System for thawing recovery of biological material and method thereof

Abstract

The application discloses a system and a method for thawing and recovering biomaterials, wherein the system comprises a carrier, a dish container and a chip. The carrier is provided with a groove near the front end, and the groove is filled with biomaterials to be thawed and a cryoprotective agent. The dish container is filled with an oil-phase thawing solution, and the groove of the carrier is immersed in the oil-phase thawing solution, so that the oil-phase thawing solution covers the groove of the carrier and thaws and recovers the biomaterials. The chip is packaged with chemicals containing the cryoprotective agent and / or a basic culture solution. The carrier is removed from the oil-phase thawing solution, and the chip covers the carrier. The chip is moved relative to the carrier, and the chemicals fully cover and contact the groove to remove the cryoprotective agent in the groove. The application can effectively standardize and automate the whole thawing and recovering process, realize effective quality control, increase processing efficiency, and reduce personnel training requirements.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to a system for thawing and recovering biological materials and a method thereof. BACKGROUND

[0002] Cell freezing preservation technology is an important technology in many biological fields, and it is also important to restore cells from a low-temperature frozen state to a normal metabolic state. The thawing and recovery process includes the process of removing and replacing the cryoprotective agent with normal culture medium.

[0003] The existing technology has high requirements for thawing and recovery processing, and it is a tedious, difficult, highly focused, stressful and difficult to standardize work. SUMMARY

[0004] The main purpose of the present application is to provide a system for thawing and recovering biological materials and a method thereof, so as to solve the problem of low efficiency of manual thawing and recovery in the prior art.

[0005] According to an embodiment of the present application, a system for thawing and recovering biological materials is provided, which comprises: a carrier, which is provided with a groove near the front end, and the groove contains biological materials to be thawed and a cryoprotective agent; a dish container, which contains an oil phase thawing solution, the oil phase thawing solution is used to cover the groove of the carrier invading therein and thaw and warm the biological materials; a chip, which is packaged with chemicals, the chemicals contain a cryoprotective agent and / or a basic culture solution; the chip moves relative to the carrier, and the chemicals fully cover and contact the groove to remove the cryoprotective agent in the groove.

[0006] The specific embodiments can include one or more of the following. The chemical contains a non-permeable cryoprotectant. The chemical includes thawing solution, dilution solution and washing solution made in the form of hydrogel; the chip is provided with multiple intervals for sequentially embedding the thawing solution hydrogel, the dilution solution hydrogel and the washing solution hydrogel, wherein the coverage area of each interval is greater than the opening area of the groove; when the chip moves relative to the carrier, the thawing solution hydrogel, the dilution solution hydrogel and the washing solution hydrogel sequentially move above the groove and sequentially make full coverage contact with the groove. The chemical includes thawing solution, dilution solution and washing solution in the form of solution; the chip is provided with a permeable film supporting the thawing solution, the dilution solution and the washing solution in the form of solution; wherein the permeable film is a perforated film, a mesh, a dialysis film or a water-soluble film. Before the chip moves relative to the carrier, the chemical covers part of the area of the carrier and does not cover the groove of the carrier, and there is a gap distance between the chemical and the groove of the carrier. The middle region of the substrate is used to place the carrier, and the length direction of the substrate is provided with two parallel tracks, which provide support and fixation for the chip, so that the lower surface of the chip keeps in contact with the upper surface of the carrier.

[0007] According to the embodiments of the present application, a system for thawing and recovering biological materials is also provided, which includes: a first carrier containing biological materials to be thawed and a cryoprotectant; a dish container containing a thawing solution, which is used to cover the first carrier and thaw and recover the biological materials to make the biological materials separate from the first carrier; a second carrier provided with a groove near the front end, and the groove of the second carrier contains a first dilution solution; a capillary for transferring the biological materials to the groove of the second carrier; a chip encapsulating chemicals containing a cryoprotectant and / or a basic culture solution; the chip moves relative to the second carrier, and the chemicals make full coverage contact with the groove of the second carrier to remove the cryoprotectant in the groove.

[0008] In some embodiments, the chemical composition comprises a non-permeable cryoprotectant. In some embodiments, the chemical composition comprises a second diluent and a washing solution, the second diluent comprising a non-permeable cryoprotectant at a concentration lower than that of the first diluent. In some embodiments, the chip is provided with a plurality of sections for sequentially embedding the second diluent hydrogel and the washing solution hydrogel, wherein each section has an area larger than the opening area of the groove. In some embodiments, the second diluent hydrogel and the washing solution hydrogel sequentially move above the groove and fully cover the groove when the chip moves relative to the carrier. In some embodiments, the chemical composition comprises a second diluent and a washing solution in solution form, wherein the second diluent comprises a non-permeable cryoprotectant at a concentration lower than that of the first diluent. In some embodiments, the chip is provided with a permeable membrane supporting the second diluent and the washing solution in solution form, wherein the permeable membrane is a perforated membrane, a mesh, a dialysis membrane, or a water-soluble membrane. In some embodiments, the chemical composition covers part of the second carrier and does not cover the groove of the second carrier before the chip moves relative to the second carrier, and there is a gap distance between the chemical composition and the groove of the second carrier. In some embodiments, the base is provided with two parallel tracks in the length direction, which support and fix the chip so that the lower surface of the chip is in contact with the upper surface of the second carrier.

[0009] According to some embodiments of the present application, a method for thawing and recovering biological materials is provided, which comprises: providing a carrier, the carrier being provided with a groove near the front end, the groove containing biological materials to be thawed and a cryoprotectant; immersing the groove of the carrier in an oil-phase thawing solution, the oil-phase thawing solution covering the groove of the carrier and thawing and recovering the biological materials; providing a chip encapsulating a chemical composition, the chemical composition comprising a cryoprotectant and / or a basic culture solution; removing the carrier from the oil-phase thawing solution and covering the carrier with the chip; moving the chip relative to the carrier, the chemical composition fully covering the groove to remove the cryoprotectant in the groove.

[0010] Particular embodiments can include one or more of the following. The chemical contains a non-permeable cryoprotectant. The chemical covers a portion of the carrier and does not cover the well of the carrier before the chip moves relative to the carrier, with a gap distance between the chemical and the well of the carrier. The chemical includes a thawing solution, a dilution solution, and a washing solution made in the form of a hydrogel; the chip is provided with a plurality of intervals for sequentially embedding the thawing solution hydrogel, the dilution solution hydrogel, and the washing solution hydrogel, wherein each interval has a coverage area greater than the opening area of the well; when the chip moves relative to the carrier, the thawing solution hydrogel, the dilution solution hydrogel, and the washing solution hydrogel sequentially move above the well and sequentially make full coverage contact with the well. The chemical includes a thawing solution, a dilution solution, and a washing solution in the form of a solution; the chip is provided with a permeable membrane that supports the thawing solution, the dilution solution, and the washing solution in the form of a solution; wherein the permeable membrane is a perforated membrane, a mesh, a dialysis membrane, or a water-soluble membrane.

[0011] According to an embodiment of the present application, a method for thawing and recovering biological material is also provided, which includes: providing a first carrier containing biological material to be thawed and a cryoprotectant; immersing the first carrier in a thawing solution that covers the first carrier and thaws and warms up the biological material; providing a second carrier with a well near the front end, the well of the second carrier containing a first dilution solution; transferring the biological material to the well of the second carrier; providing a chip encapsulating a chemical, so that the chip covers the second carrier; wherein the chemical contains a cryoprotectant and / or a basic culture solution; moving the chip relative to the second carrier, so that the chemical makes full coverage contact with the well of the second carrier to remove the cryoprotectant in the well.

