WO2023036224A1 - 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
WO2023036224A1
WO2023036224A1 PCT/CN2022/117748 CN2022117748W WO2023036224A1 WO 2023036224 A1 WO2023036224 A1 WO 2023036224A1 CN 2022117748 W CN2022117748 W CN 2022117748W WO 2023036224 A1 WO2023036224 A1 WO 2023036224A1
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Prior art keywords
carrier
solution
grid
biochip
biological tissue
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PCT/CN2022/117748
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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 CN202111063600.9A external-priority patent/CN115777683A/zh
Priority claimed from CN202111063587.7A external-priority patent/CN115777682A/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 WO2023036224A1 publication Critical patent/WO2023036224A1/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 and recovery of biological tissue which includes: a carrier, a groove or a through hole is arranged on the carrier, and a grid structure or a through hole is arranged at the opening of the groove.
  • the upper and lower ends of the through hole are respectively provided with a grid-like structure, and the grid-like structure confines the biological tissue in the groove or the through hole.
  • a device for cryopreservation or thawing and resuscitation of biological tissues which includes: a carrier, the carrier has a microchannel for the solution to flow, and the two ends of the microchannel are respectively provided with a solution inlet and a A solution outlet, the solution outlet is provided with a grid-like structure; wherein, the grid-like structure confines the biological tissue in the microchannel, preventing the biological tissue from floating out of the solution outlet.
  • the biological tissue is restricted in a certain position of the carrier by the grid structure, so as to avoid the biological tissue from floating out of the carrier, so the delivery or removal of the cryoprotectant can be carried out in an orderly manner, which effectively improves the biological Efficiency of tissue cryopreservation or thawing and recovery operations.
  • FIG. 1A is a schematic diagram of a top view of a carrier according to an embodiment of the present invention.
  • Figure 1B is a partially enlarged view in Figure 1A;
  • Fig. 1C is a schematic cross-sectional view (groove) along line 1C-1C in Fig. 1A;
  • Figure 1D is a partially enlarged view in Figure 1C;
  • Fig. 1E is a schematic cross-sectional view (through hole) along line 1C-1C in Fig. 1A;
  • Figure 1F is a partially enlarged view in Figure 1E;
  • FIG. 2A is a top view of a glass capillary passing through a grid structure according to an embodiment of the present invention
  • Fig. 2B is a partially enlarged view among Fig. 2A;
  • Figure 2C is a schematic cross-sectional view along line 2C-2C in Figure 2A;
  • Figure 2D is a partially enlarged view in Figure 2C;
  • FIG. 3 is a schematic diagram of a carrier, a chassis and a mechanical movement device according to an embodiment of the present invention
  • FIG. 4A is a schematic diagram of a top view of a biochip and a carrier according to an embodiment of the present invention.
  • Fig. 4B is a schematic cross-sectional view along line 4B-4B in Fig. 4A;
  • Figure 4C is a partially enlarged view in Figure 4B;
  • 5A is a schematic diagram of a top view of a carrier according to an embodiment of the present invention.
  • Figure 5B is a schematic cross-sectional view along line 5B-5B in Figure 5A;
  • Figure 5C is a partially enlarged view in Figure 5B;
  • Figure 5D is a partially enlarged view in Figure 5C;
  • Figure 5E is a side view of a glass capillary passing through a grid structure according to one embodiment of the present invention.
  • Figure 6 is a schematic diagram of a carrier and mechanical movement device according to one embodiment of the present invention.
  • a device for cryopreservation or thawing and resuscitation of biological tissue is provided. pole.
  • the front sheet of the carrier 11 can be made of plastic material with uniform thickness, transparent material, biocompatibility and good heat transfer, so as to ensure the applicability to hold embryos and the heat transfer speed during subsequent freezing.
  • the rear end of the carrier 11 can also be referred to as a handle or a handle, which is a position directly touched by the operator's hand.
  • the thickness of the carrier 11 may be 0.25-0.7 mm.
  • a groove 111 is provided on the front end of the carrier 11 (ie, the front end sheet), and the groove 111 is used to hold the biological tissue to be treated and the relevant solution for protecting the biological tissue.
  • a grid structure 112 is disposed at the opening of the groove 111 .
  • the depth of the groove 111 may be 0.2-0.5 mm, and the diameter of the upper surface of the groove 111 may be 0.2-1 mm.
  • a through hole 115 is provided on the front end of the carrier 11 (i.e., the front end sheet). solution.
