CN104012521B - A kind of contactless sessile drop method refrigerating plant and freezing method - Google Patents
A kind of contactless sessile drop method refrigerating plant and freezing method Download PDFInfo
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- CN104012521B CN104012521B CN201410256167.4A CN201410256167A CN104012521B CN 104012521 B CN104012521 B CN 104012521B CN 201410256167 A CN201410256167 A CN 201410256167A CN 104012521 B CN104012521 B CN 104012521B
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Abstract
A kind of contactless sessile drop method refrigerating plant and freezing method, device comprises seal cover, seal cover is connected with drop receptacle threaded engagement, a manhole is had bottom drop receptacle, freezing film is adhered to bottom drop receptacle, cryogen vessel top has a manhole, cryogen vessel is mutually bonding with drop receptacle, freezing film is made to be sandwiched between the two, drop receptacle and cryogen vessel are connected and fixed further by screw bolt and nut, freezing method is first make the cell suspension being mixed with freezing liquid with drops, be added on freezing film, it is made to be that dot matrix is arranged on the surface of freezing film, afterwards, refrigerating plant is overturn, low-temperature refrigerant is at the uniform velocity poured in the cryogen vessel of refrigerating plant, it is made to gasify at freezing film surface boiling, the present invention can realize glass freezing rapidly, ensures higher biological tissue's survival rate, prevents biological pollution, operating process is simple and clear, is easy to operation and realizes.
Description
Technical field
The invention belongs to biological cryogenic freezing and preserve field, be specifically related to a kind of contactless sessile drop method refrigerating plant and freezing method.
Background technology
Cryogenic freezing, as a kind of effective means of preserving biological cell, cell mass and biological tissue, has and uses widely and demand in biological study, medical application etc.
Traditional slow speed cryopreservation technology, often because freezing rate is not high enough, when freezen protective, intracellular fluid degree of crystallinity is higher, and the ice crystal of formation can destroy cellular tissure structure, very easily causes artifact activity of thawing to reduce.Meanwhile, its freezing flow process is complicated, consuming time longer.
In addition; the glass freezing means that some freezing rates are higher; such as sessile drop method is freezing, OPS method is freezing; Cryotop method is freezing, by allowing the cold-producing mediums such as frozen matter and liquid nitrogen directly contact, thus accelerates freezing rate; ultrahigh speed is realized freezing under the cryoprotector of relative lower concentration; obstruction ice crystal is formed, thus realizes glass freezing, weakens the negative effect of ice crystal.But directly by the refrigerant contact such as cellular tissure and liquid nitrogen, also bring some problems, such as, may there is bacterium in (1) liquid nitrogen, cause the pollution of freezing biological tissue; (2) cell of other kinds may be there is in liquid nitrogen, biological pollution is caused to the tissue after freezing; (3) freezing end artifact tissue is scattered and is not easy to collect.Therefore, these freezing methods still need certain improvement.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, the object of the present invention is to provide a kind of contactless sessile drop method refrigerating plant and freezing method, glass freezing can be realized rapidly, ensure higher biological tissue's survival rate, avoid the contact of freezing biological tissue and liquid nitrogen simultaneously, prevent biological pollution, also can collect easily freezing after biological tissue; And the refrigerating plant structure of designed employing is simple, operating process is simple and clear, is easy to operation and realizes.
To achieve these goals, present invention employs following technical scheme:
A kind of contactless sessile drop method refrigerating plant, comprise seal cover 1, seal cover 1 is connected with drop receptacle 2 threaded engagement, a manhole is had bottom drop receptacle 2, low temperature resistant glue is used to be adhered to bottom drop receptacle 2 by freezing film 5, cryogen vessel 3 top has a manhole, use low temperature resistant glue that cryogen vessel 3 top is mutually bonding with bottom drop receptacle 2, freezing film 5 is made to be sandwiched between the two, and with low temperature resistant glue by the gap sealing between three, drop receptacle 2 and cryogen vessel 3 are connected and fixed further by bolt 6 and nut 4.
Described seal cover 1, drop receptacle 2, cryogen vessel 3 are all made with low temperature material, and low temperature material comprises polytetrafluoroethylmaterial material.
Described freezing film 5 is made with highly heat-conductive material, and thermal conductivity factor is greater than 200W/ (m DEG C), and highly heat-conductive material comprises silver, gold, copper or aluminium.
The thickness of described freezing film 5 is 20-200 μm.