[0012] In some embodiments, the chemical includes a non-permeable cryoprotectant. In some embodiments, the chemical covers a portion of the second carrier and does not cover the recess of the second carrier before the chip moves relative to the carrier, and the chemical has a gap distance from the recess of the second carrier. In some embodiments, the chemical includes a second diluent and a washing solution, the second diluent including a non-permeable cryoprotectant at a concentration lower than the first diluent. In some embodiments, the chip is provided with a plurality of intervals for sequentially embedding the second diluent and the washing solution, each interval having a coverage area greater than an opening area of the recess. In some embodiments, the second diluent and the washing solution sequentially move over the recess and sequentially fully cover the recess when the chip moves relative to the carrier. In some embodiments, the chemical includes a second diluent and a washing solution in solution form, the second diluent including a non-permeable cryoprotectant at a concentration lower than the first diluent. In some embodiments, the chip is provided with a permeable membrane supporting the second diluent and the washing solution in solution form, the permeable membrane being a perforated membrane, a mesh, a dialysis membrane, or a water-soluble membrane.

[0013] According to embodiments of the present disclosure, a method for thawing and recovering biological materials is also provided. The method includes: preparing a chemical into a hydrogel structure, wherein the chemical includes a cryoprotectant and / or a basic culture solution; and contacting a biological material including a cryoprotectant with the hydrogel to diffuse the cryoprotectant from the hydrogel to remove the cryoprotectant.

[0014] In some embodiments, the chemical solution comprises a non-permeable cryoprotectant. In some embodiments, the agarose solution is prepared by dissolving agarose in the chemical solution at a temperature of 80-90 °C to obtain an agarose solution at a concentration of 0.1-6%. In some embodiments, the agarose gel is prepared by stirring, mixing, cooling, and solidifying the agarose solution. In some embodiments, the sodium alginate solution is prepared by dissolving sodium alginate in the chemical solution to obtain a sodium alginate solution at a concentration of 0.3-10%. In some embodiments, the calcium chloride solution is prepared by dissolving calcium chloride in the chemical solution to obtain a calcium chloride solution at a concentration of 0.01M-0.2M. In some embodiments, the calcium alginate hydrogel is prepared by adding the calcium chloride solution to the sodium alginate solution on the chip, and solidifying the sodium alginate solution into calcium alginate hydrogel after the calcium chloride diffuses to the bottom of the chip. In some embodiments, the gelatin solution is prepared by dissolving gelatin in the chemical solution at a temperature of 36-45 °C to obtain a gelatin solution at a concentration of 1-15%. In some embodiments, the gelatin hydrogel is prepared by adding the gelatin solution at a temperature of 36-50 °C to the chip, and solidifying the gelatin solution into gelatin hydrogel after the gelatin solution cools down. In some embodiments, the GelMA solution is prepared by dissolving GelMA in the chemical solution at a temperature of 36-50 °C to obtain a GelMA solution at a concentration of 1-15%. In some embodiments, the GelMA hydrogel is prepared by dissolving a photoinitiator in the GelMA solution at a temperature of 36-50 °C at a concentration of 0.001-2%, and irradiating the GelMA solution at a temperature of 36-50 °C for 5-30 minutes to solidify the GelMA solution into GelMA hydrogel. In some embodiments, the chemical solution comprises one or more of a thawing solution, a dilution solution, or a washing solution.

[0015] According to the technical solution of the present application, after the biological material such as an embryo is thawed, a chip encapsulating a chemical is covered on a carrier carrying the embryo, so that the chemical fully contacts the groove of the carrier to remove the cryoprotectant in the groove. The present application can effectively standardize and automate the entire thawing process, achieve effective quality control, increase processing efficiency, and reduce personnel training requirements. BRIEF DESCRIPTION OF DRAWINGS

[0016] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0017] Figure 1 is a flowchart of a method for thawing a biological material according to an embodiment of the present application;

[0018] Figure 2 is a flowchart of a method for thawing a biological material according to another embodiment of the present application;

[0019] Figure 3 is a flowchart of a method for thawing a biological material according to still another embodiment of the present application;

[0020] Figure 4is a flow chart of a method of thawing and recovering biological material according to another embodiment of the present application;

[0021] Figure 5 is a schematic diagram of a carrier and dish container according to an embodiment of the present application;

[0022] Figure 6 is a schematic diagram of a carrier according to an embodiment of the present application;

[0023] Figure 7 is a schematic diagram of a chip according to an embodiment of the present application;

[0024] Figure 8 is a schematic diagram of a biological material thawing and recovery system according to an embodiment of the present application;

[0025] Figure 9 is a schematic diagram of a base according to an embodiment of the present application;

[0026] Figure 10 is a schematic diagram of a biological material thawing and recovery system according to another embodiment of the present application;

[0027] Figure 11 is a schematic diagram of a biological material thawing and recovery system according to yet another embodiment of the present application. DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described below in detail with reference to the embodiments of the present application and the corresponding drawings. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0029] The technical solutions provided by the embodiments of the present application will be described in detail below with reference to the drawings.

[0030] Reference Figure 1 According to the method of thawing and recovering biological material according to the embodiments of the present application, the method specifically comprises the following steps:

[0031] Step S102, providing a carrier, the carrier is provided with a recess near the front end, the recess contains biological material to be thawed and a cryoprotective agent.

[0032] The carrier carrying the biological material is stored in a low-temperature liquid nitrogen environment (e.g., -196 degrees), and a cryoprotectant is provided near or around the biological material to protect the biological material. The biological material can be cells, biological tissues, oocytes or embryos, and the cryoprotectant can be a vitrification solution (VS) for protecting the biological material.

[0033] In step S104, the recess of the carrier is immersed in an oil-phase thawing solution, and the oil-phase thawing solution covers the recess of the carrier and thaws the biological material. The oil-phase thawing solution does not contain a non-permeable cryoprotectant.

[0034] The carrier is quickly moved from the low-temperature environment to a dish container containing the oil-phase thawing solution, and the recess on the carrier must be immersed in the oil-phase thawing solution to ensure the thawing speed. Because the density of the oil-phase thawing solution is lower than that of the cryoprotectant, the cryoprotectant in the recess will stay in the recess together with the embryo and will not float out of the recess. The purpose of this step is to quickly thaw the embryo / oocyte in the recess.

[0035] In step S106, a chip containing chemicals is provided, and the chemicals contain a cryoprotectant and / or a basic culture solution.

[0036] In the embodiments of the present application, the concentration of the cryoprotectant contained in the chemicals is lower than the concentration of the cryoprotectant in the recess. In actual applications, the cryoprotectant contained in the chemicals can be a non-permeable cryoprotectant or a permeable cryoprotectant. Specifically, when the chemicals are used to remove the cryoprotectant in the embryo, the chemicals can contain a cryoprotectant and / or a basic culture solution, or the chemicals can contain a non-permeable cryoprotectant and / or a basic culture solution. To achieve better removal effect, the chemicals can contain a non-permeable cryoprotectant and / or a basic culture solution. The main reason is that the non-permeable cryoprotectant will not pass through the cell membrane and is generally a non-cytotoxic molecule. The permeable cryoprotectant is generally a small molecule with cytotoxicity. In thawing applications, the non-permeable cryoprotectant is used to ensure that the osmotic pressure difference between the inside and outside of the cell is not too large. That is, when the cell is taken out of the liquid nitrogen, the inside of the cell is filled with a permeable cryoprotectant, resulting in a high osmotic pressure in the cell. If the cell is directly placed in the culture medium during thawing, the osmotic pressure difference will cause water molecules to enter the cell at a very fast speed, resulting in cell death. However, if the cell is placed in a solution containing a non-permeable cryoprotectant during thawing, the water molecules will enter the cell at a slower speed due to the small osmotic pressure difference, and the permeable cryoprotectant in the cell will diffuse out of the cell, achieving the purpose of slowly removing the cryoprotectant.

