System and method for thawing and reanimating biological material - Patents.com

Abstract

The present invention discloses a system and method for thawing and reanimating a biomaterial, comprising a carrier, a vessel and a chip. A groove is provided near the tip of the carrier, in which a biomaterial and a cryoprotectant are accommodated. The vessel contains an oil-phase thawing liquid. The oil-phase thawing liquid is impregnated into the groove of the carrier so that the oil-phase thawing liquid covers the groove of the carrier, and the biomaterial is thawed and rewarmed. The chip is filled with chemicals including a cryoprotectant and / or a basal culture medium. The carrier is transferred from the oil-phase thawing liquid so as to cover the carrier, and the chip is moved relative to the carrier, and the cryoprotectant is removed from the groove by contacting the chemicals with the groove so as to cover the entire groove. This application can effectively standardize and automate the entire thawing and reanimation process, realize effective quality control, improve processing efficiency and reduce staff training.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to the field of biotechnology technology, and more particularly to a system and method for thawing and resuscitating biological material.

[0002] [Background technology] Cell cryopreservation is an important technique in many areas of biology, as is the recovery of cells from cryogenic freezing to a normal metabolic state. Such thawing and resuscitation processes include the removal of cryoprotectants and replacement with normal media processes.

[0003] Conventional techniques make the thaw-resuscitation procedure a demanding, tedious, difficult, highly intensive, stressful and difficult to standardize task.

[0004] DISCLOSURE OF THEINVENTION SUMMARY OF THE PRESENT EMBODIMENT The primary objective of the present invention is to provide a system and method for thawing and resuscitating biological material to solve the problem of low efficiency of the prior art manual thawing and resuscitation.

[0005] According to an embodiment of the present invention, there is provided a system for thawing and resuscitating a biomaterial, comprising: a carrier having a groove near a tip in which a biomaterial requiring thawing and a cryoprotectant are contained; a vessel containing an oil-phase thawing solution for covering the groove of the impregnated carrier and for thawing and rewarming the biomaterial; and a tip containing a chemical agent including a cryoprotectant and / or a basal culture medium, which is moved relative to the carrier and contacted with the chemical agent so as to entirely cover the groove, thereby removing the cryoprotectant from the groove.

[0006] Specific embodiments may include one or more of the following: The cryoprotectant contained in the chemical is a non-permeable cryoprotectant. The chemical includes a thawing solution, a diluting solution, and a washing solution made in a hydrogel form, and the chip is provided with a plurality of compartments for sequentially embedding the thawing solution hydrogel, the diluting solution hydrogel, and the washing solution hydrogel, and the covering area of ​​each compartment is larger than the opening area of ​​the groove, and when the chip moves relative to the carrier, the thawing solution hydrogel, the diluting solution hydrogel, and the washing solution hydrogel move sequentially onto the groove and sequentially contact the groove so as to cover the entire groove. The chemical includes a thawing solution, a diluting solution, and a washing solution, and the chip supports the thawing solution, the diluting solution, and the washing solution, and is provided with a permeable film, which is a perforated film, a grid, a dialysis membrane, or a water-soluble film. Before the chip moves relative to the carrier, the chemical does not cover the groove of the carrier, covers a partial area of ​​the carrier, and has a clearance distance between the chemical and the groove of the carrier. The central region of the base is used for positioning the carrier and has two parallel tracks along the length of the base which support and fix the chip so that its underside remains in contact with the upper surface of the carrier.

[0007] According to an embodiment of the present invention, there is further provided a system for thawing and resuscitating a biomaterial, comprising: a first carrier containing a biomaterial and a cryoprotectant, a vessel containing a thawing liquid for covering the impregnated first carrier and thawing and rewarming the biomaterial so as to peel it off from the first carrier, a second carrier having a groove near a tip thereof containing a first diluent, a capillary for transporting the biomaterial to the groove of the second carrier, and a tip containing a chemical agent including a cryoprotectant and / or a basal culture medium, the tip being moved relative to the second carrier and contacting the chemical agent with the groove of the second carrier so as to cover the entirety, thereby removing the cryoprotectant from the groove.

[0008] Specific embodiments may include one or more of the following: the cryoprotectant contained in the chemical is a non-permeable cryoprotectant; the chemical includes a second diluent and a washing solution made in a hydrogel form, the non-permeable cryoprotectant contained in the second diluent has a lower concentration than the non-permeable cryoprotectant contained in the first diluent; the chip is provided with a plurality of compartments for sequentially embedding the second diluent hydrogel and the washing solution hydrogel, the covering area of ​​each compartment is larger than the opening area of ​​the groove, and when the chip moves relative to the carrier, the second diluent hydrogel and the washing solution hydrogel move sequentially onto the groove and sequentially contact the groove so as to cover the entire groove; the chemical includes a second diluent in a solution form, the non-permeable cryoprotectant contained therein is a lower concentration than the non-permeable cryoprotectant contained in the first diluent, and a washing solution; the chip is provided with a permeable film, which is a perforated film, a grid, a dialysis membrane, or a water-soluble film, supporting the second diluent and the washing solution in a solution form. Before the chip moves relative to the second carrier, the chemical does not cover the groove of the second carrier, covers a partial area of ​​the second carrier, and has a clearance distance between the groove of the second carrier. A central region of the base is used for positioning the second carrier, and is provided with two tracks parallel to each other in the length direction of the base, and the tracks support and fix the chip such that its lower surface remains in contact with the upper surface of the second carrier.

[0009] According to an embodiment of the present invention, there is further provided a method for thawing and reanimating a biomaterial, comprising: providing a carrier having a groove near a tip in which a biomaterial in need of thawing and a cryoprotectant are accommodated; impregnating the groove of the carrier with the oil-phase thawing liquid so that the oil-phase thawing liquid covers the groove of the carrier, thereby thawing and rewarming the biomaterial; providing a chip in which a chemical agent including a cryoprotectant and / or a basal culture medium is encapsulated; transferring the carrier from the oil-phase thawing liquid and covering the chip with the carrier; and moving the chip relative to the carrier and contacting the chemical agent with the groove of the carrier so as to entirely cover the groove, thereby removing the cryoprotectant from the groove.

[0010] Specific embodiments may include one or more of the following: The cryoprotectant contained in the chemical is a non-permeable cryoprotectant; Before the chip moves relative to the carrier, the chemical does not cover the groove of the carrier, covers a part of the carrier, and has a clearance distance between the groove of the carrier. The chemical includes a thawing liquid, a diluting liquid, and a washing liquid made in a hydrogel form, and the chip is provided with a plurality of compartments for sequentially embedding the thawing liquid hydrogel, the diluting liquid hydrogel, and the washing liquid hydrogel, and the covering area of ​​each compartment is larger than the opening area of ​​the groove, and when the chip moves relative to the carrier, the thawing liquid hydrogel, the diluting liquid hydrogel, and the washing liquid hydrogel sequentially move onto the groove and sequentially contact the groove so as to cover the entirety. The chemical includes a thawing liquid, a diluting liquid, and a washing liquid in a solution form, and the chip is provided with a permeable film that supports the thawing liquid, the diluting liquid, and the washing liquid in a solution form, and is a permeable film that is a perforated film, a grid, a dialysis membrane, or a water-soluble film.