Described bolt 6 and nut 4 all adopt low temperature material, and low temperature material comprises Fypro.
Described refrigerating plant can frozen cell, cell mass or other biological tissue, and freezing method is:
In refrigerating process, first to outward winding seal cover 1, drop receptacle 2 side is placed upward, then use liquid-transfering gun or cell printing technology to make the cell suspension being mixed with freezing liquid with drops, be added on the freezing film 5 at device center, make it be that dot matrix is arranged on the surface of freezing film 5;
Afterwards, refrigerating plant is overturn, make cryogen vessel 3 upward, drop receptacle 2 is placed down, afterwards, low-temperature refrigerant is at the uniform velocity poured in the cryogen vessel 3 of refrigerating plant, makes it gasify at freezing film 5 surface boiling, cold is passed to freezing film 5 opposite side, completes the glass freezing to drop;
After freezing end, the drop be frozen still stays freezing film 5 surface in drop receptacle 2, and meanwhile, refrigerating plant seal cover 1 is put into liquid nitrogen and preserved after being sealed by drop receptacle 2, also can global transfer transport.
Apparatus of the present invention have following characteristics:
1. constructional device is simple, easily realize, and freezing flow operations is simple and clear.
2. adopt the glass freezing based on drop, drop size is little, and freezing rate is fast, is convenient to form glassy solid, thus avoids the damage of biological tissue in refrigerating process as far as possible.
3. adopt high heat conduction film as heat catalysis, chilling rate is fast, low-temperature refrigerant and biological tissue is kept apart simultaneously.
4. in refrigerating process, freezing biological tissue does not directly contact with low-temperature refrigerant, can not cause biological pollution.
5., after freezing end, freezing drop, still at freezing film surface, is easy to collect.
6. after freezing end, refrigerating plant can use seal cap sealing, puts into liquid nitrogen and preserves, or global transfer transport.
7. this refrigerating plant can the other biological such as frozen cell or cell mass tissue, and not by the restriction of cell category.
Accompanying drawing explanation
Fig. 1 is the decomposing schematic representation of refrigerating plant of the present invention.
Fig. 2 is refrigerating plant overall structure schematic diagram.
Fig. 3 is the representative colored graph anyway of freezing rear cell, and wherein the white bright spot of left figure is living cells, and the white bright spot of right figure is dead cell.
Fig. 4 be freezing before, add frozen solution after and freezing after cell survival rate statistical chart.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in detail.
See figures.1.and.2, a kind of contactless sessile drop method refrigerating plant, comprise seal cover 1, seal cover 1 is connected with drop receptacle 2 threaded engagement, a manhole is had bottom drop receptacle 2, low temperature resistant glue is used to be adhered to bottom drop receptacle 2 by freezing film 5, cryogen vessel 3 top has a manhole, use low temperature resistant glue that cryogen vessel 3 top is mutually bonding with bottom drop receptacle 2, freezing film 5 is made to be sandwiched between the two, and with low temperature resistant glue by the gap sealing between three, drop receptacle 2 and cryogen vessel 3 are connected and fixed further by bolt 6 and nut 4.
Described seal cover 1, drop receptacle 2, cryogen vessel 3 are all made with low temperature material, and low temperature material comprises polytetrafluoroethylmaterial material.
Described freezing film 5 is made with highly heat-conductive material, and thermal conductivity factor is greater than 200W/ (m DEG C), and highly heat-conductive material comprises silver, gold, copper or aluminium.
The thickness of described freezing film 5 is 20-200 μm.
Described bolt 6 and nut 4 all adopt low temperature material, and low temperature material comprises Fypro.
Described refrigerating plant can frozen cell, cell mass or other biological tissue; freezing method is: after configuration cell, cell mass or microorganism cultures suspension; add dimethyl sulphoxide solution or other cryoprotectors wherein; for growing up by ice crystal in T suppression cell in refrigerating process; thus accelerate the formation of glassy solid
In refrigerating process, first to outward winding seal cover 1, drop receptacle 2 side is placed upward, then use liquid-transfering gun or cell printing technology to make the cell suspension being mixed with freezing liquid with drops, be added on the freezing film 5 at device center, make it be that dot matrix is arranged on the surface of freezing film 5;
Afterwards, overturn by refrigerating plant, make cryogen vessel 3 upward, drop receptacle 2 is placed down, and due to capillary effect, the drop on freezing film 5 will hang on freezing film 5 surface; Afterwards, low-temperature refrigerant is at the uniform velocity poured in the cryogen vessel 3 of refrigerating plant, makes it gasify at freezing film 5 surface boiling, by the high capacity of heat transmission of freezing film 5, cold is passed on drop, complete the glass freezing to drop;
After freezing end, the drop be frozen still stays freezing film 5 surface in drop receptacle 2, and meanwhile, refrigerating plant seal cover 1 is put into liquid nitrogen and preserved after being sealed by drop receptacle 2, also can global transfer transport.