[0037] The types of the chemicals include thawing solution, dilution solution and washing solution. In the embodiments of the present application, the chemicals can be in the form of solid state made of hydrogel or in the form of solution.

[0038] In the case of the chemicals in solid state, the thawing solution, the dilution solution and the washing solution are embedded in the chip in the form of hydrogel, i.e. thawing hydrogel (TS gel), dilution hydrogel (DS gel) and washing hydrogel (WS gel). The hydrogel is a solid material whose main component is liquid. Because of the three-dimensional cross-linked network in the gel, the hydrogel is non-flowing. The hydrogel can be soft or hard. The liquid in the gel contains non-permeable cryoprotectant and / or basal medium. Specifically, the thawing hydrogel contains basal medium and non-permeable cryoprotectant with very high concentration, for example, 0.5-1.5M non-permeable cryoprotectant. The dilution hydrogel contains basal medium and non-permeable cryoprotectant with lower concentration than the thawing hydrogel, for example, 0.1-0.75M non-permeable cryoprotectant. The washing hydrogel contains only basal medium.

[0039] Specifically, three intervals can be arranged on the chip in sequence, and the thawing hydrogel, the dilution hydrogel and the washing hydrogel are embedded in the intervals in sequence, wherein the coverage area of each interval is greater than the opening area of the groove of the carrier. In addition, four intervals can also be arranged on the chip in sequence, and the extra interval is used to embed a piece of dilution hydrogel, wherein the concentration of non-permeable cryoprotectant contained in the second piece of dilution hydrogel is lower than that contained in the first piece of dilution hydrogel. Because the change of osmotic pressure is relatively slow, the embryo can be less damaged by osmotic pressure.

[0040] In the case of the chemicals in solution form, the thawing solution, the dilution solution and the washing solution are enclosed in the chip by permeable membrane in the form of solution. The concentration of non-permeable cryoprotectant and basal medium contained in the chemicals in solution form is similar to that of the hydrogel, which is not described here.

[0041] Specifically, three intervals can be arranged in sequence on the chip, and the thawing solution, the dilution solution and the washing solution can be enclosed in the intervals in sequence, wherein the coverage area of each interval is greater than the opening area of the groove. In addition, four intervals can be arranged in sequence on the chip, and the extra interval is used to enclose the second dilution solution, wherein the non-permeable cryoprotectant concentration contained in the second dilution solution is lower than the non-permeable cryoprotectant concentration contained in the first dilution solution, so that the embryo can be less damaged by the change of the osmotic pressure. In practical application, a perforated membrane, a mesh or a dialysis membrane with a suitable thickness and pore size can be arranged on the lower surface of the chip, and the solution can be directly added to the interval, and the solution can be fixed in the chip by the support of the perforated membrane, the mesh, the dialysis membrane or the water-soluble film.

[0042] Step S108, the carrier is removed from the oil phase thawing solution, and the chip covers the carrier.

[0043] When the rewarming is completed (generally 2-3 seconds in the oil phase thawing solution), the chip packaged with chemicals is placed on the carrier. In order to achieve better diffusion removal effect, the thawing solution, the dilution solution and the washing solution in the form of hydrogel or solution cover part of the area of the carrier, that is, do not cover the groove of the carrier, and there is a gap distance of several millimeters between the thawing solution, the dilution solution and the washing solution and the groove of the carrier.

[0044] Step S110, the chip is moved relative to the carrier, and the chemicals fully cover the groove to remove the cryoprotectant in the groove.

[0045] In the case of fixing the chemicals in the form of solid hydrogel in the chip, when the chip is moved relative to the carrier, the thawing hydrogel, the dilution hydrogel and the washing hydrogel embedded in the chip are moved in sequence above the groove of the carrier and fully contact the biological material to be thawed in the groove in sequence. Since the chemical molecules can freely diffuse in the gel, when the groove on the carrier is contacted, the embryo and the cryoprotectant in the groove will diffuse into the hydrogel, achieving the function of removing the cryoprotectant. Specifically, under the thawing hydrogel, the embryo can remove the high-concentration toxic permeable cryoprotectant in the embryo under the lower osmotic pressure difference. Under the dilution hydrogel, water can slowly diffuse into the embryo again, and the non-permeable cryoprotectant molecules around the embryo are sequentially removed (too fast will cause the embryo to be damaged by the osmotic pressure). Under the washing hydrogel, more water can slowly diffuse into the embryo again, and the non-permeable cryoprotectant molecules around the embryo are completely removed, and the embryo is returned to the normal osmotic pressure environment again. After the embryo is treated by the three kinds of hydrogels, the embryo does not contain any cryoprotectant, and has been sequentially restored to the normal osmotic pressure environment, and the embryo can be transferred from the carrier to the culture dish and cultured in the incubator.

[0046] In the case of fixing the chemical in the form of solution in the chip, the covering of the groove by the permeable membrane is formed to avoid the embryo overflow, and the solution penetration is completed when the solution on both sides of the permeable membrane is contacted, and the diffusion exchange of the solution on both sides is completed, which can be referred to the embodiment of hydrogel.

[0047] In summary, no matter the chemical is in solid or liquid state, the embryo can be blocked in the groove while the diffusion exchange of the chemical in the groove is completed, and the chemical does not flow out of the chip. After the whole removal process is completed, the embryo is in the groove of the carrier, the position of the embryo can be quickly determined and the embryo can be captured, the embryo can be transferred from the carrier to the culture dish and cultured in the incubator for recovery. Since the medium composition of the cleaning hydrogel (or cleaning solution) is not necessarily the same as that of the user, the user can put the embryo transferred from the carrier into the user's culture medium for the last rinse, and then transfer it to the user's culture system for culture and recovery.

[0048] In Figure 1 In the embodiment shown, oil phase thawing solution is used for thawing and warming. In other embodiments, non-oil phase thawing solution, i.e., general thawing solution, can also be used for thawing and warming. Referring to Figure 2 The method for thawing and recovering biological material according to the embodiment of the present application comprises the following steps:

[0049] Step S202, providing a first carrier, the first carrier containing biological material to be thawed and cryoprotectant.

[0050] The first carrier is used to carry the biological material preserved in a low-temperature liquid nitrogen environment, and the cryoprotectant is used to protect the biological material in the vicinity or around the biological material, wherein the biological material can be cells, biological tissues, oocytes or embryos, and the cryoprotectant can be vitrification cryoprotectant. For simplicity, the biological material is described by taking the embryo as an example in the following.

[0051] In some embodiments, the front end of the first carrier can have a groove, and the biological material is contained in the groove of the first carrier.

[0052] Step S204, immersing the first carrier into thawing solution, the thawing solution covering the first carrier and thawing and warming the biological material.