[0011] According to an embodiment of the present invention, there is further provided a method for thawing and reanimating a biomaterial, comprising: providing a first carrier containing a biomaterial requiring thawing and a cryoprotectant; impregnating the first carrier with a thawing liquid so that the thawing liquid covers the first carrier, thereby thawing and rewarming the biomaterial; providing a second carrier having a groove near a tip thereof in which a first diluent is contained; transferring the biomaterial to the groove of the second carrier; providing a tip containing a chemical agent including a cryoprotectant and / or a basal culture solution and covering the second carrier; and removing the cryoprotectant from the groove by moving the tip relative to the second carrier and contacting the chemical agent with the groove of the second carrier so as to cover the entirety of the groove.

[0012] Specific embodiments may include one or more of the following: the cryoprotectant contained in the chemical is a non-permeable cryoprotectant; before the chip moves relative to the carrier, the chemical does not cover the groove of the second carrier, covers a part of the area of ​​the second carrier, and has a clearance distance between the groove of the second carrier; the chemical includes a second diluent and a washing solution made in a hydrogel state, the non-permeable cryoprotectant contained in the second diluent has a lower concentration than the non-permeable cryoprotectant contained in the first diluent, the chip is provided with a plurality of compartments for sequentially embedding the second diluent hydrogel and the washing solution hydrogel, the covering area of ​​each compartment is larger than the opening area of ​​the groove, and when the chip moves relative to the carrier, the second diluent hydrogel and the washing solution hydrogel move sequentially over the groove and contact the groove sequentially so as to cover the entire groove. The chemicals include a second diluent and a washing solution in a solution state, the second diluent containing a non-permeable cryoprotectant having a lower concentration than the non-permeable cryoprotectant contained in the first diluent, and the chip supports the second diluent and the washing solution in a solution state and is provided with a permeable film, which may be a perforated film, a grid, a dialysis membrane, or a water-soluble film.

[0013] According to an embodiment of the present invention, there is further provided a method for thawing and reanimating a biomaterial, comprising preparing a chemical agent including a cryoprotectant and / or a basal culture medium in a structural form of a hydrogel, contacting a biomaterial including a cryoprotectant with said hydrogel, and removing the diffusion of the cryoprotectant by diffusion of the cryoprotectant into the hydrogel.

[0014] Specific embodiments may include one or more of the following. The cryoprotectant contained in the above chemicals is a non-permeable cryoprotectant. Agarose is dissolved in a chemical solution at 80-90°C to prepare a 0.1-6% agarose solution, and the agarose gel containing the chemicals is obtained by stirring, mixing, cooling, and hardening. Sodium alginate is dissolved in a chemical solution to prepare a 0.3-10% sodium alginate solution, calcium chloride is dissolved in a chemical solution to prepare a 0.01M-0.2M calcium chloride solution, and the calcium chloride solution is added to the top of the sodium alginate solution in the chip, and the sodium alginate solution is hardened into a calcium alginate hydrogel by the time the calcium chloride diffuses to the bottom of the chip. Gelatin is dissolved in a chemical solution at 36-45°C to prepare a 1-15% gelatin solution, and the gelatin solution at 36-50°C is added to the chip, and the gelatin solution is cooled and hardened into a hydrogel containing the chemicals. GelMA is dissolved in a chemical solution at 36-50°C to prepare a 1-15% GelMA solution, a photoinitiator is dissolved in the 36-50°C GelMA solution at a concentration of 0.001-2%, the 36-50°C GelMA solution is added to the chip, and the 36-50°C GelMA solution is irradiated with light for 5-30 minutes to harden the GelMA solution into a hydrogel. The above chemicals include one or more of the following: thawing solution, dilution solution, or washing solution.

[0015] According to the technical solution of the present invention, after thawing and rewarming of a biological material such as an embryo, a chip containing a chemical agent can cover the carrier carrying the embryo, and the chemical agent can be brought into contact with the groove of the carrier so as to completely cover it, thereby removing the cryoprotectant from the groove. Therefore, the present application can effectively standardize and automate the entire thawing and reanimation process, achieve effective quality control, improve processing efficiency, and reduce staff training.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings described herein are provided for a better understanding of the present invention and constitute a part of this application, and the exemplary embodiments of the present invention and the description thereof are used to explain the present invention and are not to be construed as undue limitations on the present invention. The accompanying drawings are as follows:

[0017] FIG. 1 is a flow chart of a method for thawing and resuscitating biological material according to one embodiment of the present invention.

[0018] FIG. 2 is a flow chart of a method for thawing and resuscitating biological material according to another embodiment of the present invention.

[0019] FIG. 3 is a flow chart of a method for thawing and resuscitating biological material according to a further embodiment of the invention.

[0020] FIG. 4 is a flow chart of a method for thawing and resuscitating biological material according to yet another embodiment of the present invention.

[0021] FIG. 5 is a schematic diagram of a carrier and vessel according to an embodiment of the present invention.

[0022] FIG. 6 is a schematic diagram of a carrier according to an embodiment of the present invention.

[0023] FIG. 7 is a schematic diagram of a chip according to an embodiment of the present invention.

[0024] FIG. 8 is a schematic diagram of a system for thawing and resuscitating biological material according to one embodiment of the present invention.

[0025] FIG. 9 is a structural schematic diagram of a base according to an embodiment of the present invention.

[0026] FIG. 10 is a schematic diagram of a system for thawing and resuscitating biological material in accordance with another embodiment of the present invention.

[0027] FIG. 11 is a schematic diagram of a system for thawing and resuscitating biological material according to a further embodiment of the present invention.

[0028] [Mode for carrying out the invention] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described below in a clear and complete manner in accordance with the specific embodiments of the present invention and the corresponding accompanying drawings. It will be clear that the described embodiments are only a part of the embodiments of the present invention, and are not all of them. Based on the embodiments in the present invention, all other embodiments that can be obtained by a person skilled in the art without making inventive efforts are included in the scope of the claims of the present invention.

[0029] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, technical solutions according to various embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0030] Referring to FIG. 1, a method for thawing and reanimating biological material according to an embodiment of the present application is proposed, which includes the following specific steps:

[0031] In step S102, a carrier is provided having a groove near its tip in which a biomaterial that needs to be thawed and a cryoprotectant are contained.

[0032] The carrier transports and preserves the biomaterial in a low temperature liquid nitrogen environment (e.g., -196°C), and a cryoprotectant for protecting the biomaterial is in the vicinity or surroundings of the biomaterial, where the biomaterial may be a cell, a living tissue, an egg, or an embryo, and the cryoprotectant may be a vitrification solution (abbreviated as VS) used to protect the biomaterial.

[0033] In step S104, the grooves of the carrier are impregnated with the oil-phase thawing liquid so that the grooves of the carrier are covered with the oil-phase thawing liquid, and the biomaterial is thawed and rewarmed. Here, the oil-phase thawing liquid does not contain a non-permeable cryoprotectant.

[0034] The carrier is quickly transferred from the cold environment to a vessel containing oil-phase thawing solution, where the grooves of the carrier must be immersed in the oil-phase thawing solution to ensure the thawing and warming speed. Since the oil-phase thawing solution has a lower density than the cryoprotectant, the cryoprotectant in the groove remains in the groove together with the embryo and does not float out of the groove. The purpose of this step is to allow for rapid thawing and warming of the embryo / egg in the groove.

[0035] In step S106, a chip containing chemicals including a cryoprotectant and / or a basal medium is provided.

