WO2023036223A1 - 用于生物组织冷冻保存或解冻复苏的装置 - Google Patents

用于生物组织冷冻保存或解冻复苏的装置 Download PDF

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Publication number
WO2023036223A1
WO2023036223A1 PCT/CN2022/117744 CN2022117744W WO2023036223A1 WO 2023036223 A1 WO2023036223 A1 WO 2023036223A1 CN 2022117744 W CN2022117744 W CN 2022117744W WO 2023036223 A1 WO2023036223 A1 WO 2023036223A1
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Prior art keywords
solution
groove
biochip
carrier
microchannel
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PCT/CN2022/117744
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English (en)
French (fr)
Inventor
陈昊楠
舒怡玮
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深圳拜尔洛克生物技术有限公司
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Priority claimed from CN202111063574.XA external-priority patent/CN115777681A/zh
Priority claimed from CN202111061882.9A external-priority patent/CN115777680A/zh
Application filed by 深圳拜尔洛克生物技术有限公司 filed Critical 深圳拜尔洛克生物技术有限公司
Publication of WO2023036223A1 publication Critical patent/WO2023036223A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the invention relates to the technical field of bioengineering, in particular to a device for cryopreservation or thawing recovery of biological tissues.
  • Cell cryopreservation technology is an important technology in many biological fields. It is also important to restore cells from a cryogenically frozen state to a normal metabolic state.
  • the process of thawing and resuscitation includes the process of removing the cryoprotectant and replacing it with a normal medium.
  • the devices in the prior art are complicated to operate when the embryo is frozen or thawed, and the efficiency is low.
  • the main purpose of the present invention is to provide a device for cryopreservation or thawing and resuscitating of biological tissues, so as to solve the problems of low efficiency caused by complex operation of the device in the prior art when freezing or thawing embryos.
  • a device for cryopreservation or thawing recovery of biological tissue which includes: a biochip, which includes a solution inlet and a solution outlet; a carrier, which has a first groove, and the first Biological tissue is carried in the groove; wherein, the carrier is closely attached to the bottom of the biochip, and a microchannel passing through the first groove is formed between the biochip and the carrier, and the microchannel Only for solution to pass through.
  • a device for cryopreservation or thawing and recovery of biological tissue which includes: a biochip, the biochip includes a solution inlet and a solution outlet; the bottom of the biochip is provided with a permeable film , to form a microchannel between the biochip and the permeable film; a carrier, the carrier has a groove, and biological tissues are carried in the groove; wherein, the carrier is closely attached to the Below the chip, the permeable film covers the groove and confines the biological tissue in the groove, and the solution in the microchannel diffuses into the groove through the permeable film.
  • a microchannel that can only allow the solution to pass is formed above the groove of the carrier, and the biological tissue is restricted in the groove, so that the cryoprotectant can be delivered or removed in an orderly manner, effectively improving Improve the efficiency of biological tissue cryopreservation or thawing recovery operation.
  • FIG. 1A is a schematic perspective view of a device according to an embodiment of the present invention.
  • Figure 1B is a top view of the device in Figure 1A;
  • Fig. 1C is a schematic cross-sectional view along line 1C-1C in Fig. 1B;
  • Figure 1D is a partially enlarged view in Figure 1C;
  • Figure 2A is a top view of a device according to another embodiment of the present invention.
  • Fig. 2B is a schematic cross-sectional view along line 2B-2B in Fig. 2A;
  • Figure 2C is a partially enlarged view in Figure 2B;
  • Figure 3A is a top view of a device according to yet another embodiment of the present invention.
  • Fig. 3B is a schematic cross-sectional view along line 3B-3B in Fig. 3A;
  • Fig. 3C is a partially enlarged view among Fig. 3B;
  • FIG. 4A is a top view of a device according to yet another embodiment of the present invention.
  • Fig. 4B is a schematic cross-sectional view along line 4B-4B in Fig. 4A;
  • FIG. 4C is a partially enlarged view of FIG. 4B.
  • the device includes: biochip 11 and carrier 12, wherein the biochip 11 is generally rectangular parallelepiped structure, biochip Part or all of the bottom (or lower surface) of the biochip is an open area, that is to say, the entire bottom of the biochip is open, or the bottom area of the corresponding carrier of the biochip is open.
  • the biochip 11 may include two solution inlets 111 and 112 and one solution outlet 113 . The number of solution inlets of the biochip 11 can be adjusted according to actual needs. If multiple cryoprotectant solutions need to flow through the embryos sequentially, multiple solution inlets can be provided.
  • the inlet channels shown in the figure are only illustrative, and what is important is that the solutions flowing into the biochip 11 through different solution inlets can flow to the vicinity of the embryo in sequence.
  • the carrier 12 is generally a strip structure, which includes a front sheet and a rear support rod.
  • the front sheet of the carrier 12 can be made of a plastic material with uniform thickness, transparent material, biocompatibility and good heat transfer, so as to ensure the suitability for holding embryos and the heat transfer speed during subsequent freezing.