[0053] The first carrier is quickly moved from the low-temperature environment to a dish container containing the thawing solution, so that the embryo carried by the first carrier is completely immersed in the thawing solution to ensure the thawing and warming speed, and the embryo is separated from the first carrier.

[0054] Step S206: Provide a second carrier, the second carrier having a groove near its front end, the groove of the second carrier containing the first diluent.

[0055] A second carrier with a groove at the front end can be prepared in advance, and the groove of the second carrier can be filled with a first diluent, requiring that the amount of the first diluent is greater than the capacity of the groove.

[0056] Step S208: Transfer the biomaterial into the groove of the second carrier.

[0057] After the embryo is detached from the first carrier, its exact location is determined, and the embryo is transferred to the groove of the second carrier using a capillary tube (e.g., a glass capillary tube).

[0058] Step S210: Provide a chip encapsulated with chemicals, such that the chip covers the second carrier, wherein the chemicals contain a cryoprotectant and / or a basic culture medium.

[0059] In this embodiment, the concentration of cryoprotectant in the chemical is lower than the concentration of cryoprotectant in the groove. In practical applications, the cryoprotectant in the chemical can be a non-permeable cryoprotectant or a permeable cryoprotectant. Specifically, when using chemicals to remove cryoprotectant from embryos, the chemicals may contain cryoprotectant and / or basal culture medium, or the chemicals may contain non-permeable cryoprotectant and / or basal culture medium. To achieve better removal results, the chemical may contain components of non-permeable cryoprotectant and / or basal culture medium. The main reason is that non-permeable cryoprotectants do not penetrate the cell membrane and are generally non-cytotoxic molecules. Permeable cryoprotectants are generally small molecules with cytotoxicity. In thawing applications, the role of non-permeable cryoprotectants is to ensure that the osmotic pressure difference between the inside and outside of the cell is not too large. That is, in thawing applications, when cells are taken out of liquid nitrogen, the inside of the cells is filled with permeable cryoprotectant, resulting in a high osmotic pressure inside the cells. During thawing, if the cells are placed directly into the culture medium, this osmotic pressure difference will cause water molecules to enter the cells very quickly, leading to cell death. However, if the cells are placed in a solution containing a non-permeable cryoprotectant during thawing, the rate at which water molecules enter the cells can be slowed down due to the small osmotic pressure difference. At the same time, the permeable cryoprotectant inside the cells can diffuse out of the cells, thus achieving the purpose of slowly removing the cryoprotectant.

[0060] The type of the chemical includes a second dilution solution and a washing solution. The second dilution solution contains a base culture solution and a non-permeable cryoprotectant with a lower concentration than the first dilution solution. The washing solution contains only the base culture solution. Specifically, two intervals are arranged on the chip in sequence, and the second dilution solution and the washing solution are sequentially sealed in the intervals. The coverage area of each interval is greater than the opening area of the groove of the second carrier.

[0061] In the embodiments of the present application, the chemical can be in a solid state in the form of a hydrogel or in a solution form. In the case of the chemical in a solid state, the second dilution solution and the washing solution are in the form of a hydrogel, i.e., a second dilution hydrogel (DS2 gel) and a washing hydrogel (WS gel), which are embedded in the chip. In the case of the chemical in a solution form, the dilution solution and the washing solution are in a solution form and are sealed in the chip by a permeable membrane. In practical applications, a perforated membrane, a mesh or a dialysis membrane with a suitable thickness and pore size can be arranged on the lower surface of the chip, and the solution can be directly added to the interval. The solution is fixed in the chip by the support of the perforated membrane, the mesh, the dialysis membrane or the water-soluble membrane.

[0062] In step S212, the chip is moved relative to the second carrier, and the chemical fully covers the groove of the second carrier to remove the cryoprotectant in the groove.

[0063] In the case of the chemical in a solid state in the form of a hydrogel fixed in the chip, when the chip is moved relative to the carrier, the second dilution hydrogel and the washing hydrogel embedded in the chip are sequentially moved above the groove of the second carrier and sequentially fully contact the biological material to be thawed in the groove. Since the molecules of the chemical can freely diffuse in the gel, when the groove on the carrier is contacted, the embryo and the cryoprotectant in the groove will diffuse into the hydrogel, achieving the function of removing the cryoprotectant. Under the second dilution hydrogel, water can slowly diffuse into the embryo again, sequentially removing the non-permeable cryoprotectant molecules around the embryo. Under the washing hydrogel, more water can slowly diffuse into the embryo again, completely removing the non-permeable cryoprotectant molecules around the embryo, so that the embryo returns to the normal osmotic pressure environment again. The embryo treated by the hydrogel does not contain any cryoprotectant and has been sequentially restored to the normal osmotic pressure environment. The embryo can be transferred from the carrier to a culture dish and cultured in an incubator.

[0064] In the case of the chemical in a solution form fixed in the chip, the permeable membrane forms a cover for the groove to prevent the embryo from overflowing, and when the solutions on both sides of the permeable membrane are in contact, the solution penetration occurs to complete the diffusion exchange of the solutions on both sides. For details, refer to the embodiments of the hydrogel.

[0065] In summary, the chemical, whether solid or liquid, can be blocked in the recess while performing the diffusion removal of the cryoprotectant and prevent the chemical from flowing out of the chip. After the entire removal process is completed, the embryo is in the recess of the carrier, the position of the embryo can be quickly determined and the embryo can be captured. The embryo can be transferred from the carrier to a culture dish and cultured in an incubator for recovery. Because the medium composition of the washing hydrogel (or washing liquid) is not necessarily the same as that of the user, the user can place the embryo transferred from the carrier in the user's culture medium for the last rinse and then transfer it to the user's culture system for culture recovery.

[0066] When thawing and rewarming using an oil phase thawing solution, if a non-standard carrier is used to carry the embryo, the embryo needs to be transferred to a standard carrier after thawing and rewarming, so that the cryoprotectant can be removed in cooperation with the chip. Referring to Figure 3 According to the embodiments of the present application, a method for thawing and recovering biological materials is also provided, which specifically includes the following steps:

[0067] Step S302, providing a first carrier, wherein the first carrier carries biological materials to be thawed and a cryoprotectant;

[0068] Step S304, immersing the first carrier in an oil phase thawing solution, wherein the oil phase thawing solution covers the first carrier and thaws and rewarms the biological materials;

[0069] Step S306, providing a second carrier, wherein a recess is arranged near the front end of the second carrier, and the recess of the second carrier carries a first dilution liquid.

[0070] The second carrier can work and be used in cooperation with the chip.

[0071] Step S308, transferring the biological materials to the recess of the second carrier.

[0072] Step S310, providing a chip encapsulating a chemical, wherein the chip covers the second carrier; and the chemical contains a cryoprotectant and / or a basic culture solution.

[0073] The chemical can contain a cryoprotectant and / or a basic culture solution, or the chemical can contain a non-permeable cryoprotectant and / or a basic culture solution. Preferably, the chemical contains a non-permeable cryoprotectant and / or a basic culture solution. Moreover, the chemical covers part of the area of the carrier and does not cover the recess of the carrier, and there is a gap distance between the chemical and the recess of the carrier.

[0074] Step S312, moving the chip relative to the carrier, the chemical makes full coverage contact with the groove of the second carrier to remove the cryoprotectant in the groove.

[0075] The explanations of the terms in this embodiment can refer to the same or similar parts of the previous embodiments, which will not be described here again.