[0036] In the embodiment of the present application, the cryoprotectant contained in the above chemical has a lower concentration than the cryoprotectant in the groove. In practical applications, the cryoprotectant contained in the chemical may be a non-permeable cryoprotectant or a permeable cryoprotectant. Specifically, when using a chemical to remove the cryoprotectant in the embryo, the chemical may contain a cryoprotectant and / or a basal culture medium, or may contain a non-permeable cryoprotectant and / or a basal culture medium. In order to obtain a better removal effect, a chemical whose components contain a non-permeable cryoprotectant and / or a basal culture medium may be used. This is because a non-permeable cryoprotectant is a molecule that does not pass through the cell membrane and generally does not have cytotoxicity. A permeable cryoprotectant is generally a small molecule that has cytotoxicity. In thawing applications, the role of the non-permeable cryoprotectant is to prevent the osmotic pressure difference between the inside and outside of the cell from becoming too large. That is, in the case of thawing, when the cells are taken out of liquid nitrogen, the inside of the cells is filled with a permeable cryoprotectant, so the osmotic pressure inside the cells becomes very high, and if the cells are put into a culture medium in the case of thawing, the osmotic pressure difference causes water molecules to rapidly enter the cells, causing the cells to die. However, when the cells are put into a solution containing a non-permeable cryoprotectant during thawing, the osmotic pressure difference is small, so the water molecules enter the cells at a slower rate, and at the same time, the permeable cryoprotectant inside the cells can diffuse out of the cells, so that the purpose of slowly removing the cryoprotectant can be achieved.

[0037] Here, the types of the above chemicals include thawing liquid, dilution liquid and cleaning liquid. In the embodiment of the present application, the above chemicals can be in the form of a solid state made into a hydrogel or in the form of a solution.

[0038] When the chemicals are in a solid state, the thawing solution, the dilution solution and the washing solution are embedded in the chip in the form of hydrogels, i.e., thawing hydrogel (TS gel), dilution hydrogel (DS gel) and washing hydrogel (WS gel). Here, the hydrogel is a solid material mainly composed of liquid, and has no fluidity because of the presence of a three-dimensional crosslinked network inside. The hydrogel may be soft or hard. The liquid in the hydrogel contains a non-permeable cryoprotectant and / or a basal culture medium. Specifically, the thawing hydrogel contains a basal culture medium and a very high concentration of a non-permeable cryoprotectant, such as 0.5-1.5M of a non-permeable cryoprotectant. The dilution hydrogel contains a basal culture medium and a lower concentration of a non-permeable cryoprotectant than the thawing hydrogel, such as 0.1-0.75M of a non-permeable cryoprotectant. The washing hydrogel contains only a basal culture medium.

[0039] Specifically, three compartments arranged in sequence on the chip are provided, and the thawed hydrogel, the diluted hydrogel (i.e., the first diluted hydrogel), and the washing hydrogel are sequentially embedded in the compartments, where the coverage area of ​​each compartment is larger than the opening area of ​​the groove of the carrier.Furthermore, four compartments arranged in sequence on the chip may be provided, and an additional compartment may be used to embed a second diluted hydrogel, where the non-permeable cryoprotectant contained in the second diluted hydrogel has a lower concentration than the non-permeable cryoprotectant contained in the first diluted hydrogel, and the change in osmotic pressure is relatively gradual, so that the osmotic pressure damage to the embryo can be less.

[0040] When the chemicals are in solution, the thawing solution, dilution solution and washing solution are sealed in solution in the chip by a permeable film. The basal medium and non-permeable cryoprotectant contained in the chemicals in solution form have the same concentration as the hydrogel, so they are not repeated here.

[0041] Specifically, three compartments arranged in sequence on the chip may be provided, and the thawing solution, dilution solution and washing solution may be sequentially sealed in the compartments, where the covering area of ​​each compartment is greater than the opening area of ​​the groove.Furthermore, four compartments arranged in sequence on the chip may be provided, and an additional compartment may be used to seal the second dilution solution, where the non-permeable cryoprotectant contained in the second dilution solution has a lower concentration than the non-permeable cryoprotectant contained in the first dilution solution, and the change in osmotic pressure is relatively gradual, so that the osmotic pressure damage to the embryo can be reduced.In practical application, a perforated film, grid or dialysis membrane with appropriate thickness and pore size may be selected and placed on the underside of the chip, and the solution may be directly added to the compartment, so that the solution is fixed on the chip by the support of the perforated film, grid, dialysis membrane or water-soluble film for the solution.

[0042] In step S108, the carrier is removed from the oil phase thawing liquid so that the chips cover the carrier.

[0043] After the rewarming is completed (usually, it can be immersed in the oil-phase thawing liquid for 2-3 seconds), the chip with the chemical encapsulated is placed on the carrier. In order to achieve a better diffusion removal effect, the hydrogel or solution-like thawing liquid, diluting liquid and cleaning liquid should cover a part of the carrier, i.e., not cover the groove of the carrier, and the clearance distance between the thawing liquid, diluting liquid and cleaning liquid and the groove of the carrier should be several millimeters.

[0044] In step S110, the chip is moved relative to the carrier to remove cryoprotectant from the grooves by contacting the chemical to completely cover the grooves.

[0045] When the solid hydrogel-like chemical is fixed on the chip, when the chip moves relative to the carrier, the thawing hydrogel, dilution hydrogel and washing hydrogel embedded in the chip move sequentially over the groove of the carrier and contact the biomaterial in the groove that needs to be thawed sequentially to cover the whole. Since the chemical molecules diffuse freely in the hydrogel, when they contact the groove of the carrier, the embryo and the cryoprotectant in the groove diffuse into the hydrogel, which serves to remove the cryoprotectant. Specifically, under the thawing hydrogel, the embryo can remove the high concentration of toxic permeating cryoprotectant in the embryo in response to the low permeating pressure difference. Under the dilution hydrogel, the water can slowly diffuse back into the embryo and orderly remove the non-permeating cryoprotectant molecules around the embryo (removal too quickly will cause permeating pressure damage to the embryo). Under the washing hydrogel, more water slowly diffuses back into the embryo, completely removing the non-permeating cryoprotectant molecules around the embryo and allowing the embryo to return to a normal osmotic environment. Embryos treated with these three hydrogels were already free of any cryoprotectant and neatly restored to a normal osmotic environment, allowing the embryos to be transferred from the carrier to a culture dish and cultured in an incubator.

[0046] When a chemical agent in solution is immobilized inside the chip, a permeable film is formed to cover the groove to prevent the embryo from spilling out. When the solutions on both sides of the permeable film come into contact with each other, the solution permeates, completing the diffusion and exchange of the solutions on both sides; see the examples of hydrogel for details.

[0047] That is, when the chemical agent, whether solid or liquid, removes the diffusion of the cryoprotectant, it can complete the diffusion exchange with the chemical agent in the groove while blocking the embryo in the groove, so that the chemical agent does not flow out to the chip. After the entire removal process is completed, the embryo can be quickly positioned and captured because it is in the groove of the carrier, transferred from the carrier to a culture dish, and cultured in an incubator for resuscitation. Since the composition of the medium of the washing hydrogel (or washing solution) is not necessarily identical to the composition of the user's medium, the user can place the embryo transferred from the carrier in the user's medium for final washing as needed, and then transfer it to the user's culture system for culture resuscitation.

