CN115226707A - Method for freezing and slicing tissue stored at low temperature and application - Google Patents
Method for freezing and slicing tissue stored at low temperature and application Download PDFInfo
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- CN115226707A CN115226707A CN202211073313.0A CN202211073313A CN115226707A CN 115226707 A CN115226707 A CN 115226707A CN 202211073313 A CN202211073313 A CN 202211073313A CN 115226707 A CN115226707 A CN 115226707A
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000008014 freezing Effects 0.000 title claims abstract description 14
- 238000007710 freezing Methods 0.000 title claims abstract description 14
- 238000012546 transfer Methods 0.000 claims abstract description 37
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 7
- 235000011089 carbon dioxide Nutrition 0.000 claims abstract description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- 239000011324 bead Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 5
- 230000032258 transport Effects 0.000 claims 4
- 230000004656 cell transport Effects 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 10
- 238000003860 storage Methods 0.000 abstract description 8
- 238000013467 fragmentation Methods 0.000 abstract description 5
- 238000006062 fragmentation reaction Methods 0.000 abstract description 5
- 238000004043 dyeing Methods 0.000 abstract description 4
- 238000011067 equilibration Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 6
- 238000010186 staining Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000010827 pathological analysis Methods 0.000 description 2
- 230000002411 adverse Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of frozen tissue sections, and particularly relates to a method for freezing and storing tissues at low temperature for frozen section and application, wherein the method comprises procedural rewarming, embedding and slicing, and comprises preparing a transfer box in advance, and precooling the transfer box in a refrigerator at minus 80 ℃ for one night for standby; taking out the tissue frozen at low temperature, placing the tissue in a transfer box, placing the transfer box in a refrigerator at minus 80 ℃ for 0.5 to 1 hour, and placing the transfer box in a refrigerator at minus 40 ℃ for 0.5 to 1 hour; the tissue was removed on dry ice for embedding and sectioned after equilibration in an ice cutter for 0.5-1 h. The invention takes out the non-embedded tissue frozen at low temperature from the storage place, carries out procedural rewarming, and then carries out frozen embedding and slicing, thereby effectively solving the problems of tissue fragmentation and damage when the non-embedded tissue frozen at low temperature is applied to frozen tissue slice experiments, and the frozen tissue slice processed by rewarming has good integrity, good shape and uniformity; after dyeing, the shape is good, and the tissue structure is clearly displayed.
Description
Technical Field
The invention belongs to the technical field of frozen tissue sections, and particularly relates to a method for freezing and storing tissues at low temperature to perform frozen section and application.
Background
The frozen section is a pathological experimental technology which freezes or embeds living tissues to a certain hardness by low-temperature freezing and then performs sectioning, and is widely applied to clinic and scientific research. The frozen section technology can be used for quickly preparing section specimens and better storing the enzyme activity in cells. Meanwhile, the frozen section has small damage to antigen and no obvious contraction of tissues, can preserve the activity of fat, lipoid and various enzymes, is widely applied to immunohistochemistry or immunofluorescence experiments, and has irreplaceable effect in scientific research experiments. To produce high-quality frozen sections, the technical key needs to be fully mastered in each link of material taking, tissue freezing embedding, section and staining, and each operation step needs to be well controlled. The quality of the frozen section directly affects the experimental result, so that the key for obtaining the high-quality experimental result is to manufacture the high-quality frozen section. In the process of freezing the tissue, due to the influence of water content, freezing time, temperature and the like, the manufactured frozen section can generate the problems of ice crystal formation, section folding, section falling, poor dyeing and the like, and the storage condition of the tissue specimen can also generate certain influence on the antigen in the tissue and the tissue form of the frozen section.
At present, frozen sections are generally sliced by using tissues subjected to frozen embedding, and an embedding agent can be OCT or glue. The frozen embedded tissue is generally sliced immediately, or the embedded tissue sample is stored in an ultra-low temperature refrigerator at minus 80 ℃, the sample is taken out during slicing, and is transferred to a ice cutter to be buffered for 30min and then sliced. However, sometimes some tissues can not be frozen and embedded in time because frozen sections are not considered before experiments or because of time urgency or large sampling amount and other reasons, and are directly wrapped by a freezing tube or tinfoil paper and stored in a liquid nitrogen or an ultra-low temperature refrigerator at minus 80 ℃. Therefore, if the tissue frozen at low temperature is directly taken out for embedding the section, the condition that the tissue section is torn and damaged is easy to occur, the tissue form is influenced, the accuracy of a staining result is interfered, the wrong judgment is easy to cause, and certain adverse effect is caused to pathological diagnosis or scientific research.
