WO2020147604A1 - Ultralow-temperature freezing and sealing type preservation method - Google Patents
Ultralow-temperature freezing and sealing type preservation method Download PDFInfo
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- WO2020147604A1 WO2020147604A1 PCT/CN2020/070482 CN2020070482W WO2020147604A1 WO 2020147604 A1 WO2020147604 A1 WO 2020147604A1 CN 2020070482 W CN2020070482 W CN 2020070482W WO 2020147604 A1 WO2020147604 A1 WO 2020147604A1
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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
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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
- 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
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- cold sources such as commercially available liquid nitrogen may be contaminated by pathogenic microorganisms such as bacteria, viruses, and fungi during production, transportation, and long-term storage in liquid nitrogen tanks and other storage tanks.
- Pathogenic microorganisms can tolerate low temperatures and have the risk of contamination and cross-infection after resuscitation, so embryos stored in open vitrification have potential risks of contamination.
- closed pull straws CPS, closed pulled straws
- straw-in-straw straw-in-straw
- Rapid-i closed vitrification methods are used in assisted reproduction clinics , But the survival rate is poor and it is not widely used.
- the aseptic container is subjected to aseptic treatment before use, for example, treatment with high temperature, ultraviolet light and/or ethylene oxide.
- the material of the aseptic container includes but is not limited to metal, plastic, foam, glass, etc.
- the present invention also provides an embodiment in which the sterile liquid air prepared by the method of the present invention is used as a cold source to achieve long-term aseptic preservation of embryos, including the following steps: an outer tube is used as a sterile container, wherein the outer tube may be made of metal, Such as medical stainless steel, a precision screw is set at the opening, and the freezing rod loaded with embryos is put into the outer tube to complete vitrification and then tightened with the corresponding precision nut to achieve a spiral seal (both internal or external rotation), and transfer to Long-term storage in conventional liquid nitrogen tanks can realize safe, sterile, and long-term storage of biological samples without cross contamination.
- the outer sleeve may also be made of plastic material.
- the liquid nitrogen in the liquid nitrogen tank is not clean (commercial liquid nitrogen contains bacteria, viruses, fungi and other pathogenic microorganisms), the embryos are vitrified and frozen in sterile liquid air and stored in the liquid nitrogen tank. Therefore, the external non-clean liquid nitrogen will not affect the sterile state of the frozen embryos.
- Step 3 A step of removing moisture or mist at the opening 10 of the sterile container. Specifically, during the process of preparing a sterile cold source, water vapor may be deposited at the opening 10. Before the opening 10 is sealed, a heat source should be used close to the opening 10 or a sterile gauze should be used to wipe the first opening 10, etc. The water vapor is removed so as to seal the first opening 10 easily.
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Abstract
Provided is an ultralow-temperature freezing and sealing type preservation method, comprising the following steps: 1) providing an aseptic container with an opening; 2) placing the aseptic container in an initial cold source, wherein the height of the opening of the aseptic container is higher than a liquid level layer of the initial cold source; 3) forming a liquefied gas as an aseptic refrigerant in the aseptic container; 4) placing a sample to be frozen, supported on a carrier, into the aseptic refrigerant for vitrification freezing; and 5) sealing the aseptic container, and performing ultralow-temperature preservation of the sealed aseptic container, together with the vitrification fronzen sample sealed therein.
Description
本发明涉及生物样品保存领域，尤其涉及一种超低温冷冻及封闭式保存方法。The invention relates to the field of biological sample preservation, in particular to an ultra-low temperature freezing and closed preservation method.
医学、生命科学(包括辅助生殖)、食品、化工等领域均需要用到超低温保存。超低温保存需要使用冷源，冷源包括但不限于液氮等常规制冷剂。Medical, life sciences (including assisted reproduction), food, chemical and other fields require ultra-low temperature storage. Ultra-low temperature storage requires the use of cold sources, which include but are not limited to conventional refrigerants such as liquid nitrogen.
以人辅助生殖领域为例，该领域需要进行胚胎、卵子等细胞样本的冷冻保存，液氮是主要的冷源。玻璃化冷冻因为降温速率快(20,000℃/min以上)、复苏存活率高(≥95％)成为目前人胚胎和卵子冷冻的主要方法。然而，玻璃化冷冻技术潜在的污染风险一直困扰着辅助生殖从业人员。在玻璃化冷冻过程中，需要将装载在冷冻载杆上的胚胎样本与液氮等冷源直接接触。这是因为，只有直接接触液氮才能达到超快速降温，从而保证复苏存活率。而市售液氮等冷源在生产、运输以及液氮罐等其他储存罐内长期保存等环节均有可能被细菌、病毒、真菌等病原微生物污染。致病微生物可以耐受低温并在复苏后具有污染和交叉感染风险，所以开放式玻璃化冻存的胚胎具有潜在的污染风险。为了避免污染或交叉污染，封闭式拉细麦管(CPS，closed pulled straws)、麦管套麦管(straw-in-straw)和Rapid-i等封闭式玻璃化冷冻方法被应用于辅助生殖临床，但存活率欠佳，未能广泛使用。CPS、麦管套麦管和Rapid-i等封闭式玻璃化冷冻方法均是将装载胚胎的冷冻载体***外套管封闭后再投入液氮实现样本冻结，降温速率慢(仅约2,000℃/min左右)，导致解冻存活率低。Take the field of human assisted reproduction as an example. This field requires cryopreservation of cell samples such as embryos and eggs, and liquid nitrogen is the main cold source. Vitrification has become the main method of freezing human embryos and eggs due to its fast cooling rate (over 20,000°C/min) and high survival rate (≥95%). However, the potential pollution risk of vitrification technology has been plagued by assisted reproduction practitioners. In the process of vitrification, it is necessary to directly contact the embryo sample loaded on the freezing rod with a cold source such as liquid nitrogen. This is because only direct contact with liquid nitrogen can achieve ultra-fast cooling, thereby ensuring the survival rate of resuscitation. However, cold sources such as commercially available liquid nitrogen may be contaminated by pathogenic microorganisms such as bacteria, viruses, and fungi during production, transportation, and long-term storage in liquid nitrogen tanks and other storage tanks. Pathogenic microorganisms can tolerate low temperatures and have the risk of contamination and cross-infection after resuscitation, so embryos stored in open vitrification have potential risks of contamination. In order to avoid contamination or cross-contamination, closed pull straws (CPS, closed pulled straws), straw-in-straw (straw-in-straw) and Rapid-i closed vitrification methods are used in assisted reproduction clinics , But the survival rate is poor and it is not widely used. Closed vitrification methods such as CPS, straw-in-a-straw, and Rapid-i are all methods of inserting the embryo-loaded freezing carrier into the outer tube and then putting liquid nitrogen into it to freeze the sample. The cooling rate is slow (only about 2,000°C/min) ), resulting in low thaw survival rate.
中国专利(申请号201720870512.2)提供一种在液氮工厂生产无菌液氮的设备和方法。虽然生产的液氮无菌，但液氮在运输、储存和使用过程中维持无菌的状态极其困难。而且，细菌或病毒感染患者的胚胎还可能在液氮罐内间接将病原传递给其他“健康”胚胎，导致胚胎发生交叉感染。The Chinese patent (application number 201720870512.2) provides an equipment and method for producing sterile liquid nitrogen in a liquid nitrogen factory. Although the produced liquid nitrogen is sterile, it is extremely difficult to maintain the sterile state during transportation, storage and use of liquid nitrogen. Moreover, the embryos of patients infected by bacteria or viruses may indirectly pass the pathogen to other "healthy" embryos in the liquid nitrogen tank, causing cross-infection of the embryos.
