant forms that can play an interception role.

[0162] The intersection presents a square shape. The width of the intersection is wider than the width of the main channel 4. The intersection is symmetrically arranged. The intersection and the main channel 4 are in a smooth transition form.

[0163] Such as Figure 1As shown, the dam body 2 is provided with a groove 3. The dam body 2 presents a shape roughly like the letter 'n' and is arranged at the bottom end of the intersection.

[0164] The upper end height of the dam body 2 does not exceed the height of the main channel 4. For example, the height of the dam body 2 accounts for more than 50% of the height of the main channel 4, and preferably, the height of the dam body 2 accounts for 85 - 95% of the height of the main channel 4. Since the interception effect of the dam body 2 is negatively correlated with the space size, the higher the dam body 2, the narrower the space between it and the upper wall of the channel, and the better the interception effect. Therefore, in the present utility model, it is preferably that the height of the dam body 2 accounts for 85 - 95% of the height of the main channel 4.

[0165] The dam body 2 and the liquid channel of the microfluidic chip can be formed in an integrally molded manner. Or, the dam body 2 and the liquid channel of the microfluidic chip are arranged at the bottom end of the intersection by bonding.

[0166] The material of the dam body 2 and the microfluidic chip can be the same, or the dam body 2 is made of PDMS material. The Chinese name of PDMS is polydimethylsiloxane.

[0167] One side of the dam body 2 has two supporting feet, and a groove 3 is formed between the two supporting feet. The groove 3 is located at the position on the microfluidic chip opposite to the auxiliary channel 5 so that the reagent 2 in the auxiliary channel 5 can reach the groove 3.

[0168] The two supporting feet of the dam body 2 are symmetrically arranged, and the width between the two supporting feet of the dam body 2 exceeds half of the width of the entire dam body 2.

[0169] An elastic film 7 is also arranged on the microfluidic chip. The elastic film 7 is arranged on the end face of the microfluidic chip on the side where the main channel 4 and the auxiliary channel 5 open, and there is a gap between the elastic film 7 and the dam body 2.

[0170] The elastic film 7 can only cover the intersection, or the elastic film 7 covers all the main channels 4 and the auxiliary channels 5; or the elastic film 7 covers the entire range of the microfluidic chip.

[0171] The thickness of the elastic film 7 is 50 to 250 microns, preferably 75 - 150 microns. Because at 75 - 150 microns, it is not only convenient for manufacturing but also can have better elasticity and higher response sensitivity in control. The elastic film 7 is made of PDMS material.

[0172] Such as Figure 16After the arrangement shown, reagent 1 is more likely to pass through the first leg 31 and merge with reagent 2, and then pass through the second leg 32. The pressure increases gradually during the whole process, avoiding the situation that the pressure of reagent 1 is high when passing through the first leg 31, the convergence process is too sudden, and the convergence occurs locally on the side of the groove 3 and bubbles are formed at the top of the groove 3. The second leg is higher than the first leg, so that after reagent 1 passes through the first leg and merges with reagent 2, the second leg will form a barrier to the fluid again due to its higher height, so that reagent 1 and reagent 2 can be more fully integrated, and the mixed gas that may be generated by the convergence can be further discharged. In actual use, it is preferred that the height of the second leg is greater than 1 / 10 of the height of the first leg, which has a significant effect.

[0173] According to the experimental comparison of multiple groups of solutions, the effects of the solutions with different leg heights are as follows:

[0174]

[0175] The microfluidic chip also includes an air path layer 6, which has an air path channel connected to an external air pressure source. The air path layer 6 and the elastic film 7 cooperate to form a pneumatic microvalve, which includes a first pneumatic microvalve and a second pneumatic microvalve. The dam body 2 is roughly in the shape of the letter n. The dam body 2 has two legs, and a groove 3 is formed between the two legs. The groove 3 is located in the microfluidic chip facing the auxiliary channel 5. The two legs are respectively a first leg 31 and a second leg 32. The first leg 31 is located on the side where the reagent 1 flows in, and the second leg 32 is located on the side where the reagent 1 and the reagent 2 converge and flow out. The first pneumatic microvalve controls the main channel 4. The first pneumatic microvalve only covers a part of the first leg 31; the second pneumatic microvalve controls the auxiliary channel 5, and the second pneumatic microvalve covers a part of the groove 3. The second pneumatic microvalve does not intercept the entire auxiliary channel 5 and the cross section of the groove 3, so as to prevent the reagent 1 from being sealed when the second pneumatic microvalve is closed; in order to accommodate the two pneumatic microvalves in space, the first leg 31 is extended; in the confluence step, the reagent 1 is introduced into the main channel 4, and when the reagent 1 in the main channel 4 moves to the dam body 2, a positive pressure is applied to the first pneumatic microvalve to close the first pneumatic microvalve, and the elastic film 7 of the first pneumatic microvalve contacts the dam body 2 downward, thereby closing the main channel 4;

[0176] The reagent 2 is introduced into the auxiliary channel 5, and when the reagent 2 moves to the dam body 2, a positive pressure is applied to the second pneumatic microvalve, the second pneumatic microvalve is closed, and the driving pressure of the reagent 2 is stopped;

[0177] Apply a negative pressure to the pneumatic microvalve corresponding to the position of the dam body 2. The elastic film 7 of the first pneumatic microvalve bulges upward (alternatively, remove the positive pressure of the first pneumatic microvalve, and the elastic film 7 of the first pneumatic microvalve returns to the planar state), driving the reagent 1 to continue advancing over the dam body 2. When the reagent reaches the second pneumatic microvalve, remove the positive pressure of the second pneumatic microvalve (alternatively, apply a negative pressure to the second pneumatic microvalve, and the elastic film 7 of the second pneumatic microvalve bulges upward). When the reagent 1 passes above the reagent 2, confluence is completed with it;

[0178] When the confluent reagent crosses the dam body 2 and enters the waste liquid channel, remove the negative pressure of the pneumatic microvalve corresponding to the position of the dam body 2, and continue to fill the waste liquid channel to complete the pre-charging.

[0179] The gas path layer 6 is provided with a concave portion in the area corresponding to the intersection, and the external air pressure source can be communicated through this concave portion. The area of the concave portion of the gas path layer 6 is not less than the area of the intersection region. The shape of the concave portion of the gas path layer 6 is the same as the shape of the intersection. The preferred shape of the concave portion of the gas path layer 6 is rectangular or circular.

[0180] The position of the pneumatic microvalve corresponds to the position of the dam body 2. When a negative pressure is applied to the gas path channel in the gas path layer 6, the elastic film 7 under the gas path channel bends upward and contracts in the direction of the space of the gas path channel in the gas path layer 6, making the passage between the dam body 2 and the elastic film 7 larger.

[0181] By controlling the flow of the reagents in the main channel 4 and the auxiliary channel 5, the reagent 2 input from the auxiliary channel 5 first reaches the intersection. There is a dam body 2 at the intersection. The reagent 2 is intercepted first by using the dam body 2, so that the reagent 2 cannot cross the groove 3 of the dam body 2; subsequently, the reagent 1 input from the main channel 4 also reaches the intersection; when the reagent 1 passes above the groove 3, it contacts the reagent 2 in the groove 3 to complete the confluence, and then the two reagents are confluent to form a synthetic flow, and the synthetic flow finally crosses the dam body 2 and continues to flow downstream.

[0182] The length L1 of the groove 3 is greater than 0% of the length L2 of the dam body 2, and the length L1 of the groove 3 is less than 100% of the length L2 of the dam body 2; preferably, the length L1 of the groove 3 accounts for 20 - 80% of the length L2 of the dam body 2.

[0183] As an embodiment of this pre-charging method, there are two auxiliary channels 5, and the dam body 2 is provided with two grooves 3. The two auxiliary channels 5 are respectively located on the left and right sides of the main channel 4. One groove 3 corresponds to one of the auxiliary channels 5, and the other groove 3 corresponds to the other auxiliary channel 5. By controlling the flow of the reagents in the main channel 4 and the two auxiliary channels 5, the reagent 2 input from the two auxiliary channels 5 first reaches the intersection. There is a dam body 2 at the intersection. The reagent 2 is intercepted first by using the two grooves 3 of the dam body 2, so that the reagent 2 cannot cross the two grooves 3 of the dam body 2;

[0184] Subsequently, the reagent 1 input through the main channel 4 also reaches the intersection;

[0185] When the reagent 1 passes over the two grooves 3, it contacts the reagent 2 in the two grooves 3 to complete the confluence, and then the confluent flow is formed. The confluent flow finally crosses the dam body 2 and continues to flow downstream.

