WO2014166389A1 - 一种生物样本玻璃化冷冻载体及其用途 - Google Patents
一种生物样本玻璃化冷冻载体及其用途 Download PDFInfo
- Publication number
- WO2014166389A1 WO2014166389A1 PCT/CN2014/074987 CN2014074987W WO2014166389A1 WO 2014166389 A1 WO2014166389 A1 WO 2014166389A1 CN 2014074987 W CN2014074987 W CN 2014074987W WO 2014166389 A1 WO2014166389 A1 WO 2014166389A1
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- WO
- WIPO (PCT)
- Prior art keywords
- biological sample
- carrier
- frozen
- sealing cap
- sample
- Prior art date
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Classifications
-
- 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
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
-
- 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
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
- A01N1/0257—Stationary or portable vessels generating cryogenic temperatures
-
- 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
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
- A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
Definitions
- This invention relates to the field of biological sample freezing devices and, in particular, to providing a safe, efficient and easy to operate closed cryopreservation for use in sterile vitrification of frozen embryos or egg cells in the field of assisted reproductive technology. Background technique
- the Leiden Frost effect produced by the room temperature material and the extremely low temperature coolant, such as liquid nitrogen, will form a heat-insulating vapor film on the surface of the frozen material and slow down the required frozen material.
- Cooling speed generally need to increase the concentration of cryoprotectant to prevent the formation of ice crystals during the freezing process of biological samples, while high concentration cryoprotectants are toxic to biological samples, resulting in reduced cell quality after resuscitation, therefore, the pursuit of fast frozen glass
- the refrigeration technology has been developed.
- the vitrification and freezing carriers at home and abroad are generally open, and there are a stretched straw method, a quartz capillary method, a Cryoloop method, a Cryotip method, and the like.
- Kuwayama proposed a new method of increasing the cooling rate by minimizing the volume of the solution, a Cryotop method, etc., in which the frozen carrier carrying the sample was directly immersed in liquid nitrogen.
- Patent application (200710192245. 9) discloses a secret that prevents foreign pathogenic substances from contaminating biological samples. Sealed vitrification freezer, but despite the closed system that is isolated from the coolant, the operation of this type of refrigerated carrier is complicated by the need for various steps such as thermoplastic closure.
- the invention provides a high-efficiency vitrification and easy-to-operate biological sample sealed storage device.
- the invention provides a biological sample vitrification and freezing carrier, wherein the carrier comprises a body and a sealing cap, wherein the proximal end of the body and the bottom of the sealing cap are sealed to each other to form a sealed frozen sample placing area, the frozen sample
- the placement area has a frozen sample placement platform.
- the body is provided with a coolant circulation passage
- the sealing cap is provided with a flow guiding hole corresponding to the coolant circulation passage, so that when the biological sample is frozen, the coolant flowing in the passage is The outer surface of the frozen sample placement zone contacts and cools the biological sample within the frozen sample placement zone.
- the frozen liquid circulating in the channel is in direct contact with the outer surface of the frozen sample placement platform to cool the biological sample placed on the frozen sample placement platform.
- the cryogen flowing outside the vitrified frozen carrier is in direct contact with the outer surface of the frozen sample placement platform.
- the biological sample placed on the frozen sample placement platform is thereby cooled.
- a sealing gasket is further disposed between the proximal end of the body and the sealing cap.
- the frozen sample placement platform is a flat surface of a suitable sample size; and (a) is disposed on the top of the proximal end of the body or is fixed to the proximal end of the body.
- the top portion; or (b) is disposed at the bottom of the sealing cap (especially the inner bottom surface); or is fixed to the bottom of the sealing cap.
- the frozen sample placement platform is at the same level as the top surface of the body proximal end; or the frozen sample placement platform is lower than the top surface of the proximal end of the body.
- the height of the frozen sample placement zone is 0. 5-5mm
- the placing platform forming method comprises integrally forming, bonding forming, and fastening forming with the carrier body or the sealing cap.
- the biological sample comprises: a cell, a tissue, an organ.
- the cells include egg cells, embryo cells.
- the body further includes a grip portion at the distal end of the body and a proximal end and a distal end of the body An engaging portion for sealing engagement with the sealing cap.
- the passage includes a distal opening at the grip and a side opening provided in the side wall of the body, and the distal opening is in communication with the side opening.