  • the rear end of the carrier 12 can also be referred to as a handle or a handle, which is a position directly touched by the operator's hand. In the drawings in this application, only the front sheet part of the carrier 12 is shown, and the rear part of the carrier 12 is not shown as a key part.
  • the front end of the carrier 12 (front end sheet) has a groove 121, the groove 121 is used to hold the biological tissue to be treated and the related solution for protecting the biological tissue, and the surface of the groove 121 can be circular.
  • the biological tissue may be biological materials such as embryos, eggs, cells, etc., which is not limited in the present application, and the following description will be made by taking the biological tissue as an embryo as an example.
  • the thickness of the carrier 12 may be 0.25-0.7 mm
  • the depth of the groove 121 may be 0.2-0.5 mm
  • the diameter of the groove 121 may be 0.2-1 mm.
  • a microchannel 13 is formed between the chip 11 and the carrier 12 to pass through the groove 121 and only allow solution (such as freezing liquid or thawing liquid) to pass through.
  • the height (or width) of the microchannel 13 is smaller than the diameter of the embryo.
  • the microchannel 13 includes a solution micro-inlet 131, a solution micro-outlet 132, a solution inlet channel and a solution outlet channel, wherein the solution inlet channel is between the solution micro-inlet 131 and the groove 121, and the solution inlet channel is between the groove 121 and the solution micro-outlet 132.
  • the space is the solution outlet channel.
  • the height (or width) of the solution micro-inlet 131, the solution micro-outlet 132, the solution inlet channel and the solution outlet channel are equal or approximately equal, and the height (or width) is smaller than the diameter of the embryo. In this way, during the freezing and/or thawing operation, the microchannel 13 can only allow the solution to pass through but not the embryo, so that the embryo is confined in the groove 121 and will not float out of the groove 121 .
  • the biochip 11 When cryopreservation is performed, the biochip 11 is placed on the carrier 12 and pressed tightly, wherein the groove 121 of the carrier 12 carries embryos, and a microchannel is formed between the biochip 11 and the carrier 12 for the solution to pass through.
  • a biochip solution pusher such as a syringe pump or an air pump
  • the freezing solution is slowly poured into the interior of the biochip 11 from the solution inlet 111 and/or the solution inlet 112 .
  • the solution flows to the groove 121 through the microchannel 13, and the cryoprotectant in the solution can diffuse into the embryo to achieve the purpose of delivering the cryoprotectant, replacing the existing manual operation scheme.
  • the solution leaves the biochip 11 through the solution outlet 113.
  • the solution outlet 113 can be connected to a flexible pipe to drain the waste liquid to the waste liquid collection pipe, or a groove can be designed near the solution outlet 113 to collect the waste liquid flowing out (generally several ten to hundreds of microliters of solution).
  • the user separates the carrier 12 from the biochip 11, and puts the carrier 12 into liquid nitrogen for freezing.
  • the carrier 12 When thawing and resuscitating, the carrier 12 is taken out from the liquid nitrogen environment, and the transparent sheet at the front end of the carrier 12 is pressed onto a hot stage that has been preheated to 37 degrees, and then the biochip 11 that is also preheated to 37 degrees is used. Press the transparent sheet part of the front end of the carrier 12, because the carrier 12 is very thin, it can also reach a sufficient rate of temperature rise on the hot stage.
  • the biochip 11 and the carrier 12 are closely attached, and the biochip solution is used to push the instrument (such as a syringe pump or an air pump), and solutions such as thawing solution are slowly poured into the interior of the biochip 11 from the solution inlet 111 and/or the solution inlet 112 .
  • the solution flows to the groove 121 through the microchannel 13, and the cryoprotectant in and around the embryo is taken away by the thawing liquid to achieve the purpose of thawing and resuscitating, replacing the existing manual operation scheme.
  • the solution leaves the biochip 11 through the solution outlet 113.
  • the solution outlet 113 can be connected to a flexible pipe to drain the waste liquid to the waste liquid collection pipe, or a groove can be designed near the solution outlet 113 to collect the waste liquid flowing out (generally several ten to hundreds of microliters of solution).
  • the carrier 12 and the biochip 11 are separated, and the embryos in the groove 121 are taken out using a glass capillary, and placed in a petri dish to continue culturing.
  • biochip 21 may include two solution inlet 211 and 212 and a solution outlet 213.
  • the front end of the carrier 12 (front sheet) has a groove 221.
  • the bottom of the biochip 21 has a protruding part 215, and the protruding part 215 makes the bottom of the biochip 21 form a segmented structure.
  • the protrusion 215 is opposite to the groove 221 , and generally the length of the protrusion 215 is smaller than the length of the groove 221 .
  • a microchannel 23 that passes through the groove 221 and can only pass through the groove 221 is formed between the protruding part 215 and the groove 221, wherein the width of the microchannel 23 (or height) less than the diameter of the embryo.
  • the microchannel 23 includes a solution micro-inlet 231 and a solution micro-outlet 232, wherein the width (or height) of the solution micro-inlet 231 and the solution micro-outlet 232 is equal or approximately equal, and the width (or height) of the microchannel 23 actually means The width (or height) of the solution micro-inlet 231 and the solution micro-outlet 232, that is to say the width (or height) of the solution micro-inlet 231 and the solution micro-outlet 232 is smaller than the diameter of the embryo.