[0048] In the example shown in Figure 1, an oil-phase thawing liquid is used for thawing and rewarming. In other examples, a non-oil-phase thawing liquid, i.e., a general thawing liquid, can also be used for thawing and rewarming. Referring to Figure 2, a method for thawing and resuscitating a biomaterial according to an embodiment of the present application includes the following steps:

[0049] In step S202, a first carrier is provided, containing a biomaterial that needs to be thawed and a cryoprotectant.

[0050] The first carrier is used to transport and store the biomaterial in a low temperature liquid nitrogen environment, and a cryoprotectant for protecting the biomaterial is in the vicinity or surroundings of the biomaterial, where the biomaterial can be a cell, a living tissue, an egg or an embryo, and the cryoprotectant can be a vitrification liquid. Hereinafter, the biomaterial will be described by taking an embryo as an example for simplicity.

[0051] In some embodiments, the tip of the first carrier may have a groove, and the biomaterial is contained in the groove of the first carrier.

[0052] In step S204, the first carrier is impregnated with the thawing liquid so that the thawing liquid covers the first carrier, and the biomaterial is thawed and rewarmed.

[0053] The first carrier is quickly transferred from the low-temperature environment to a vessel containing thawing solution, and the embryos carried in the first carrier are completely immersed in the thawing solution to ensure the thawing and warming speed, and the embryos are detached from the first carrier.

[0054] In step S206, a second carrier is provided having a groove proximate a tip thereof in which a first diluent is contained.

[0055] A second carrier may be pre-prepared at its tip, the second carrier including a groove filled with the first diluent such that the volume of the first diluent is greater than the volume of the groove.

[0056] In step S208, the biomaterial is transferred to the groove of the second carrier.

[0057] After the embryo is detached from the first carrier, the exact location of the embryo is identified and the embryo is transferred to the groove of the second carrier with a capillary tube (eg, a glass capillary tube).

[0058] In step S210, a chemical-encapsulated chip is provided that covers the second carrier, where the chemical includes a cryoprotectant and / or a basal medium.

[0059] In the embodiment of the present application, the cryoprotectant contained in the above chemical has a lower concentration than the cryoprotectant in the groove. In practical applications, the cryoprotectant contained in the chemical may be a non-permeable cryoprotectant or a permeable cryoprotectant. Specifically, when using a chemical to remove the cryoprotectant in the embryo, the chemical may contain a cryoprotectant and / or a basal culture medium, or may contain a non-permeable cryoprotectant and / or a basal culture medium. In order to obtain a better removal effect, a chemical whose components contain a non-permeable cryoprotectant and / or a basal culture medium may be used. This is because a non-permeable cryoprotectant is a molecule that does not pass through the cell membrane and generally does not have cytotoxicity. A permeable cryoprotectant is generally a small molecule that has cytotoxicity. In thawing applications, the role of the non-permeable cryoprotectant is to prevent the osmotic pressure difference between the inside and outside of the cell from becoming too large. That is, in the case of thawing, when the cells are taken out of liquid nitrogen, the inside of the cells is filled with a permeable cryoprotectant, so the osmotic pressure inside the cells becomes very high, and if the cells are put into a culture medium in the case of thawing, the osmotic pressure difference causes water molecules to rapidly enter the cells, causing the cells to die. However, when the cells are put into a solution containing a non-permeable cryoprotectant during thawing, the osmotic pressure difference is small, so the water molecules enter the cells at a slower rate, and at the same time, the permeable cryoprotectant inside the cells can diffuse out of the cells, so that the purpose of slowly removing the cryoprotectant can be achieved.

[0060] Here, the types of chemicals include a second diluent composed of a basal culture medium and a non-permeable cryoprotectant at a lower concentration than the first diluent, and a washing solution composed of only the basal culture medium. Specifically, two compartments arranged in sequence on the chip may be provided, and the second diluent and the washing solution may be sequentially sealed in the compartments, where the covering area of ​​each compartment is larger than the opening area of ​​the groove of the second carrier.

[0061] In the embodiment of the present application, the above chemicals can be in the form of a solid state made in hydrogel or in the form of a solution encapsulated in the chip. When the chemicals are in the form of a solid state, the second dilution liquid and the washing liquid are embedded in the chip in the form of hydrogel, i.e., the second dilution hydrogel (DS2 gel) and the washing hydrogel (WS gel). When the chemicals are in the form of a solution, the dilution liquid and the washing liquid are encapsulated in the chip in the form of a solution by a permeable film. In practical application, a perforated film, a grid or a dialysis membrane with a suitable thickness and pore size can be selected and placed on the underside of the chip, and the solution can be directly added to the compartment, so that the solution is fixed on the chip by the support of the perforated film, the grid, the dialysis membrane or the water-soluble film for the solution.

[0062] In step S212, the tip is moved relative to the second carrier to bring the chemical into contact with the grooves of the second carrier so as to completely cover the grooves, thereby removing the cryoprotectant from the grooves.

[0063] When the solid hydrogel-like chemical is fixed on the chip, when the chip moves relative to the carrier, the second dilution hydrogel and the washing hydrogel embedded in the chip move sequentially over the groove of the second carrier and contact the biomaterial in the groove that needs to be thawed sequentially so as to cover the entirety. Since the chemical molecules are freely diffusing in the hydrogel, when they contact the groove of the carrier, the embryo and the cryoprotectant in the groove diffuse into the hydrogel, which serves to remove the cryoprotectant. Under the second dilution hydrogel, the water can slowly diffuse back into the embryo and orderly remove the non-permeable cryoprotectant molecules around the embryo. Under the washing hydrogel, more water can slowly diffuse back into the embryo and completely remove the non-permeable cryoprotectant molecules around the embryo, allowing the embryo to return to a normal osmotic environment. The embryo treated with the hydrogel is already free of any cryoprotectant and has been orderly restored to a normal osmotic environment, and the embryo can be transferred from the carrier to a culture dish and cultured in an incubator.

[0064] When a chemical agent in solution is immobilized inside the chip, a permeable film is formed to cover the groove to prevent the embryo from spilling out. When the solutions on both sides of the permeable film come into contact with each other, the solution permeates, completing the diffusion and exchange of the solutions on both sides; see the examples of hydrogel for details.

[0065] That is, when the chemical agent, whether solid or liquid, removes the diffusion of the cryoprotectant, it can complete the diffusion exchange with the solution in the groove while blocking the embryo in the groove, so that the chemical agent does not flow out to the chip. After the entire removal process is completed, the embryo can be quickly positioned and captured because it is in the groove of the carrier, transferred from the carrier to a culture dish, and cultured in an incubator for resuscitation. Since the composition of the medium of the washing hydrogel (or washing solution) is not necessarily identical to the composition of the user's medium, the user can place the embryo transferred from the carrier in the user's medium for final washing as needed, and then transfer it to the user's culture system for culture resuscitation.

[0066] When an oil-phase thawing solution is used for thawing and rewarming, when the embryo is carried in a non-standardized carrier, the cryoprotectant can be removed in conjunction with the chip by transferring the embryo to a standardized carrier after thawing and rewarming. Referring to FIG. 3, according to an embodiment of the present application, a method for thawing and resuscitating a biomaterial is further provided, specifically including the following steps:

[0067] In step S302, a first carrier is provided, containing a biomaterial that needs to be thawed and a cryoprotectant.