因此，亟需一种既能快速降温，又能避免冷源对样品污染的保存方法。Therefore, there is an urgent need for a preservation method that can not only quickly cool down the temperature, but also avoid the contamination of the sample by the cold source.
发明内容Summary of the invention
本发明所要解决的技术问题是，提供一种超低温冷冻及封闭式保存方法，其能够按照开放式载体的操作方法实现生物样本的无菌保存，彻底避免开放式载体玻璃化冷冻的污染或交叉污染风险，并且不影响降温速率、不会降低胚胎存活率。The technical problem to be solved by the present invention is to provide an ultra-low temperature freezing and closed storage method, which can realize the aseptic storage of biological samples according to the operation method of the open carrier, and completely avoid the contamination or cross contamination of the vitrification of the open carrier Risk, and does not affect the cooling rate, will not reduce the embryo survival rate.
为了解决上述问题，本发明提供了一种超低温冷冻及封闭式保存方法，其包括如下步骤：In order to solve the above problems, the present invention provides an ultra-low temperature freezing and closed storage method, which includes the following steps:
1)提供一具有开口的无菌容器；1) Provide a sterile container with an opening;
2)将所述无菌容器置于初始冷源中，且所述无菌容器开口的高度高于所述初始冷源的液面层；2) Place the aseptic container in the initial cold source, and the height of the opening of the aseptic container is higher than the liquid level of the initial cold source;
3)在所述无菌容器中形成液化气体，作为无菌制冷剂；3) Forming liquefied gas in the aseptic container as a sterile refrigerant;
4)将承载在载体上的待冷冻样品置于所述无菌制冷剂中进行玻璃化冷冻；4) Place the sample to be frozen on the carrier in the sterile refrigerant for vitrification;
5)密封所述无菌容器，并将所述密封无菌容器与密封在其中的玻璃化冷冻样品一起超低温保存。5) Seal the aseptic container, and store the sealed aseptic container together with the vitrified frozen sample sealed in it at an ultra-low temperature.
在一个优选的实施方案中，所述无菌容器的开口高于所述初始冷源所形成的蒸汽层。In a preferred embodiment, the opening of the aseptic container is higher than the vapor layer formed by the initial cold source.
在一个更优选的实施方案中，所述初始冷源是液氮、液氦或者其他超低温冷源。In a more preferred embodiment, the initial cold source is liquid nitrogen, liquid helium or other ultra-low temperature cold sources.
在一个更优选的实施方案中，所述无菌容器是管状容器；在更优选的实施方案中，所述管状容器一端开口；在更优选的实施方案中，所述管状容器具有侧面开口；在更优选的方案中，在需要***初始冷源液面的一端，由密度较大的材料制得或放置有密度较大的物体，以使该管状容器能***初始冷源液面以下；在更优选的实施方案中，所述管状容器的主体部分是圆柱形；在更优选的实施方案中，所述管状容器的内径为1mm至30mm，优选1.5mm至20mm，更优选2mm至10mm，更优选为2.5mm至6mm，更优选为3至5mm，更优选为3.5至4.5mm；在更优选的实施方案中，所述管状容器的长度为2cm至40cm，优选5cm至30cm，更优选10cm至29cm，更优选为14cm至28cm，最优选为14cm或28cm。在一个更优选的实施方案中，所述管状容器在密封后的长度不变。In a more preferred embodiment, the sterile container is a tubular container; in a more preferred embodiment, the tubular container is open at one end; in a more preferred embodiment, the tubular container has a side opening; In a more preferred solution, at the end that needs to be inserted into the initial cold source liquid level, it is made of a denser material or placed with a denser object, so that the tubular container can be inserted below the initial cold source liquid level; In a preferred embodiment, the main body of the tubular container is cylindrical; in a more preferred embodiment, the inner diameter of the tubular container is 1 mm to 30 mm, preferably 1.5 mm to 20 mm, more preferably 2 mm to 10 mm, more preferably 2.5mm to 6mm, more preferably 3 to 5mm, more preferably 3.5 to 4.5mm; in a more preferred embodiment, the length of the tubular container is 2cm to 40cm, preferably 5cm to 30cm, more preferably 10cm to 29cm , More preferably 14cm to 28cm, most preferably 14cm or 28cm. In a more preferred embodiment, the length of the tubular container remains unchanged after sealing.
所述无菌容器在使用前预先经过无菌处理，例如，经过高温、紫外线和/或环氧乙烷等处理。所述无菌容器的材质包括但不限于金属、塑料、泡沫、玻璃等。The aseptic container is subjected to aseptic treatment before use, for example, treatment with high temperature, ultraviolet light and/or ethylene oxide. The material of the aseptic container includes but is not limited to metal, plastic, foam, glass, etc.
在另一个优选的实施方案中，所述无菌容器在开口处装有空气无菌滤器，外部气体通过滤器净化后进入该无菌容器中，并在容器外部初始冷源的作用下液化形成无菌冷源。在更优选的实施方案中，所述空气无菌滤器包含0.22μm或0.45μm孔径的滤膜。In another preferred embodiment, the aseptic container is equipped with an air aseptic filter at the opening, and the external air enters the aseptic container after being purified by the filter, and is liquefied to form an airless container under the action of the initial cold source outside the container. Bacteria cold source. In a more preferred embodiment, the air sterile filter comprises a filter membrane with a pore size of 0.22 μm or 0.45 μm.
在另一个优选的实施方案中，上述步骤5)中的超低温保存是指将所述密封无菌容器与密封在其中的玻璃化冷冻样品一起置于液氮、液氦或其他超低温冷源中保存。In another preferred embodiment, the ultra-low temperature storage in step 5) above refers to storing the sealed sterile container together with the vitrified frozen sample sealed in it in liquid nitrogen, liquid helium or other ultra-low temperature cold source .
在另一个优选的实施方案中，密封所述开口的方法包括但不限于超声封口、螺旋封口或热封口；在更优选的实施方案中，在密封所述开口前还包括除去空气无菌滤器的步骤。In another preferred embodiment, the method of sealing the opening includes, but is not limited to, ultrasonic sealing, spiral sealing or heat sealing; in a more preferred embodiment, it also includes removing the sterile air filter before sealing the opening. step.
在另一个优选的实施方案中，在密封所述开口步骤之前还包括去除所述开口处湿气或雾气的步骤。In another preferred embodiment, before the step of sealing the opening, a step of removing moisture or mist from the opening is further included.
在另一个优选的实施方案中，所述承载待冷冻样品的载体是冷冻载杆；在更优选的实施方案中，所述冷冻载杆包括样品承载端、手持端和位于所述样品承载端和手持端之间的连接部分。In another preferred embodiment, the carrier carrying the sample to be frozen is a freezing carrier rod; in a more preferred embodiment, the freezing carrier rod includes a sample carrying end, a hand-held end, and a The connection part between the handheld terminals.
在另一个优选的实施方案中，所述冷冻样品包括但不限于人类或动物的胚胎、合子、卵子、***、组织(如卵巢组织、睾丸组织等)等需要长期冷冻保存的细胞或组织。In another preferred embodiment, the frozen sample includes, but is not limited to, human or animal embryos, zygotes, eggs, sperm, tissues (such as ovarian tissue, testicular tissue, etc.) and other cells or tissues that require long-term cryopreservation.