[0186] As an embodiment of this pre-filling method, the intercepting structure is a single-channel dam body 17. There are two single-channel dam bodies 17, namely the first single-channel dam body and the second single-channel dam body. The first single-channel dam body is arranged on the auxiliary channel 5, and the second single-channel dam body is arranged on the main channel 4. In the confluence step, by controlling the flow of the reagents in the main channel 4 and the auxiliary channel 5, the reagent 2 input through the auxiliary channel 5 is intercepted by the first single-channel dam body before reaching the intersection, so that the reagent 2 cannot cross the first single-channel dam body;

[0187] The reagent 1 input through the main channel 4 is intercepted by the second single-channel dam body before reaching the intersection, so that the reagent 2 cannot cross the second single-channel dam body;

[0188] Control the reagent 1 and the reagent 2 to cross the first single-channel dam body and the second single-channel dam body at the same time, so that the reagent 1 and the reagent 2 reach the intersection at the same time. After the reagent 1 contacts the reagent 2, they are confluent to form a confluent flow, and the confluent flow continues to flow downstream.

[0189] The single-channel dam body is used to be arranged on the main channel 4 or the auxiliary channel 5 for interception.

[0190] The microfluidic chip further includes a gas path layer 6. The gas path layer 6 has a gas path channel connected to an external air pressure source. The gas path layer 6 and the elastic film 7 cooperate to form a pneumatic microvalve. There are two pneumatic microvalves. One pneumatic microvalve corresponds to the position of the first single-channel dam body, and the other pneumatic microvalve corresponds to the position of the second single-channel dam body. In the confluence step, the reagent 1 is introduced into the main channel 4. When the reagent 1 travels to the front of the second single-channel dam body, a positive pressure is applied to the pneumatic microvalve corresponding to the position of the second single-channel dam body to close the pneumatic microvalve corresponding to the position of the second single-channel dam body. The elastic film 7 of the pneumatic microvalve corresponding to the position of the second single-channel dam body contacts the second single-channel dam body downward, thereby closing the main channel 4;

[0191] The reagent 2 is introduced into the auxiliary channel 5. When the reagent 2 travels to the front of the first single-channel dam body, a positive pressure is applied to the pneumatic microvalve corresponding to the position of the first single-channel dam body to close the pneumatic microvalve corresponding to the position of the first single-channel dam body. The elastic film 7 of the pneumatic microvalve corresponding to the position of the first single-channel dam body contacts the first single-channel dam body downward, thereby closing the auxiliary channel 5;

[0192] Remove the positive pressure of the pneumatic micro-valve corresponding to the position of the first single-channel dam and the pneumatic micro-valve corresponding to the position of the second single-channel dam, so that the elastic film 7 of the pneumatic micro-valve corresponding to the position of the first single-channel dam returns to the planar state and the elastic film 7 of the pneumatic micro-valve corresponding to the position of the second single-channel dam returns to the planar state. Or, apply negative pressure to the pneumatic micro-valve corresponding to the position of the first single-channel dam and the pneumatic micro-valve corresponding to the position of the second single-channel dam, so that the elastic film 7 of the pneumatic micro-valve corresponding to the position of the first single-channel dam bulges upward and the elastic film 7 of the pneumatic micro-valve corresponding to the position of the second single-channel dam bulges upward. Then control the reagent 1 and the reagent 2 to cross the first single-channel dam and the second single-channel dam simultaneously, so that the reagent 1 and the reagent 2 reach the intersection at the same time. After the reagent 1 and the reagent 2 come into contact, they merge together to form a synthetic flow, and the synthetic flow continues to flow downstream to complete the priming.

[0193] The single-channel dam 17 is in a long strip shape.

[0194] The upper end height of the first single-channel dam does not exceed the height of the auxiliary channel 5, and the upper end height of the second single-channel dam does not exceed the height of the main channel 4; for example, the upper end height of the first single-channel dam accounts for more than 50% of the height of the auxiliary channel 5, and the upper end height of the second single-channel dam accounts for more than 50% of the height of the main channel 4; preferably, the upper end height of the first single-channel dam accounts for 85-95% of the height of the auxiliary channel 5, and the upper end height of the second single-channel dam accounts for 85-95% of the height of the main channel 4.

[0195] The single-channel dam 17 is made of the same material as the microfluidic chip, and preferably the single-channel dam 17 is made of PDMS material.

[0196] As an embodiment of step (1) of the present invention, in step (1): load the second reagent, the third reagent, and the third reagent for backwashing into the grooves 3 of the cross micro-dam 2 at the first intersection M1, the second intersection M2, and the third intersection M3 respectively through the corresponding channels (for example, the corresponding channels are the second channel 12, the third channel 14, and the backwashing channel 15), and then reduce the driving pressure or directly remove the driving pressure and close the corresponding pneumatic micro-valves (for example, the second pneumatic micro-valve V2, the third pneumatic micro-valve V3, and the sixth pneumatic micro-valve V6); due to the obstruction of the cross micro-dam 2, the reagent can be loaded into the liquid channel and stay in the groove 3 under the driving of an appropriate pressure. At this time, the second channel 12, the third channel 14, and the backwashing channel 15 are the auxiliary channels;

[0197] Close the corresponding pneumatic micro-valves (e.g., the fourth pneumatic micro-valve V4 and the fifth pneumatic micro-valve V5), load the first reagent into the first channel 11. During the downstream flow of the first reagent, negative pressure is applied to the gas path channels at the first intersection M1, the second intersection M2, and the third intersection M3 in sequence, causing the elastic film 7 above the cross micro-dam 2 to arch, increasing the reagent passage area. The first reagent converges with the second reagent, the third reagent, and the third reagent for backwashing at the first intersection M1, the second intersection M2, and the third intersection M3 in sequence during the process from the inlet of the first channel 11 to the outlet of the waste liquid channel 16. When the first reagent passes above the groove 3, it contacts the second reagent, the third reagent, and the third reagent for backwashing to complete the convergence, and then further fills the area above the entire cross micro-dam 2, and finally completely crosses the cross micro-dam 2 and continues to flow downstream; the entire convergence process fundamentally eliminates the generation of bubbles; at this time, the liquid path channel from the first channel 11 to the waste liquid channel 16 is the main channel;

[0198] Close and open the corresponding pneumatic micro-valves (e.g., close the seventh pneumatic micro-valve V7 and open the fourth pneumatic micro-valve V4), and pour a small amount of the first reagent into the cell loading channel 8.

[0199] The step (2) includes:

[0200] First, inject cells into the loading channel 8 of the microfluidic chip: A single-channel dam 17 is provided in the cell loading channel 8 of the microfluidic chip, and an elastic film 7 is provided above the cell loading channel 8. Inject the reagent containing cells from the cell loading channel 8 of the microfluidic chip, and the reagent can pass through the single-channel dam 17 while the cells are blocked by the single-channel dam 17;

[0201] Second, load the cells into the cell processing channel 9: When the elastic film 7 bends upward, the passage between the single-channel dam 17 and the elastic film 7 becomes larger, and the cells flow over the single-channel dam 17 with the reagent and enter the cell processing channel 9.

[0202] There is a cell processing area T in the cell processing channel 9, where the cell processing area T refers to various implementation forms such as components, positions, or regions that can temporarily locate and fix the relative positions of cells, so as to determine the positions of cells and achieve cell processing during subsequent cell processing.

[0203] There is a gap between the elastic film 7 and the single-channel dam 17. When the elastic film 7 remains flat, that is, when the elastic film 7 does not bend upward, the reagent flow can pass above the single-channel dam 17, and the cells are blocked by the single-channel dam 17, preventing the cells from entering the cell processing channel 9, dividing the cell loading process into two segments, which are controlled by the single-channel dam 17 and the elastic film 7.

[0204] The single-channel dam 17 is in the shape of a long strip and is disposed at the bottom end of the cell loading channel 8 .

[0205] The microfluidic chip also includes an air path layer 6 having an air path channel connected to an external air pressure source. The air path layer 6 and the elastic film 7 cooperate to form a pneumatic microvalve, and the position of the pneumatic microvalve corresponds to the position of the single-channel dam 17 .

[0206] Negative pressure is applied to the gas path in the gas path layer 6, and the elastic film 7 under the gas path bends upward, so that the passage between the single channel dam 17 and the elastic film 7 becomes larger, so that the cells pass through the single channel dam 17 and enter the cell processing channel 9 along with the reagent flow.

[0207] Positive pressure is applied to the gas channel in the gas channel layer 6 , and the elastic film 7 under the gas channel bends downward, so that the elastic film 7 contacts the single channel dam 17 , thereby isolating the cell loading channel 8 .

[0208] As an embodiment of step (2), step (2) includes:

[0209] Close and open the corresponding pneumatic microvalve (for example, close the first pneumatic microvalve V1 and open the seventh pneumatic microvalve V7), inject the cells into the cell loading hole, and the mechanical arm 501 moves the microscope module to the cell loading hole position to ensure that the cells are observed to enter the hole;

[0210] The robot arm 501 connects the cell loading interface to the cell loading transmission interface, and then moves the microscopic module to the corresponding pneumatic microvalve (for example, the fourth pneumatic microvalve V4);

[0211] Apply air pressure to send the cells into the cell loading channel 8. At this time, the cells will be blocked by the single-channel dam 17 at the corresponding pneumatic microvalve (e.g., the fourth pneumatic microvalve V4). When the microscopic module detects that the cells enter the field of view of the corresponding pneumatic microvalve (e.g., the fourth pneumatic microvalve V4), the corresponding pneumatic microvalve (e.g., the fourth pneumatic microvalve V4) switches to negative pressure, and the cells continue to move downstream.