[0076] Reference Figure 4 According to the embodiments of the present application, a method for thawing and recovering biological materials is also provided, which includes the following steps:

[0077] Step S402, preparing the chemical into a hydrogel structure, wherein the chemical contains (first) cryoprotectant and / or basic culture solution;

[0078] Preferably, the chemical contains non-permeable cryoprotectant and / or basic culture solution.

[0079] Step S404, contacting the biological material containing (second) cryoprotectant with the hydrogel, and removing the (second) cryoprotectant by diffusion of the (second) cryoprotectant to the hydrogel.

[0080] The chemical includes one or more of thawing solution, dilution solution or washing solution.

[0081] The preparation of the gel can be prepared by a physical hydrogel preparation method, such as a sodium alginate hydrogel, a gelatin hydrogel or an agarose gel preparation method, or a chemical hydrogel preparation method, such as a PEGDA hydrogel or a GelMA hydrogel preparation method. In addition, although the thawing solution is prepared into a hydrogel in this embodiment, the operation of pouring the vitrification cryoprotectant into the embryo is completed at one time. However, in use, according to the specific situation, such as the concentration of the thawing solution, the thawing solution can also be prepared into multiple hydrogels with different concentrations of thawing solution, and then the ordered and controllable removal of the cryoprotectant is completed by using multiple hydrogels with different concentrations of thawing solution, so as to complete the removal operation of the cryoprotectant. The preparation methods of various forms of hydrogels are described in detail below.

[0082] Further, the method for preparing agarose gel includes:

[0083] Dissolve agarose in a chemical solution at 80-90°C to prepare an agarose solution with a concentration of 0.1-6%; after stirring, mixing, cooling and solidification, an agarose gel containing the chemical is obtained.

[0084] Further, the method for preparing the sodium alginate hydrogel comprises:

[0085] The sodium alginate is dissolved in the thawing solution to form a 0.3-10% sodium alginate solution; the calcium chloride is dissolved in the thawing solution to form a 0.01M-0.2M calcium chloride solution; the calcium chloride solution is slowly added to the sodium alginate solution in the chip; and the calcium alginate hydrogel is formed after the calcium chloride diffuses to the bottom of the chip.

[0086] Further, the method for preparing the gelatin hydrogel comprises:

[0087] The gelatin is dissolved in the thawing solution at 36-45°C to form a 1-15% gelatin solution; the gelatin solution at 36-50°C is added to the chip; and the gelatin solution is cooled and solidified to form the hydrogel containing the thawing solution.

[0088] Further, the method for preparing the GelMA hydrogel comprises:

[0089] The GelMA is dissolved in the thawing solution at 36-50°C to form a 1-15% GelMA solution; the photoinitiator is dissolved in the GelMA solution at 36-50°C at a concentration of 0.001-2%; the GelMA solution at 36-50°C is added to the chip; and the GelMA solution at 36-50°C is irradiated with light for 2-30 minutes to solidify the GelMA solution into the hydrogel.

[0090] The system for thawing and recovering biomaterials according to the embodiments of the present application is provided in combination with reference to Figures 5 to 11 The system comprises a carrier 1, a dish container 5 and a chip 2.

[0091] With reference to Figure 6 The carrier 1 is provided with a groove 11 near the front end, and the groove 11 contains the biomaterials to be thawed and the cryoprotective agent (VS) for protecting the biomaterials to be thawed, wherein the biomaterials can be cells, biological tissues, ova or embryos, and the cryoprotective agent can be vitrification cryoprotective solution. The carrier 1 is in a strip structure, comprising a handle 11, a sheet 12 and a groove 13. The groove 13 is located in the sheet 12 near the front end, and the groove 13 contains the biomaterials to be thawed and the cryoprotective agent (VS) for protecting the biomaterials to be thawed, wherein the biomaterials can be cells, biological tissues, ova or embryos, and the cryoprotective agent can be vitrification cryoprotective solution. The size of the groove 13 can be adjusted according to the number and size of the embryos to be treated and the amount of solution to be contained.

[0092] In the embodiment of the present application, the carrier 1 can be in a strip structure to facilitate the thawing and warming operation of the embryo by the system and improve the compatibility of the carrier. Meanwhile, the handle part of the carrier is designed to have a sufficient width to facilitate the setting of a label for marking the relevant information of the embryo to be processed. The sheet is made of a plastic material with uniform thickness, transparent material, biological compatibility and good heat transfer performance to ensure the applicability to the embryo and the heat transfer speed during the subsequent thawing. Similarly, in other embodiments, the carrier can also be designed in other structure forms with grooves, such as a flat plate structure, according to different use conditions and requirements.

[0093] The dish container 5 contains an oil phase thawing solution which does not contain a non-permeable cryoprotectant. The carrier is immersed in the oil phase thawing solution, so that the groove of the carrier is immersed below the liquid level. The oil phase thawing solution can thaw and warm the embryo, so that the embryo is warmed to room temperature or 37°C. Since the cryoprotectant and the oil phase thawing solution cannot be mixed together, and the density of the oil phase thawing solution is lower than that of the cryoprotectant, the cryoprotectant in the groove together with the embryo will always stay in the groove, that is, during the thawing and warming process, the cryoprotectant in the groove together with the embryo to be thawed will stay in the groove and will not float out of the carrier.

[0094] Reference Figure 7 The chip 2 adopts a plate frame structure, and a plurality of intervals 22 are arranged on the chip 2 in sequence to fix or package chemicals respectively. In the embodiment, the chip 2 adopts a frame structure composed of two support plates 20 and two partition plates 21. The two support plates 20 are kept in parallel, the two partition plates 21 are kept in parallel, and the two partition plates 21 are located between the two support plates 20 to divide the area between the two support plates 20 into three independent intervals 22 for setting different chemicals to be poured.

[0095] Optionally, the partition plate 21 and the support plate 20 in the chip 2 can be designed as a movable connection, so that the size of the interval 22 can be freely adjusted. For example, a sliding groove is arranged on the opposite surface of the two support plates 20, and the end of the partition plate 21 is inserted into the sliding groove, so that the position of the partition plate 21 in the sliding groove can be freely adjusted to adjust the size of the interval and meet the bearing of different pouring amount of chemicals.

[0096] The types of chemicals on the chip include thawing solution, dilution solution and cleaning solution. The chemicals encapsulated on the chip can be in solid or liquid form. In the case of solid form, the chemicals are made into hydrogels, i.e. thawing hydrogel, dilution hydrogel and cleaning hydrogel are embedded in the chip. In the case of solution form, the thawing solution, dilution solution and cleaning solution are enclosed in the chip by permeable membrane in solution form. In practical application, a perforated membrane, mesh or dialysis membrane with appropriate thickness and pore size can be arranged on the lower surface of the chip, and the solution is directly added into the interval, and the solution is fixed in the chip by the support of the perforated membrane, mesh, dialysis membrane or water-soluble membrane.

[0097] Reference Figure 8 The chip 2 encapsulating chemicals is covered on the carrier 1, at this time the chemicals only cover part of the area of the carrier and do not cover the grooves of the carrier, and there is a gap distance between the chemicals and the grooves of the carrier. The chip is moved relative to the carrier, and the thawing solution, dilution solution and cleaning solution in solid or liquid form are sequentially moved above the grooves of the carrier and sequentially make full coverage contact with the biological materials to be thawed in the grooves, so that the cryoprotective agent in the grooves can be removed. During the removal process, the embryo is blocked in the groove while completing the diffusion exchange with the chemicals in the groove, and the chemicals do not flow out of the chip. After the whole removal process is completed, the embryo has already been removed of any cryoprotective agent and has been sequentially restored to the normal osmotic pressure environment, at this time the embryo stays in the groove of the carrier, the position of the embryo can be quickly determined and the embryo can be captured, the embryo can be transferred from the carrier to a culture dish and cultured in an incubator.