本发明还提供了用于上述超低温冷冻及封闭式保存方法的套件，其包含：具有一开口的无菌容器；以及可置于所述无菌容器中的冷冻载体，用于承载需要冷冻的样品。The present invention also provides a kit for the above-mentioned ultra-low temperature freezing and closed storage method, which comprises: a sterile container with an opening; and a freezing carrier that can be placed in the sterile container for carrying samples that need to be frozen .
在一个更优选的实施方案中，所述无菌容器是管状容器；在更优选的实施方案中，所述管状容器一端开口；在更优选的实施方案中，所述管状容器具有侧面开口；在更优选的方案中，需要***初始冷源液面的一端由密度较大的材料制得或放置有密度较大的物体，以使该管状容器能***初始冷源液面以下，而不易漂浮；在更优选的实施方案中，所述管状容器的主体部分是圆柱形；在更优选的实施方案中，所述管状容器的所述管状容器的内径为1mm至30mm，优选1.5mm至20mm，更优选2mm至10mm，更优选为2.5mm至6mm，更优选为3至5mm，更优选为3.5至4.5mm；在更优选的实施方案中，所述管状容器的长度为所述管状容器的长度为2cm至40cm，优选5cm至30cm，更优选10cm至29cm，更优选为14cm至28cm，最优选为14cm或28cm。在一个更优选的实施方案中，所述管状容器在密封后的长度不变。In a more preferred embodiment, the sterile container is a tubular container; in a more preferred embodiment, the tubular container is open at one end; in a more preferred embodiment, the tubular container has a side opening; In a more preferred solution, the end that needs to be inserted into the liquid surface of the initial cold source is made of a denser material or is placed with a denser object, so that the tubular container can be inserted below the liquid level of the initial cold source without floating easily; In a more preferred embodiment, the main body of the tubular container is cylindrical; in a more preferred embodiment, the inner diameter of the tubular container of the tubular container is 1mm to 30mm, preferably 1.5mm to 20mm, more Preferably 2mm to 10mm, more preferably 2.5mm to 6mm, more preferably 3 to 5mm, more preferably 3.5 to 4.5mm; in a more preferred embodiment, the length of the tubular container is 2 cm to 40 cm, preferably 5 cm to 30 cm, more preferably 10 cm to 29 cm, more preferably 14 cm to 28 cm, most preferably 14 cm or 28 cm. In a more preferred embodiment, the length of the tubular container remains unchanged after sealing.
在本发明优选实施方案中，所述无菌容器外部的空气为经过其他净化设备净化过的空气，本发明对净化方式不进行限定。在另一个优选的实施方案中，所述无菌容器在开口处装有空气无菌滤器。在更优选的实施方案中，所述空气无菌滤器包含0.22μm或0.45μm孔径的滤膜。In a preferred embodiment of the present invention, the air outside the aseptic container is air purified by other purification equipment, and the present invention does not limit the purification method. In another preferred embodiment, the sterile container is equipped with an air sterile filter at the opening. In a more preferred embodiment, the air sterile filter comprises a filter membrane with a pore size of 0.22 μm or 0.45 μm.
在其中的一个实施方案中，所述管状容器可以被密封。本发明并不对密封方式进行限定。密封方式可以包括但不限于螺旋封口、热封口、超声封口等，只要在冷冻载杆***外套管后，能够实现开口处的封闭即可。在一优选方案中，所述外套管开口可用超声封口机封口。在另一个优选的实施方案中，所述管状容器的开口位于其中一端，当承载有样品的冷冻载体置于所述内腔中，所述开口被锁合，以使所述外套管密闭；在更优选的实施方案中，在所述开口的内侧壁设置有一内螺纹或者在所述开口的外侧壁设置有一外螺纹，以螺帽与所述内螺纹或者所述外螺纹配合，以密封所述内腔；在更优选的实施方案中，所述开口的侧壁具 有至少一朝向所述密封部中心延伸的切口，所述开口的侧壁以所述切口为分界线贴合，以使所述开口被锁合；在更优选的实施方案中，所述开口的侧壁上具有两个朝向所述密封部中心延伸的切口，两个所述切口对称设置，所述开口的侧壁以所述切口为分界线贴合，以使所述开口被锁合。In one of these embodiments, the tubular container may be sealed. The present invention does not limit the sealing method. The sealing method may include, but is not limited to, spiral sealing, heat sealing, ultrasonic sealing, etc., as long as the opening can be closed after the frozen carrier rod is inserted into the outer sleeve. In a preferred solution, the opening of the outer sleeve can be sealed with an ultrasonic sealer. In another preferred embodiment, the opening of the tubular container is located at one end, and when the freezing carrier carrying the sample is placed in the inner cavity, the opening is locked to seal the outer sleeve; In a more preferred embodiment, an internal thread is provided on the inner side wall of the opening or an external thread is provided on the outer side wall of the opening, and a nut is matched with the internal thread or the external thread to seal the In a more preferred embodiment, the side wall of the opening has at least one incision extending toward the center of the sealing portion, and the side wall of the opening is attached with the incision as the dividing line so that the The opening is locked; in a more preferred embodiment, the side wall of the opening has two slits extending toward the center of the sealing portion, and the two slits are arranged symmetrically, and the side wall of the opening is The incision is attached to the dividing line so that the opening is locked.
在另一个优选的实施方案中，所述管状容器的开口位于靠近其中一端的侧面；在更优选的实施方案中，在所述管状容器一端的端面或侧面设置有标贴，用于标记所述冷冻载体承载的样品的信息。In another preferred embodiment, the opening of the tubular container is located on the side close to one end; in a more preferred embodiment, a label is provided on the end or side of one end of the tubular container for marking the Information about the sample carried by the freezing carrier.
在优选的实施方案中，所述承载待冷冻样品的载体是冷冻载杆；在更优选的实施方案中，所述冷冻载杆包括样品承载端、手持端和位于所述样品承载端和手持端之间的连接部分。In a preferred embodiment, the carrier that carries the sample to be frozen is a freezing rod; in a more preferred embodiment, the freezing rod includes a sample carrying end, a hand-held end, and the sample carrying end and the hand-held end. The connecting part between.
本发明的优点在于：The advantages of the present invention are:
1)在无菌容器中制得的无菌制冷剂的温度可以达到-195℃，能够满足液氮工作场景所需要的超低温(液氮温度-196℃)；1) The temperature of the sterile refrigerant prepared in the sterile container can reach -195°C, which can meet the ultra-low temperature (liquid nitrogen temperature -196°C) required by the liquid nitrogen work scene;
2)既可以实现“封闭式载体”无菌、无交叉污染风险的长期超低温保存，在玻璃化冷冻时又可以达到“开放式载体”的超快速降温(降温速率≥20,000℃/min)，复苏存活率和“开放式载体”相同(存活率≥95％)；以人第三天胚胎为例，复苏存活率大于99％。2) It can not only realize the "closed carrier" aseptic and long-term ultra-low temperature storage without the risk of cross-contamination, and achieve the ultra-fast cooling of the "open carrier" during vitrification (cooling rate ≥20,000°C/min) and recovery The survival rate is the same as the "open carrier" (survival rate ≥95%); taking human embryos on the third day as an example, the recovery survival rate is greater than 99%.