[0212] Move the microscope module to the cell processing area T. When the cells safely enter the cell processing area T, close and open the corresponding pneumatic microvalves (for example, close the fourth pneumatic microvalve V4, open the first pneumatic microvalve V1 and the second pneumatic microvalve V2), and enable the microscope module to observe the cells. At this time, the piezoelectric objective lens positioner and the AI ​​module work together to achieve automatic focus.

[0213] The cell processing area T refers to various forms of components, positions, or areas that can temporarily locate cells and fix their relative positions, so that in the subsequent cell processing process, the position of the cells can be determined and the cells can be processed.

[0214] In step (2), when the cells enter the cell loading channel 8, the gas path above the single-channel dam 17 is not pressurized, the liquid flow can pass over the single-channel dam 17, and the cells will be intercepted by the single-channel dam 17. The cells can be observed to be intercepted at the single-channel dam 17, and precise control of the stroke of the loaded cells can be achieved.

[0215] The step (3) comprises:

[0216] The first processing step: closing and opening the corresponding pneumatic microvalve (for example, closing the fourth pneumatic microvalve V4, opening the first pneumatic microvalve V1 and the second pneumatic microvalve V2), driving the first reagent and the second reagent to treat the cells, while keeping the total flow of the first reagent and the second reagent unchanged, the flow of the first reagent is gradually reduced from 100% to 0, and the flow of the second reagent is increased from 0 to 100%; the total processing time of the first reagent and the second reagent is 15 minutes, including 9 minutes of gradient processing and 6 minutes of subsequent processing of the second reagent.

[0217] Table 1

[0218]

[0219] The second processing step: close and open the corresponding pneumatic microvalve (for example, close the first pneumatic microvalve V1 and the second pneumatic microvalve V2, and open the third pneumatic microvalve V3), drive the third reagent to treat the cells, and impact the cells at a short-term high speed of 100-120 μl / min for 20-40 seconds, during which obvious shrinkage of the cells can be observed.

[0220] The step (4) comprises:

[0221] Step A: After the reagent is input through the corresponding input port (for example, the reagent can enter the microfluidic chip through the cell loading channel 8 or other channels and ports), the microfluidic chip is pre-filled so that the reagent is blocked by the single channel dam 17 at the cell recovery channel 10 while filling other fluid channels of the microfluidic chip and thus cannot enter the cell recovery channel 10;

[0222] Step B: loading cells from the cell loading channel 8 into the microfluidic chip, for example, into the cell processing area T, and performing solution treatment;

[0223] Step C: An elastic film 7 is provided above the single-channel dam 17, so that the elastic film 7 bends upward, and the passage between the single-channel dam 17 and the elastic film 7 becomes larger, driving the recoil reagent to flow from the recoil channel 15 to the cell recovery channel 10, and the recoil reagent carries the cells through the single-channel dam 17 and enters the cell recovery channel 10.

[0224] In step A, only the gas in the microfluidic chip is allowed to freely enter the cell recovery channel 10;

[0225] When a solution reaches the inlet end of the cell recovery channel 10, the elastic film 7 bends downward, contacts the single-channel dam body 17, and shields the single-channel dam body 17, thereby closing the inlet end of the cell recovery channel 10, so that the reagent cannot enter the cell recovery channel 10 or only a trace amount of the solution can enter the cell recovery channel 10.

[0226] In the step A, after the reagent is input via the cell loading channel 8 or the cell recovery channel 10, a suitable driving pressure is maintained, so that the reagent fills other liquid path channels of the microfluidic chip while being intercepted by the single-channel dam body 17 at the cell recovery channel 10 and thus fails to enter the cell recovery channel 10.

[0227] In the step C, a negative pressure is applied to the gas path channel in the gas path layer 6, and the elastic film 7 under the gas path channel bends upward, so that the passage between the single-channel dam body 17 and the elastic film 7 becomes larger, driving the backflush reagent to flow from the backflush channel 15 to the cell recovery channel 10. The backflush reagent entrains the cells and completes cell recovery through the single-channel dam body 17 via the cell recovery channel 10.

[0228] In the step A, a positive pressure is applied to the gas path channel in the gas path layer 6, and the elastic film 7 under the gas path channel bends downward, so that the elastic film 7 contacts the single-channel dam body 17, thereby closing the cell recovery channel 10 and preventing the reagent from entering the cell recovery channel 10.

[0229] In step (4), the reagent is intercepted by the single-channel dam body 17, so that the reagent cannot enter the cell recovery channel 10. When performing cell recovery, the volume of the reagent entraining the cells during recovery is controlled by adjusting the pressure and time during backflush, and this operation has good reproducibility.

[0230] As an embodiment of step (4), in the step (4), the corresponding pneumatic microvalves are closed and opened (for example, the third pneumatic microvalve V3 and the seventh pneumatic microvalve V7 are closed, and the sixth pneumatic microvalve V6 and the fifth pneumatic microvalve V5 are opened), and the third reagent for backflush is driven to backflush the cells and recovered through the cell recovery channel 10.

[0231] In the step (4), when the preset recovery time is reached, the electromagnetic switch in the transmission module is triggered to drive the cantilever to pop up the cryotube upward, and at the same time, the backflush is stopped. At this time, the cryotube is separated from the backflush solution, and the volume of the solution entraining the cells in the cryotube no longer increases. The vitrification freezing treatment is completed.

[0232] The present utility model also discloses a vitrification freezing treatment device, which includes a microfluidic chip, a transmission module, a pressure regulating device, and a microscopic module. Cells complete vitrification freezing treatment in the microfluidic chip. The transmission module is used to realize the transmission of substances and energy between the microfluidic chip and the outside. The pressure regulating device is docked with the transmission module and is used to deliver air pressure to the microfluidic chip. The microscopic module is used to observe the cell state.

[0233] I. The microfluidic chip of the present utility model can realize a pre-charging function, specifically as follows:

[0234] The microfluidic chip includes a liquid path layer 1;

[0235] Liquid path channels are provided in the liquid path layer 1. Reagents in the microfluidic chip flow through the liquid path channels. The liquid path channels have a main channel 4 and an auxiliary channel 5 that intersect.

[0236] An interception structure is used to intercept reagents and / or cells in the liquid path channels. The interception structure is arranged at the intersection of the two liquid path channels, so that any reagent has a certain cross-overlap with the channel where the other reagent is located before confluence without blocking it.

[0237] As an embodiment of the microfluidic chip, the interception structure is a dam body 2, and the dam body 2 is provided with a groove 3, and the groove 3 corresponds to the auxiliary channel 5.

[0238] The intersection presents a square shape. The width of the intersection is wider than the width of the main channel 4, and the intersection is symmetrically arranged. The intersection and the main channel 4 are smoothly transitioned.

[0239] The length L1 of the groove 3 is greater than 0% of the length L2 of the dam body 2, and the length L1 of the groove 3 is less than 100% of the length L2 of the dam body 2; preferably, the length L1 of the groove 3 accounts for 20 - 80% of the length L2 of the dam body 2.

[0240] The dam body 2 generally presents the shape of the letter n, and the dam body 2 is arranged at the bottom end of the intersection.

[0241] The upper height of the dam body 2 does not exceed the height of the main channel 4. For example, the height of the dam body 2 accounts for more than 50% of the height of the main channel 4, and preferably, the height of the dam body 2 accounts for 85 - 95% of the height of the main channel 4.

[0242] The dam body 2 and the liquid path channels of the microfluidic chip can be formed by an integral molding method. Or, the dam body 2 and the liquid path channels of the microfluidic chip are arranged at the bottom end of the intersection by an adhesive method.

[0243] The material of the dam body 2 and the microfluidic chip can be the same, or the dam body 2 is made of PDMS material.

[0244] One side of the dam body 2 has two feet, and a groove 3 is formed between the two feet. The groove 3 is located at a position on the microfluidic chip opposite to the auxiliary channel 5, so that the reagent 2 in the auxiliary channel 5 can reach the groove 3.

[0245] The two feet of the dam body 2 are symmetrically arranged, and the width between the two feet of the dam body 2 exceeds half of the width of the entire dam body 2.

[0246] An elastic film 7 is also provided on the microfluidic chip. The elastic film 7 is arranged on the end face of the opening side of the main channel 4 and the auxiliary channel 5 of the microfluidic chip, and there is a gap between the elastic film 7 and the dam body 2.

[0247] The elastic film 7 can only cover the intersection, or the elastic film 7 covers all the main channels 4 and auxiliary channels 5; or the elastic film 7 covers the entire range of the microfluidic chip.