  • the upper end and the lower end of the through hole 115 are respectively provided with a grid structure 116 .
  • the depth of the through hole 115 may be 0.25-0.7 mm, and the diameter of the upper surface/lower surface of the through hole 115 may be 0.2-1 mm.
  • 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 grid structure 112 includes a plurality of flexible grid bars, and the distance between every two grid bars in the plurality of flexible grid bars is smaller than the diameter of the embryo and larger than the diameter of the solution molecule, wherein the The distance of the gap is 0.05-0.08 mm.
  • the grid structure just can limit embryo in groove or through hole, carry out cryopreservation or thaw recovery operation of embryo.
  • the flexible grid bars of the grid-like structure are elastic, and the grid-like structure can be deformed when it contacts with foreign objects (such as glass capillaries), so that the foreign objects can pass through the grid-like structure through the slit between the two flexible grid bars.
  • foreign objects such as glass capillaries
  • the glass capillary 15 leaves the groove 111 and the grid structure 112 returns to its original state. Since the grid pitch of the grid structure 112 is smaller than the diameter of the embryo, the embryo can be restricted in the groove 111 and will not follow The flow of liquid leaves the carrier 11 . Similarly, the same method can also be used to insert the glass capillary 15 into the groove 111, suck the embryos into the capillary 15, and transfer the embryos to other places.
  • the process of using the glass capillary 15 to inject embryos into the through hole 115 is similar to the above description and will not be repeated here.
  • the difference is that the through hole 115 has the advantage that the molecules can diffuse inward or outward from the upper and lower ends, which can speed up the delivery and removal of the cryoprotectant compared to the embodiment in which the groove can only diffuse to one end.
  • a device for cryopreservation or thawing and resuscitation of biological tissue which includes: a carrier 11 provided with a grid-like structure, a chassis 14 and a mechanical movement device.
  • a carrier 11 provided with a grid-like structure
  • a chassis 14 and a mechanical movement device.
  • the mechanical motion device includes a first motion shaft 161 that moves up and down and a second motion shaft 162 that moves left and right, wherein the first motion shaft 161 is connected with the carrier 11 for driving the carrier 11 to move up and down; the second motion shaft 162 It is connected with the chassis 14 and is used to drive the chassis 14 to move left and right.
  • the container moves to the bottom of the carrier, and then the mechanical movement device immerses the transparent sheet part of the front of the carrier into the vitrification liquid again, and controls the chassis to move slowly left and right, so that the carrier moves with it in the container, which can ensure high-concentration cryoprotection
  • the delivery speed of the agent is faster, the processing time of the embryo is accelerated, and the toxicity damage of high-concentration cryoprotectant is reduced.
  • the user puts the carrier into liquid nitrogen for storage.
  • the mechanical movement device When performing thawing application operations, pre-prepare the thawing liquid that has been preheated to 37 degrees, and add the diluent and cleaning liquid into the container and place it on the chassis of the mechanical movement device. Then take the carrier out of the liquid nitrogen, immerse the transparent sheet at the front end of the carrier in the thawing solution that has been preheated to 37 degrees, after 50 seconds, install the carrier on the mechanical movement device, and immerse the transparent sheet at the front end of the carrier After 3 minutes, the mechanical movement device will take out the carrier from the top of the container, and move the chassis to move the container containing the cleaning solution to the bottom of the carrier, and then the mechanical movement device will immerse the transparent sheet part of the front end of the carrier in the cleaning solution again.
  • the mechanical movement device pulls the carrier out from the top of the container, and moves the chassis to move another container containing the cleaning solution to the bottom of the carrier, and then the mechanical movement device immerses the transparent sheet part of the front end of the carrier in the cleaning solution again, and controls The chassis is slowly moved from side to side to ensure that the cryoprotectant is removed from the embryos. Finally, the user uses the glass capillary to pass through the grid structure of the carrier to recover the embryos from the groove, and put them in a petri dish for further cultivation.
  • a device for cryopreservation or thawing recovery of biological tissue includes: a carrier 21 and a biochip 22, wherein the front end of the carrier 21 (ie, the front end sheet) is set There is a groove 211, and the groove 211 is used to contain the biological tissue to be treated and the relevant solution for protecting the biological tissue.
  • a grid structure 212 is disposed at the opening of the groove 211 .
  • the biochip 22 generally has a cuboid structure, including two solution inlets 221 , 222 and a solution outlet 223 .