Below for frozen cell, freezing film 5 adopts silver film, is described in detail to freezing method.A freezing method for contactless sessile drop method refrigerating plant, comprises the following steps:
1) cell freezing
1.1) configure cell suspension, make its concentration be 1 × 10
4-1 × 10
6cells/ml;
1.2) after refrigerating plant being sterilized, air-dry, wait cell to be added;
1.3) configure cryoprotection solution, composition is: 95% (v/v) cell culture fluid+5% (v/v) dimethyl sulfoxide (DMSO) (DMSO);
1.4) get the above-mentioned cell suspension prepared of 2ml, abandon supernatant, add frozen solution 2ml after centrifugal, concussion makes it mix, and is configured to frozen cell suspension;
1.5) to outward winding seal cover 1, drop receptacle 2 side is placed upward;
1.6) dripped on the freezing film 5 of refrigerating plant with drops by frozen cell suspension with liquid-transfering gun or cell printing machine, droplet size is 5 μ l, and freezing film 5 arranges with latticed form;
1.7) refrigerating plant is inverted, make cryogen vessel 3 upward, low-temperature refrigerant (liquid nitrogen), is at the uniform velocity poured in cryogen vessel 3 down by drop receptacle 2, make it at freezing film 5 surface boiling, complete the cell freezing to freezing film 5 another side;
1.8) after being sealed with seal cover 1 by the drop receptacle 2 of refrigerating plant, put into liquid nitrogen and store, refrigerating plant also can global transfer or transport;
2) cell recovery
2.1) to outward winding seal cover 1, and drop receptacle 2 side of refrigerating plant is placed upward;
2.2) refrigerating plant is placed in water-bath, in 37 DEG C of warm water, cell drop is thawed, when it becomes mixture of ice and water, shift out water-bath;
2.3) the drop liquid-transfering gun after dissolving on freezing film 5 is transferred in vitro, in vitro add 1ml culture fluid simultaneously, shake with dilution refrigeration liquid;
2.4), by centrifugal for the test tube containing cell, after abandoning supernatant, add 45 μ l culture fluids, obtain the cell suspension after thawing.
In order to set forth the advantage of described refrigerating plant and freezing method further, on the basis of above-mentioned frozen cell method, noncontact freezing experiment has been done to NIH-3T3 cell, and after freezing end, fluorescent dye is used to carry out life or death dyeing to cell, and at fluorescence microscopy Microscopic observation statistics cell quantity anyway.The survival rate of the cell using this freezing method to obtain is 84% ± 1%.Fig. 3 gives the representative colored graph anyway of freezing rear cell, and wherein the white bright spot of left figure is living cells, and the white bright spot of right figure is dead cell.Fig. 4 then give freezing before, add frozen solution after and freezing after cell survival rate statistical chart.
Claims (4)
1. a contactless sessile drop method refrigerating plant, comprise seal cover (1), it is characterized in that: seal cover (1) is connected with drop receptacle (2) threaded engagement, drop receptacle (2) bottom has a manhole, low temperature resistant glue is used freezing film (5) to be adhered to drop receptacle (2) bottom, cryogen vessel (3) top has a manhole, use low temperature resistant glue that cryogen vessel (3) top is mutually bonding with drop receptacle (2) bottom, freezing film (5) is made to be sandwiched between the two, and with low temperature resistant glue by the gap sealing between three, drop receptacle (2) and cryogen vessel (3) are connected and fixed further by bolt (6) and nut (4),
Described freezing film (5) is made with highly heat-conductive material, and thermal conductivity factor is greater than 200W/ (m DEG C), and highly heat-conductive material comprises silver, gold, copper or aluminium;
The thickness of described freezing film (5) is 20-200 μm.
2. the contactless sessile drop method refrigerating plant of one according to claim 1, it is characterized in that: described seal cover (1), drop receptacle (2), cryogen vessel (3) are all made with low temperature material, and low temperature material comprises polytetrafluoroethylmaterial material.