  • the microchannel 23 can only allow the solution to pass through but not the embryo, so that the embryo is confined in the groove 221 and will not float out of the groove 221 .
  • the specific freezing and thawing process please refer to the previous description, and will not repeat them here.
  • 3A to 3C also provides a biological tissue cryopreservation or thawing recovery device, the device includes: a biochip 31 and a carrier 32, the biochip 31 may include two solution inlets 311 and 312 and a solution outlet 313.
  • the biochip 31 and the carrier 32 and the biochip 11 and the carrier 12 in the above-described embodiments will not be repeated, and only the differences will be described below.
  • the carrier 32 includes a first groove 321 and a second groove 322 , the first groove 321 is located inside the second groove 322 and the first groove 321 may be located in the middle of the second groove 322 .
  • the first groove 321 is used to contain the biological tissue to be treated and related solutions for protecting the biological tissue, and the surface of the first groove 321 may be circular.
  • the length of the second groove 322 is greater than the length of the first groove 321
  • the depth of the second groove 322 is smaller than the depth of the first groove 321 .
  • the thickness of the carrier 32 may be 0.25-0.7 mm
  • the depth of the first groove may be 0.2-0.5 mm
  • the diameter of the first groove may be 0.2-1 mm.
  • the depth of the second groove can be 0.01-0.08mm, and the length of the second groove can be 2-10mm.
  • the bottom of the biochip 31 has a protruding part 315, and the protruding part 315 makes the bottom of the biochip 31 form a segmented structure.
  • the length of the protrusion 315 is less than the length of the second groove 322 and greater than the length of the first groove 321, for example, the length of the protrusion 315 can be 1-6 mm.
  • a microchannel 33 that passes through the first groove 321 and can only pass through the solution (such as freezing liquid or thawing liquid) is formed between the protrusion 315 and the second groove 322.
  • the height of the microchannel 33 is (or width) less than the diameter of the embryo.
  • the microchannel 33 includes a solution micro-inlet 331, a solution micro-outlet 332, a solution inlet channel and a solution outlet channel, wherein the solution inlet channel is between the solution micro-inlet 331 and the first groove 321, and the first groove 21 to the solution Between the micro-outlets 332 is a solution outlet channel.
  • the heights (or widths) of the solution micro-inlet 331, the solution micro-outlet 332, the solution inlet channel and the solution outlet channel are equal or approximately equal, and the height (or width) is smaller than the diameter of the embryo.
  • the microchannel 33 can only allow the solution to pass through but not the embryo, so that the embryo is confined in the first groove 321 and will not float out of the first groove 321 .
  • the specific freezing and thawing process please refer to the previous description, and will not repeat them here.
  • a microchannel that only allows the solution to pass through but not the embryo is formed between the biochip and the carrier, and the embryo is restricted in the groove of the carrier during cryopreservation or thawing and recovery operations, so it can be orderly
  • the cryoprotectant is delivered or removed, which effectively improves the efficiency of embryo cryopreservation or thawing and recovery operations.
  • a device for cryopreservation or thawing and recovery of biological tissues includes: a biochip 41 and a carrier 42, wherein the biochip 41 is generally in a rectangular parallelepiped structure, and the biochip 41 may include two solution inlets 411 and 412 and one solution outlet 413 .
  • the number of solution inlets of the biochip 41 can be set according to requirements. If multiple different cryoprotectant solutions are required to flow through the embryo sequentially, then multiple solution inlets can be set.
  • the inlet channels shown in the figure are only illustrative, and what is important is that the solutions flowing into the biochip 41 through different solution inlets can flow to the vicinity of the embryo in sequence.
  • the carrier 42 is generally a strip structure, which includes a front sheet and a rear support rod.
  • the front sheet of the carrier 42 can be made of plastic material with uniform thickness, transparent material, biocompatibility and good heat transfer, so as to ensure the suitability for holding embryos and the heat transfer speed during subsequent freezing.
  • the rear end of the carrier 42 can also be called a handle or a handle, which is a position where the operator's hand directly touches and operates. In the drawings in this application, only the front sheet part of the carrier is shown, and the rear part of the carrier is not shown as the key part.
  • the surface of the groove 421 can be circular.
  • the biological tissue may be biological materials such as embryos, eggs, cells, etc., which is not limited in the present application, and the following description will be made by taking the biological tissue as an embryo as an example.
  • the thickness of the carrier 42 may be 0.25-1 mm
  • the depth of the groove 421 may be 0.2-0.8 mm
  • the diameter of the groove 421 may be 0.2-1 mm.
  • the bottom (or lower surface) of the biochip 41 is provided with a permeable film 43, and the permeable film 43 seals the bottom of the biochip so as to form a microchannel 44 between the permeable film 43 and the biochip 41, and the microchannel 44 It is connected with solution inlet and solution outlet.
  • the permeable film 43 covers the carrier 42 and seals the groove 421 .
  • the permeable membrane 43 has a plurality of micropores, and the diameter (pore) of the micropores is smaller than the diameter of the embryo but larger than the diameter of the solution (freezing liquid or thawing liquid) molecules.