  • the number of solution inlets of the biochip 22 can be set according to requirements. If multiple different cryoprotectant solutions are required to flow through the embryo sequentially, 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 22 through different inlets can flow to the vicinity of the embryo in sequence.
  • part or all of the bottom (or lower surface) of the biochip 22 is an open area, that is to say, the bottom of the biochip 22 is completely open or the bottom area of the biochip 22 corresponding to the carrier 21 is open.
  • the carrier 21 seals the open area at the bottom of the biochip 22, and forms a microchannel 23 passing through the groove 211 between the biochip 22 and the carrier 21, and the solution can pass through the solution inlet 221 and 222 enter the microchannel 23 and exit the microchannel 23 through the solution outlet 223 .
  • the solution After the solution enters the microchannel 23 through the solution inlets 221 and 222, the solution will pass above the groove 211, and the solution will pass through the grid structure 212 and diffuse to the In the groove 211, to carry out cryopreservation or thawing recovery operation of the embryo.
  • the carrier 21 and the biochip 22 are in a separated state at the beginning, and the user first uses a glass capillary to pass through the grid structure 212 of the carrier 21 to inject embryos into the groove 211, and then place the biochip 22 On the top of the carrier 21 and pressed tightly, the biochip 22 and the carrier 21 are closely attached, and a microchannel 23 for the solution to pass is formed between the biochip 22 and the carrier 21 .
  • 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 22 from the solution inlet 221 and/or the solution inlet 222 .
  • the solution flows in the microchannel 23.
  • the cryoprotectant in the solution can pass through the grid structure 212 and enter the groove 211 and diffuse into the embryo to achieve delivery of the cryoprotectant. the goal of.
  • the solution leaves the biochip 22 through the solution outlet 223, and the solution outlet 223 can be connected to the flexible pipe to drain the waste liquid to the waste liquid collection pipe, or a groove can be designed near the solution outlet 223 to collect the waste liquid flowing out (generally tens to hundreds of microliters of solution).
  • the user separates the carrier 21 from the biochip 22 and puts the carrier 21 into liquid nitrogen for freezing.
  • the carrier 21 When thawing the application operation, the carrier 21 is taken out from the liquid nitrogen environment, and the transparent sheet part at the front end of the carrier 21 is pressed onto a hot stage that has been preheated to 37 degrees, and then the biochip 22 that is also preheated to 37 degrees is used Press the transparent sheet portion at the front end of the carrier 21. Since the carrier 21 is very thin, a sufficient rate of temperature rise can also be achieved on the hot stage.
  • the biochip 22 and the carrier 21 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 221 and/or the solution inlet.
  • the solution 222 is poured into the inside of the biochip 22.
  • the solution flows in the microchannel 23.
  • the solution will pass through the grid structure 212 and enter the groove 211, and the cryoprotectant molecules in the embryo body and near the embryo will be taken away by the thawing solution. , to achieve the purpose of thawing and recovery.
  • the solution leaves the biochip 22 through the solution outlet 223, and the solution outlet 223 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 223 to collect the waste liquid flowing out (generally several ten to hundreds of microliters of solution).
  • the carrier 21 and the biochip 22 are separated, and the embryos in the groove 211 are taken out using a glass capillary, and placed in a petri dish to continue culturing.
  • the grid structure restricts the embryos in the groove or through hole of the carrier and cannot float out, and the freezing liquid or thawing liquid can pass through the grid structure and the embryos In this way, the cryoprotectant can be delivered or removed in an orderly manner, which effectively improves the efficiency of embryo cryopreservation or thawing and recovery operations.
  • the device includes a carrier 31, the carrier 31 is generally a long strip structure, which includes a front sheet and a rear end load rod.
  • the front sheet of the carrier 31 can be made of plastic material with uniform thickness, transparent material, biocompatibility and good heat transfer, so as to ensure the suitability for containing biological tissues and the heat transfer speed during subsequent freezing.
  • the rear end of the carrier 31 can also be referred to as a handle or a handle, which is a position where the operator's hand directly touches and operates.
  • the thickness of the carrier 31 may be 0.25-0.7 mm.
  • the front end of the carrier 31 (that is, the front sheet) has a microchannel 311 for the solution to flow.
  • the width of the microchannel can be 0.5 mm, the height can be 0.2-0.5 mm, and the length can be 20 mm.