3)玻璃化冷冻完成后，可以将完成冷冻的样品直接密封于外套管内，解冻时从液氮罐内取出外套管，外套管一次性使用，使用后即可丢弃，这样带来的技术效果包括：第一，公知的胚胎冷冻外套管一般放置3根以上冷冻载杆，胚胎解冻(一次解冻一根)后需将外套管重新放回液氮罐，本发明为一次性使用套装，避免了样品取放过程中的再次污染等问题；第二，本发明一次性使用的外套管胚胎解冻后即可丢弃，释放了液氮罐空间，增加了液氮罐的空间使用率2倍以上；第三，本发明套装解冻后丢弃，彻底避免了公知外套管解冻后放回液氮罐内位置放错的情况发生。3) After the vitrification is completed, the frozen sample can be directly sealed in the outer tube, and the outer tube is taken out of the liquid nitrogen tank when thawing. The outer tube is used once and can be discarded after use. The technical effects brought by this include : First, the well-known embryo freezing outer tube is generally placed with more than 3 freezing rods. After the embryo is thawed (one at a time), the outer tube needs to be put back into the liquid nitrogen tank. The present invention is a disposable set, which avoids samples. Problems such as re-contamination during the pick-and-place process; second, the disposable outer tube embryos of the present invention can be discarded after thawing, which frees up the space of the liquid nitrogen tank and increases the space utilization rate of the liquid nitrogen tank by more than 2 times; third; , The suit of the present invention is thawed and discarded, which completely avoids the situation that the known outer sleeve is put back into the liquid nitrogen tank after being thawed.
4)本发明制备液态空气用于细胞玻璃化冷冻，可以做到即制即用，不受场地、时间和其他制备条件的限制。4) The liquid air prepared by the present invention is used for cell vitrification, which can be prepared and used immediately, and is not restricted by site, time and other preparation conditions.
在本发明的实施方案中，创造性地将无菌制冷剂的制备、玻璃化冷冻以及超低温储存在同一个容器中进行，并取得了预料不到的技术效果。为实施这一方案，本发明设计了专用于本发明的套件，其包含具有一开口的无菌容器；以及可置于所述无菌容器中的冷冻载体，用 于承载需要冷冻的样品；冷冻载体装载胚胎后置于无菌容器内，利用无菌容器内已经制得的无菌液态空气(-195℃)实现玻璃化冻结；再将无菌容器(即外套管)封闭，整体置于液氮罐内长期保存。在本发明中，无菌容器的尺寸是关键的因素之一。如果容器太大，大体积无菌容器制备制冷剂需要的时间久，且更难以保证进入容器的空气是无菌的，从而难以保证制得的无菌制冷剂符合无菌要求。另一方面，如果容器太小不能容纳冷冻载杆也无价值，且太小的无菌容器制备的无菌制冷剂的量达不到要求，也无法实现快速玻璃化冷冻，从而无法实现本发明目的。经过发明人的反复实验验证，得到了本发明实施方案中的尺寸。In the embodiment of the present invention, the preparation of aseptic refrigerant, vitrification and ultra-low temperature storage are creatively carried out in the same container, and unexpected technical effects have been achieved. In order to implement this solution, the present invention designs a kit dedicated to the present invention, which includes a sterile container with an opening; and a freezing carrier that can be placed in the sterile container for carrying samples that need to be frozen; After the carrier is loaded with embryos, it is placed in a sterile container, and the sterile liquid air (-195°C) that has been prepared in the sterile container is used to achieve vitrification; then the sterile container (ie outer tube) is sealed and the whole is placed in the liquid Long-term storage in nitrogen tank. In the present invention, the size of the sterile container is one of the key factors. If the container is too large, it will take a long time for the large-volume aseptic container to prepare the refrigerant, and it will be more difficult to ensure that the air entering the container is sterile, so that it is difficult to ensure that the prepared aseptic refrigerant meets the sterility requirements. On the other hand, if the container is too small to contain the freezing rod, it is of no value, and the amount of aseptic refrigerant prepared by the aseptic container is too small to meet the requirements, and rapid vitrification cannot be achieved, thus the present invention cannot be realized. purpose. After repeated experiments and verification by the inventor, the size in the embodiment of the present invention was obtained.
本发明超低温冷冻及封闭式保存方法提供一种简单、方便的采用无菌制冷剂冷冻及保存样品的方法。其可应用在辅助生殖领域内生物样本(胚胎、卵子、***等)的长期超低温无菌保存。另外，其应用并不局限于辅助生殖领域，食品、化工、医学、生命科学等需要用到超低温保存(如液氮保存)或超低温治疗(如冷冻消融)的领域均可使用该方法自制无菌或洁净液态空气。The ultra-low temperature freezing and closed preservation method of the present invention provides a simple and convenient method for freezing and preserving samples using sterile refrigerants. It can be used for long-term ultra-low temperature aseptic preservation of biological samples (embryos, eggs, sperm, etc.) in the field of assisted reproduction. In addition, its application is not limited to the field of assisted reproduction. Food, chemical industry, medicine, life sciences and other fields that require ultra-low temperature storage (such as liquid nitrogen storage) or ultra-low temperature treatment (such as cryoablation) can be used to make sterile products. Or clean liquid air.
本发明还提供以本发明的方法制备的无菌液态空气作为冷源实现胚胎长期无菌保存的实施方案，包括如下步骤：将一外套管作为无菌容器，其中，外套管可以是金属材质，如医用不锈钢，开口处设置精密螺旋，装载有胚胎的冷冻载杆投入外套管完成玻璃化冻结后，用对应的精密螺帽拧紧，实现螺旋式封口(内旋或外旋均可)，转移至常规液氮罐内长期保存，即可实现生物样本安全、无菌、无交叉污染的长期保存。所述外套管也可为塑料材质，此时除了螺旋式封口实现封闭外，还可以用超声封口机对开口处封口实现封闭。若封闭前，外套管开口处产生的水汽、雾气等不利于超声封口、热封口或螺旋封口，此时可采用热源靠近或无菌纱布擦拭开口处等方式去除水汽、雾气后再封口。其中，外套管需要封闭后保存在液氮罐内才能确保胚胎与液氮长期彻底隔绝，实现胚胎的无菌冷冻保存。此时尽管液氮罐内的液氮非洁净(市售液氮含有细菌、病毒、真菌等致病微生物)，但是胚胎是在无菌液态空气内玻璃化冻结并封闭后保存在液氮罐内，因此，外部非洁净的液氮并不会影响冻存的胚胎一直处于无菌状态。The present invention also provides an embodiment in which the sterile liquid air prepared by the method of the present invention is used as a cold source to achieve long-term aseptic preservation of embryos, including the following steps: an outer tube is used as a sterile container, wherein the outer tube may be made of metal, Such as medical stainless steel, a precision screw is set at the opening, and the freezing rod loaded with embryos is put into the outer tube to complete vitrification and then tightened with the corresponding precision nut to achieve a spiral seal (both internal or external rotation), and transfer to Long-term storage in conventional liquid nitrogen tanks can realize safe, sterile, and long-term storage of biological samples without cross contamination. The outer sleeve may also be made of plastic material. In this case, in addition to the spiral seal to achieve sealing, an ultrasonic sealing machine can also be used to seal the opening to achieve sealing. If the water vapor and mist generated at the opening of the outer sleeve are not conducive to ultrasonic sealing, heat sealing or spiral sealing before sealing, the water vapor and mist can be removed by means of close to the heat source or wipe the opening with sterile gauze before sealing. Among them, the outer tube needs to be sealed and stored in a liquid nitrogen tank to ensure that the embryos are completely isolated from the liquid nitrogen for a long time and realize the aseptic cryopreservation of the embryos. At this time, although the liquid nitrogen in the liquid nitrogen tank is not clean (commercial liquid nitrogen contains bacteria, viruses, fungi and other pathogenic microorganisms), the embryos are vitrified and frozen in sterile liquid air and stored in the liquid nitrogen tank. Therefore, the external non-clean liquid nitrogen will not affect the sterile state of the frozen embryos.