[0068] In step S304, the first carrier is impregnated with the oil-phase thawing liquid so that the oil-phase thawing liquid covers the first carrier, and the biomaterial is thawed and rewarmed.

[0069] In step S306, a second carrier is provided having a groove proximate a tip thereof in which a first diluent is contained.

[0070] The second carrier is operable and usable in conjunction with the chip.

[0071] In step S308, the biomaterial is transferred to the groove of the second carrier. In step S310, a chemical-encapsulated chip is provided that covers the second carrier, where the chemical includes a cryoprotectant and / or a basal medium.

[0072] Here, the chemical agent may include a cryoprotectant and / or a basal culture medium, or may include a non-permeable cryoprotectant and / or a basal culture medium. Preferably, a chemical agent whose components include a non-permeable cryoprotectant and / or a basal culture medium may be used. And, the chemical agent does not cover the groove of the carrier, covers a part of the carrier, and has a clearance distance between the groove of the carrier.

[0073] In step S312, the cryoprotectant is removed from the grooves of the second carrier by moving the chip relative to the carrier and contacting the chemical so as to completely cover the grooves.

[0074] The explanations of the terms in this embodiment may refer to the same or similar parts as in the previous embodiment, and will not be repeated here.

[0075] Referring to FIG. 4, according to an embodiment of the present application, there is further provided a method for thawing and reanimating a biological material, comprising the following steps:

[0076] In step S402, a (first) cryoprotectant and / or chemicals including a basal medium are prepared into a hydrogel structural form.

[0077] Here, the chemicals may include a permeable cryoprotectant and / or a basal medium, or may include a non-permeable cryoprotectant and / or a basal medium. Preferably, a chemical whose components include a non-permeable cryoprotectant and / or a basal medium may be used.

[0078] In step S404, the biomaterial containing the (second) cryoprotectant is contacted with said hydrogel, and diffusion of the (second) cryoprotectant into the hydrogel removes the diffusion of the (second) cryoprotectant.

[0079] Here, the chemical agent includes one or more of a thawing solution, a dilution solution, or a cleaning solution.

[0080] Here, the preparation of the hydrogel may be carried out by a preparation method of a physical hydrogel, such as a preparation method of a sodium alginate hydrogel, a gelatin hydrogel, or an agarose gel, or may be carried out by a preparation method of a chemical hydrogel, such as a preparation method of a PEGDA hydrogel or a GelMA hydrogel. Furthermore, in this embodiment, the thawing solution is directly prepared as a one-part hydrogel, so that the cryoprotectant can be removed from the embryo at once (e.g., vitrification freezing solution). However, when used, the thawing solution is made into a hydrogel of multiple parts of thawing solution with different concentrations according to specific circumstances, such as the concentration of the thawing solution used, and then the cryoprotectant is removed in an orderly and controllable manner by the multiple parts of hydrogel with different concentrations, thereby completing the whole operation of removing the cryoprotectant. The preparation methods of various forms of hydrogels are described in detail below.

[0081] Additionally, a method for preparing an agarose gel includes It involves dissolving agarose in a chemical solution at 80-90°C to prepare a 0.1-6% agarose solution, and then stirring, mixing, cooling, and hardening to obtain an agarose gel containing the chemicals.

[0082] Further, a method for preparing a sodium alginate hydrogel comprises: The process involves dissolving sodium alginate in a thaw solution to make a 0.3-10% sodium alginate solution, dissolving calcium chloride in the thaw solution to make a 0.01M-0.2M calcium chloride solution, and slowly adding the calcium chloride solution on top of the sodium alginate solution in the chip to allow the entire sodium alginate solution to harden into a calcium alginate hydrogel by the time the calcium chloride has diffused to the bottom of the chip.

[0083] Further, the method for preparing a gelatin hydrogel comprises: The method involves dissolving gelatin in a thawing liquid solution at 36-45°C to prepare a 1-15% gelatin solution, and then adding the 36-50°C gelatin solution to the chip and cooling it to harden it into a hydrogel containing the thawing liquid.

[0084] Furthermore, the method for preparing GelMA hydrogels includes: The process involves dissolving GelMA in a 36-50°C thaw solution to make a 1-15% GelMA solution, dissolving a photoinitiator in the 36-50°C GelMA solution at a concentration of 0.001-2%, adding the 36-50°C GelMA solution to the chip, and irradiating the 36-50°C GelMA solution with light for 2-30 minutes to harden the GelMA solution into a hydrogel.

[0085] According to an embodiment of the present invention, a system for thawing and resuscitating a biological material is provided, and referring to FIGS. 5 to 11, said system includes a carrier 1, a vessel 5 and a tip 2.

[0086] Referring to FIG. 6, the carrier 1 is provided with a groove 13 near the tip, in which a biomaterial that needs to be thawed and a cryoprotectant (VS) that protects the biomaterial that needs to be thawed are accommodated, where the biomaterial can be a cell, a biological tissue, an egg, or an embryo, and the cryoprotectant can be a vitrification liquid. The carrier 1 is a strip-shaped structure composed of a handle 11, a sheet 12, and a groove 13. Here, the groove 13 is located near the tip of the sheet 12, and accommodates a biomaterial that needs to be thawed and a cryoprotectant (VS) that protects the biomaterial that needs to be thawed, where the biomaterial can be a cell, a biological tissue, an egg, or an embryo, and the cryoprotectant can be a vitrification liquid. The size of the groove 13 can be adjusted and set according to the number and size of the embryos to be processed and the amount of solution to be held.

[0087] In the embodiment of the present application, the carrier 1 can be in a band-like structure to facilitate the thawing and rewarming process of the embryo in conjunction with the system and to improve the adaptability of the carrier. At the same time, the handle part of the carrier is preferentially designed to have a structure with a sufficient width to facilitate the installation of a label that marks the relevant information about the embryo to be processed, and the sheet is made of a plastic material with a uniform thickness, transparent material, biocompatible material, and good heat transfer properties, thereby ensuring the adaptability of the contained embryo and the heat transfer rate during the subsequent thawing. Similarly, in other embodiments, the carrier can also be designed into other structural forms with grooves, such as a flat plate structure, according to different use conditions and requirements.

[0088] The vessel 5 is a container that contains an oil-phase thawing liquid inside, and its shape and structure are not particularly limited. It can be any container that has the function of containing an oil-phase thawing liquid, but preferably it can be a dish-shaped container. The oil-phase thawing liquid does not contain a non-permeable cryoprotectant. The carrier is impregnated with the oil-phase thawing liquid so that the grooves of the carrier are impregnated below the liquid level, and the embryo can be thawed and rewarmed with the oil-phase thawing liquid so that it is rewarmed to room temperature or 37°C. The cryoprotectant does not fuse with the oil-phase thawing liquid, and the density of the oil-phase thawing liquid is lower than that of the cryoprotectant, so that the cryoprotectant in the groove remains in the groove together with the embryo, that is, the cryoprotectant in the groove remains in the groove together with the embryo that needs to be thawed during the thawing and rewarming process, and does not float out of the carrier.

[0089] 7, the chip 2, sometimes called a biochip, adopts a plate-like frame structure and is provided with a plurality of sequentially arranged compartments 22 for respectively fixing or encapsulating chemicals. In this embodiment, the chip 2 adopts a frame structure consisting of two support plates 20 and two partition plates 21. Here, the two support plates 20 are held in a parallel relationship, the two partition plates 21 are held in a parallel relationship, and the two partition plates 21 are located between the two support plates 20, dividing the area between the two support plates 20 into three mutually independent compartments 22 for respectively arranging different administration chemicals.