  • the two ends of the microchannel 311 have a solution inlet 312 and a solution outlet 313 respectively.
  • the solution inlet 312 is provided with a raised portion 314, and the raised portion 314 protrudes from the carrier 31 to facilitate insertion of the pipette into the solution, wherein the height of the raised portion 314 can be 1-3 millimeters, and the solution inlet 312 is a circular hole, and the diameter of the hole can be 1 millimeter.
  • the microchannel 311 has a solution outlet 313, the solution outlet 313 is a through hole, and the diameter of the through hole can be 0.5-1 mm.
  • the openings at the upper and lower ends of the through hole are respectively provided with a grid-like structure 315.
  • the grid-like structure 315 includes a plurality of grid bars, and there are slits between every two grid bars in these multiple grid bars. The distance between these slits is The (grid pitch) is smaller than the diameter of the biological tissue and larger than the diameter of the solution molecules, wherein the distance between the slits can be 0.05-0.08 mm. In this way, the grid structure 315 can confine the biological tissue in the microchannel 311 to prevent the biological tissue from floating out of the solution outlet 313 .
  • 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 bars of the grid-like structure 315 are elastic, and the grid-like structure 315 can be deformed when in contact with a foreign object (such as a glass capillary), so that the foreign object can pass through the grid-like structure through the slit between the two grid bars.
  • a foreign object such as a glass capillary
  • the grid-like structure 315 will be deformed, continue to insert the glass capillary 32 downward, and the glass capillary 32 will be Enter the microchannel 311 through the grid structure 315, so that the embryos in the glass capillary 32 can be dropped into the microchannel 311.
  • the glass capillary 32 leaves the microchannel 311, and the grid structure 315 returns to its original state. Since the grid pitch of the grid structure 315 is smaller than the diameter of the embryo, the embryo can be confined in the microchannel 311 without Leaves the carrier 31 with the flow of liquid.
  • the device for cryopreservation or thawing and resuscitating of biological tissue further includes: a mechanical movement device for controlling the pipetting pump into a container loaded with various solutions, drawing the solution and The operation of releasing the solution.
  • the mechanical movement device includes a first movement shaft 331 for moving the pipette pump up and down, a second movement shaft 332 for moving the chassis left and right, and a third movement shaft 332 for moving the pipette pump forward and backward. Shaft 333.
  • the mechanical movement device is provided with a pipetting pump 334 and a chassis 335, and the chassis 335 includes a fixing groove for fixing a carrier and a plurality of containers, such as petri dishes, solution tubes, multi-well plates and the like.
  • the three axes of motion can make the pipette pump suck out the required solution (freezing solution during freezing and thawing solution during thawing) from the container, and pour it into the microchannel of the carrier 31 through the solution inlet 312 (not shown). shown), the solution flows through the microchannel to the position of the solution outlet (not shown) where the embryo is located, and the cryoprotectant is delivered to the embryo or removed from the embryo to achieve the function of freezing or thawing treatment.
  • the embryos are blocked in the outlet by the grid-like structure (not shown), and will not leave the carrier with the solution.
  • the pipette pump rises up, leaves the carrier, and moves into the container containing the diluent.
  • the solution inlet is slowly poured into the microchannel of the carrier.
  • the pipette pump rises, leaves the carrier, and moves to the container containing the cleaning solution.
  • the pump sucks out the cleaning solution and moves to the microchannel of the carrier again.
  • the cleaning solution is poured into the entrance, and after 5 minutes, the pipette pump leaves the carrier.
  • the user uses a glass capillary to pass through the grid structure of the carrier to recover the embryos from the microchannel and put them in a petri dish for further cultivation.
  • the grid structure restricts the embryos in the microchannel of the carrier and cannot float out, and the freezing liquid or thawing liquid can pass through the grid structure and contact the embryos, so that The cryoprotectant can be delivered or removed in an orderly manner, effectively improving the efficiency of embryo cryopreservation or thawing and recovery operations.