  • the permeable membrane 43 can only allow the solution to pass through, but not the embryo, so that the embryo is confined in the groove 421 and will not float out of the groove 421 .
  • the permeable film 43 can be a perforated film, a grid-shaped film, a dialysis film, or a water-soluble film with selective permeability.
  • the biochip 41 When performing cryopreservation application operations, the biochip 41 is placed on the carrier 42 and pressed tightly, wherein the groove 421 of the carrier 42 carries embryos. A microchannel 44 that only allows solutions to pass is formed between the biochip 41 and the permeable film 43 disposed at the bottom thereof.
  • a biochip solution pushing device such as a syringe pump or an air pump
  • the freezing solution is slowly poured into the interior of the biochip 41 from the solution inlet 411 and/or the solution inlet 412 .
  • the solution flows in the microchannel 44.
  • the cryoprotectant in the solution can pass through the permeable film 43 and enter the groove 421 and diffuse into the embryo to achieve delivery of cryoprotection.
  • the solution leaves the biochip 41 through the solution outlet 413.
  • the solution outlet 413 can be connected to a flexible pipe to drain the waste liquid to the waste liquid collection pipe, or a groove can be designed near the solution outlet 413 to collect the waste liquid flowing out (generally several ten to hundreds of microliters of solution).
  • the user separates the carrier 42 from the biochip 41 and puts the carrier 42 into liquid nitrogen for freezing.
  • the carrier 42 When carrying out the thawing application operation, the carrier 42 is taken out from the liquid nitrogen environment, and the transparent sheet part at the front end of the carrier 42 is pressed onto a hot stage that has been preheated to 37 degrees, and then the biochip 41 that is also preheated to 37 degrees is used The transparent sheet portion at the front end of the carrier 42 is pressed. Since the carrier 42 is very thin, a sufficient rate of temperature rise can also be achieved on the hot stage.
  • the biochip 41 and the carrier 42 are closely attached, and the biochip solution is used to push the instrument (such as a syringe pump or an air pump), and solutions such as thawing solution (such as diluent, cleaning solution) are slowly released from the solution inlet 411 and/or the solution inlet.
  • the solution 412 is poured into the inside of the biochip 41.