[0248] The thickness of the elastic film 7 is 50 to 250 microns, preferably 75 to 150 microns, and the elastic film 7 is made of PDMS material.

[0249] The microfluidic chip further includes a gas path layer 6. The gas path layer 6 has a gas path channel connected to an external air pressure source, and the gas path layer 6 and the elastic film 7 cooperate to form a pneumatic microvalve.

[0250] The gas path layer 6 is provided with a recess in the area corresponding to the intersection, and the external air pressure source can be communicated through the recess. The area of the recess of the gas path layer 6 is not less than the area of the intersection area. The shape of the recess of the gas path layer 6 is the same as the shape of the intersection. The preferred shape of the recess of the gas path layer 6 is rectangular or circular.

[0251] The position of the pneumatic microvalve corresponds to the position of the dam body 2. When a negative pressure is applied to the gas path channel in the gas path layer 6, the elastic film 7 under the gas path channel bends upward and contracts in the direction of the space of the gas path channel in the gas path layer 6, so that the passage between the dam body 2 and the elastic film 7 becomes larger.

[0252] As Figure 2 shown, as an embodiment of the liquid path layer 1, there are two auxiliary channels 5 and two grooves 3. The two auxiliary channels 5 are respectively located on the left and right sides of the main channel 4, and one groove 3 corresponds to one auxiliary channel 5, and the other groove 3 corresponds to the other auxiliary channel 5.

[0253] As Figure 3 shown, as another embodiment of the liquid path layer 1, there are two auxiliary channels 5. The two auxiliary channels 5 are located on the same side of the main channel 4, and the groove 3 corresponds to the two auxiliary channels 5.

[0254] During operation, the reagent input through the auxiliary channel 5 stays at the groove 3. When the reagent input through the main channel 4 passes above the groove 3, it contacts the reagent in the groove 3 to complete confluence. Then, the two reagents merge to form a synthetic flow, and the synthetic flow finally crosses the dam body 2 and continues to flow downstream. Since the reagents in the main channel 4 and the auxiliary channel 5 can be fused into a synthetic flow at the dam body 2, gas introduction caused by the confluence of different liquids in two different channels is avoided, thus completely eliminating the mixing of gas generated by the confluence of different solutions. In principle, the generation of bubbles is prevented during the entire confluence process.

[0255] As Figure 4 shown, the microfluidic chip further includes an elastic film 7. Since the height of the dam body 2 does not exceed the height of the liquid path channels in the liquid path layer 1, there is a certain gap between the elastic film 7 and the dam body 2. When the elastic film 7 is arranged on the upper side of the liquid path layer 1, it does not interfere with the flow of the reagent liquid in each liquid path channel of the liquid path layer 1.

[0256] As Figure 5 shown, the microfluidic chip further includes a gas path layer 6. The gas path layer 6 can be connected to an external controllable gas pressure source to provide a certain pressure to the gas path layer 6, such as positive pressure or negative pressure. An air-operated microvalve is formed by the cooperation of the gas path layer 6 and the elastic film 7, and the air-operated microvalve is used to control the opening and closing of each liquid path channel of the liquid path layer 1.

[0257] As another embodiment of the microfluidic chip, as Figure 6 shown, the intercepting structure is a single-channel dam body 17. There are two single-channel dam bodies 17, namely the first single-channel dam body and the second single-channel dam body. The first single-channel dam body is arranged on the auxiliary channel 5, and the second single-channel dam body is arranged on the main channel 4.

[0258] The single-channel dam body 17 is in a long strip shape.

[0259] The upper end height of the first single-channel dam body does not exceed the height of the auxiliary channel 5, and the upper end height of the second single-channel dam body does not exceed the height of the main channel 4. For example, the upper end height of the first single-channel dam body accounts for more than 50% of the height of the auxiliary channel 5, and the upper end height of the second single-channel dam body accounts for more than 50% of the height of the main channel 4. Preferably, the upper end height of the first single-channel dam body accounts for 85 - 95% of the height of the auxiliary channel 5, and the upper end height of the second single-channel dam body accounts for 85 - 95% of the height of the main channel 4.

[0260] The single-channel dam body 17 is made of the same material as the microfluidic chip. Preferably, the single-channel dam body 17 is made of PDMS material.

[0261] The microfluidic chip further includes a gas path layer 6. The gas path layer 6 has a gas path channel connected to an external air pressure source. The gas path layer 6 and the elastic film 7 cooperate to form a pneumatic microvalve. There are two pneumatic microvalves, one of which corresponds to the position of the first single-channel dam body, and the other corresponds to the position of the second single-channel dam body.

[0262] Principle of the pneumatic microvalve: When a negative pressure is applied to the gas path channel in the gas path layer 6, the elastic film 7 under the gas path channel bends upward and contracts in the direction of the space of the gas path channel in the gas path layer 6, which can make the passage between the dam body 2 or the single-channel dam body 17 in the liquid path layer and the elastic film 7 larger, so that more fluids and cells that are convenient to process can pass through; when a positive pressure is applied to the gas path channel in the gas path layer 6, the elastic film 7 under the gas path channel bends downward, squeezing the liquid path channel under the elastic film 7; when the positive pressure is removed, the elastic film 7 returns, thus realizing the control of the pneumatic microvalve.

[0263] The present utility model uses the dam body 2 or the single-channel dam body 17 to prevent the generation of bubbles when reagents converge, thereby avoiding the interference of bubbles on the cell processing process.

[0264] When the microfluidic chip of the present utility model is pre-filled, the generation of bubbles is avoided, thus ensuring the safety of cells.

[0265] II. The microfluidic chip of the present utility model can realize the cell loading function, specifically as follows:

[0266] As Figure 7 and Figure 8 shown, the liquid path layer 1 is provided with a cell loading channel 8. A single-channel dam body 17 is provided in the cell loading channel 8. The single-channel dam body 17 is used to intercept cells in the cell loading channel 8.

[0267] The elastic film 7 is arranged above the cell loading channel 8. The elastic film 7 covers the single-channel dam body 17. There is a gap between the elastic film 7 and the single-channel dam body 17. The elastic film 7 maintains a planar state. The cells in the cell loading channel 8 will be blocked by the single-channel dam body 17, and the reagents in the cell loading channel 8 can pass through the single-channel dam body 17; when the elastic film 7 bends upward, the passage between the single-channel dam body 17 and the elastic film 7 becomes larger, and the cells can flow over the single-channel dam body 17 along with the reagent.

[0268] The liquid path layer 1 further includes a cell processing channel 9. The cell processing channel 9 is communicated with the cell loading channel 8. When the elastic film 7 bends upward, the passage between the single-channel dam body 17 and the elastic film 7 becomes larger, and the cells can flow over the single-channel dam body 17 along with the solution and enter the cell processing channel 9. The single-channel dam body 17 is in a long strip shape.

[0269] The microfluidic chip also includes an air path layer 6, which has an air path channel connected to an external air pressure source. The air path layer 6 and the elastic film 7 cooperate to form a pneumatic microvalve, and the position of the pneumatic microvalve corresponds to the position of the single-channel dam 17; when negative pressure is applied to the air path channel in the air path layer 6, the elastic film 7 under the air path channel bends upward, so that the passage between the single-channel dam 17 and the elastic film 7 becomes larger, so that the cells pass through the single-channel dam 17 with the reagent flow; when positive pressure is applied to the air path channel in the air path layer 6, the elastic film 7 under the air path channel bends downward, so that the elastic film 7 contacts the single-channel dam 17, thereby achieving isolation of the cell loading channel 8.

[0270] 3. The microfluidic chip of the utility model can realize the cell recovery function, specifically:

[0271] like Figure 9 As shown, the liquid circuit layer 1 is provided with a cell recovery channel 10, and the cell recovery channel 10 is disposed at an inlet end facing the inside of the microfluidic chip and is provided with a valve, which enables cells to pass through, thereby enabling cells to enter the cell recovery channel 10 from the inlet end.

[0272] The valve has a single-channel dam 17 , which is used to intercept cells and reagents and allows gas to freely enter the cell recovery channel 10 .

[0273] The valve also includes an elastic film 7, which is arranged above the single-channel dam 17. When the elastic film 7 bends upward, the passage between the single-channel dam 17 and the elastic film 7 becomes larger, and the recoil reagent entrains the cells through the single-channel dam 17 and the cell recovery channel 10 to complete cell recovery.

[0274] The microfluidic chip also includes an air path layer 6, which has an air path channel connected to an external air pressure source. The air path layer 6 and the elastic film 7 cooperate to form a pneumatic valve, and the position of the pneumatic valve corresponds to the position of the single-channel dam 17; when negative pressure is applied to the air path channel in the air path layer 6, the elastic film 7 under the air path channel bends upward, so that the passage between the single-channel dam 17 and the elastic film 7 becomes larger, so that the cells flow over the single-channel dam 17 into the cell recovery channel 10 with the solution; when positive pressure is applied to the air path channel in the air path layer 6, the elastic film 7 under the air path channel bends downward, so that the elastic film 7 contacts the single-channel dam 17, thereby closing the cell recovery channel 10 and preventing the reagent from entering the cell recovery channel 10.