[0090] Optionally, the partition plate 21 and the support plate 20 in the chip 2 may be movably connected to each other so that the size of the compartment 22 can be freely adjusted. For example, by providing slide grooves on the opposing surfaces of the two support plates 20 and inserting the ends of the partition plate 21 into the slide grooves, the position of the partition plate 21 can be freely adjusted within the slide grooves, thereby realizing adjustment of the size of the compartment according to the difference in the amount of the chemical agent to be administered.

[0091] The types of chemicals in the chip include thawing liquid, dilution liquid and washing liquid. Here, the chemicals to be encapsulated in the chip can be in solid form or liquid form, and when in solid form, they are made into hydrogels, i.e., thawing hydrogel, dilution hydrogel and washing hydrogel, and embedded in the chip. When the chemicals are in solution form, the thawing liquid, dilution liquid and washing liquid are encapsulated in the chip in solution form by a permeable film. In practical application, a perforated film, grid or dialysis membrane with appropriate thickness and pore size can be selected and placed on the underside of the chip, and the solution can be directly added to the compartment, so that the solution is fixed on the chip by the support of the perforated film, grid, dialysis membrane or water-soluble film for the solution.

[0092] Referring to FIG. 8, the chip 2 containing the chemical agent is covered by the above-mentioned carrier 1, and the chemical agent does not cover the groove of the carrier, but only covers a part of the area of ​​the carrier, and has a clearance distance between the groove of the carrier. By moving the chip relative to the carrier, the thawing solution, dilution solution and washing solution in solid or liquid form can move on the groove of the carrier in sequence, and sequentially contact the biomaterial in the groove that needs to be thawed so as to cover the whole, and the cryoprotectant can be removed from the groove. In the removal process, the embryo completes the diffusion exchange with the chemical agent in the groove while being blocked in the groove, and does not flow out of the chip. When the entire removal process is completed, it is already free of any cryoprotectant and has been orderly restored to a normal osmotic pressure environment, and at this time, the embryo remains in the groove of the carrier, so that it can be quickly positioned and captured, transferred from the carrier to a culture dish, and cultured in an incubator to be revived.

[0093] In this embodiment, three compartments are provided on the chip to respectively place solutions containing different concentrations of cryoprotectants, so that the concentration relationship of different solutions administered to the embryos in sequence can be precisely controlled and the administration accuracy of the solutions can be ensured. In some other embodiments, the number of compartments on the chip can be appropriately adjusted according to the type, number and concentration control requirements of the chemicals administered, so that the concentration gradient between the chemicals in the adjacent compartments can be precisely controlled and the administration accuracy of the chemicals can be ensured.

[0094] It should be noted that in this example, the carrier provided has only one groove, however in other examples, it is entirely possible to have multiple grooves on the carrier at the same time depending on the number of embryos to be processed, so that multiple embryos can be processed simultaneously on the same carrier, improving processing efficiency.

[0095] 9 and 10, the above-mentioned system for thawing and resuscitating biological materials may further include a base 3 which is a channel steel structure, in which the central region of the base 3 is used for supporting and fixing the carrier 1, and the two side walls along its length are used as tracks 31 for supporting and adsorbing and fixing the support plate 20 of the chip 2, and the positional relationship between the chip 2 and the upper surface of the carrier 1 can be adjusted by changing the height of the tracks 31, thereby ensuring effective contact between the hydrogel and the solution in the groove.

[0096] In another embodiment, one guide step can be provided for each of the two tracks of the base 3. In this way, when the chip is disposed between two guide steps, it is also possible to form a guide effect on the chip during movement by using the guide steps, and the accuracy of the movement direction of the chip on the base can be improved.

[0097] A removable fixed connection can be formed between the track 31 and the chip 2 by magnetic attraction. For example, the track 31 is made of a ferromagnetic metal, and a magnet 24 is provided at the corresponding position of the support plate 20, so that a fixed connection by magnetic attraction is formed between the track 31 and the chip 2. More preferably, the track can adopt the form of an electromagnet structure, so that quick connection and release between the base and the chip can be realized by electrical control, and the convenience of operation can be improved.

[0098] By installing the base, for example, through an adhesive or engagement connection between the base and the carrier, it is possible to provide support and fixation for the carrier, ensure the positional stability of the carrier during the entire operation process, as well as provide support and fixation for the chip, maintain effective contact between the chip and the carrier, and avoid the chip and the carrier from unintentionally peeling off during relative movement to affect normal operation; at the same time, the base can also collect the solution flowing out of the groove to avoid contamination of the surrounding environment caused by the solution overflowing.

[0099] 9, the base 3 is also provided with a light-transmitting area 32 at the center, that is, the area of ​​the base 3 corresponding to the groove 13 is formed as a light-transmitting area by selecting a light-transmitting material. At this time, the carrier and the chip as a whole are moved to a microscope through the base by using the transmitted light through the light-transmitting area for direct observation, thereby realizing real-time observation of the chemical dosing process and accurately controlling the progress of the chemical dosing.

[0100] In addition, according to the requirements of actual operation, the light-transmitting region can be selected from traditional light-transmitting materials to only meet the requirements of light transmission, or can be selected from light-transmitting materials with heating function, such as heating glass materials, to simultaneously meet the purposes of light transmission and temperature control.

[0101] Furthermore, although only one carrier and one chip are provided on the base in this embodiment, in the actual operation process, multiple carriers can be arranged side by side on the base at the same time depending on the number of embryos to be processed and the width size of the chip compartment, i.e., the hydrogel coating width, so that during one movement of the chip, the operation of administering chemicals to embryos in multiple carriers can be performed simultaneously, improving the efficiency of the operation.

[0102] 11, the system for thawing and reanimating the above-mentioned biological material may further include a base 4 for directly supporting and fixing the base 3, to which a driving unit consisting of a stepping motor 41, a screw 42 and a push rod 43 is attached. Here, the base 3 is located on the base 4 and kept parallel to the screw 42, and the push rod 43 is covered by the screw 42 and can be reciprocated along the screw 42 by driving the stepping motor 41, and is fixedly connected to the tip 2 at the same time.

[0103] At this time, by driving the push rod 43 to reciprocate horizontally along the screw 42 by the stepping motor 41, the chip 2 can be reciprocated horizontally relative to the carrier 1, and the hydrogels in different compartments of the chip 2 can be controlled to be contacted sequentially with the solution in the groove 13, thereby realizing automated control. Furthermore, by controlling the operation of the stepping motor 41, the contact time between the different hydrogels in the chip 2 and the solutions in the groove 13 can also be precisely controlled, so that the administration time of the solutions in the hydrogels and the contact time between the embryo and the different solutions can be precisely controlled, and the administration accuracy of the different solutions to the embryo can be further improved.

[0104] Furthermore, in another embodiment, instead of a screw and push rod, a track slider structure is selected to form the drive unit, and the reciprocating movement of the slider along the track drives the chip to move relative to the carrier.

[0105] 11, the base 4 is also provided with one optical axis 44, which is a smooth cylindrical shaft. The optical axis 44 is kept parallel to the screw 42 and connected to the free end of the push rod 43, providing auxiliary guidance for the reciprocating movement of the push rod 43, improving the stability of the movement of the tip 2 driven by the push rod 43, and improving the stability of the contact process between the hydrogel and the solution in the groove.