  • the solution flows in the microchannel 44, and when the solution flows to the position above the groove 421, the cryoprotectant in and around the embryo passes through the permeable membrane 43 and diffuses into the thawing solution in the microchannel 44 and is absorbed by the thawing solution. Take it away and remove it to achieve the purpose of thawing and resuscitating, replacing the existing manual operation scheme.
  • the solution leaves the biochip 41 through the solution outlet 413.
  • the solution outlet 413 can be connected to a flexible pipe to drain the waste liquid to the waste liquid collection pipe, or a groove can be designed near the solution outlet 113 to collect the waste liquid flowing out (generally tens of to hundreds of microliters of solution).
  • the carrier 42 and the biochip 41 are separated, and the embryos in the groove 421 are taken out using a glass capillary, and placed in a petri dish to continue culturing.
  • a microchannel is formed between the biochip and the permeable film that only allows the solution to pass through and does not allow the embryo to pass through.
  • the permeable film restricts the embryo to the carrier In the groove without floating out, the freezing liquid or thawing liquid flows in the microchannel and enters the groove through the permeable film to contact the embryo, so the cryoprotectant can be delivered or removed in an orderly manner, effectively improving the It improves the efficiency of embryo cryopreservation or thawing recovery operation.

Abstract

一种用于生物组织冷冻保存或解冻复苏的装置，其包括：生物芯片，所述生物芯片包括溶液入口和溶液出口；载体，其具有第一凹槽，所述第一凹槽内承载有生物组织；其中，所述载体紧密贴合在所述生物芯片的下方，所述生物芯片与所述载体之间形成经过所述第一凹槽的微通道，所述微通道仅供溶液通过。所述装置能够有序进行冷冻保护剂的投送或移除，有效提高了生物组织冷冻保存或解冻复苏操作的效率。

Description

用于生物组织冷冻保存或解冻复苏的装置 技术领域
本发明涉及生物工程技术领域，尤其涉及一种用于生物组织冷冻保存或解冻复苏的装置。
背景技术
细胞冷冻保存技术在很多生物领域是一项重要的技术，把细胞从低温冷冻状态恢复到正常代谢状态也同样重要，解冻复苏的过程包含了把冷冻保护剂移除替换成正常培养基的过程。
现有技术的装置在胚胎冷冻或解冻时操作复杂，效率较低。
发明内容
本发明的主要目的在于提供一种用于生物组织冷冻保存或解冻复苏的装置，以解决现有技术的装置在进行胚胎冷冻或解冻时操作复杂所导致效率较低等问题。
根据本发明实施例提出一种用于生物组织冷冻保存或解冻复苏的装置，其包括：生物芯片，所述生物芯片包括溶液入口和溶液出口；载体，其具有第一凹槽，所述第一凹槽内承载有生物组织；其中，所述载体紧密贴合在所述生物芯片的下方，所述生物芯片与所述载体之间形成经过所述第一凹槽的微通道，所述微通道仅供溶液通过。
根据本发明实施例还提出一种用于生物组织冷冻保存或解冻复苏的装置，其包括：生物芯片，所述生物芯片包括溶液入口和溶液出口；所述生物芯片的底部设置有通透性薄膜，以在所述生物芯片与所述通透性薄膜之间形成微通道；载体，所述载体具有凹槽，所述凹槽内承载有生物组织；其中，所述载体紧密贴合在所述芯片的下方，所述通透性薄膜覆盖所述凹槽并将生物组织限制在所述凹槽内，所述微通道内的溶液穿过所述通透性薄膜扩散至所述凹槽内。
根据本发明的技术方案，在载体的凹槽上方形成只能够允许溶液通过的微通道，而将生物组织限制在凹槽内，因此能够有序进行冷冻保护剂的投送或移除，有效提高了生物组织冷冻保存或解冻复苏操作的效率。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1A是根据本发明一个实施例的装置的立体示意图；
图1B是图1A中装置的俯视图；
图1C是沿图1B中1C-1C线的剖面示意图；
图1D是图1C中的局部放大图；
图2A是根据本发明另一实施例的装置的俯视图；
图2B是沿图2A中2B-2B线的剖面示意图；
图2C是图2B中的局部放大图；
图3A是根据本发明又一实施例的装置的俯视图；
图3B是沿图3A中3B-3B线的剖面示意图；
图3C是图3B中的局部放大图；