[0098] In this embodiment, three intervals are opened on the chip for placing solutions with different concentrations of cryoprotective agent, so as to accurately control the concentration relationship of sequentially delivering different solutions to the embryo and ensure the accuracy of delivering the solution. In other embodiments, according to the number of types of chemicals to be delivered and the concentration control requirements, the number of intervals on the chip can be arbitrarily adjusted, the concentration gradient between the chemicals in adjacent intervals is accurately controlled, and the accuracy of delivering the chemicals is ensured.

[0099] It should be noted that although only one groove is arranged on the carrier in this embodiment, in other embodiments, according to the number of embryos to be processed, multiple grooves can be arranged on the carrier at the same time, so that multiple embryos can be processed on the same carrier at the same time, improving the processing efficiency.

[0100] Reference Figure 9 and Figure 10The system for thawing and recovering the biomaterials can further comprise a base 3, which is in the shape of a channel steel. The middle region of the base 3 is used to support and fix the carrier 1, and the two sidewalls along the length direction of the base 3 can be used as rails 31 to support and adsorptively fix the support plate 20 of the chip 2. By changing the height of the rails 31, the positional relationship between the chip 2 and the upper surface of the carrier 1 can be adjusted, thereby ensuring the effective contact between the gel and the solution in the groove.

[0101] In other embodiments, a guide step can be arranged on each of the two rails of the base 3. In this way, when the chip is placed between the two guide steps, the guide steps can guide the movement of the chip, thereby improving the directional accuracy of the movement of the chip on the base.

[0102] The rails 31 and the chip 2 are connected in a detachable manner by magnetic adsorption. For example, the rails 31 are made of ferromagnetic metal and a magnet 24 is arranged at the corresponding position on the support plate 20, thereby forming the magnetic adsorptive connection between the rails 31 and the chip 2. Further preferably, the rails can also be in the form of an electromagnet, so that the base and the chip can be quickly connected and separated by electrical control, thereby improving the convenience of operation.

[0103] By arranging the base, the carrier can be supported and fixed, for example by pasting or buckling between the base and the carrier, thereby ensuring the stability of the position of the carrier during the entire operation process. In addition, the chip can also be supported and fixed, thereby maintaining the effective contact between the chip and the carrier and avoiding the accidental separation of the chip and the carrier during the relative movement, which can affect the normal operation. In addition, the base can also collect the solution flowing out of the groove, thereby avoiding the pollution of the surrounding environment caused by the overflow of the solution.

[0104] Reference Figure 9 A light-transmitting region 32 is arranged at the middle position of the base 3, that is, the region of the base 3 corresponding to the groove 13 is formed into a light-transmitting region by selecting a light-transmitting material. At this time, by penetrating the light provided by the light-transmitting region, the carrier and the chip can be moved as a whole to the microscope through the base for direct observation, thereby realizing the real-time observation of the chemical dispensing process and accurately controlling the dispensing progress of the chemicals.

[0105] Further, according to the actual operation needs, the light-transmitting region can be selected from conventional light-transmitting materials to meet the light-transmitting requirement alone, or can be selected from light-transmitting materials with heating function, for example, a heating glass material, thereby meeting the light-transmitting and temperature control purposes at the same time.

[0106] In addition, although only one carrier and one chip are arranged on the substrate in the embodiment, in actual operation, according to the number of embryos to be treated and the interval width size of the chip, i.e. the covering width of the gel, a plurality of carriers can be arranged side by side on the substrate, so that the chemical dispensing operation on the embryos on the plurality of carriers can be completed simultaneously in a single movement of the chip, thereby improving the operation efficiency.

[0107] Reference Figure 11 The system for thawing and recovering the biological material can further include a base 4 for directly supporting and fixing the substrate 3, and a driving unit composed of a stepping motor 41, a lead screw 42 and a pushing rod 43 is installed on the base 4. The substrate 3 is arranged on the base 4 and parallel to the lead screw 42, and the pushing rod 43 is sleeved on the lead screw 42 and can reciprocate along the lead screw 42 under the driving of the stepping motor 41, and the pushing rod 43 is fixedly connected with the chip 2.

[0108] At this time, the horizontal reciprocating movement of the pushing rod 43 along the lead screw 42 driven by the stepping motor 41 can drive the chip 2 to reciprocate horizontally relative to the carrier 1, so as to control the gel in different intervals of the chip 2 to be in contact with the solution in the groove 13 in turn, thereby realizing automatic control. Further, by controlling the action of the stepping motor 41, the contact time of different gels in the chip 2 with the solution in the groove 13 can be accurately controlled, so as to accurately control the dispensing time of the solution in the gel and the contact time of the embryo with different solutions, thereby further improving the dispensing precision of different solutions for the embryo.

[0109] In addition, in other embodiments, a track slider structure can be used instead of the lead screw and the pushing rod to form the driving unit, and the chip is driven to move relative to the carrier by the reciprocating movement of the slider along the track.

[0110] Continuing to refer to Figure 11 The base 4 is further provided with an optical axis 44 which is parallel to the lead screw 42 and connected with the free end of the pushing rod 43, for providing auxiliary guidance for the reciprocating movement of the pushing rod 43, improving the stability of the movement of the pushing rod 43 driving the chip 2, and improving the stability of the contact process of the gel with the solution in the groove.

[0111] As shown in the figure, a hollow area 45 is further provided at the middle position of the base 4, i.e. the area corresponding to the light-transmitting area of the chip 3 is in a hollow structure. In this way, the light can smoothly pass through the base 4 and be projected to the light-transmitting area of the chip 3, ensuring the normal use of the microscope. Similarly, according to the actual use in different environments, the hollow area can also be made of light-transmitting material, such as light-transmitting glass, to achieve the purpose of light transmission. In addition, light-transmitting material with heating function, such as heating glass, can also be selected to simultaneously achieve the purposes of light transmission and temperature control.

[0112] In addition, although the above embodiments all take the operation of dispensing different chemical solutions in the process of thawing embryos as an example to introduce the technical solutions of the present application, for those skilled in the art, according to the concept of the present application, the chemical dispensing system can be applied to the operation of accurately dispensing chemicals to other biological materials or base solutions, for example, when dispensing powdered chemicals into a solution, the single-time dispensing amount of chemicals can be temporarily supported by means of a water-soluble film, and in the process of moving the chip relative to the carrier, when the water-soluble film is in contact with the solution and dissolves, the quantitative chemicals are directly released into the solution, thereby completing the accurate dispensing of the chemicals.

[0113] According to the present application, a system for thawing and recovering biological materials is also provided, which comprises a first carrier, a dish container, a second carrier, a capillary tube and a chip. Specifically:

[0114] The first carrier contains biological materials to be thawed and cryoprotectants. The present application does not limit the shape and structure of the first carrier, that is, the first carrier can have a groove and use the groove to carry the biological materials, or the first carrier does not have a groove and uses other ways to carry the biological materials.