[0106] As shown in the figure, a hollow area 45 is also provided at the center position of the base 4, that is, the corresponding area of ​​the light-transmitting area in the chip 3 is a hollow structure. In this way, it can ensure that the light passes through the base 4 and is smoothly projected onto the light-transmitting area of ​​the chip 3, and ensure the normal use of the microscope. Similarly, according to the actual use under different environments, the hollow area can be made of light-transmitting materials such as light-transmitting glass to achieve the purpose of light transmission, or can be selected from light-transmitting materials with heating function such as heating glass to simultaneously meet the purpose of light transmission and temperature control.

[0107] Furthermore, in the above embodiments, the technical solution of the present invention is introduced by taking the administration operation of different chemical solutions in the embryo thawing process as an example, but according to the concept of the present invention, it is completely possible for those skilled in the art to apply the chemical delivery system to the precise delivery of chemicals to other biological materials or basic solutions. For example, when administering a powdered chemical into a solution, a single dose of the chemical is temporarily supported by a water-soluble film, and when the water-soluble film comes into contact with the solution and dissolves during the movement of the chip relative to the carrier, a quantitative amount of the chemical is directly released into the solution, thereby completing the precise administration of the chemical.

[0108] According to an embodiment of the present application, there is further provided a system for thawing and reanimating a biological material, including a first carrier, a vessel, a second carrier, a capillary tube, and a tip, specifically comprising: The first carrier contains the biomaterial that needs to be thawed and the cryoprotectant. The present application does not limit the shape or result of the first carrier, i.e., the first carrier can have a groove and carry the biomaterial in the groove, or it can have no groove and carry the biomaterial by other means.

[0109] the vessel contains a thawing solution that coats the impregnated first carrier and thaws and rewarms the biomaterial to detach the biomaterial from the first carrier; a groove containing a first diluent is provided near a tip of the second carrier; the capillary is used to transfer the biomaterial to the groove of the second carrier; The chip is filled with chemicals including a cryoprotectant and / or a basic culture medium, and the chip is moved relative to the second carrier, the chemicals are brought into contact with the grooves of the second carrier so as to completely cover the grooves, and the cryoprotectant is removed from the grooves.

[0110] In the embodiment of the present application, the cryoprotectant contained in the above chemical has a lower concentration than the cryoprotectant in the groove. In practical application, the cryoprotectant contained in the chemical may be a non-permeable cryoprotectant or a permeable cryoprotectant. Specifically, the chemical may contain a cryoprotectant and / or a basal culture medium, or a non-permeable cryoprotectant and / or a basal culture medium, when the cryoprotectant in the embryo is removed by the chemical. In order to obtain a better removal effect, a chemical may be used whose components contain a non-permeable cryoprotectant and / or a basal culture medium.

[0111] Here, the types of chemicals include a second diluent consisting of a basal culture medium and a non-permeable cryoprotectant at a lower concentration than the first diluent, and a washing solution consisting of only the basal culture medium. The chemicals can be sealed in the chip in a solid state or a solution state. If the chemicals are in a solid state, the second diluent and the washing solution are fixed in a hydrogel state, 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 move sequentially over the groove and contact the groove sequentially so as to cover the entire groove. If the chemicals are in a solution state, the diluent and the washing solution are sealed in the chip by a permeable film. In practical applications, a perforated film, grid, or dialysis membrane with a suitable thickness and pore size can be selected and placed on the underside of the chip, and the solution can be directly added to the compartment so as to be fixed in the chip by the support of the perforated film, grid, dialysis membrane, or water-soluble film for the solution.

[0112] The shape and structure of the second carrier and the chip in this example are similar to those described in the above examples and will not be repeated here. In this example, the embryo is immersed in a non-oil phase thawing solution by the first carrier, thawed and rewarmed, and since the first carrier cannot be used in combination with the chip (and other components in the system), it is necessary to transfer the embryo to a second carrier and use the second carrier in combination with the chip (and other components in the system) to remove the cryoprotectant.

[0113] It should be noted that the system in this embodiment may also include components such as a base and a pedestal, which will not be repeated here.

[0114] The operational steps of the method of the present invention correspond to the structural features of the system and may be cross-referenced and will not be repeated here.

[0115] The above technical solutions of the present invention have the following advantageous effects:

[0116] (1) The present invention proposes a standardized process driven by a machine to adjust the removal rate of the cryoprotectant, as opposed to the existing manual thawing. The thawing and reanimation process is stable every time, and the flow is simple. When combined with the freezing method previously applied for by the applicant, the embryologist does not need to add the embryo to the carrier, and only needs to move the embryo from the carrier to the culture dish before the machine completes the thawing process. The embryo is in one fixed position on the carrier during the entire thawing and reanimation process, so the embryologist does not need to search for the embryo in a large volume of liquid, and the throughput is high.

[0117] (2) In contrast to existing microfluidics, the carrier of the present invention has no dead volume and the position of the embryo on the carrier allows the embryologist to directly retrieve the embryo using a glass capillary tube.

[0118] (3) The throughput of the present invention is higher than that of existing robots (which mimic the human hand), the cost is lower because the core function only requires one linear motor platform, and no machine vision is required for embryo identification.

[0119] It should be understood by those skilled in the art that the embodiments of the present invention may be provided as a method, a system, or a computer program product. Thus, the present invention may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment that is a combination of software and hardware. Furthermore, the present invention may take the form of a computer program product embodied on one or more computer usable storage media (including, but not limited to, disk storage, CD-ROM, optical memory, etc.) that contains computer usable program code therein.

[0120] The above description is merely one embodiment of the present invention, and is not intended to limit the present invention, and various modifications and variations are possible for those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention are intended to be included in the scope of the claims of the present invention. [Brief description of the drawings]

[0121] [Figure 1] FIG. 1 is a flow chart of a method for thawing and resuscitating biological material according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a flow chart of a method for thawing and resuscitating biological material according to another embodiment of the present invention. [Diagram 3] FIG. 3 is a flow chart of a method for thawing and resuscitating biological material according to a further embodiment of the invention. [Figure 4] FIG. 4 is a flow chart of a method for thawing and resuscitating biological material according to yet another embodiment of the present invention. [Diagram 5] FIG. 5 is a schematic diagram of a carrier and vessel according to an embodiment of the present invention. [Figure 6] FIG. 6 is a schematic diagram of a carrier according to an embodiment of the present invention. [Figure 7] FIG. 7 is a schematic diagram of a chip according to an embodiment of the present invention. [Figure 8] FIG. 8 is a schematic diagram of a system for thawing and resuscitating biological material according to one embodiment of the present invention. [Figure 9] FIG. 9 is a structural schematic diagram of a base according to an embodiment of the present invention. [Figure 10] FIG. 10 is a schematic diagram of a system for thawing and resuscitating biological material in accordance with another embodiment of the present invention. [Figure 11] FIG. 11 is a schematic diagram of a system for thawing and resuscitating biological material according to a further embodiment of the present invention.

Claims

1. a carrier having a groove near the tip in which biomaterial that needs to be thawed and a cryoprotectant are contained; a vessel covering the grooves of the impregnated carrier and containing an oil-phase thawing solution for thawing and rewarming the biomaterial; and a tip in which a chemical agent including a cryoprotectant and / or a basal culture medium is enclosed, the tip being moved relative to the carrier and contacting the chemical agent with the groove so as to entirely cover the groove, thereby removing the cryoprotectant from the groove. A system for thawing and resuscitating biological material.