图4A是根据本发明再一实施例的装置的俯视图；
图4B是沿图4A中4B-4B线的剖面示意图；
图4C是图4B中的局部放大图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明具体实施例及相应的附图对本发明技术方案进行清楚、完整地描述。显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有 其他实施例，都属于本发明保护的范围。
以下结合附图，详细说明本发明各实施例提供的技术方案。
参考图1A至1D，根据本发明实施例提供了一种用于生物组织冷冻保存或解冻复苏的装置，所述装置包括：生物芯片11和载体12，其中生物芯片11大体呈长方体结构，生物芯片的底部(或下表面)的部分或全部的为敞开式区域，也就是说生物芯片的底部的全部敞口，或者生物芯片对应载体的底部区域敞口。生物芯片11可包括两个溶液入口111和112以及一个溶液出口113。生物芯片11的溶液入口的数量可以根据实际需求进行调整，如果需要多种不同的冷冻保护剂溶液顺序流过胚胎，那么就可以设置多个溶液入口。图中所示的入口通道只是示例性说明，重要的是通过不同的溶液入口流进生物芯片11的溶液能够按顺序流到胚胎的附近。
载体12大体为长条状结构，其包括前端薄片和后端载杆。载体12的前端薄片可采用厚度均匀、材质透明、生物兼容且拥有良好传热性的塑料材料制成，保证对盛放胚胎的适用性以及后续冷冻时的热传递速度。载体12的后端载杆也可称为手柄或手持部，是操作者的手直接接触操作的位置。在本申请中的附图中仅示出了载体12的前端薄片部分，载体12的后端载杆部分不作为重点部分而未示出。载体12(前端薄片)的前端具有凹槽121，凹槽121用于盛放待处理的生物组织以及保护生物组织的相关溶液，凹槽121的表面可以呈圆形。其中，所述生物组织可以是胚胎、卵子、细胞等生物材料，本申请对此不进行限制，下面以生物组织是胚胎为例进行描述。在本申请实施例中，载体12的厚度可为0.25-0.7毫米，凹槽121的深度可为0.2-0.5毫米，凹槽121的直径可为0.2-1毫米。
在进行冷冻或解冻操作时，由于生物芯片11的底部的全部或部分区域为敞开式设计，当生物芯片11跟载体12贴合时，载体12把生物芯片11底部的敞开式区域封闭，在生物芯片11与载体12之间形成经过凹槽121、且仅供溶液(例如冷冻液或解冻液)通过的微通道13，该微通道13的高度(或宽度)小于胚胎的直径。具体地，微通道13包括溶液微入口131、溶液微出口132、溶液入通道和溶液出通道，其中溶液微入口131到凹槽121之间为溶液入通道，凹槽121到溶液微出口132之间为溶液出通道。并且，溶液微 入口131、溶液微出口132、溶液入通道和溶液出通道的高度(或宽度)相等或近似相等、且该高度(或宽度)小于胚胎的直径。这样，在冷冻和/或解冻操作过程中，微通道13仅能够允许溶液通过而不允许胚胎通过，从而胚胎被限制在凹槽121内，而不会飘出凹槽121以外。
当进行冷冻保存操作时，将生物芯片11放置在载体12的上方并压紧，其中载体12的凹槽121内承载有胚胎，生物芯片11与载体12之间形成仅供溶液通过的微通道。使用生物芯片溶液推动仪器(例如注射泵或者气压泵)，将冷冻液慢慢从溶液入口111和/或溶液入口112灌入生物芯片11的内部。溶液经过微通道13流动到凹槽121，此时溶液内的冷冻保护剂就可以扩散到胚胎内部，达到投送冷冻保护剂的目的，替代现有的人工操作的方案。之后溶液经过溶液出口113离开生物芯片11，溶液出口113可以连接软管将废液引流到废液收集管，也可以在溶液出口113的附近设计一个凹槽，收集流出的废液(一般为几十到几百微升的溶液)。最后使用者把载体12和生物芯片11分开，并将载体12投入至液氮冷冻。
当进行解冻复苏操作时，将载体12从液氮环境中取出，将载体12前端的透明薄片部分压到已经预热到37度的热台上，然后使用同样预热到37度的生物芯片11压住载体12前端的透明薄片部分，由于载体12很薄，在热台上也能达到足够的升温速度。将生物芯片11和载体12紧密贴合，使用生物芯片溶液推动仪器(例如注射泵或者气压泵)，将解冻液等溶液慢慢从溶液入口111和/或溶液入口112灌入生物芯片11的内部。溶液经过微通道13流动到凹槽121，胚胎体内和胚胎附近的冷冻保护剂被解冻液带走移除，达到解冻复苏的目的，替代现有的人工操作的方案。之后溶液经过溶液出口113离开生物芯片11，溶液出口113可以连接软管将废液引流到废液收集管，也可以在溶液出口113的附近设计一个凹槽，收集流出的废液(一般为几十到几百微升的溶液)。最后将载体12和生物芯片11分开，使用玻璃毛细管将凹槽121内的胚胎取出，放置到培养皿内继续培养。
参考图2A至2C，根据本发明实施例还提供一种用于生物组织冷冻保存或解冻复苏的装置，所述装置包括：生物芯片21和载体22，生物芯片21可包括二个溶液入口211和212以及一个溶液出口213。载体12(前端薄片) 的前端具有凹槽221，生物芯片21和载体22与以上描述的实施例中的生物芯片11和载体12的类似之处不再赘述，下面仅描述不同之处。
生物芯片21的底部具有突出部215，突出部215使得生物芯片21的底部形成一个分段结构。其中，突出部215与凹槽221相对，一般地突出部215的长度小于凹槽221的长度。在本申请实施例中，突出部215与凹槽221之间形成一个经过凹槽221、且仅能够供溶液(例如冷冻液或解冻液)通过的微通道23，其中微通道23的宽度(或高度)小于胚胎的直径。具体地，微通道23包括溶液微入口231和溶液微出口232，其中溶液微入口231和溶液微出口232的宽度(或高度)相等或近似相等，微通道23的宽度(或高度)实际是指溶液微入口231和溶液微出口232的宽度(或高度)，也就是说溶液微入口231和溶液微出口232的宽度(或高度)小于胚胎的直径。这样，在冷冻和/或解冻操作过程中，微通道23仅能够允许溶液通过而不允许胚胎通过，从而胚胎被限制在凹槽221内，而不会飘出凹槽221以外。具体地冷冻和解冻过程请参考之前的描述，此处不再赘述。