3. the contactless sessile drop method refrigerating plant of one according to claim 1, is characterized in that: described bolt (6) and nut (4) all adopt low temperature material, and low temperature material comprises Fypro.
4. the contactless sessile drop method refrigerating plant of one according to claim 1, is characterized in that: described refrigerating plant can frozen cell, cell mass or other biological tissue, and freezing method is:
In refrigerating process, first to outward winding seal cover (1), drop receptacle (2) side is placed upward, then liquid-transfering gun or cell printing technology is used to make the cell suspension being mixed with freezing liquid with drops, be added on the freezing film (5) at device center, make it be that dot matrix is arranged on the surface of freezing film (5);
Afterwards, refrigerating plant is overturn, make cryogen vessel (3) upward, drop receptacle (2) is placed down, afterwards, low-temperature refrigerant is at the uniform velocity poured in the cryogen vessel (3) of refrigerating plant, makes it gasify at freezing film (5) surface boiling;
After freezing end, the drop be frozen still stays freezing film (5) surface in drop receptacle (2), meanwhile, after drop receptacle (2) seals with seal cover (1) by refrigerating plant, put into liquid nitrogen to preserve, also can global transfer transport.
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| WO2017143162A1 (en) * | 2016-02-19 | 2017-08-24 | Regents Of University Of Minnesota | Cryoprotection compositions and methods |
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| US11311008B2 (en) | 2016-04-19 | 2022-04-26 | Regents Of The University Of Minnesota. | Cryopreservation compositions and methods involving nanowarming |
| CN107318827B (en) * | 2017-07-03 | 2020-12-01 | 上海理工大学 | Medical speed-regulating and cooling device with communication function |
| CN108328082B (en) | 2018-02-13 | 2019-07-05 | 京东方科技集团股份有限公司 | A kind of biological sheet storage device |
| CN108917255A (en) * | 2018-07-09 | 2018-11-30 | 成都酷卓生命科技有限公司 | A kind of superelevation prompt drop Thawing Methods and device |
| WO2024199368A1 (en) * | 2023-03-29 | 2024-10-03 | Light Innovation Technology Limited | Sample vessel with fep thin film for holding biological sample in optical microscopy inspection |
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| US6982172B2 (en) * | 2000-01-04 | 2006-01-03 | University Of Connecticut | Oocyte vitrification technique |
| CN100348096C (en) * | 2004-09-15 | 2007-11-14 | 中国科学院海洋研究所 | Vitrifiation freezing preservation method for genine porgy embryo particles |
| DE102006055331B4 (en) * | 2006-01-27 | 2010-12-09 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Sample carriers and sample storage for the cryopreservation of biological samples |
| US20100216230A1 (en) * | 2006-09-28 | 2010-08-26 | Cornell Research Foundation, Inc. | Systems for Increased Cooling and Thawing Rates of Protein Solutions and Cells for Optimized Cryopreservation and Recovery |
| CN100459861C (en) * | 2007-06-18 | 2009-02-11 | 北京锦绣大地农业股份有限公司 | Livestock embryo vitrifying freeze process on metal surface |
| CN101779623B (en) * | 2010-03-11 | 2012-02-22 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| DE102010053461A1 (en) * | 2010-12-03 | 2012-06-06 | Eberhard Lampeter | Cryopreserving biological material, comprises conveying cell suspension by pump to nozzle, applying direct current between nozzle and electrode, which is produced by high voltage generator and introducing droplets into cryogenic vessel |
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Effective date of registration: 20230718 Address after: 215129 South half of floor 2, building 3, No. 599, Taishan Road, high tech Zone, Suzhou, Jiangsu Province Patentee after: Suzhou DianAn Biotechnology Co.,Ltd. Address before: 215129 Four Blocks, 66 Wangmi Street, Suzhou High-tech Zone, Jiangsu Province Patentee before: Suzhou Fulai Enterprise Management Consulting Partnership (L.P.) Effective date of registration: 20230718 Address after: 215129 Four Blocks, 66 Wangmi Street, Suzhou High-tech Zone, Jiangsu Province Patentee after: Suzhou Fulai Enterprise Management Consulting Partnership (L.P.) Address before: 710049 No. 28, Xianning Road, Xi'an, Shaanxi Patentee before: XI'AN JIAOTONG University |
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