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Abstract

一种用于生物组织冷冻保存或解冻复苏的装置，其包括：载体，所述载体上设置有凹槽或通孔，所述凹槽的开口处设置有栅状结构或所述通孔的上下两端分别设置有栅状结构，所述栅状结构将生物组织限制在所述凹槽或所述通孔中。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：载体，所述载体上设置有凹槽或通孔，所述凹槽的开口处设置有栅状结构或所述通孔的上下两端分别设置有栅状结构，所述栅状结构将生物组织限制在所述凹槽或所述通孔中。
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：载体，所述载体具有供溶液流动的微通道，所述微通道的两端分别设有溶液入口和溶液出口，所述溶液出口设置有栅状结构；其中，所述栅状结构将生物组织限制在所述微通道中，避免所述生物组织从所述溶液出口飘出。
根据本发明的技术方案，通过栅状结构将生物组织限制在载体的一定位置内，以避免生物组织从载体飘出，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载体的俯视的示意图；
图1B是图1A中的局部放大图；
图1C是沿图1A中1C-1C线的剖面示意图(凹槽)；
图1D是图1C中的局部放大图；
图1E是沿图1A中1C-1C线的剖面示意图(通孔)；
图1F是图1E中的局部放大图；
图2A是根据本发明一个实施例的玻璃毛细管穿过栅状结构的俯视图；
图2B是图2A中的局部放大图；
图2C是沿图2A中2C-2C线的剖面示意图；
图2D是图2C中的局部放大图；
图3是根据本发明一个实施例的载体、底盘和机械运动装置的示意图；
图4A是根据本发明一个实施例的生物芯片和载体的俯视的示意图；
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；
图5A是根据本发明一个实施例的载体的俯视的示意图；
图5B是沿图5A中5B-5B线的剖面示意图；
图5C是图5B中的局部放大图；
图5D是图5C中的局部放大图；
图5E是根据本发明一个实施例的玻璃毛细管穿过栅状结构的侧视图；
图6是根据本发明一个实施例的载体和机械运动装置的示意图。
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至图1F，根据本发明实施例提供一种用于生物组织冷冻保存或解冻复苏的装置，该装置包括载体11，载体11大体为长条状结构，其包括前端薄片和后端载杆。载体11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11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11的后端载杆部分不作为重点部分而未示出。在本申请实施例中，载体11的厚度可为0.25-0.7毫米。
在本申请的一些实施例中，如图1D所示，载体11的前端(即前端薄片)上设置有凹槽111，凹槽111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111的开口处设置有栅状结构112。在本申请实施例中，凹槽111的深度可为0.2-0.5毫米，凹槽111的上表面的直径可为0.2-1毫米。
在本申请的另一些实施例中，如图1F所示，载体11的前端(即前端薄片)上设置有通孔115，通孔115用于盛放待处理的生物组织和保护生物组织的相关溶液。通孔115的上端和下端分别设置有栅状结构116。在本申请实施例中，通孔115的深度可为0.25-0.7毫米，通孔115的上表面/下表面的直径可为0.2-1毫米。
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，栅状结构112包括多个柔性栅条，多个柔性栅条中的每两个栅条之间的间隙的距离小于胚胎的直径且大于溶液分子的直径，其中所述间隙的距离为0.05-0.08毫米。这样，栅状结构就能够将胚胎限制在凹 槽或通孔中的情况下，进行胚胎的冷冻保存或解冻复苏操作。
栅状结构的柔性栅条具有弹性，栅状结构可在与外物(例如玻璃毛细管)接触时发生形变从而可使外物由两个柔性栅条之间的细缝穿过栅状结构。参考图2A至图2D，使用玻璃毛细管15向凹槽111中投放胚胎时，当玻璃毛细管15触碰到栅状结构112时，栅状结构112会发生形变，继续向下插入玻璃毛细管15，则玻璃毛细管15就能够穿过栅状结构112的细缝进入凹槽111中，而将玻璃毛细管15内的胚胎投入到凹槽111中。待胚胎投放后，玻璃毛细管15离开凹槽111则栅状结构112恢复成初始状态，由于栅状结构112的栅距小于胚胎的直径，胚胎可以被限制在凹槽111中，并不会随着液体的流动离开载体11。同样地，也可以使用同样的方法将玻璃毛细管15插入凹槽111，将胚胎吸入毛细管15内，将胚胎转移到其他地方。