- the distal opening extends from the grip portion toward the center of the body to the proximal end of the body, and together with the frozen sample placement platform, the distal end is provided with an opening and is hollow Ontology.
- a side opening of the body sidewall is located between the frozen sample placement area and the meshing portion, and more preferably, a side opening of the body sidewall is closely attached to the frozen sample. Area.
- the side walls of the body side wall have at least two, more preferably, 4-6.
- the engaging portion surface is provided with a movable fastening member for hermetic engagement with the sealing cap.
- the movable fastening member includes a screw fastening member, a socket fastening member, and a fastening fastening member.
- the outer top surface or the side surface of the sealing cap is provided with the same information mark area.
- the sealing caps are of different colors to distinguish embryos of different developmental cycles, different classes of cells or tissues.
- the platform has a diameter of from 1 to 15, preferably from 3 to 12, more preferably from 5 to 10; optimally, from 6 to 8; and/or
- the thickness of the platform is 0. 06-0. 15mm, preferably 0. 07-0. 12mm, more preferably 0. 08-0. 10mm; and / or
- the height of the biological sample placement zone is 0.5 - 5 let, preferably 1-4, more preferably 2 - 3 let.
- the carrier has a freezing rate of at least 10,000-20000 after placing the biological sample.
- °C/min Preferably, it is 30000-50000 ° C / min; more preferably, 60000-100000 ° C / min.
- the body and the sealing cap material are the same or different and are selected from the group consisting of: a polymer material, a metal; and/or
- the carrier platform material is selected from the group consisting of polymer materials and metals.
- the polymer material comprises PE (polyethylene), HDPE (high density polyethylene);
- PP polypropylene
- PET polyethylene terephthalate
- the metal comprises stainless steel or a titanium alloy.
- the polymer material or metal is a medical polymer material or a medical metal.
- the holding portion has a diameter of 6-15, the distal opening diameter of 2 to 10; and/or the body side opening has a size of l-3 mm X l-3 mm; and / or
- the size of the sealing cap of the sealing cap is 2 - 5 mm X 2 - 5 mm.
- the chilled liquid flow passage has a stepped shape and a topped cylindrical shape.
- the body side opening may be a circular hole, an elliptical hole, a shaped hole or a flat hole.
- the body side opening is at least two, more preferably 4-6, and the sealing cap has at least two, more preferably 4-6.
- a closed vitrification method for a biological sample comprising the steps of: a) providing a vitrified frozen carrier for a biological sample according to the first aspect of the invention, wherein the carrier comprises a body and a sealing cap, And placing the biological sample on the frozen sample placement platform of the biological sample vitrification freezing carrier, thereby forming a carrier body containing the biological sample;
- step b) engaging and tightening the carrier body of the biological sample containing step a) with the sealing cap to form a sealed carrier containing the biological sample;
- step b) labeling the sealed carrier containing the biological sample of step b) in the marking area;
- step d) Place the sealed carrier, which has been labeled with biological sample information in step c), in a coolant.
- the closed vitrification method of the biological sample further comprises: marking the sealed carrier containing the biological sample; and/or marking the frozen carrier, placing the biological sample and sealing, thereby obtaining The labeled biological sample seals the carrier.
- a biological sample cryopreservation assembly comprising: i) one or more carriers according to the first aspect of the invention; ⁇ ) liquid nitrogen; and iii) liquid nitrogen storage Device.
- the interior of the liquid nitrogen storage device is further provided with a bracket for movably connecting the liquid nitrogen storage device for accommodating the carrier.
- the stent is of one or more layers, and the stent can accommodate from 5 to 120 of the carriers, preferably from 10 to 100, more preferably. , for 20-80.
- the size of the stent may vary depending on the internal volume of the liquid nitrogen storage device.
- the present invention also provides a use of the biological sample closed vitrification and freezing carrier according to the first aspect of the present invention, which is used for hermetic cryopreservation of a biological sample. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS
- Fig. 1 is a schematic view showing the assembly of a vitrified frozen carrier 1 of the biological sample of the present invention.
- Fig. 2 is a schematic view showing the assembly of the vitrified frozen carrier 2 of the biological sample of the present invention.
- Fig. 3 is a schematic view showing the assembly of the vitrification and freezing carrier 3 of the biological sample of the present invention.
- Figure 4 shows a photograph of a stent in the biological sample cryoprecipitator of the present invention.