[0275] like Figure 10As shown in the figure, the microfluidic chip includes a liquid channel layer 1. The liquid channel layer 1 is provided with liquid channels. A dam 2 is provided at the intersection of two liquid channels. The microfluidic chip further includes a gas channel layer 6 and an elastic film 7 sandwiched between the gas channel layer 6 and the liquid channel layer 1. A pneumatic microvalve is formed by the cooperation of the gas channel layer 6 and the elastic film 7. The pneumatic microvalve is used to control the opening and closing of each liquid channel in the liquid channel layer 1. Principle of the pneumatic microvalve: When a positive pressure is applied to the gas channel in the gas channel layer 6, the elastic film 7 under the gas channel bends downward, squeezing the channel below the elastic film 7; when the positive pressure is removed, the elastic film 7 returns to its original state, thereby realizing the control of the pneumatic microvalve.

[0276] There is a gap between the elastic film 7 and the dam 2.

[0277] The liquid channels include a cell loading channel 8, a cell processing channel 9, a cell recovery channel 10, a first channel 11, a second channel 12, a mixing channel 13, a third channel 14, a backflush channel 15, and a waste liquid channel 16. The pneumatic microvalves include a first pneumatic microvalve V1, a second pneumatic microvalve V2, a third pneumatic microvalve V3, a fourth pneumatic microvalve V4, a fifth pneumatic microvalve V5, a sixth pneumatic microvalve V6, and a seventh pneumatic microvalve V7. The intersections include a first intersection M1, a second intersection M2, and a third intersection M3. The dam 2 is provided at each of the first intersection M1, the second intersection M2, and the third intersection M3. A cell processing area T is provided in the cell processing channel 9.

[0278] The first channel 11 is provided with the first pneumatic microvalve V1, the second channel 12 is provided with the second pneumatic microvalve V2, and the intersection position of the first channel 11 and the second channel 12 forms the first intersection M1.

[0279] One end of the mixing channel 13 is connected to the first intersection M1, the other end of the mixing channel 13 is connected to the cell processing channel 9, the third channel 14 is connected to the mixing channel 13, and the intersection position of the third channel 14 and the mixing channel 13 forms the second intersection M2. The third channel 14 is provided with the third pneumatic microvalve V3.

[0280] The waste liquid channel 16, the cell processing channel 9, and the cell recovery channel 10 are connected in sequence. The waste liquid channel 16 is provided with the seventh pneumatic microvalve V7, and the cell recovery channel 10 is provided with the fifth pneumatic microvalve V5.

[0281] The backflush channel 15 is connected to the waste liquid channel 16, and the intersection position of the backflush channel 15 and the waste liquid channel 16 forms the third intersection M3. The backflush channel 15 is provided with the sixth pneumatic microvalve V6.

[0282] The cell loading channel 8 is connected to the cell processing channel 9, and the cell loading channel 8 is provided with the fourth pneumatic microvalve V4.

[0283] In the gas path layer 6, there are gas path channels corresponding to the positions of the first intersection M1, the second intersection M2, and the third intersection M3 respectively. When negative pressure is applied to the three gas path channels, the elastic film 7 above the first intersection M1, the second intersection M2, and the third intersection M3 bulges, and the reagent passage area increases.

[0284] A single-channel dam 17 is provided in the liquid path channel. The number of single-channel dams 17 is the same as the number of pneumatic microvalves, and one single-channel dam 17 is provided corresponding to each pneumatic microvalve position.

[0285] Such as Figure 11 , 12 As shown in the figure, the transmission module includes a device main body, a liquid storage container 201, a first chip interface, a cell loading container 206, a first interface, and a cell recovery device 213. The liquid storage container 201 is installed on the device main body. One end of the first chip interface is communicated with the liquid storage container 201, and the other end of the first chip interface is used to be communicated with the liquid path channel of the microfluidic chip;

[0286] The cell loading container 206 is installed on the device main body. One end of the first interface is communicated with the cell loading container 206, and the other end of the first interface is used to be communicated with the cell loading channel 8 of the microfluidic chip;

[0287] The cell recovery device 213 is installed on the device main body. The cell recovery device 213 is detachably connected to the device main body, and the cell recovery device 213 is used to be communicated with the cell recovery channel 10 of the microfluidic chip.

[0288] The transmission module further includes a waste liquid hole 202. The waste liquid hole 202 is provided on the device main body, and the waste liquid hole 202 is used to be communicated with the waste liquid channel 16 of the microfluidic chip;

[0289] The transmission module further includes a second chip interface. One end of the second chip interface is communicated with the waste liquid hole 202, and the other end of the second chip interface is used to be communicated with the waste liquid channel 16 of the microfluidic chip.

[0290] The transmission module further includes a first sealing ring. The first sealing ring is installed at the connection between the other end of the first chip interface and the liquid path channel, so as to ensure the airtightness of the connection between the other end of the first chip interface and the liquid path channel.

[0291] The transmission module further includes a second sealing ring and a third sealing ring. The second sealing ring is installed at the connection between the other end of the second chip interface and the waste liquid channel 16, so as to ensure the airtightness of the connection between the other end of the second chip interface and the waste liquid channel 16. The third sealing ring is installed at the connection between the other end of the first interface and the cell loading channel 8, so as to ensure the airtightness of the connection between the other end of the first interface and the cell loading channel 8.

[0292] The transmission module further includes a connection mechanism for connecting the device main body and the microfluidic chip together.

[0293] The device main body is a clamp.

[0294] The clamp includes an upper clamp body 203 and a lower clamp body 205.

[0295] The liquid storage container 201, the cell loading container 206 and the cell recovery device 213 are installed on the upper clamp body 203.

[0296] The microfluidic chip is arranged on the lower clamp body 205.

[0297] The cell loading container 206 is internally provided with a slope.

[0298] The cell loading container 206 is in a funnel shape with a wider upper part and a narrower lower part.

[0299] As Figure 17 and Figure 18 shown, the cell recovery device includes a bracket 207. The device main body is provided with a recovery hole 208. The bracket 207 is provided with a moving part, and the moving part can perform a lifting movement on the bracket 207. The moving part is provided with a connecting part for detachably connecting with a cryogenic tube 211.

[0300] The connecting component provided on the moving component can be a hole, a groove, a clamping mechanism, etc., as long as the cryopreservation tube 211 can be fixed on the connecting component and can be removed from the connecting component. For example, the cryopreservation tube 211 is placed in the hole of the moving component, and the cryopreservation tube 211 is stuck in the hole. At the same time, the cryopreservation tube 211 can also be pulled out from the hole; similarly, the clamping mechanism can clamp the cryopreservation tube 211, and the cryopreservation tube 211 can also be taken out from the clamping mechanism. For example, the connecting component is an opening or a slot provided on the moving component, and the opening or the slot cooperates with the cryopreservation tube 211 to realize the fixation and placement of the cryopreservation tube 211. For example, the connecting component is a through hole provided on the moving component, and the through hole cooperates with the cryopreservation tube 211 to realize the fixation and placement of the cryopreservation tube 211. The through hole can be in a conical shape with a larger upper part and a smaller lower part, so as to cooperate with the cryopreservation tube 211 to realize the fixation and placement of the cryopreservation tube 211. The through hole can also have a shape consistent with the outer shape of the cryopreservation tube 211 for adapting to the cryopreservation tube 211, so as to better fix and place the cryopreservation tube 211.

[0301] The moving component can perform lifting movement on the bracket 207 through a mechanical structure and an artificial method. As a preferred embodiment of the present invention, the microfluidic chip cell recovery device further includes a driving mechanism for driving the moving component to perform lifting movement on the bracket 207.

[0302] In some embodiments, the driving mechanism includes a solenoid valve switch, the moving component is a cantilever 212, the cantilever 212 is connected to the valve core of the solenoid valve switch, and the valve core of the solenoid valve switch drives the cantilever 212 to lift. A cryopreservation tube 211 is placed on the connecting component of the cantilever 212, and the lifting of the cantilever 212 drives the cryopreservation tube 211 to rise, so that the cryopreservation tube 211 is separated from the cell recovery channel.

[0303] The microfluidic chip cell recovery device further includes a control device, and the control device can control the solenoid valve switch. As its first embodiment: the control device can control the solenoid valve switch to perform a lifting action with a predetermined stroke within a predetermined time; as its second embodiment: the control device can control the solenoid valve switch to perform a rising action with a predetermined stroke within a predetermined time.

[0304] The transmission module further includes a second interface. One end of the second interface is communicated with the cell recovery device 213, and the other end of the second interface is used to be communicated with the cell recovery channel 10 of the microfluidic chip.

[0305] The transmission module further includes a third interface. One end of the third interface is communicated with the recovery hole, and the other end of the second chip interface is used to be communicated with the cell recovery channel 10 of the microfluidic chip.