更特别的，通过本发明的方法，彻底解决了目前配子和胚胎等生殖样本无菌保存的难题。More specifically, the method of the present invention completely solves the current problem of aseptic preservation of reproductive samples such as gametes and embryos.
图1是本发明超低温冷冻及封闭式保存方法的装置的立体示意图；Figure 1 is a three-dimensional schematic diagram of the device of the ultra-low temperature freezing and enclosed storage method of the present invention;
图2是本发明超低温冷冻及封闭式保存方法的装置的截面示意图；2 is a schematic cross-sectional view of the device of the ultra-low temperature freezing and closed storage method of the present invention;
图3是本发明无菌制冷剂污染测试结果。其中图3A是对照组血平板菌落生长情况；图3B是本发明制得的无菌制冷剂的血平板菌落生长情况。Figure 3 shows the results of the contamination test of the sterile refrigerant of the present invention. Figure 3A shows the growth of the colony on the blood plate of the control group; Figure 3B shows the growth of the colony on the blood plate of the sterile refrigerant prepared by the present invention.
下面结合附图1及2对本发明提供的一种超低温冷冻及封闭式保存方法的具体实施方式做详细说明。The specific implementation of the ultra-low temperature freezing and closed storage method provided by the present invention will be described in detail below in conjunction with accompanying drawings 1 and 2.
实施例1：制备无菌制冷剂Example 1: Preparation of sterile refrigerant
步骤1、提供一容器B。所述容器B为一敞口结构，其中所述容器B的敞口即为第二开口11。所述容器B的尺寸也可根据需求进行选择，本发明对此不进行限定。在本实施例中，所述容器B为长260mm*宽150mm*深115mm无盖泡沫箱。所述容器B内盛放有一初始冷源12。在本实施例中，所述容器B内盛放的所述初始冷源12为市售的液氮。所述初始冷源12具有一液态层12A及一位于所述液态层12A上方的蒸汽层12B，其中，所述蒸汽层12B为液态层12A的液体挥发形成。为了清楚显示两者的区别，在附图中采用不同的阴影线绘示所述液态层12A及所述蒸汽层12B。具体地说，所述容器B的深度是115mm，所述容器B内初始冷源12的液态层12A的深度为90mm，蒸汽层12B的厚度为25mm，即蒸汽层12B的厚度等于容器B的深度与液态层12A的深度之差。Step 1. Provide a container B. The container B has an open structure, and the opening of the container B is the second opening 11. The size of the container B can also be selected according to requirements, and the present invention does not limit this. In this embodiment, the container B is an uncovered foam box with a length of 260 mm * a width of 150 mm * a depth of 115 mm. An initial cold source 12 is placed in the container B. In this embodiment, the initial cold source 12 contained in the container B is commercially available liquid nitrogen. The initial cooling source 12 has a liquid layer 12A and a vapor layer 12B located above the liquid layer 12A, wherein the vapor layer 12B is formed by the volatilization of the liquid of the liquid layer 12A. In order to clearly show the difference between the two, the liquid layer 12A and the vapor layer 12B are drawn with different hatching in the drawings. Specifically, the depth of the container B is 115mm, the depth of the liquid layer 12A of the initial cold source 12 in the container B is 90mm, and the thickness of the vapor layer 12B is 25mm, that is, the thickness of the vapor layer 12B is equal to the depth of the container B The difference with the depth of the liquid layer 12A.
步骤2、提供一无菌容器A，其具有一第一开口10。所述无菌容器A为塑料管，其内底部具有金属配重块；所述容器开口位于顶端。所述管状容器的直径(内径)分别为2.5mm、3.5mm、9.5mm和28mm(见下表1)。塑料管开口处与Millipore 0.22um滤器连接。Step 2. Provide a sterile container A with a first opening 10. The aseptic container A is a plastic tube with a metal weight at the inner bottom; the container opening is located at the top. The diameter (inner diameter) of the tubular container is 2.5mm, 3.5mm, 9.5mm and 28mm respectively (see Table 1 below). The opening of the plastic tube is connected to a Millipore 0.22um filter.
步骤3、将所述无菌塑料管(外套管)插在试管架后置于盛有液氮的泡沫盒(容器B)内，利用液氮冷却制得无菌液态空气。在进行的对比实验中，部分塑料管开口10的高度高于所述初始冷源12的蒸汽层12B，即所述开口10突出于所述蒸汽层12B的界面，其并未被所述蒸汽层12B覆盖(即无菌容器A开口处高于容器B的上沿)；而剩余部分塑料管开口10的高度低于所述初始冷源12的蒸汽层12B，但高于所述初始冷源12的液态层12A(即无菌容器A开口处位于液面层12A与容器B上沿之间的蒸汽层12B内)。在该步骤中，所述初始冷源12接触所述塑料管的外侧壁，使得所述塑料管内壁的温度与所述初始冷源12的温度无限接近，所述塑料管内的空气(主要是氮气和氧气)在该温度下会冷凝液化，则随着空气的液化，所述塑料管内会形成负压，外部空气或12B蒸汽层内的氮气会不断进入所述塑料管 内，并液化，以形成无菌制冷剂13。其中，所述空气为经过净化的空气。Step 3. Insert the sterile plastic tube (outer tube) into the test tube rack and place it in a foam box (container B) containing liquid nitrogen, and use liquid nitrogen to cool the sterile liquid air. In the comparative experiment, the height of the opening 10 of part of the plastic tube is higher than the vapor layer 12B of the initial cold source 12, that is, the opening 10 protrudes from the interface of the vapor layer 12B, which is not affected by the vapor layer 12B. 12B cover (that is, the opening of the aseptic container A is higher than the upper edge of the container B); and the height of the remaining part of the plastic tube opening 10 is lower than the vapor layer 12B of the initial cold source 12, but higher than the initial cold source 12 The liquid layer 12A (that is, the opening of the sterile container A is located in the vapor layer 12B between the liquid surface layer 12A and the upper edge of the container B). In this step, the initial cold source 12 contacts the outer side wall of the plastic tube, so that the temperature of the inner wall of the plastic tube is infinitely close to the temperature of the initial cold source 12, and the air (mainly nitrogen gas) in the plastic tube And oxygen) will condense and liquefy at this temperature. As the air liquefies, negative pressure will be formed in the plastic pipe, and the external air or nitrogen in the 12B vapor layer will continue to enter the plastic pipe and liquefy to form Bacteria refrigerant 13. Wherein, the air is purified air.
下表1为使用不同规格塑料管和开口处于不同位置时制得无菌制冷剂的情况。Table 1 below shows the use of different specifications of plastic tubes and openings in different positions to prepare sterile refrigerants.