- the inventors have for the first time provided a carrier and device for the closed cryopreservation of biological samples through extensive and intensive research. Tests have shown that the designed coolant circulation channel, coolant diversion hole and biological sample freezing platform of the carrier of the invention can achieve high-efficiency vitrification freezing effect while sealing the biological sample and the freezing liquid, and after rewarming The embryo has a high survival rate and is easy to operate. On this basis, the present invention has been completed.
- the proximal end of the body of the present invention and the inner bottom surface of the sealing cap of the present invention are sealed to each other to form a sealed frozen sample placing area, and the frozen sample placing area is provided with a frozen sample placing platform.
- the platform may be fixedly coupled to a top surface of the proximal end of the body, and together with the proximal end of the body, form a hollow body structure that is closed at the proximal end.
- the plane of the platform may be at the same level as the top surface of the proximal end of the body, or lower than the top surface of the proximal end of the body.
- the platform can be fixed to the top surface of the body by various conventional fixed connection methods.
- the platform can be molded with the body, connected to the proximal end of the body by a polymer bonding substance, or fastened by a fastening connection. At the proximal end of the body.
- a preferred method of attachment is compression molding with the body.
- the platform may also be fixedly coupled to the inner bottom surface of the sealing cap. After the platform is coupled to the sealing cap, the body is inverted into the sealing cap and fitted into the platform by the proximal end of the body and forms a sealed frozen sample placement zone.
- a sealing ring is provided for sealing the gap between the proximal end of the body and the sealing cap, ensuring the sealing of the frozen sample placement area, effectively isolating the biological sample and cooling The contact of the agent, thereby preventing cross-contamination and poisoning of biological samples by toxic substances such as unknown pathogens in the refrigerant.
- the material of the frozen sample placement platform of the invention is non-toxic and can withstand the sudden cooling and the sudden temperature change Use polymer materials, metals.
- materials that can be used in the frozen sample placement platform of the present invention include PE, HDPE, PET, PP, medical stainless steel, medical titanium alloys.
- the frozen sample placement platform of the present invention is characterized in that the platform has a diameter of from 1 to 15 mm, preferably from 3 to 12, more preferably from 5 to 10 mm; most preferably from 6 to 8 mm; and/or a carrier
- the thickness of the platform is 0. 06-0. 15mm, preferably 0. 07-0. 12mm, more preferably 0. 08-0. 10mm.
- the size of the vitrified frozen carrier of the biological sample of the present invention may vary depending on the specific size of the biological sample to be frozen and the removal of the excess cryoprotectant.
- the freezer carrier platform may have a diameter of from 1 to 15 mm and a height of from 0.5 to 5 mm.
- the coolant flow passage that can be used in the present invention includes the following parts:
- the distal opening and the side opening communicate with each other via a center of the body, and correspond to a flow guiding hole located at a sidewall of the sealing cap.
- the side opening is located at the proximal end of the body, and more preferably, the side opening is in close contact with the frozen sample placement platform.
- the side opening and the flow guiding hole are at least two, more preferably four or more.
- the sealing cap and the body when closed and sealed to each other, they are placed in the frozen liquid, and the frozen liquid can be circulated through the freezing liquid circulation passage from the distal opening-side opening-drainage hole, so that the coolant flows.
- the surface of the biological sample carrier platform can be pre-cooled. Due to the thinness of the carrier platform, the le idenfrost effect is reduced, and the tissue sample located on the frozen placement platform is rapidly cooled.
- the body side opening has a diameter of l-3 mm, or 2 X 3 mm elliptical transverse holes
- the sealing cap has a diameter of 2 to 5 mm X 2 - 5 mm.
- the three-dimensional shape of the distal opening or the body side opening or the diversion hole which can be used in the present invention is not particularly limited, and may be any shape which is compatible with each other to facilitate the circulation of the refrigerant, and generally, each opening or the orifice in the present invention
- the three-dimensional shape is stepped and topped.
- the method for rapidly freezing a biological sample in the present invention comprises the steps of:
- the carrier comprising a body and a sealing cap, and placing the biological sample on a frozen sample placing platform of the biological sample vitrification freezing carrier, Thereby forming a carrier body containing the biological sample; b) engaging and tightening the carrier body of the biological sample containing step a) with the sealing cap to form a sealed carrier containing the biological sample;
- step b) labeling the sealed carrier containing the biological sample of step b) in the marking area;
- step c) Place the sealed carrier, which has been labeled with biological sample information, in step c) in a coolant.