[0306] The transmission module further includes a fourth sealing ring, which is installed at the connection between the other end of the second interface and the cell recovery channel 10, so as to ensure the airtightness of the connection between the other end of the second interface and the cell recovery channel 10;

[0307] The transmission module further includes a fifth sealing ring, which is installed at the connection between the other end of the third interface and the cell recovery channel, so as to ensure the airtightness of the connection between the other end of the third interface and the cell recovery channel 10.

[0308] An air path interface 209 is provided on the device body.

[0309] The device body is provided with an observation window 210.

[0310] The liquid storage container 201 is used to store reagents and, when air pressure is applied, to realize the input of reagents into the liquid path channel of the microfluidic chip.

[0311] During operation, the microfluidic chip is placed in the fixture, fixed tightly by the fixture, a certain amount of reagent is added to the liquid storage container 201, a positive pressure is applied to the liquid storage container 201, the reagent is injected into the liquid path channel of the microfluidic chip, and finally flows out through the waste liquid hole 202.

[0312] The cell loading container 206 is used for cell loading. There is a ramp inside the cell loading container 206. Preferably, the cell loading container 206 is in the shape of a funnel with a wider upper part and a narrower lower part, which is convenient for cells to slide into the first interface and enter the cell loading channel 8 of the microfluidic chip under the action of gravity. During operation, the microfluidic chip is placed in the fixture, fixed tightly by the fixture, a reagent containing cells is added to the cell loading container 206. When the cells enter the cell loading container 206, a positive pressure is applied to the cell loading container 206 or a negative pressure is applied to the waste liquid channel to push or suck the cells into the cell loading channel 8 of the microfluidic chip to complete cell loading.

[0313] During cell recovery, the microfluidic chip is placed in the fixture and fixed tightly by the fixture; the cryotube 211 is installed on the connecting part of the cantilever 212, the cryotube 211 extends into the recovery hole 208. In the non-powered state, the valve core of the solenoid valve switch is in a lower position, and the end of the cryotube 211 is connected to the cell recovery channel of the microfluidic chip; due to capillary action, cells and solution enter the cryotube 211. When the solenoid valve switch is powered on, the valve core drives the cantilever 212 to lift, so that the cryotube 211 is separated from the cell recovery channel and no longer sucks the solution. By controlling the timing of the lifting of the cantilever 212, the volume of the cell recovery solution can be controlled; at this time, the cells are located in the cryotube 211 and are wrapped by a specified small amount of solution; then the cryotube 211 is taken out.

[0314] Such asFigure 13 As shown in Figure 13 , the pressure regulating device includes a switching mode between constant positive pressure and constant negative pressure, a switching mode for finely adjusting the positive pressure and atmospheric pressure, a switching mode for finely adjusting the negative pressure and atmospheric pressure, and a switching mode between constant positive pressure, constant negative pressure, and atmospheric pressure, and a switching mode for finely adjusting positive pressure, finely adjusting negative pressure, and atmospheric pressure.

[0315] The first pneumatic micro-valve V1, the second pneumatic micro-valve V2, the third pneumatic micro-valve V3, the sixth pneumatic micro-valve V6, and the seventh pneumatic micro-valve V7 of the microfluidic chip adopt the switching mode between constant positive pressure and constant negative pressure; for example, when the first pneumatic micro-valve V1 is in the constant positive pressure mode, then the elastic film 7 bends downward to block the liquid passage, and at this time the first pneumatic micro-valve V1 is called closed; when the first pneumatic micro-valve V1 is in the constant negative pressure mode, then the elastic film 7 bends upward to enlarge the liquid passage, and at this time the first pneumatic micro-valve V1 is called open.

[0316] The first channel 11, the second channel 12, and the backflush channel 15 adopt the switching mode for finely adjusting the positive pressure and atmospheric pressure.

[0317] The cell loading channel 8 adopts the switching mode for finely adjusting the positive pressure and atmospheric pressure, or the cell loading channel 8 adopts the switching mode for finely adjusting the negative pressure and atmospheric pressure.

[0318] The fourth pneumatic micro-valve V4 and the fifth pneumatic micro-valve V5 adopt the switching mode between constant positive pressure, constant negative pressure, and atmospheric pressure; for example, when the fifth pneumatic micro-valve V5 is in the constant positive pressure mode, then the elastic film 7 bends downward to block the liquid passage, and at this time the fifth pneumatic micro-valve V5 is called closed; when the fifth pneumatic micro-valve V5 is in the constant negative pressure mode, then the elastic film 7 bends upward to enlarge the liquid passage, and at this time the fifth pneumatic micro-valve V5 is called open; when the fifth pneumatic micro-valve V5 is in the atmospheric pressure mode, then the elastic film 7 neither bends downward nor upward, and the elastic film 7 returns to the planar state, and at this time the fifth pneumatic micro-valve V5 is in the non-pressurized gear.

[0319] The third channel 14 adopts the switching mode for finely adjusting the positive pressure, finely adjusting the negative pressure, and atmospheric pressure.

[0320] The pressure regulating device includes a pneumatic generating module 110, a pneumatic distribution module 120, and a pneumatic output interface 130. The pneumatic distribution module 120 includes a regulating valve. The input end of the regulating valve is communicated with the pneumatic generating module 110 through a pipeline, and the output end of the regulating valve is communicated with the pneumatic output interface 130 through a pipeline.

[0321] The air pressure output interface 130 is docked with the transmission module. The air pressure output interface 130 includes an air pressure output interface board and a cell loading air pressure input interface. Among them, the air pressure output interface board integrates interfaces for docking with the air path interface 209 and the liquid storage container 201; the cell loading air pressure input interface is docked with the cell loading container 206.

[0322] The air pressure generating module 110 includes a constant positive pressure module 111, a constant negative pressure module 112, a positive pressure source module 113, a negative pressure source module 114, a fine-tuning positive pressure module 115, and a fine-tuning negative pressure module 116. The constant positive pressure module 111 is used to generate a constant positive air pressure, the constant negative pressure module 112 is used to generate a constant negative air pressure, the positive pressure source module 113 is used to generate a positive air pressure, the positive pressure source module 113 is connected to the fine-tuning positive pressure module 115, and the fine-tuning positive pressure module 115 is used to adjust the positive air pressure output by the positive pressure source module 113. The negative pressure source module 114 is used to generate a negative air pressure, the negative pressure source module 114 is connected to the fine-tuning negative pressure module 116, and the fine-tuning negative pressure module 116 is used to adjust the negative air pressure output by the negative pressure source module 114.

[0323] The regulating valves include a first regulating valve 121, a second regulating valve 122, a third regulating valve 123, a fourth regulating valve 124, and a fifth regulating valve 125. The air pressure output interface 130 includes a first air pressure output interface, a second air pressure output interface, a third air pressure output interface, a fourth air pressure output interface, and a fifth air pressure output interface;

[0324] For the first regulating valve 121, the first interface of the first regulating valve 121 is communicated with the constant positive pressure module 111, the second interface of the first regulating valve 121 is communicated with the constant negative pressure module 112, the third interface of the first regulating valve 121 is communicated with the first air pressure output interface, and the first regulating valve 121 is used to implement the switching mode between constant positive pressure and constant negative pressure;

[0325] The first interface of the second regulating valve 122 is communicated with the fine-tuning positive pressure module 115, the second interface of the second regulating valve 122 is communicated with the external atmospheric pressure, the third interface of the second regulating valve 122 is communicated with the second air pressure output interface, and the second regulating valve 122 is used to implement the switching mode between fine-tuning positive pressure and atmospheric pressure;

[0326] The first interface of the third regulating valve 123 is communicated with the fine-tuning negative pressure module 116, the second interface of the third regulating valve 123 is communicated with the external atmospheric pressure, the third interface of the third regulating valve 123 is communicated with the third air pressure output interface, and the third regulating valve 123 is used to implement the switching mode between fine-tuning negative pressure and atmospheric pressure;

[0327] The first interface of the fourth regulating valve 124 is communicated with the constant positive pressure module 111, the second interface of the fourth regulating valve 124 is communicated with the constant negative pressure module 112, the third interface of the fourth regulating valve 124 is communicated with the external atmospheric pressure, the fourth interface of the first regulating valve 121 is communicated with the fourth air pressure output interface, and the fourth regulating valve 124 is used to realize the switching modes of constant positive pressure, constant negative pressure, and atmospheric pressure;

[0328] The first interface of the fifth regulating valve 125 is communicated with the fine-tuning positive pressure module 115, the second interface of the fifth regulating valve 125 is communicated with the fine-tuning negative pressure module 116, the third interface of the fifth regulating valve 125 is communicated with the external atmospheric pressure, the fourth interface of the fifth regulating valve 125 is communicated with the fifth air pressure output interface, and the fifth regulating valve 125 is used to realize the switching modes of fine-tuning positive pressure, fine-tuning negative pressure, and atmospheric pressure.