表1：制备无菌制冷剂Table 1: Preparation of sterile refrigerant
从上表可看出，当塑料管开口在所述蒸汽层内时，制备制冷剂的效率较低，而所述开口高度高于所述蒸汽层时，能够大大提高无菌制冷剂的制备效率。这一效果是预料不到的。通 常认为，液氮蒸汽层中仍然含有大量的氮气，如果塑料管的开口位于蒸汽层内，蒸汽层中的氮气能够进入塑料管中，从而更快速地在塑料管内形成制冷剂。然而，上述对比实验结果与此恰好相反。这有可能是因为蒸汽层中的液氮蒸汽阻挡了蒸汽层外部的空气，使得空气不能顺利进入塑料管中，降低了无菌制冷剂的制备效率。It can be seen from the above table that when the plastic tube is opened in the vapor layer, the efficiency of preparing refrigerant is low, and when the height of the opening is higher than the vapor layer, the efficiency of preparing the sterile refrigerant can be greatly improved . This effect is unexpected. It is generally believed that the liquid nitrogen vapor layer still contains a large amount of nitrogen. If the opening of the plastic tube is located in the vapor layer, the nitrogen in the vapor layer can enter the plastic tube, thereby forming a refrigerant in the plastic tube more quickly. However, the above-mentioned comparative experiment results are exactly the opposite. This may be because the liquid nitrogen vapor in the vapor layer blocks the air outside the vapor layer, so that the air cannot enter the plastic pipe smoothly, which reduces the efficiency of aseptic refrigerant preparation.
此外，从上表中还可看出，制冷剂的制备效率与塑料管的直径密切相关。直径较小的塑料管能更快地制得深度满足玻璃化冷冻样品所需的制冷剂。更为重要的是，在塑料管放置在液氮中一定时间后，塑料管内的制得的制冷剂的量即达到平衡状态，不再增加。实际应用中，胚胎冷冻放在玻璃化冷冻平衡液内的时间一般为10分钟左右，而3.5mm内径的外套管10分钟即可产生40mm深度的液态空气，足以满足玻璃化冻结之需求。In addition, it can be seen from the above table that the efficiency of refrigerant preparation is closely related to the diameter of the plastic pipe. A plastic tube with a smaller diameter can quickly produce a refrigerant with a depth that meets the requirements for vitrified frozen samples. More importantly, after the plastic tube is placed in liquid nitrogen for a certain period of time, the amount of the produced refrigerant in the plastic tube reaches an equilibrium state and does not increase. In practical applications, the embryo freezing time in the vitrification freezing balance solution is generally about 10 minutes, and the outer tube of 3.5mm inner diameter can produce 40mm deep liquid air in 10 minutes, which is sufficient to meet the requirements of vitrification.
实施例2：无菌制冷剂成分及温度测定Example 2: Aseptic refrigerant composition and temperature measurement
将实施例1中塑料管中制得的液态空气置于室温下，将气化后的空气导入已抽真空的铝箔袋中，气相色谱仪进样测制得液态空气中氮气的占比。气相色谱仪采用Agilent 6890，配TCD检测器，碳分子筛填充柱，2m×2mmID，载气为氮气，载气流速：20mL/min，柱温40℃，运行时间5min。进样方式：气体阀进样，定量管体积1mL，充样时间0.5min，进样时间0.5min。The liquid air prepared in the plastic tube in Example 1 was placed at room temperature, the vaporized air was introduced into an evacuated aluminum foil bag, and the gas chromatograph was used to measure the proportion of nitrogen in the liquid air. The gas chromatograph adopts Agilent 6890, equipped with TCD detector, carbon molecular sieve packed column, 2m×2mmID, carrier gas is nitrogen, carrier gas flow rate: 20mL/min, column temperature 40℃, running time 5min. Sampling mode: gas valve injection, loop volume 1mL, sample filling time 0.5min, sample injection time 0.5min.
测定的结果是，制得的液态空气中，氮气含量约85.5％。超低温温度计测得液态空气的温度约-195℃。使用液态空气空作为制冷剂，就可以随时随地以液氮作为冷源，制备无菌制冷剂。The result of the measurement is that the nitrogen content in the prepared liquid air is about 85.5%. The temperature of the liquid air measured by the ultra-low temperature thermometer is about -195°C. Using liquid air as a refrigerant, liquid nitrogen can be used as a cold source to prepare sterile refrigerants anytime and anywhere.
实施例3：无菌制冷剂污染测试Example 3: Aseptic refrigerant contamination test
按实施例1所述制备无菌液态空气，用无菌棉签反复多次蘸取液态空气后，将棉签蘸取的标本(实验组)密涂于哥伦比亚血平板上，置35℃，5％CO
2培养箱中培养48小时，观察细菌生长情况。取用于制备该无菌液态空气的初始冷源液氮10毫升做对照(对照组)。结果显示，对照组血平板上有黄色和灰白色两种菌落生长(图3A)，而实验组无菌落生长(附图3B)。进一步地，对实验组的两个菌落进行分离纯化，革兰染色和分子鉴定，结果显示黄色菌落细菌镜下为革兰阳性球菌，鉴定为Neomicrococcus aestuarii strain(七叶树新微球菌菌)；灰白色菌落细菌镜下为革兰阴性杆菌，鉴定为Moraxella osloensis(奥斯陆莫拉菌)。
Prepare aseptic liquid air as described in Example 1. After repeatedly dipping the liquid air with a sterile cotton swab, the specimen (experimental group) dipped in the cotton swab is densely smeared on a Colombian blood plate and placed at 35°C, 5% CO 2 Cultivate in an incubator for 48 hours and observe the growth of bacteria. Take 10 ml of initial cold source liquid nitrogen used to prepare the sterile liquid air as a control (control group). The results showed that there were yellow and off-white colonies growing on the blood plate in the control group (Figure 3A), while the experimental group was growing aseptic colonies (Figure 3B). Furthermore, the two colonies in the experimental group were separated and purified, gram stained and molecularly identified. The results showed that the yellow colonies were gram-positive cocci under the microscope, which was identified as Neomicrococcus aestuarii strain (Aesculus aestuarii strain); gray-white colonies of bacteria Under the microscope, it was a gram-negative bacillus, which was identified as Moraxella osloensis (Moraxella osloensis).
实施例4：冷冻样品Example 4: Frozen samples
实施例4.1一般步骤Example 4.1 General Procedure
步骤1、根据实施例1，使用液氮作为初始冷源，用内径为3.5mm直径的塑料管制备无菌制冷剂。其中所述塑料管开口高于所述液氮的蒸汽层。Step 1. According to Example 1, liquid nitrogen is used as the initial cold source, and a plastic tube with an inner diameter of 3.5 mm is used to prepare a sterile refrigerant. The opening of the plastic pipe is higher than the vapor layer of the liquid nitrogen.
步骤2、将一承载有冷冻样品的载体15投入所述无菌制冷剂13中进行玻璃化冷冻。在附图中采用虚线绘示所述承载有样品的载体15。在本实施例中，所述承载有样品的载体15是冷冻载杆。Step 2. Put a carrier 15 carrying a frozen sample into the sterile refrigerant 13 for vitrification. In the drawings, dotted lines are used to depict the carrier 15 carrying the sample. In this embodiment, the carrier 15 carrying the sample is a frozen carrier rod.