类似地，使用玻璃毛细管15向通孔115投放胚胎的过程与以上描述类似，不再赘述。不同的是，通孔115的优势在于分子可以从上下两端向内或外扩散，相比于凹槽仅能够向一端扩散的实施例，可以加快冷冻保护剂的投送和移除的速度。
参考图3，根据本申请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，该装置包括：设置有栅状结构的载体11、底盘14和机械运动装置。其中载体11的具体结构和功能请参考上面的描述，此处不再赘述。
具体地，底盘14上设置并固定有承载溶液的多个容器，例如培养皿，溶液管等等。机械运动装置包括进行上下移动的第一运动轴161和进行左右移动的第二运动轴162，其中，第一运动轴161与载体11连接，用于驱动载体11的上下移动；第二运动轴162与底盘14连接，用于驱动底盘14的左右移动。
当进行冷冻应用操作时，首先利用玻璃毛细管穿过载体的栅状结构将胚胎投放到凹槽内，然后将载体装在机械运动装置上，并把装有平衡液(ES)和玻璃化冷冻液(VS)的容器放到底盘上，机械运动装置将载体前端的透明薄片部分浸入平衡液里，十分钟后，机械运动装置移动载体向上由容器中取出，并移动底盘将装有玻璃化冷冻液的容器移动到载体的下方，然后机械运动装置再次将载体前端的透明薄片部分浸入玻璃化冷冻液，并控制底盘慢慢 左右移动，使载体在容器内与其随动，这样可以确保高浓度冷冻保护剂的投送速度更快，加快胚胎的处理时间，减低高浓度冷冻保护剂的毒性伤害。完成处理后，使用者将载体投入液氮保存。
当进行解冻应用操作时，预先准备已经预热到37度的解冻液，并将稀释液和清洗液加入到容器内并放在机械运动装置的底盘上。然后将载体从液氮取出，将载体前端的透明薄片部分浸入已经预热到37度的解冻液里，50秒后，将载体装于机械运动装置上，并将载体前端的透明薄片部分浸入稀释液里，3分钟后，机械运动装置将载体由容器上方取出，并移动底盘将装有清洗液的容器移动到载体的下方，然后机械运动装置再次将载体前端的透明薄片部分浸入清洗液里，5分钟后，机械运动装置将载体由容器上方抽出，并移动底盘将装有清洗液的另外一个容器移动到载体的下方，然后机械运动装置再次把载体前端的透明薄片部分浸入清洗液，并控制底盘慢慢左右移动，确保把胚胎内的冷冻保护剂移除干净。最后使用者利用玻璃毛细管穿过载体的栅状结构将胚胎从凹槽内回收，并放到培养皿内继续培养。
参考图4A至图4C，根据本申请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，该装置包括：载体21和生物芯片22，其中载体21的前端(即前端薄片)设置有凹槽211，凹槽211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211的开口处设置有栅状结构212。
生物芯片22大体呈长方体结构，包括两个溶液入口221、222以及一个溶液出口223。在一些实施例中，生物芯片22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，则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，重要的是可以使通过不同入口流进生物芯片22的溶液能够按顺序流到胚胎的附近。
根据本发明实施例，生物芯片22的底部(或下表面)的部分或全部为敞开式区域，也就是说生物芯片22的底部全部敞口或者生物芯片22对应载体21的底部区域敞口。当生物芯片22与载体21贴合时，载体21将生物芯片22底部的敞开式区域封闭，在生物芯片22与载体21之间形成经过凹槽211上方的微通道23，溶液可以通过溶液入口221和222进入微通道23并通过溶液出口223离开微通道23。当溶液通过溶液入口221和222进入微通道23 后，溶液会经过凹槽211的上方，由于栅状结构212的细缝间距大于溶液分子的直径，这样溶液就会经过栅状结构212并扩散至凹槽211内，以进行胚胎的冷冻保存或解冻复苏操作。
当进行冷冻保存操作时，开始时载体21和生物芯片22处于分离的状态，使用者首先利用玻璃毛细管穿过载体21的栅状结构212将胚胎注入到凹槽211内，然后将生物芯片22放置在载体21的上方并压紧，使生物芯片22与载体21紧密贴合，生物芯片22与载体21之间形成有供溶液通过的微通道23。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221和/或溶液入口222灌入生物芯片22的内部。溶液在微通道23内流动，当溶液流动到凹槽211上方的位置时，溶液内的冷冻保护剂就可以穿过栅状结构212进入凹槽211并扩散到胚胎内部，达到投送冷冻保护剂的目的。之后溶液经过溶液出口223离开生物芯片22，可以使溶液出口223连接软管将废液引流到废液收集管，也可以在溶液出口223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21和生物芯片22分开，并将载体21投入至液氮冷冻。