[0329] There are five first air pressure output interfaces. One ends of the five first air pressure output interfaces are connected in parallel with the third interface of the first regulating valve 121, and the other ends of the five first air pressure output interfaces are respectively communicated with the air passage channels of the air passage layer 6 of the first pneumatic micro-valve V1, the second pneumatic micro-valve V2, the third pneumatic micro-valve V3, the sixth pneumatic micro-valve V6, and the seventh pneumatic micro-valve V7, so as to realize the switching of constant positive pressure and constant negative pressure of the first pneumatic micro-valve V1, the second pneumatic micro-valve V2, the third pneumatic micro-valve V3, the sixth pneumatic micro-valve V6, and the seventh pneumatic micro-valve V7;

[0330] There are three second air pressure output interfaces. One ends of the three second air pressure output interfaces are connected in parallel with the third interface of the second regulating valve 122, and the other ends of the three second air pressure output interfaces are respectively communicated with the first channel 11, the second channel 12, and the backflush channel 15;

[0331] The cell loading channel 8 is communicated with the second air pressure output interface, or the waste liquid channel 16 is communicated with the third air pressure output interface;

[0332] There are two fourth air pressure output interfaces. One ends of the two fourth air pressure output interfaces are connected in parallel with the fourth interface of the fourth regulating valve 124, and the other ends of the two fourth air pressure output interfaces are respectively communicated with the air passage channels of the air passage layer 6 of the fourth pneumatic micro-valve V4 and the fifth pneumatic micro-valve V5, so as to realize the switching of constant positive pressure, constant negative pressure, and atmospheric pressure of the fourth pneumatic micro-valve V4 and the fifth pneumatic micro-valve V5;

[0333] The fifth air pressure output interface is communicated with the third channel 14.

[0334] The pressure regulating device of the present utility model realizes complex operations of the microfluidic chip by modularly integrating various air pressure output forms, and at the same time maintains good scalability.

[0335] As Figure 14 shown, the vitrification freezing treatment device further includes an automatic operation device, which includes a robotic arm 501, a camera, and a controller. A pick-and-place mechanism is provided at the end of the robotic arm 501. The pick-and-place mechanism is used to pick up and put down items. The camera is used to collect images of the items, determine what kind of items they are through the images, manipulate the robotic arm 501 to move through the controller, and control the pick-and-place mechanism to complete the pick-up or put-down action through the controller.

[0336] The pick-and-place mechanism can pick up and put down items by means of a suction cup, a magnet, etc. Preferably, the pick-and-place mechanism is a mechanical clamp 502, and the mechanical clamp 502 can adjust the grasping radius and the grasping force.

[0337] The mechanical clamp 502 includes a first clamping plate 503 and a second clamping plate 504. The first clamping plate 503 and the second clamping plate 504 are symmetrically arranged, and the first clamping plate 503 and the second clamping plate 504 can perform opening and closing actions.

[0338] Anti-slip pads are provided on the inner surfaces of the first clamping plate 503 and the second clamping plate 504 to prevent the items from slipping through the anti-slip pads.

[0339] The camera is installed on the robotic arm 501.

[0340] The automatic operation device further includes a machine table 505, and the robotic arm 501 is installed on the machine table 505.

[0341] The automatic operation device further includes a first driving mechanism and a second driving mechanism. The controller is connected to the first driving mechanism, and the controller is used to issue instructions to the first driving mechanism. The first driving mechanism drives the robotic arm 501 to move; the controller is connected to the second driving mechanism, and the controller is used to issue instructions to the second driving mechanism. The second driving mechanism drives the pick-and-place mechanism to complete the pick-up or put-down action.

[0342] The first driving mechanism includes a first motor and a first transmission mechanism. The controller is connected to the first motor, the first motor is connected to the first transmission mechanism, and the first transmission mechanism is connected to the robotic arm 501; the second driving mechanism includes a second motor and a second transmission mechanism. The controller is connected to the second motor, the second motor is connected to the second transmission mechanism, and the second transmission mechanism is connected to the pick-and-place mechanism.

[0343] The objects that the mechanical clamp 502 can grasp include: reagent bottles, pressure regulating interfaces, microscopic modules, and cryotubes.

[0344] During operation, the robotic arm 501 is in the default position. Place the microfluidic chip on the workbench and start the cell processing procedure. According to the requirements of step (3), the robotic arm 501 and the mechanical clamp 502 sequentially: grasp the reagent bottle, pour the reagent into the corresponding liquid storage container 201, and then put the reagent bottle back; grasp the pneumatic output interface 130, connect it to the pneumatic interface 209 of the transmission module, and then return to the original position; grasp the pneumatic output interface 130 and connect it to the cell loading container 206, and then return to the original position; grasp the cryotube, place it into the cell recovery device 213, and then return to the original position. After the cells enter the microfluidic chip, grasp the microscopic module, position it to a specific field of view as needed, and adjust the height in the z-axis direction to complete microscopic focusing. After the cells leave the microfluidic chip, put the microscopic module back and then return to the original position.

[0345] The automated operation device of the present utility model can identify objects and perform corresponding object handling operations, realizing the automated operation of each component, with high efficiency and meeting user requirements.

[0346] As Figure 15 shown, the microscopic module includes a mechanical positioning device, a lens 301, and a camera 302 located above the lens 301. The mechanical positioning device is used to move the microscopic module and / or the workbench 300, so as to realize the relative displacement of the microscopic module and the workbench 300 in the x, y, and z directions. Among them, the movement in the x and y directions is used to track the cell displacement, and the z-axis direction is used for microscopic focusing.

[0347] The mechanical positioning device can realize the relative displacement of the microscopic module and the workbench 300 in the x, y, and z directions by moving either the microscopic module or the workbench 300.

[0348] The mechanical positioning device is a robotic arm 501, an electric guide rail, other mechanical devices that can perform positioning functions, and combinations of these devices.

[0349] The lens 301 is a lens with adjustable magnification, so as to expand the observation range or improve the ability to observe details as needed.

[0350] The lens 301 can also be a group, switching between different magnifications or different technologies; for specific technologies, corresponding optical components are also included.

[0351] The microscopic module further includes a light source, and the light source, the lens 301, and the first camera 302 are connected as a whole.

[0352] The microscopic module is perpendicular to the workbench 300.

[0353] The utility model collects cell images through a lens 301 and a first camera 302, judges the cell image information through image recognition software, and drives the microscope module and / or the workbench 300 to move through a mechanical positioning device, so as to realize microscopic tracking observation of cells in a microfluidic chip.

[0354] The microscope module has a large field of view and is used to track and observe whether the cells safely reach the designated position.

[0355] The microscope module further includes an objective lens, a piezoelectric objective lens positioner, an inverted DIC microscopic imaging system, a second light source and a second camera. The piezoelectric objective lens positioner is responsible for realizing the autofocus function.

[0356] The field of view of the microscope module is fixed in the cell processing area, has a high magnification and resolution, and cooperates with DIC microscopic imaging technology, so that the details of the collected images are richer.

[0357] The vitrification freezing treatment device further includes an AI module, which makes real-time decisions based on the data collected during the operation process, and outputs instructions to the machine in subsequent steps or provides reference suggestions to the operator.

[0358] The AI module includes: an item recognition algorithm, a cell recognition algorithm, and a cell processing parameter algorithm.

[0359] Among them, the item recognition algorithm is based on the image data collected by the camera in the automatic operation device to assist the robotic arm 501 to grab objects; the cell recognition algorithm gives a judgment on the cell state based on the image data collected by the microscope module, and assists in completing automatic cell manipulation, including autofocus; the cell processing parameter algorithm is based on the image data collected by the microscope module during the cell processing process, and gives cell processing parameters as a reference according to the change characteristics of the cell shape during the processing process.

[0360] The vitrification freezing treatment device further includes a controller, which is used to control the electromagnetic switch in the transmission module, the air pressure source switch in the pressure regulating device, the solenoid valve group switch group, the robotic arm 501 in the automatic operation device, and the piezoelectric objective lens positioner in the microscope module. The industrial computer is used to receive image data, run software and algorithms, and issue instructions to the controller.

[0361] The above content is a further detailed description of the present utility model in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present utility model is only limited to these descriptions. For those of ordinary skill in the technical field to which the present utility model belongs, without departing from the concept of the present utility model, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present utility model.

Claims

1. A vitrification freezing treatment device with a microfluidic chip, Characterized in that: The microfluidic chip includes a liquid path layer (1); Liquid path channels, the liquid path channels are arranged in the liquid path layer (1), and the reagents in the microfluidic chip flow through the liquid path channels. The liquid path channels have a main channel (4) and an auxiliary channel (5) that cross; The microfluidic chip further includes an interception structure for intercepting the reagents and / or cells in the liquid path channels, and the interception structure is arranged at the intersection of the two liquid path channels.

2. The vitrification freezing treatment device with a microfluidic chip according to claim 1, Characterized in that: The vitrification freezing treatment device further includes a microscopy module.