- the closed vitrification method of the biological sample further comprises labeling the sealed carrier containing the biological sample; and/or labeling the frozen carrier, placing the specimen and sealing to obtain a labeled sealed carrier.
- a cooling rate of 2000 CTC / min can be achieved, and more preferably, a cooling rate of 4 oooo-iooooo ° c / min, depending on the material or size of the carrier platform The cooling rate and vitrification effect of the biological sample.
- the carrier of the present invention is used after sterilizing treatment when applied to an experiment or a diagnosis, and maintains a sterile environment during the operation.
- the materials which can be used to prepare the vitrified frozen carrier of the biological sample of the present invention are non-toxic, aseptically treated, and can withstand high temperature materials and metals which are subject to quenching and rapid changes in temperature.
- the polymer material comprises PE (polyethylene), HDPE (high density polyethylene), PP (polypropylene), and PET (polyethylene terephthalate).
- the metal comprises stainless steel or a titanium alloy.
- the polymer material or metal is a medical polymer material or a medical metal.
- the body and the sealing cap are interlocked and sealed with each other in various movable socket manners such as mutual fastening or screw rotation connection.
- the fastening or threaded rotary connecting device can achieve the sealing effect by various conventional methods, such as increasing the sealing ring which can withstand the sudden temperature change of the quenching and the sudden heating, such as changing the shape or angle of the thread, etc., to ensure the sealing.
- the device is hermetically self-locking during the quenching and heating process.
- the shape of the body and the sealing cap in the present invention is not particularly limited, and may be any matching cylindrical appearance such as a cylindrical shape or a hexagonal column shape, thereby facilitating the operator to hold and the carrier when placing biological samples such as embryos and eggs. Stable.
- the body and sealing cap of the present invention can be used in a variety of different colors to distinguish the carrier for cryopreservation of different biological samples, tissues, cells, and the like.
- the carrier and method of the invention are used for isolating and confining biological samples from frozen liquids, safe and pollution-free, and reducing the potential toxicity risk of frozen liquids on biological samples.
- the freezing rate is fast, and the high-efficiency vitrification freezing is achieved: the carrier and the method of the invention can be used.
- the loooo-ioooocrc/min freezing rate depending on the material or size of the carrier platform, results in different cooling rates and vitrification effects of the biological sample. This enables rapid vitrification and avoids the effects of ice crystal damage during the freezing of biological samples.
- the carrier of the invention adopts a sealing method such as screw fastening or socket fastening, fastening fastening, etc., only need to place the biological sample on the platform and then tighten the sealing cap, then it can be placed in liquid nitrogen storage instead of A complex procedure for traditional thermoplastic sealing.
- a sealing method such as screw fastening or socket fastening, fastening fastening, etc.
- the experimental animals were clean Kunming mice (purchased from Shanghai Institute of Biological Products), and the females were 9 weeks old, 29-33 g/only, a total of 80.
- the males were 9 weeks old and weighed 38-45 g/head, a total of 20, and the light was alternated with 12 hours of light and dark.
- Frozen carrier 80 sets of sterilized frozen carrier 1 (Fig. 1), 2 (Fig. 2) and 3 (Fig. 3) of the present invention are sterilized, and the selected pronuclear embryos are loaded and frozen in liquid nitrogen, 3 After the day, take it out, rewarmed it, culture it, and observe its embryonic division.
- the number of 2 cell embryos is the number of surviving 2 dividing cell embryos obtained one day after fertilization of the fertilized cells; 4 the number of cells is the number of surviving 4 dividing cell embryos obtained 48 hours after the culture of the fertilized cells; the number of blastocysts is after fertilization The number of cells that entered the blastocyst stage obtained in 96 hours.
- 2 cell rate 2 cell embryos / number of pronuclear embryos
- 4 cell rate 4 cell embryos / 2 cell embryos
- blastocyst rate number of blastocysts / 2 number of cell embryos.