参考图3A至3C，根据本发明实施例还提供一种用于生物组织冷冻保存或解冻复苏的装置，所述装置包括：生物芯片31和载体32，生物芯片31可包括二个溶液入口311和312以及一个溶液出口313。生物芯片31和载体32与以上描述的实施例中的生物芯片11和载体12的类似之处不再赘述，下面仅描述不同之处。
所述载体32包括第一凹槽321和第二凹槽322，第一凹槽321位于第二凹槽322之内并且第一凹槽321可位于第二凹槽322的中间位置。第一凹槽321用于盛放待处理的生物组织以及保护生物组织的相关溶液，第一凹槽321的表面可以呈圆形。在本申请实施例中，第二凹槽322的长度大于第一凹槽321的长度，且第二凹槽322的深度小于第一凹槽321的深度。例如，载体32的厚度可为0.25-0.7毫米，第一凹槽的深度可为0.2-0.5毫米，第一凹槽的直径可为0.2-1毫米。第二凹槽的深度可为0.01-0.08毫米，第二凹槽的长度可为2-10毫米。
生物芯片31的底部具有突出部315，突出部315使得生物芯片31的底部形成一个分段结构。突出部315的长度小于第二凹槽322的长度且大于第 一凹槽321的长度，例如突出部315的长度可为1-6毫米。在本申请实施例中，突出部315与第二凹槽322之间形成经过第一凹槽321、且仅能够供溶液(例如冷冻液或解冻液)通过的微通道33，微通道33的高度(或宽度)小于胚胎的直径。具体地，微通道33包括溶液微入口331、溶液微出口332、溶液入通道和溶液出通道，其中溶液微入口331到第一凹槽321之间为溶液入通道，第一凹槽21到溶液微出口332之间为溶液出通道。并且，溶液微入口331、溶液微出口332、溶液入通道和溶液出通道的高度(或宽度)相等或近似相等、且该高度(或宽度)小于胚胎的直径。这样，在冷冻和/或解冻操作过程中，微通道33仅能够允许溶液通过而不允许胚胎通过，从而胚胎被限制在第一凹槽321内，而不会飘出第一凹槽321以外。具体地冷冻和解冻过程请参考之前的描述，此处不再赘述。
通过上述实施例，在生物芯片和载体之间形成只允许溶液通过而不允许胚胎通过的微通道，在进行冷冻保存或解冻复苏操作时，将胚胎限制在载体的凹槽内，因此能够有序进行冷冻保护剂的投送或移除，有效提高了胚胎冷冻保存或解冻复苏操作的效率。
参考图4A至4C，根据本发明实施例还提供一种用于生物组织冷冻保存或解冻复苏的装置，所述装置包括：生物芯片41和载体42，其中生物芯片41大体呈长方体结构，生物芯片41可包括两个溶液入口411和412以及一个溶液出口413。生物芯片41的溶液入口的数量可以按照需求设置，如果需要多种不同的冷冻保护剂溶液顺序流过胚胎，那么就可以设置多个溶液入口。图中所示的入口通道只是示例性说明，重要的是通过不同的溶液入口流进生物芯片41的溶液能够按顺序流到胚胎的附近。
载体42大体为长条状结构，其包括前端薄片和后端载杆。载体42的前端薄片可采用厚度均匀、材质透明、生物兼容且拥有良好传热性的塑料材料制成，保证对盛放胚胎的适用性以及后续冷冻时的热传递速度。载体42的后端载杆也可称为手柄或手持部，是操作者的手直接接触操作的位置。在本申请中的附图中仅示出了载体的前端薄片部分，载体的后端载杆部分不作为重点部分而未示出。在载体42(前端薄片)的前端具有凹槽421，凹槽421用于盛放待处理的生物组织和保护生物组织的相关溶液，凹槽421的表面可以 呈圆形。其中，所述生物组织可以是胚胎、卵子、细胞等生物材料，本申请对此不进行限制，下面以生物组织是胚胎为例进行描述。在本申请实施例中，载体42的厚度可为0.25-1毫米，凹槽421的深度可为0.2-0.8毫米，凹槽421的直径可为0.2-1毫米。
生物芯片41的底部(或下表面)设置有通透性薄膜43，通透性薄膜43封住生物芯片的底部从而在通透性薄膜43和生物芯片41之间形成微通道44，微通道44与溶液入口和溶液出口相连通。通透性薄膜43覆盖在载体42的上方并封闭凹槽421。在本申请实施例中，通透性薄膜43具有多个微孔，该微孔的直径(孔隙)小于胚胎的直径但大于溶液(冷冻液或解冻液)分子的直径，在冷冻和/或解冻操作过程中，通透性薄膜43仅能够允许溶液通过，而不允许胚胎通过，从而使得胚胎被限制在凹槽421内，而不会飘出凹槽421以外。其中通透性薄膜43可为穿孔膜、网格状薄膜、透析膜、或水溶性薄膜等具有选择透过性的薄膜。
当进行冷冻保存应用操作时，将生物芯片41放置在载体42的上方并压紧，其中载体42的凹槽421内承载有胚胎。生物芯片41与设置在其底部的通透性薄膜43之间形成仅允许溶液通过的微通道44。使用生物芯片溶液推动仪器(例如注射泵或者气压泵)，将冷冻液慢慢从溶液入口411和/或溶液入口412灌入生物芯片41内部。溶液在微通道44内流动，当溶液流动到凹槽421上方的位置时，溶液内的冷冻保护剂就可以穿过通透性薄膜43进入凹槽421并扩散到胚胎内部，达到投送冷冻保护剂的目的，替代现有的人工操作的方案。之后溶液经过溶液出口413离开生物芯片41，溶液出口413可以连接软管将废液引流到废液收集管，也可以在溶液出口413的附近设计一个凹槽，收集流出的废液(一般为几十到几百微升的溶液)。最后使用者将载体42和生物芯片41分开，并将载体42投入至液氮冷冻。
当进行解冻应用操作时，将载体42从液氮环境中取出，将载体42前端的透明薄片部分压到已经预热到37度的热台上，然后使用同样预热到37度的生物芯片41压住载体42前端的透明薄片部分。由于载体42很薄，在热台上也能达到足够的升温速度。将生物芯片41和载体42紧密贴合，使用生物芯片溶液推动仪器(例如注射泵或者气压泵)，将解冻液等溶液(例如稀释 液、清洗液)慢慢从溶液入口411和/或溶液入口412灌入生物芯片41的内部。溶液在微通道44内流动，当溶液流动到凹槽421上方的位置时，胚胎体内和胚胎附近的冷冻保护剂穿过通透性薄膜43扩散到微通道44内的解冻液中并被解冻液带走移除，达到解冻复苏的目的，替代现有的人工操作的方案。之后溶液经过溶液出口413离开生物芯片41，溶液出口413可以连接软管将废液引流到废液收集管，也可以溶液出口113的附近设计一个凹槽，收集流出的废液(一般为几十到几百微升的溶液)。最后将载体42和生物芯片41分开，使用玻璃毛细管将凹槽421内的胚胎取出，放置到培养皿内继续培养。