步骤3、去除所述无菌容器开口10处的湿气或雾气的步骤。具体地说，制备无菌冷源过程中，在所述开口10处可能会有水汽等沉积，在密封所述开口10之前，需采用热源靠近开口10或无菌纱布擦拭第一开口10处等方式去除水汽，以便于密封所述第一开口10。Step 3. A step of removing moisture or mist at the opening 10 of the sterile container. Specifically, during the process of preparing a sterile cold source, water vapor may be deposited at the opening 10. Before the opening 10 is sealed, a heat source should be used close to the opening 10 or a sterile gauze should be used to wipe the first opening 10, etc. The water vapor is removed so as to seal the first opening 10 easily.
步骤4、密封所述无菌容器的开口10，并将密封的所述无菌容器A置于一含有制冷剂的存储罐中存储，以使得样品能够进行长期存储，待需要使用时再取出，解冻后使用。Step 4. Seal the opening 10 of the aseptic container, and place the sealed aseptic container A in a storage tank containing a refrigerant for storage, so that the sample can be stored for a long time and be taken out when needed. Use after thawing.
实施例4.2冷冻胚胎Example 4.2 Freezing embryos
体外受精治疗中捐赠的(签署知情同意书)异常受精的day3胚胎经ES液(玻璃化冷冻平衡液)和VS液(玻璃化液)处理后，装载于冷冻载杆Strawtop。去除塑料管开口处连接的滤膜过滤器，将装载有胚胎的Strawtop直接投入制得的无菌液态空气中实现玻璃化冻结。采用三组研究，并检测冷冻后胚胎的解冻效果。The abnormally fertilized day3 embryos donated in the IVF treatment (signed informed consent) are treated with ES solution (vitrification cryobalance solution) and VS solution (vitrification solution), and then loaded on the frozen carrier Strawtop. Remove the membrane filter connected at the opening of the plastic tube, and directly put the Strawtop loaded with embryos into the prepared sterile liquid air to achieve vitrification. Three groups of studies were used to test the thawing effect of frozen embryos.
方法1：胚胎投入液态空气实现玻璃化冷冻后，即时解冻，观察复苏情况，即1次冷冻/解冻；Method 1: After the embryos are put into liquid air to achieve vitrification, thaw them immediately and observe the recovery, that is, one freeze/thaw;
方法2：胚胎投入液态空气实现玻璃化冷冻后，即时解冻，存活的胚胎连续冷冻解冻3次观察复苏情况，即3次冷冻/解冻；Method 2: After the embryos are put into liquid air to achieve vitrification, they are immediately thawed, and the surviving embryos are frozen and thawed continuously for 3 times to observe the recovery, that is, 3 times of freezing/thawing;
方法3：胚胎投入液态空气实现玻璃化冷冻后，用超声封口机将塑料外套管封闭，液氮罐内保存1周后解冻，观察复苏情况。Method 3: After the embryos are put into liquid air to achieve vitrification, the plastic outer tube is sealed with an ultrasonic sealer, and stored in a liquid nitrogen tank for 1 week and then thawed to observe the recovery.
每组研究均设市售液氮为自制制冷剂(液态空气)的对照组。结果请参见表2，结果显示，以自制无菌液态空气和市售液氮作为制冷剂，三种方法玻璃化冷冻胚胎的存活率均为100％，方法1的卵裂球溶解率分别为1.3％和1.2％；方法2的卵裂球溶解率分别为0.8％和1.8％；方法3的卵裂球溶解率分别为0％和1.0％，不同制冷剂的胚胎存活率和卵裂球溶解率均无统计学差异。所以，采用本发明方法制备的无菌液态空气可以获得和液氮相同的胚胎存活率和卵裂球完整率。Each group of studies set up a control group with commercially available liquid nitrogen as a self-made refrigerant (liquid air). The results are shown in Table 2. The results show that using homemade sterile liquid air and commercially available liquid nitrogen as refrigerants, the survival rate of vitrified embryos in the three methods is 100%, and the blastomere dissolution rate of method 1 is 1.3 respectively. % And 1.2%; the blastomere dissolution rate of method 2 is 0.8% and 1.8%, respectively; the blastomere dissolution rate of method 3 is 0% and 1.0%, respectively, the embryo survival rate and blastomere dissolution rate of different refrigerants There was no statistical difference. Therefore, the sterile liquid air prepared by the method of the present invention can obtain the same embryo survival rate and blastomere integrity rate as liquid nitrogen.
表2 液态空气玻璃化冷冻胚胎的存活率和卵裂球溶解率Table 2 Survival rate and blastomere dissolution rate of liquid air vitrified embryos
实施例4.3保存稀少和单个***Example 4.3 Preservation of rare and single sperm
用超薄片(其结构请参见中国专利授权公告号CN 205143336 U)装载稀少/单个***，液氮蒸汽熏蒸法冷冻，待冷冻液微滴冻结后，去除塑料管开口处连接的滤膜过滤器，将超薄片投入塑料管，超声封口机封闭塑料管，转移至液氮罐长期保存。此时尽管液氮罐内的液氮非洁净(市售液氮含有细菌、病毒、真菌等致病微生物)，但是***是在无菌液态空气内封闭保存，所以可维持超低温无菌状态。Use ultra-thin sheets (please refer to Chinese Patent Authorization Announcement No. CN 205143336 U) to load rare/single sperm and freeze them by liquid nitrogen vapor fumigation. After the frozen liquid droplets freeze, remove the membrane filter connected to the opening of the plastic tube , Put the super flakes into the plastic tube, the ultrasonic sealing machine seals the plastic tube, and transfer it to the liquid nitrogen tank for long-term storage. At this time, although the liquid nitrogen in the liquid nitrogen tank is not clean (commercial liquid nitrogen contains bacteria, viruses, fungi and other pathogenic microorganisms), the sperm is sealed and stored in sterile liquid air, so the ultra-low temperature and sterile state can be maintained.
实施例5 无菌容器实施例Example 5 Aseptic container example
实施例5.1 14cm长的无菌容器Example 5.1 Aseptic container with a length of 14 cm
所述管状容器总长度14cm，开口处位于离下端(需要***液面一端)9cm处，开口长度2.5cm；在开口处下方留有用于封口机封闭的1cm长度，开口上方长度2.5cm标记用。在最 下端具有1cm长度的金属重物。The total length of the tubular container is 14cm, the opening is located 9cm from the lower end (the end that needs to be inserted into the liquid surface), and the length of the opening is 2.5cm; below the opening, there is a length of 1cm for sealing by the sealing machine, and the length above the opening is 2.5cm for marking. There is a metal weight with a length of 1 cm at the bottom end.
开口上方2.5cm的标记部分可为中空或实心结构。为了增加整个管状容器的重量，本实施例采用实心结构，起类似于管底重物的作用，保存在液氮罐内时不容易浮起来。The marking part 2.5 cm above the opening can be hollow or solid. In order to increase the weight of the entire tubular container, this embodiment adopts a solid structure, which acts like a weight at the bottom of the tube, and is not easy to float when stored in a liquid nitrogen tank.
实施例5.2 28cm长的无菌容器Example 5.2 28cm long aseptic container
所述外套管总长度为28cm，离下端(需要***液面一端)9cm处，开口长度5cm；在开口处下方留有用于封口机封闭的1cm长度，开口上方长度14cm标记用。在最下端具有1cm长度的金属重物。The total length of the outer sleeve is 28cm, 9cm from the lower end (the end that needs to be inserted into the liquid surface), and the opening length is 5cm; below the opening, a length of 1cm for sealing by the sealing machine is reserved, and the length above the opening is 14cm for marking. There is a metal weight with a length of 1 cm at the bottom end.