当进行解冻应用操作时，将载体21从液氮环境中取出，将载体21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22压住载体21前端的透明薄片部分。由于载体21很薄，在热台上也能达到足够的升温速度。将生物芯片22和载体21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221和/或溶液入口222灌入生物芯片22的内部。溶液在微通道23内流动，当溶液流动到凹槽211上方的位置时，溶液会穿过栅状结构212进入凹槽211，胚胎体内和胚胎附近的冷冻保护剂分子被解冻液带走移除，达到解冻复苏的目的。之后溶液经过溶液出口223离开生物芯片22，溶液出口223可以连接软管把废液引流到废液收集管，也可以在溶液出口223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21和生物芯片22分开，使用玻璃毛细管将凹槽211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，在进行冷冻保存或解冻复苏操作时，栅状结构将胚 胎限制在载体的凹槽或通孔内而不能够飘出，冷冻液或解冻液能够穿过栅状结构与胚胎接触，这样就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5A至图5E，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，该装置包括载体31，载体31大体为长条状结构，其包括前端薄片和后端载杆。载体31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，保证对盛放生物组织的适用性以及后续冷冻时的热传递速度。载体31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31的后端载杆部分不作为重点部分而未示出。在本申请实施例中，载体31的厚度可为0.25-0.7毫米。
在本申请的实施例中，载体31的前端(即前端薄片)内部具有供溶液流动的微通道311，微通道的宽度可为0.5毫米、高度可为0.2-0.5毫米、长度可为20毫米。微通道311两端分别具有溶液入口312和溶液出口313。其中，溶液入口312设有凸起部314，凸起部314凸出于载体31以方便移液管插入其中并灌入溶液，其中，凸起部314的高度可为1-3毫米，溶液入口312为圆形孔，孔的直径可为1毫米。
所述微通道311的一端具有溶液出口313，溶液出口313为一通孔，通孔的直径可为0.5-1毫米。在通孔的上下两端的开口处分别设置有栅状结构315，栅状结构315包括多个栅条，这些多个栅条中的每两个栅条之间具有细缝，这些细缝的间距(栅距)小于生物组织的直径且大于溶液分子的直径，其中所述细缝间距可为0.05-0.08毫米。这样，栅状结构315能够将生物组织限制在微通道311中，以避免生物组织从溶液出口313飘出。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。
栅状结构315的栅条具有弹性，栅状结构315可在与外物(例如玻璃毛细管)接触时发生形变从而可使外物由两个栅条之间的细缝穿过栅状结构。参考图5E，使用玻璃毛细管32向载体31中投放胚胎时，当玻璃毛细管32触碰到栅状结构315时，栅状结构315会发生形变，继续将玻璃毛细管32 向下插入，玻璃毛细管32就穿过栅状结构315进入微通道311中，这样就可以将玻璃毛细管32内的胚胎投入到微通道311中。待胚胎投放后，玻璃毛细管32离开微通道311，栅状结构315就恢复到原来的状态，由于栅状结构315的栅距小于胚胎的直径，胚胎可以被限制在微通道311中，并不会随着液体的流动离开载体31。
参考图6，根据本申请实施例，所述用于生物组织冷冻保存或解冻复苏的装置还包括：机械运动装置，用于控制移液泵到装载有多种溶液的容器内，进行吸取溶液和放出溶液的操作。具体地，所述机械运动装置包括用于使移液泵上下移动的第一运动轴331、用于使底盘左右移动的第二运动轴332、以及用于使移液泵前后移动的第三运动轴333。其中，所述机械运动装置设有移液泵334和底盘335，底盘335包括用于固定载体和多个容器的固定槽，容器例如培养皿、溶液管、多孔板等等。所述三个运动轴可以使移液泵将所需的溶液(冷冻应用时为冷冻液，解冻应用时为解冻液)从容器吸出，并由溶液入口312灌入到载体31的微通道(未示出)中，溶液经过微通道流到胚胎所在的溶液出口(未示出)的位置，将冷冻保护剂投送到胚胎里，或者从胚胎移除，达到冷冻或者解冻处理的功能。在上述过程中胚胎被栅状结构(未示出)阻挡在出口内，并不会随着溶液离开载体。