[0115] The dish container contains a thawing solution, which is used to cover the first carrier that invades into the dish container and thaw and recover the biological materials, so that the biological materials are separated from the first carrier;

[0116] The second carrier is provided with a groove near the front end, and the groove of the second carrier contains a first dilution solution;

[0117] The capillary tube is used to transfer the biological materials into the groove of the second carrier;

[0118] The chip is packaged with chemicals, and the chemicals contain cryoprotectants and / or base culture solution; the chip moves relative to the second carrier, and the chemicals fully cover the groove of the second carrier to remove the cryoprotectants in the groove.

[0119] In the present application, the concentration of the cryoprotectants contained in the chemicals is less than the concentration of the cryoprotectants in the groove. In actual application, the cryoprotectants contained in the chemicals can be non-permeable cryoprotectants or permeable cryoprotectants. Specifically, when the chemicals are used to remove the cryoprotectants in the embryos, the chemicals can contain cryoprotectants and / or base culture solution, or the chemicals can contain non-permeable cryoprotectants and / or base culture solution. In order to achieve better removal effect, the chemicals can contain non-permeable cryoprotectants and / or base culture solution.

[0120] The types of the chemicals include a second diluent and a washing solution, the second diluent containing a base culture solution and a non-permeable cryoprotectant with a lower concentration than the first diluent, and the washing solution containing only the base culture solution. The chemicals can be in a solid state or a solution state and are packaged in the chip. In the case of the chemicals in the solid state, the second diluent and the washing solution are fixed in the form of hydrogels, i.e., the second diluent hydrogel and the washing hydrogel are embedded in the chip. When the chip moves relative to the carrier, the second diluent hydrogel and the washing hydrogel are sequentially moved above the groove and sequentially make full-contact with the groove. In the case of the chemicals in the solution state, the diluent and the washing solution are sealed in the chip by a permeable film. In actual applications, a perforated film, a mesh or a dialysis film with a suitable thickness and pore size can be arranged on the lower surface of the chip, and the solution is directly added into the interval, and the solution is fixed in the chip by the perforated film, the mesh, the dialysis film or the water-soluble film.

[0121] The shape and structure of the second carrier and the chip in the embodiment are similar to those described in the above embodiments, and will not be described here. In the embodiment, the embryo is thawed and warmed in the non-oil phase thawing solution by the first carrier. Since the first carrier cannot be used in combination with the chip (and other components in the system), the embryo needs to be transferred to the second carrier, so that the second carrier can be used in combination with the chip (and other components in the system) to remove the cryoprotectant.

[0122] It should be noted that the system in the embodiment can also include a base and a pedestal, and the like, which will not be described here.

[0123] The operation steps of the method of the application correspond to the structural features of the system, and can be referred to each other, and will not be described one by one.

[0124] According to the above technical scheme of the application, the following beneficial effects are achieved:

[0125] (1) Compared with the existing manual thawing, the application proposes a standardized process, which is driven by a machine, and the removal rate of the cryoprotectant can be adjusted. The process of each thawing and recovery is stable; the process is simple, and if the freezing method applied by the applicant before is used, the embryologist does not need to add the embryo to the carrier, but only needs to wait for the machine to complete the thawing process and then move the embryo from the carrier to the culture dish; the embryo is on the carrier in a fixed position during the entire thawing and recovery process, and the embryologist does not need to search for the embryo in a large volume of liquid, so the throughput is high.

[0126] (2) Compared with the existing microfluidic, the carrier of the application has no dead volume, and the position of the embryo on the carrier can allow the embryologist to directly recover the embryo with a glass capillary tube.

[0127] (3) Compared with the existing robot (analog human hand type), the flux of the present application is high, and the core function only needs a linear moving motor platform, the cost is low, and machine vision is not needed to distinguish the embryos.

[0128] Those skilled in the art will appreciate that embodiments of the present application can be provided as methods, systems or computer program products. Accordingly, the present application can take the form of an entirely hardware embodiment, an entirely software embodiment or an embodiment combining software and hardware aspects. Furthermore, the present application can take the form of a computer program product on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROMs, optical storage devices, etc.) embodying computer readable program code thereon for use by or in connection with an instruction execution system.

[0129] The above description is merely that of embodiments of the present application, but is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modified, equivalent, improved and the like, which are made within the spirit and principle of the present application, should be included in the scope of the claims of the present application.

Claims

1. A system for thawing and recovery of biological material, characterized by, The application relates to a device for thawing biological materials, comprising: a carrier provided with a recess near the front end, the recess containing biological materials to be thawed and a cryoprotectant; a dish container containing an oil-phase thawing solution for covering the recess of the carrier and thawing the biological materials; a chip provided with a plurality of intervals, each interval having a coverage area greater than the opening area of the recess; the chip is packaged with chemicals containing a cryoprotectant and / or a basic culture solution; the chip moves relative to the carrier, and the chemicals fully cover the recess to remove the cryoprotectant in the recess; wherein the chemicals are fixed in the intervals in the form of hydrogel; or the lower end of the interval is provided with a permeable membrane, and the chemicals are sealed in the interval in the form of solution.

2. The system of claim 1, wherein, The cryoprotectant contained in the chemicals is a non-permeable cryoprotectant.

3. The system of claim 2, wherein, The chemicals contain thawing solution, dilution solution and cleaning solution in the form of hydrogel; the chip is provided with a plurality of intervals for sequentially embedding thawing solution hydrogel, dilution solution hydrogel and cleaning solution hydrogel; when the chip moves relative to the carrier, the thawing solution hydrogel, the dilution solution hydrogel and the cleaning solution hydrogel move above the recess in sequence and fully cover the recess in sequence.

4. The system of claim 2, wherein, The chemicals contain thawing solution, dilution solution and cleaning solution in the form of solution; the chip is provided with a permeable membrane supporting the thawing solution, the dilution solution and the cleaning solution in the form of solution; wherein the permeable membrane is a perforated membrane, a grid, a dialysis membrane or a water-soluble membrane.

5. The system of claim 3 or 4, wherein, Before the chip moves relative to the carrier, the chemicals cover part of the carrier and do not cover the recess of the carrier, and there is a gap distance between the chemicals and the recess of the carrier.

6. The system of claim 3 or 4, wherein, Further comprising: a base, the middle region of the base is used for placing the carrier, and the length direction of the base is provided with two parallel tracks for supporting and fixing the chip, so that the lower surface of the chip is kept in contact with the upper surface of the carrier.

7. A system for thawing and recovery of biological material, characterized by The application relates to a device for thawing biological materials, comprising: a first carrier containing biological materials to be thawed and a cryoprotectant; a dish container containing a thawing solution for covering the first carrier and thawing the biological materials to make the biological materials separate from the first carrier; a second carrier provided with a recess near the front end, the recess of the second carrier containing a first dilution solution; a capillary for transferring the biological materials into the recess of the second carrier; a chip provided with a plurality of intervals, each interval having a coverage area greater than the opening area of the recess; the chip is packaged with chemicals containing a cryoprotectant and / or a basic culture solution; the chip moves relative to the second carrier, and the chemicals fully cover the recess of the second carrier to remove the cryoprotectant in the recess. The chemical is fixed in the interval in the form of hydrogel; or the lower end of the interval is provided with a permeable membrane, and the chemical is enclosed in the interval in the form of solution by the permeable membrane.