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

3. The chemicals include a thawing liquid, a diluent, and a cleaning liquid, each of which is made in a hydrogel state; the chip is provided with a plurality of compartments for successively embedding the thawing liquid hydrogel, the diluent hydrogel, and the cleaning liquid hydrogel; the covering area of ​​each compartment is larger than the opening area of ​​the groove; and when the chip moves relative to the carrier, the thawing liquid hydrogel, the diluent hydrogel, and the cleaning liquid hydrogel move successively onto the groove and come into contact with the groove successively so as to cover the entire groove. The system of claim 2.

4. The chemicals include a thawing solution, a diluting solution, and a washing solution, and the chip is provided with a permeable film that supports the thawing solution, the diluting solution, and the washing solution and is a perforated film, a grid, a dialysis membrane, or a water-soluble film. The system of claim 2.

5. Before the chip is moved relative to the carrier, the chemical does not cover the groove of the carrier, covers a part of the carrier, and has a clearance distance between the chemical and the groove of the carrier.

5. A system according to claim 3 or 4.

6. The central region is used for placing the carrier, and has two tracks parallel to each other in the length direction, and the tracks further include a base for supporting and fixing the chip so that the lower surface of the chip remains in contact with the upper surface of the carrier.

5. A system according to claim 3 or 4.

7. a first carrier containing a biological material in need of thawing and a cryoprotectant; a vessel containing a thawing liquid for coating the first carrier and for thawing and rewarming the biomaterial so as to detach the biomaterial from the first carrier; a second carrier having a groove near a tip thereof in which a first diluent is contained; a capillary tube for transferring the biomaterial to the groove of the second carrier; a tip containing a chemical agent including a cryoprotectant and / or a basal culture medium, the tip being moved relative to the second carrier and contacting the chemical agent with the groove of the second carrier so as to completely cover the groove, thereby removing the cryoprotectant from the groove; Characterized in that it comprises A system for thawing and resuscitating biological material.

8. The cryoprotectant contained in the chemical is a non-permeable cryoprotectant. The system of claim 1 .

9. The chemicals include a second diluent and a washing solution, which are made in a hydrogel state, and the non-permeable cryoprotectant contained in the second diluent has a lower concentration than the non-permeable cryoprotectant contained in the first diluent; the chip is provided with a plurality of compartments for sequentially embedding the second diluent hydrogel and the washing solution hydrogel, and the covering area of ​​each compartment is larger than the opening area of ​​the groove; and when the chip moves relative to the carrier, the second diluent hydrogel and the washing solution hydrogel move sequentially onto the groove and sequentially come into contact with the groove so as to entirely cover it. The system of claim 8.

10. 9. The system of claim 8, wherein the chemicals include a second diluent in a solution having a lower concentration of a non-permeable cryoprotectant than the non-permeable cryoprotectant contained in the first diluent, and a washing solution, and the chip supports the second diluent in a solution and the washing solution and is provided with a permeable film that is a perforated film, a grid, a dialysis membrane, or a water-soluble film.

11. Before the chip is moved relative to the second carrier, the chemical does not cover the groove of the second carrier, covers a partial area of ​​the second carrier, and has a clearance distance between the groove of the second carrier and the chemical.

11. A system according to claim 9 or 10.

12. The system described in claim 9 or 10, characterized in that the central area is used for positioning the second carrier and is provided with two tracks parallel to each other in the longitudinal direction, and the tracks further include a base for supporting and fixing the chip so that its lower surface remains in contact with the upper surface of the second carrier.

13. providing a carrier having a groove near the tip in which the biomaterial to be thawed and a cryoprotectant are contained; impregnating the grooves of the carrier with the oil-phase thawing liquid so that the grooves of the carrier are covered with the oil-phase thawing liquid, and thawing and rewarming the biomaterial; Providing a chip in which chemicals including a cryoprotectant and / or a basal culture medium are encapsulated; removing the carrier from the oil phase thawing solution and allowing the carrier to cover the chip; removing cryoprotectant from the grooves of the carrier by moving the tip relative to the carrier and contacting the chemical agent so as to completely cover the grooves; Characterized in that it comprises A method for thawing and reanimating biological material.

14. The cryoprotectant contained in the chemical is a non-permeable cryoprotectant. The method of claim 13.

15. Before the chip is moved relative to the carrier, the chemical does not cover the groove of the carrier, covers a part of the carrier, and has a clearance distance between the chemical and the groove of the carrier. The method of claim 14.

16. The chemicals include a thawing liquid, a diluent, and a cleaning liquid, each of which is made in a hydrogel state; the chip is provided with a plurality of compartments for successively embedding the thawing liquid hydrogel, the diluent hydrogel, and the cleaning liquid hydrogel; the covering area of ​​each compartment is larger than the opening area of ​​the groove; and when the chip moves relative to the carrier, the thawing liquid hydrogel, the diluent hydrogel, and the cleaning liquid hydrogel move successively onto the groove and come into contact with the groove successively so as to cover the entire groove. The method of claim 14.

17. The chemicals include a thawing solution, a diluting solution, and a washing solution, and the chip is provided with a permeable film that supports the thawing solution, the diluting solution, and the washing solution and is a perforated film, a grid, a dialysis membrane, or a water-soluble film. The method of claim 14.

18. providing a first carrier containing a biological material in need of thawing and a cryoprotectant; impregnating the first carrier with the thawing liquid so that the thawing liquid covers the first carrier, and thawing and rewarming the biomaterial; providing a second carrier having a groove adjacent a tip thereof in which a first diluent is contained; transferring said biomaterial into a groove of said second carrier; providing a chip containing chemicals including a cryoprotectant and / or a basal medium, the chip covering the second carrier; removing cryoprotectant from the grooves of the second carrier by moving the tip relative to the second carrier and contacting the chemical agent so as to completely cover the grooves; Characterized in that it comprises A method for thawing and reanimating biological material.

19. The cryoprotectant contained in the chemical is a non-permeable cryoprotectant.

20. The method of claim 18.

20. Before the chip is moved relative to the carrier, the chemical does not cover the groove of the second carrier, covers a partial area of ​​the second carrier, and has a clearance distance between the groove of the second carrier and the chemical.

20. The method of claim 19.

21. The chemicals include a second diluent and a washing solution, which are made in a hydrogel state, and the non-permeable cryoprotectant contained in the second diluent has a lower concentration than the non-permeable cryoprotectant contained in the first diluent; the chip is provided with a plurality of compartments for sequentially embedding the second diluent hydrogel and the washing solution hydrogel, and the covering area of ​​each compartment is larger than the opening area of ​​the groove; and when the chip moves relative to the carrier, the second diluent hydrogel and the washing solution hydrogel move sequentially onto the groove and sequentially come into contact with the groove so as to entirely cover it.

20. The method of claim 19.

22. The chemicals include a second diluent and a washing solution in a liquid state, the second diluent containing a non-permeable cryoprotectant having a lower concentration than the non-permeable cryoprotectant contained in the first diluent, and the chip is provided with a permeable film that supports the second diluent and the washing solution in a liquid state and is a perforated film, a grid, a dialysis membrane, or a water-soluble film.

20. The method of claim 19.

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