当进行冷冻应用操作时，首先利用玻璃毛细管穿过载体的栅状结构将胚胎投放到载体微通道的出口内，然后将载体放在机械运动装置的固定槽内，并将装有平衡液(ES)和玻璃化冷冻液(VS)的容器放到机器的固定槽上，机械运动装置按照自动程序通过移液泵将平衡液吸出，并缓慢灌入载体微通道的溶液入口，十分钟后，移液泵向上升起，离开载体，并移动到装有玻璃化冷冻液的容器里，移液泵将玻璃化冷冻液吸出，并再次移动到载体微通道的溶液入口处灌入玻璃化冷冻液，大概60秒后，移液泵离开载体，使用者将载体投入液氮保存。
当进行解冻应用操作时，先准备一盘已经预热到37度的解冻液，并将解冻液、稀释液和清洗液加到容器内并放在机械运动装置的固定槽上，然后把载体从液氮取出，将载体前端的透明薄片部分浸入已经预热到37度的解冻液里，3-5秒后，将载体放在机械运动装置的载体固定槽内，机器按照自动 程序通过移液泵将解冻液吸出，并缓慢灌入载体微通道的入口，50秒后，移液泵向上升起，离开载体，并移动到装有稀释液的容器里，移液泵把稀释液吸出，并由溶液入口缓慢灌入载体微通道内，3分钟后，移液泵升起，离开载体，并移动到装有清洗液的容器里，移液泵将清洗液吸出，并再次移动到载体微通道的入口处灌入清洗液，5分钟后，移液泵离开载体，最后使用者利用玻璃毛细管穿过载体的栅状结构把胚胎从微通道内回收，并放到培养皿内继续培养。
通过本申请实施例，在进行冷冻保存或解冻复苏操作时，栅状结构将胚胎限制在载体的微通道内而不能够飘出，冷冻液或解冻液能够穿过栅状结构与胚胎接触，这样就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 (15)

  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：
    载体，所述载体上设置有凹槽或通孔，所述凹槽的开口处设置有栅状结构或所述通孔的上下两端分别设置有栅状结构，所述栅状结构将生物组织限制在所述凹槽或所述通孔中。
  2. 根据权利要求1所述的装置，其特征在于，所述装置还包括：
    生物芯片，所述生物芯片包括溶液入口和溶液出口；
    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有经过所述凹槽的微通道，所述微通道内的溶液经过所述栅状结构扩散至所述凹槽内。
  3. 根据权利要求2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，所述载体紧密贴合在所述生物芯片的下方以封闭所述生物芯片的敞开式区域。
  4. 根据权利要求2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。
  5. 根据权利要求2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连通。
  6. 根据权利要求1所述的装置，其特征在于，还包括：
    底盘，其上设置有多个容器，每个所述容器中分别承载有溶液；
    机械运动装置，其包括进行上下移动的第一运动轴和进行左右移动的第二运动轴，其中，所述第一运动轴与所述载体连接，所述第二运动轴与所述底盘连接。
  7. 根据权利要求6所述的装置，其特征在于，所述底盘设置在所述载体的下方。
  8. 根据权利要求1所述的装置，其特征在于，所述栅状结构包括多个相互平行设置的柔性栅条，多个柔性栅条中的每两个栅条之间的距离小于所述生物组织的直径且大于溶液分子的直径。
  9. 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：
    载体，所述载体具有供溶液流动的微通道，所述微通道的两端分别设有溶液入口和溶液出口，所述溶液出口设置有栅状结构；
    其中，所述栅状结构将生物组织限制在所述微通道中，避免所述生物组织从所述溶液出口飘出。
  10. 根据权利要求9所述的装置，其特征在于，所述溶液出口包括上下开口，所述上下开口分别设置有所述栅状结构。
  11. 根据权利要求9所述的装置，其特征在于，所述溶液入口处设有凸起部。
  12. 根据权利要求11所述的装置，其特征在于，所述凸起部的宽度大于所述微通道的宽度和高度。
  13. 根据权利要求9所述的装置，其特征在于，还包括：
    机械运动装置，其用于移动移液泵到装载有多种溶液的容器内，进行吸取溶液和放出溶液的操作。
  14. 根据权利要求13所述的装置，其特征在于，所述机械运动装置包括用于使移液泵上下移动的第一运动轴、用于使底盘左右移动的第二运动轴、以及用于使移液泵前后移动的第三运动轴。
  15. 根据权利要求9所述的装置，其特征在于，所述栅状结构包括多个栅条，所述多个栅条中的每两个栅条之间的距离小于所述生物组织的直径且大于溶液分子的直径。
PCT/CN2022/117748 2021-09-10 2022-09-08 用于生物组织冷冻保存或解冻复苏的装置 WO2023036224A1 (zh)

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