3. The vitrification freezing treatment device with a microfluidic chip according to claim 1, Characterized in that: The vitrification freezing treatment device further includes a transmission module, and the vitrification freezing treatment device further includes a pressure regulating device.

4. The vitrification freezing treatment device with a microfluidic chip according to claim 1, Characterized in that: The interception structure is a dam body (2).

5. The vitrification freezing treatment device with a microfluidic chip according to claim 4, Characterized in that, The dam body (2) is provided with a groove (3).

6. The vitrification freezing treatment device with a microfluidic chip according to claim 4, Characterized in that, The dam body (2) is arranged at the bottom end of the intersection.

7. The vitrification freezing treatment device with a microfluidic chip according to claim 4, Characterized in that, The upper end height of the dam body (2) does not exceed the height of the main channel (4), and the height of the dam body (2) accounts for more than 50% of the height of the main channel (4).

8. The vitrification freezing treatment device with a microfluidic chip according to claim 7, Characterized in that: The height of the dam body (2) accounts for 85 - 95% of the height of the main channel (4).

9. The vitrification freezing treatment device with a microfluidic chip according to claim 4, Characterized in that: The dam body (2) and the liquid path channels of the microfluidic chip are an integral structure.

10. The vitrification freezing treatment device with a microfluidic chip according to claim 4, Characterized in that: One side of the dam body (2) has two feet, and a groove (3) is formed between the two feet. The groove (3) is located at a position in the microfluidic chip facing the auxiliary channel (5).

11. The vitrification freezing treatment device with a microfluidic chip according to claim 10, Characterized in that: The width between the two feet of the dam body (2) exceeds half of the width of the entire dam body (2).

12. The vitrification freezing treatment device with a microfluidic chip according to claim 1, Characterized in that: An elastic film (7) is further arranged on the microfluidic chip, and the elastic film (7) is arranged on the end face of the microfluidic chip on the opening side of the main channel (4) and the auxiliary channel (5).

13. The vitrification freezing treatment device with a microfluidic chip according to claim 12, Characterized in that: There is a gap between the elastic film (7) and the interception structure.

14. The vitrification freezing treatment device with a microfluidic chip according to claim 12, Characterized in that: The thickness of the elastic film (7) is 50 to 250 micrometers.

15. The vitrification freezing treatment device with a microfluidic chip according to claim 14, characterized in that: The thickness of the elastic film (7) is 75 to 150 micrometers.

16. The vitrification freezing treatment device with a microfluidic chip according to any one of claims 12 to 15, characterized in that: The microfluidic chip further includes a gas path layer (6), the gas path layer (6) has a gas path channel connected to an external air pressure source, and the gas path layer (6) and the elastic film (7) cooperate to form a pneumatic microvalve.

17. The vitrification freezing treatment device with a microfluidic chip according to claim 16, characterized in that: The gas path layer (6) is provided with a recess in the corresponding area at the intersection, and an external air pressure source is connected to the recess.

18. The vitrification freezing treatment device with a microfluidic chip according to claim 10 or 11, characterized in that: The two feet are respectively a first foot (31) and a second foot (32), the height of the first foot (31) is lower than the height of the second foot (32), the first foot (31) is located on the side where reagent 1 flows in, and the second foot (32) is located on the side where reagent 1 and reagent 2 converge and flow out.

19. The vitrification freezing treatment device with a microfluidic chip according to claim 1, characterized in that: The liquid path layer (1) is provided with a cell recovery channel (10), and a valve is provided at the inlet end of the cell recovery channel (10) facing the inside of the microfluidic chip.

20. The vitrification freezing treatment device with a microfluidic chip according to claim 16, characterized in that: The gas path layer (6) is provided with a gas path channel corresponding to the position of the intersection.

21. The vitrification freezing treatment device with a microfluidic chip according to claim 16, characterized in that: The gas path layer (6) is respectively provided with gas path channels corresponding to the positions of the first intersection (M1), the second intersection (M2) and the third intersection (M3).

22. The vitrification freezing treatment device with a microfluidic chip according to claim 3, characterized in that: The transmission module includes a device main body, a liquid storage container (201), a first chip interface, a cell loading container (206), a first interface, and a cell recovery device (213). The liquid storage container (201) is installed on the device main body, one end of the first chip interface is communicated with the liquid storage container (201), and the other end of the first chip interface is used for communicating with the liquid path channel of the microfluidic chip; The cell loading container (206) is installed on the device main body, one end of the first interface is communicated with the cell loading container (206), and the other end of the first interface is used for communicating with the cell loading channel (8) of the microfluidic chip; The cell recovery device (213) is installed on the device main body, the cell recovery device (213) is detachably connected to the device main body, and the cell recovery device (213) is used for communicating with the cell recovery channel (10) of the microfluidic chip.

23. The vitrification freezing treatment device with a microfluidic chip according to claim 22, It is characterized in that: The transmission module further includes a waste liquid hole (202) provided on the device main body, and the waste liquid hole (202) is used to communicate with the waste liquid channel (16) of the microfluidic chip.

24. The vitrification freezing treatment device with a microfluidic chip according to claim 23, It is characterized in that: The transmission module further includes a second chip interface. One end of the second chip interface communicates with the waste liquid hole (202), and the other end of the second chip interface is used to communicate with the waste liquid channel (16) of the microfluidic chip.

25. The vitrification freezing treatment device with a microfluidic chip according to claim 3, It is characterized in that: The pressure regulating device includes a pneumatic generating module (110), a pneumatic distribution module (120), and a pneumatic output interface (130); The pneumatic distribution module (120) includes a regulating valve. The input end of the regulating valve is communicated with the pneumatic generating module (110) through a pipeline, and the output end of the regulating valve is communicated with the pneumatic output interface (130) through a pipeline; The pneumatic output interface (130) is used to connect to the transmission module.

26. The vitrification freezing treatment device with a microfluidic chip according to claim 25, It is characterized in that: The pneumatic generating module (110) includes a constant positive pressure module (111), a constant negative pressure module (112), a positive pressure source module (113), a negative pressure source module (114), a fine-tuning positive pressure module (115), and a fine-tuning negative pressure module (116). The constant positive pressure module (111) is used to generate a constant positive air pressure, the constant negative pressure module (112) is used to generate a constant negative air pressure, the positive pressure source module (113) is used to generate a positive air pressure, the positive pressure source module (113) is connected to the fine-tuning positive pressure module (115), and the fine-tuning positive pressure module (115) is used to adjust the positive air pressure output by the positive pressure source module (113). The negative pressure source module (114) is used to generate a negative air pressure, the negative pressure source module (114) is connected to the fine-tuning negative pressure module (116), and the fine-tuning negative pressure module (116) is used to adjust the negative air pressure output by the negative pressure source module (114).

27. The vitrification freezing treatment device with a microfluidic chip according to claim 1, It is characterized in that: It further includes an automatic operation device. The automatic operation device includes a robotic arm (501), a camera, and a controller. A picking and placing mechanism is provided at the end of the robotic arm (501). The picking and placing mechanism is used to pick up and place items. The camera is used to collect images of the items, determine what kind of items they are through the images, control the movement of the robotic arm (501) through the controller, and control the picking and placing mechanism to complete the picking or placing action through the controller.

28. The vitrification freezing treatment device with a microfluidic chip according to claim 27, It is characterized in that: The picking and placing mechanism is a mechanical clamp (502).

29. The vitrification freezing treatment device with a microfluidic chip according to claim 27, It is characterized in that: The camera is installed on the robotic arm (501).

30. The vitrification freezing treatment device with a microfluidic chip according to claim 2, characterized in that: the microscopic module includes a mechanical positioning device, a lens (301), and a first camera (302) located above the lens (301), and the mechanical positioning device is used to move the microscopic module and / or the workbench (300).

31. The vitrification freezing treatment device with a microfluidic chip according to claim 30, characterized in that: the mechanical positioning device is a robotic arm (501) or an electric guide rail.

32. The vitrification freezing treatment device with a microfluidic chip according to claim 30, characterized in that: the microscopic module further includes a first light source, and the first light source, the lens (301), and the first camera (302) are connected as a whole.

33. The vitrification freezing treatment device with a microfluidic chip according to claim 30, characterized in that: the microscopic module further includes an objective lens, a piezoelectric objective lens positioner, an inverted DIC microscopic imaging system, a second light source, and a second camera.

34. The vitrification freezing treatment device with a microfluidic chip according to claim 27, characterized in that: the vitrification freezing treatment device further includes an AI module, and the AI module includes an item recognition algorithm, a cell recognition algorithm, and a cell processing parameter algorithm, the item recognition algorithm is based on the image data collected by the camera in the automated operation device and assists the robotic arm (501) in grasping an object; the cell recognition algorithm gives a judgment on the cell state based on the image data collected by the microscopic module and assists in completing automated cell manipulation; the cell processing parameter algorithm is based on the image data collected by the microscopic module during the cell processing process, and gives cell processing parameters as a reference according to the change characteristics of the cell shape during the processing process.

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