8. The system of claim 7, wherein, The chemical contains a non-permeable cryoprotectant.

9. The system of claim 8, wherein, The chemical includes a second diluent and a cleaning solution in the form of hydrogel, the second diluent contains a non-permeable cryoprotectant with a concentration lower than that of the first diluent; the chip is provided with a plurality of intervals for sequentially embedding the second diluent hydrogel and the cleaning solution hydrogel; when the chip moves relative to the carrier, the second diluent hydrogel and the cleaning solution hydrogel sequentially move above the groove and sequentially make full coverage contact with the groove.

10. The system of claim 8, wherein, The chemical includes a second diluent and a cleaning solution in the form of solution; the second diluent contains a non-permeable cryoprotectant with a concentration lower than that of the first diluent; the chip is provided with a permeable membrane, and the permeable membrane supports the second diluent and the cleaning solution in the form of solution; the permeable membrane is a perforated membrane, a grid, a dialysis membrane, or a water-soluble membrane.

11. The system of claim 9 or 10, wherein, Before the chip moves relative to the second carrier, the chemical covers part of the area of the second carrier and does not cover the groove of the second carrier, and there is a gap distance between the chemical and the groove of the second carrier.

12. The system of claim 9 or 10, wherein, Further comprising: a base, the middle area of the base is used to place the second carrier, the length direction of the base is provided with two parallel tracks, the tracks support and fix the chip, and the lower surface of the chip keeps in contact with the upper surface of the second carrier.

13. A method for biomaterial thawing recovery of a system according to claim 1, characterized in that, Comprising: providing a carrier, the carrier is provided with a groove near the front end, the groove contains biological materials to be thawed and a cryoprotectant; immersing the groove of the carrier into an oil-phase thawing solution, the oil-phase thawing solution covers the groove of the carrier and thaws and warms up the biological materials; providing a chip encapsulating a chemical, the chemical contains a cryoprotectant and / or a basic culture solution; removing the carrier from the oil-phase thawing solution, and covering the carrier with the chip; moving the chip relative to the carrier, and the chemical makes full coverage contact with the groove of the carrier to remove the cryoprotectant in the groove.

14. The method of claim 13, wherein, The chemical contains a non-permeable cryoprotectant.

15. The method of claim 14, wherein, Before the chip moves relative to the carrier, the chemical covers part of the area of the carrier and does not cover the groove of the carrier, and there is a gap distance between the chemical and the groove of the carrier.

16. The method of claim 14, wherein, The chemical includes thawing solution, dilution solution and washing solution made in hydrogel form; the chip is provided with multiple intervals for sequentially embedding thawing solution hydrogel, dilution solution hydrogel and washing solution hydrogel, wherein the coverage area of each interval is greater than the opening area of the groove; when the chip moves relative to the carrier, the thawing solution hydrogel, the dilution solution hydrogel and the washing solution hydrogel sequentially move above the groove and sequentially make full coverage contact with the groove.

17. The method of claim 14, wherein, The chemical includes thawing solution, dilution solution and washing solution in solution form; the chip is provided with a permeable film supporting the thawing solution, the dilution solution and the washing solution in solution form; wherein the permeable film is a perforated film, a mesh, a dialysis membrane or a water-soluble film.

18. A method for biomaterial thawing recovery of a system of claim 7, characterized in that, The method comprises: providing a first carrier containing biological material to be thawed and a cryoprotective agent; immersing the first carrier in a thawing solution, which covers the first carrier and thaws the biological material; providing a second carrier provided with a groove near the front end, and the first dilution solution is contained in the groove of the second carrier; transferring the biological material into the groove of the second carrier; providing a chip containing chemicals, so that the chip covers the second carrier; wherein the chemicals contain a cryoprotective agent and / or a basic culture solution; moving the chip relative to the second carrier, and the chemicals make full coverage contact with the groove of the second carrier to remove the cryoprotective agent in the groove.

19. The method of claim 18, wherein, The cryoprotective agent contained in the chemicals is a non-permeable cryoprotective agent.

20. The method of claim 19, wherein, Before the chip moves relative to the carrier, the chemicals cover part of the second carrier and do not cover the groove of the second carrier, and there is a gap distance between the chemicals and the groove of the second carrier.

21. The method of claim 19, wherein, The chemicals include second dilution solution and washing solution made in hydrogel form, and the concentration of non-permeable cryoprotective agent contained in the second dilution solution is lower than that contained in the first dilution solution; the chip is provided with multiple intervals for sequentially embedding second dilution solution hydrogel and washing solution hydrogel, wherein the coverage area of each interval is greater than the opening area of the groove; when the chip moves relative to the carrier, the second dilution solution hydrogel and the washing solution hydrogel sequentially move above the groove and sequentially make full coverage contact with the groove.

22. The method of claim 19, wherein, The chemicals include second dilution solution and washing solution in solution form; wherein the concentration of non-permeable cryoprotective agent contained in the second dilution solution is lower than that contained in the first dilution solution; the chip is provided with a permeable film supporting the second dilution solution and the washing solution in solution form; wherein the permeable film is a perforated film, a mesh, a dialysis membrane or a water-soluble film.

23. A method for thawing and reviving biological materials, characterized in that, The method comprises: The chemical is prepared into a structure of hydrogel, including one or more of the following: a thawing hydrogel, a dilution hydrogel, and a washing hydrogel, wherein the thawing hydrogel contains a basic culture solution and a non-permeable cryoprotectant, the dilution hydrogel contains a basic culture solution and a non-permeable cryoprotectant with a lower concentration than the thawing hydrogel, and the washing hydrogel contains a basic culture solution; The thawing hydrogel, the dilution hydrogel, and the washing hydrogel are sequentially embedded in the interval of the chip; The thawing hydrogel, the dilution hydrogel, and the washing hydrogel are sequentially contacted with a biomaterial containing a permeable cryoprotectant, and the permeable cryoprotectant is diffused into the hydrogel to remove the permeable cryoprotectant by diffusion.

24. The method of claim 23, wherein, The step of preparing the chemical into a structure of hydrogel includes: Dissolving agarose in a chemical solution at 80-90°C to obtain an agarose solution with a concentration of 0.1-6%; After stirring, mixing, cooling, and solidification, an agarose gel containing the chemical is obtained.

25. The method of claim 23, wherein, The step of preparing the chemical into a structure of hydrogel includes: Dissolving sodium alginate in a chemical solution to obtain a sodium alginate solution with a concentration of 0.3-10%; Dissolving calcium chloride in a chemical solution to obtain a calcium chloride solution with a concentration of 0.01M-0.2M; Adding the calcium chloride solution to the sodium alginate solution in the chip; After the calcium chloride diffuses to the bottom of the chip, the sodium alginate solution is solidified into a calcium alginate hydrogel.

26. The method of claim 23, wherein, The step of preparing the chemical into a structure of hydrogel includes: Dissolving gelatin in a chemical solution at 36-45°C to obtain a gelatin solution with a concentration of 1-15%; Adding the gelatin solution at 36-50°C to the chip; Allowing the gelatin solution to cool and solidify into a hydrogel containing the chemical.

27. The method of claim 23, wherein, The step of preparing the chemical into a structure of hydrogel includes: Dissolving GelMA in a chemical solution at 36-50°C to obtain a GelMA solution with a concentration of 1-15%; Dissolving a photoinitiator in the GelMA solution at 36-50°C with a concentration of 0.001-2%; Adding the GelMA solution at 36-50°C to the chip; Irradiating the GelMA solution at 36-50°C with light for 5-30 minutes to solidify the GelMA solution into a hydrogel.

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