- the blastocyst rate is about 50% to 70%. From the results of the present example, it can be seen that the blastocyst rate of the vitrified frozen embryos using the vector of the present invention is 51.67%, 66.15% and 73.13%, respectively, and the results are compared with the blastocyst rate of other devices and methods in the art. It is similar or even better. Further, the carrier of the present invention has the advantages of simple operation and high safety, and therefore, it can be substituted for the conventional device and method, and is suitable for a wide range of applications.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP14783085.5A EP2984928B1 (en) | 2013-04-09 | 2014-04-09 | Biological sample vitrification carrier and usage thereof |
CN201480020485.9A CN105163579B (zh) | 2013-04-09 | 2014-04-09 | 一种生物样本玻璃化冷冻载体及其用途 |
JP2016506769A JP6099808B2 (ja) | 2013-04-09 | 2014-04-09 | 生物試料のガラス化冷凍担体およびその使用 |
US14/783,562 US10306882B2 (en) | 2013-04-09 | 2014-04-09 | Biological sample vitrification carrier and usage thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310121801.9A CN103190393B (zh) | 2013-04-09 | 2013-04-09 | 一种生物样本玻璃化冷冻载体及其用途 |
CN201310121801.9 | 2013-04-09 |
Publications (1)
Publication Number | Publication Date |
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WO2014166389A1 true WO2014166389A1 (zh) | 2014-10-16 |
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ID=48713362
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Application Number | Title | Priority Date | Filing Date |
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PCT/CN2014/074987 WO2014166389A1 (zh) | 2013-04-09 | 2014-04-09 | 一种生物样本玻璃化冷冻载体及其用途 |
Country Status (5)
Country | Link |
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US (1) | US10306882B2 (zh) |
EP (1) | EP2984928B1 (zh) |
JP (1) | JP6099808B2 (zh) |
CN (2) | CN103190393B (zh) |
WO (1) | WO2014166389A1 (zh) |
Families Citing this family (15)
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CN103190393B (zh) | 2013-04-09 | 2015-05-13 | 上海安久生物科技有限公司 | 一种生物样本玻璃化冷冻载体及其用途 |
DE102016005075A1 (de) | 2016-04-27 | 2017-11-02 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Verfahren und Vorrichtung zur Temperaturüberwachung einer kryokonservierten biologischen Probe |
DE102016005070A1 (de) | 2016-04-27 | 2017-11-02 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Vorrichtung und Verfahren zur Temperaturüberwachung einer kryokonservierten biologischen Probe |
DE102016005078A1 (de) * | 2016-04-27 | 2017-11-02 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Vorrichtung und Verfahren zur Temperaturüberwachung einer kryokonservierten biologischen Probe |
DE102016005133A1 (de) | 2016-04-27 | 2017-11-02 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Verfahren und Vorrichtung zur Temperaturüberwachung einer kryokonservierten biologischen Probe |
EP3287775B1 (en) * | 2016-08-26 | 2019-04-03 | Leica Mikrosysteme GmbH | Modular specimen holders for high pressure freezing and x-ray crystallography of a specimen |
US11352262B2 (en) | 2017-12-18 | 2022-06-07 | Praxair Technology, Inc. | Methods for automatic filling, charging and dispensing carbon dioxide snow block |
CN109362706B (zh) * | 2018-10-17 | 2021-06-11 | 北京大学深圳医院 | 胚胎冷冻载杆装置 |
CN109628297B (zh) * | 2018-12-14 | 2022-02-18 | 浙江大学宁波理工学院 | 一种微流控高通量生物样品滴冻保存装置 |
CN110521720B (zh) * | 2019-01-18 | 2020-06-16 | 上海廪典实业有限公司 | 一种超低温冷冻及封闭式保存方法 |
KR102146379B1 (ko) * | 2019-03-04 | 2020-08-21 | 고려대학교 산학협력단 | 조직의 급속동결 보관장치 및 조직의 급속동결 보관방법 |
CN110521719B (zh) * | 2019-07-08 | 2023-08-11 | 浙江大学 | 一种玻璃化冷冻装置及使用方法 |
CN111700061A (zh) * | 2020-06-12 | 2020-09-25 | 山东大学齐鲁医院 | 一种封闭式玻璃化冷冻载体及其应用 |
US12038133B2 (en) * | 2020-07-23 | 2024-07-16 | Coopersurgical, Inc. | Canister caps for cryopreservation applications |
WO2023036223A1 (zh) * | 2021-09-10 | 2023-03-16 | 深圳拜尔洛克生物技术有限公司 | 用于生物组织冷冻保存或解冻复苏的装置 |
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US10306882B2 (en) | 2019-06-04 |
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