通过本申请实施例，在生物芯片和通透性薄膜之间形成只允许溶液通过而不允许胚胎通过的微通道，在进行冷冻保存或解冻复苏操作时，通透性薄膜将胚胎限制在载体的凹槽内而不会飘出，冷冻液或解冻液在微通道内流动并穿过通透性薄膜进入凹槽与胚胎接触，因此能够有序进行冷冻保护剂的投送或移除，有效提高了胚胎冷冻保存或解冻复苏操作的效率。
以上所述仅为本发明的实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的权利要求范围之内。

Claims (13)

  1. 一种用于生物组织冷冻保存或解冻复苏的装置，其特征在于，包括：
    生物芯片，所述生物芯片包括溶液入口和溶液出口；
    载体，其具有第一凹槽，所述第一凹槽内承载有生物组织；
    其中，所述载体紧密贴合在所述生物芯片的下方，所述生物芯片与所述载体之间形成经过所述第一凹槽的微通道，所述微通道仅供溶液通过。
  2. 根据权利要求1所述的装置，其特征在于，所述生物芯片的底部的至少一部分为敞开式区域，所述载体紧贴在所述生物芯片的下方并封闭所述敞开式区域，所述生物芯片与所述载体之间的所述微通道的高度或宽度小于生物组织的大小。
  3. 根据权利要求1所述的装置，其特征在于，所述生物芯片的底部具有突出部，所述突出部与所述第一凹槽之间形成包括溶液微入口和溶液微出口的微通道，其中所述溶液微入口和所述溶液微出口的宽度小于生物组织的大小。
  4. 根据权利要求3所述的装置，其特征在于，所述突出部与所述第一凹槽相对，所述第一凹槽的长度大于所述突出部的长度。
  5. 根据权利要求1所述的装置，其特征在于，所述载体还设置有长度大于所述第一凹槽的第二凹槽，所述第一凹槽设置在所述第二凹槽之内；所述生物芯片的底部具有突出部，所述突出部与所述第二凹槽之间形成所述微通道，其中所述微通道的高度或宽度小于生物组织的大小。
  6. 根据权利要求5所述的装置，其特征在于，所述突出部的长度小于所述第二凹槽的长度且大于所述第一凹槽的长度。
  7. 根据权利要求1所述的装置，其特征在于，所述生物芯片包括二个溶液入口和一个溶液出口。
  8. 一种用于生物组织冷冻保存或解冻复苏的装置，其特征在于，包括：
    生物芯片，所述生物芯片包括溶液入口和溶液出口；所述生物芯片的底部设置有通透性薄膜，以在所述生物芯片与所述通透性薄膜之间形成微通道；
    载体，所述载体具有凹槽，所述凹槽内承载有生物组织；
    其中，所述载体紧密贴合在所述芯片的下方，所述通透性薄膜覆盖所述凹槽并将生物组织限制在所述凹槽内，所述微通道内的溶液穿过所述通透性薄膜扩散至所述凹槽内。
  9. 根据权利要求8所述的装置，其特征在于，所述通透性薄膜设有多个微孔，所述微孔的直径小于所述生物组织的直径，所述微孔的直径大于溶液分子的直径。
  10. 根据权利要求8或9所述的装置，其特征在于，所述通透性薄膜为穿孔膜、网格状薄膜、透析膜、或水溶性薄膜中的一种。
  11. 根据权利要求8所述的装置，其特征在于，所述生物芯片包括二个溶液入口和一个溶液出口。
  12. 根据权利要求8或11所述的装置，其特征在于，所述生物芯片的溶液入口和溶液出口分别与所述微通道相连通。
  13. 根据权利要求8所述的装置，其特征在于，所述载体包括前端薄片和后端载杆，所述凹槽设置在所述前端薄片上。
PCT/CN2022/117744 2021-09-10 2022-09-08 用于生物组织冷冻保存或解冻复苏的装置 WO2023036223A1 (zh)

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CN103451090A (zh) * 2013-09-18 2013-12-18 上海理工大学 微流体细胞处理芯片及其应用方法
CN203446410U (zh) * 2013-05-28 2014-02-26 武汉大学 用于超快速冷冻精子的微流控芯片
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CN105831105A (zh) * 2016-04-12 2016-08-10 上海理工大学 微流体细胞处理芯片及其应用方法
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CN107629941A (zh) * 2017-10-27 2018-01-26 中国科学院理化技术研究所 一种基于微流控芯片的细胞冻存装置及其应用
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CN103842493A (zh) * 2011-10-03 2014-06-04 株式会社北里生物制药 活细胞冷冻保存用具
US20160057992A1 (en) * 2013-04-09 2016-03-03 Wei Lou Biological sample vitrification carrier and usage thereof
CN203446410U (zh) * 2013-05-28 2014-02-26 武汉大学 用于超快速冷冻精子的微流控芯片
CN103451090A (zh) * 2013-09-18 2013-12-18 上海理工大学 微流体细胞处理芯片及其应用方法
US20160270388A1 (en) * 2013-10-15 2016-09-22 The Regents Of The University Of Michigan Vitrification of biological material
CN105831105A (zh) * 2016-04-12 2016-08-10 上海理工大学 微流体细胞处理芯片及其应用方法
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