开口上方14cm的标记部分可为中空或实心结构。为了增加整个管状容器的重量，本实施例采用实心结构，起类似于管底重物的作用，保存在液氮罐内时不容易浮起来。The marking part 14cm above the opening can be hollow or solid. In order to increase the weight of the entire tubular container, this embodiment adopts a solid structure, which acts like a weight at the bottom of the tube, and is not easy to float when stored in a liquid nitrogen tank.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as This is the protection scope of the present invention.
Claims (10)
- 一种超低温冷冻及封闭式保存方法，其包括如下步骤：An ultra-low temperature freezing and closed storage method, which includes the following steps:1)提供一具有开口的无菌容器；1) Provide a sterile container with an opening;2)将所述无菌容器置于初始冷源中，且所述无菌容器开口的高度高于所述初始冷源的液面层；2) Place the aseptic container in the initial cold source, and the height of the opening of the aseptic container is higher than the liquid level of the initial cold source;3)在所述无菌容器中形成液化气体，作为无菌制冷剂；3) Forming liquefied gas in the aseptic container as a sterile refrigerant;4)将承载在载体上的待冷冻样品置于所述无菌制冷剂中进行玻璃化冷冻；4) Place the sample to be frozen on the carrier in the sterile refrigerant for vitrification;5)密封所述无菌容器，并将所述密封无菌容器与密封在其中的玻璃化冷冻样品一起超低温保存。5) Seal the aseptic container, and store the sealed aseptic container together with the vitrified frozen sample sealed in it at an ultra-low temperature.
- 根据权利要求1的超低温冷冻及封闭式保存方法，其中所述无菌容器的开口高于所述初始冷源所形成的蒸汽层。The ultra-low temperature freezing and closed storage method according to claim 1, wherein the opening of the aseptic container is higher than the vapor layer formed by the initial cold source.
- 根据权利要求1或2的超低温冷冻及封闭式保存方法，其中所述无菌容器是管状容器内径为1mm至30mm，优选1.5mm至20mm，更优选2mm至10mm，更优选为2.5mm至6mm，更优选为3-5mm，更优选为3.5至4.5mm；其长度为2cm至40cm，优选5cm至30cm，更优选10cm至29cm，更优选为14cm至28cm，最优选为14cm或28cm。The ultra-low temperature freezing and closed storage method according to claim 1 or 2, wherein the aseptic container is a tubular container with an inner diameter of 1mm to 30mm, preferably 1.5mm to 20mm, more preferably 2mm to 10mm, more preferably 2.5mm to 6mm, It is more preferably 3-5 mm, more preferably 3.5 to 4.5 mm; its length is 2 cm to 40 cm, preferably 5 cm to 30 cm, more preferably 10 cm to 29 cm, more preferably 14 cm to 28 cm, most preferably 14 cm or 28 cm.
- 根据前述任一项权利要求的超低温冷冻及封闭式保存方法，其中所述无菌容器在开口处装有空气无菌滤器，外部空气通过滤器净化后进入该无菌容器中，并在容器外部初始冷源的作用下液化形成无菌冷源。The ultra-low temperature freezing and closed storage method according to any one of the preceding claims, wherein the sterile container is equipped with an air sterile filter at the opening, and the external air enters the sterile container after being purified by the filter, and initially Under the action of the cold source, it is liquefied to form a sterile cold source.
- 根据前述任一项权利要求的超低温冷冻及封闭式保存方法，其中所述冷冻样品是人类或动物的胚胎、合子、卵子、***、组织，所述初始冷源是液氮、液氦或者其他超低温冷源。The ultra-low temperature freezing and enclosed storage method according to any one of the preceding claims, wherein the frozen sample is a human or animal embryo, zygote, egg, sperm, tissue, and the initial cold source is liquid nitrogen, liquid helium or other ultra-low temperature Cold source.
- 根据前述任一项权利要求的超低温冷冻及封闭式保存方法，其中所述步骤5)中的超低温保存是指将所述密封无菌容器与密封在其中的玻璃化冷冻样品一起置于液氮、液氦或其他超低温冷源中保存。The ultra-low temperature freezing and closed storage method according to any one of the preceding claims, wherein the ultra-low temperature storage in step 5) refers to placing the sealed sterile container together with the vitrified frozen sample sealed in it in liquid nitrogen, Store in liquid helium or other ultra-low temperature cold sources.
- 用于根据前述任一项权利要求的超低温冷冻及封闭式保存方法的套件，其包含：具有一开口的无菌容器；以及可置于所述无菌容器中的冷冻载体，用于承载需要冷冻的样品。A kit for the ultra-low temperature freezing and closed storage method according to any one of the preceding claims, comprising: a sterile container with an opening; and a freezing carrier that can be placed in the sterile container for carrying the required freezing sample.
- 根据权利要求7的套件，其中所述无菌容器是管状容器，内径为1mm至30mm，优选1.5mm至20mm，更优选2mm至10mm，更优选为2.5mm至6mm，更优选为3-5mm，更优选为3.5至4.5mm；其长度为2cm至40cm，优选5cm至30cm，更优选10cm至29cm，更优 选为14cm至28cm，最优选为14cm或28cm。The kit according to claim 7, wherein the sterile container is a tubular container with an inner diameter of 1mm to 30mm, preferably 1.5mm to 20mm, more preferably 2mm to 10mm, more preferably 2.5mm to 6mm, more preferably 3-5mm, It is more preferably 3.5 to 4.5 mm; its length is 2 cm to 40 cm, preferably 5 cm to 30 cm, more preferably 10 cm to 29 cm, more preferably 14 cm to 28 cm, most preferably 14 cm or 28 cm.
- 根据权利要求7或8的套件，其中所述无菌容器在开口处装有空气无菌滤器，且所述无菌容器可以被密封。The kit according to claim 7 or 8, wherein the sterile container is equipped with an air sterile filter at the opening, and the sterile container can be sealed.
- 根据权利要求7-9任一项的套件，其中所述承载待冷冻样品的载体是冷冻载杆。The kit according to any one of claims 7-9, wherein the carrier carrying the sample to be frozen is a freezing carrier rod.
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| CN104719283A (en) * | 2015-03-24 | 2015-06-24 | 阳艳群 | Closed freezing carrier and use method thereof |
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| CN205143336U (en) * | 2015-05-19 | 2016-04-13 | 上海交通大学医学院附属第九人民医院 | Freezing carrier of preserving of trace sperm |
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| CN104719283A (en) * | 2015-03-24 | 2015-06-24 | 阳艳群 | Closed freezing carrier and use method thereof |
| CN205695269U (en) * | 2016-04-26 | 2016-11-23 | 同济大学苏州研究院 | A kind of Dual-closed type ovum or embryo vitrifying freeze save set |
| CN107372470A (en) * | 2017-09-11 | 2017-11-24 | 马博 | A kind of ovum embryo vitrifying freeze save set |
| CN208159973U (en) * | 2018-03-05 | 2018-11-30 | 河北医科大学第二医院 | A kind of novel closed-type glass freezing load bar |
| CN110521720A (en) * | 2019-01-18 | 2019-12-03 | 上海廪典实业有限公司 | A kind of superfreeze and closed store method |
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