Disclosure of Invention
Aiming at the problems that in the prior art, the tissue is frozen and preserved in liquid nitrogen or a refrigerator at minus 80 ℃ without being embedded, and the tissue is directly frozen and embedded and sliced after being taken out when in use, so that the tissue slices are cracked, damaged and stained unevenly. The invention provides a method for performing programmed rewarming on an un-embedded low-temperature frozen tissue and then embedding a section and application, namely, after the un-embedded tissue which is frozen at a low temperature is taken out, a series of programmed rewarming steps are performed before embedding, and then the frozen section is performed, so that the problems of tissue fragmentation and damage when the un-embedded tissue which is frozen at a low temperature is applied to a frozen tissue section experiment can be effectively solved.
In order to realize the aim, the method for freezing and storing the tissue at low temperature to carry out frozen section comprises the following steps of procedural rewarming, embedding and slicing:
(1) Procedural rewarming:
a. preparing a transfer box in advance, and precooling the transfer box in a refrigerator at minus 80 ℃ for one night for later use;
b. taking out the low-temperature frozen tissues and putting the low-temperature frozen tissues into a pretreated transfer box;
c. placing the transfer box in a refrigerator at-80 ℃ for 0.5-1h;
d. placing the transfer box in a refrigerator at the temperature of-40 ℃ for 0.5-1h;
(2) Embedding: taking out the tissue subjected to programmed rewarming, and embedding the tissue on dry ice by using an embedding agent;
(3) Slicing: the embedded tissue is placed in an ice cutting machine and is balanced for 0.5 to 1 hour and then sliced.
The principle of procedural rewarming is as follows: by gradually changing the tissue storage environment, the tissue temperature is gradually and uniformly changed and cannot be sharply increased due to the change of the environment. The temperature of the non-embedded tissues stored at low temperature in the rewarming process is strictly controlled in the process, so that the temperature of the non-embedded tissues is uniformly increased, and the problems of ice crystal formation and the like caused by rapid temperature rise (overlarge temperature span) or non-uniform temperature rise are avoided, so that the tissues can have better shapes after being sliced.
Generally, for tissues frozen at low temperature and not embedded and frozen, the tissues are directly transferred from the low-temperature storage place to dry ice for embedding, and a rewarming step is not needed in the middle, so that the problems of fragmentation and damage of frozen sections, non-uniform subsequent staining and the like can be caused by the method. In order to solve the problems and achieve the purpose of good shape and uniform dyeing of the low-temperature frozen non-embedded frozen tissues after frozen tissue slicing, the inventor finds that through a large number of experiments, the non-embedded low-temperature frozen tissues are taken out from a storage place and then subjected to programmed rewarming, the frozen tissues are firstly put into a pre-processed transfer box after being taken out, the transfer box is put into a refrigerator at minus 80 ℃ for a period of time, then the transfer box is put into the refrigerator at minus 40 ℃ for a period of time, and then embedding and slicing are carried out, the frozen tissues do not form ice crystals and the like due to sudden change of environmental temperature through the programmed rewarming process, the problems can be effectively solved, the obtained frozen tissues are complete in section and good in shape, the tissue structure is clear after dyeing, and the effect of directly freezing and embedding the tissues and immediately slicing can be basically achieved.
Further, in the above technical scheme, the low-temperature frozen tissue is non-embedded tissue stored in liquid nitrogen or non-embedded tissue stored at-80 ℃.
Further, in the technical scheme, when the low-temperature frozen tissue is the non-embedded tissue stored in liquid nitrogen, the pre-treated transfer box is placed in the liquid nitrogen for treatment for 1.5-2.5 hours before the tissue is transferred. According to the technical scheme, the cell transfer box is placed into liquid nitrogen for pretreatment before use, so that the environment of the cell transfer box is closer to the liquid nitrogen, and the frozen tissue is not influenced due to rapid environmental change after being taken out.
Furthermore, in the above technical scheme, the temperature change rate of the transfer box is less than or equal to 1 ℃/min, and the transfer box is a cell transfer box or a magnetic bead transfer box. The invention selects the transfer box with slower temperature change rate, so that the temperature change in the rewarming process of the frozen tissues is slow and uniform, and the change of the internal structure of the tissues cannot be influenced by the rapid change of the temperature.
Further, in the above technical solution, the embedding medium is OCT.
Further, in the above technical scheme, the slice thickness is 6-10 μm.
The invention also provides the application of the improved method in the application of the non-embedded tissues frozen at low temperature to frozen tissue sections. The improved method can also be used for other non-embedded tissues frozen at low temperature, and is not limited to the non-embedded tissues stored in liquid nitrogen or at-80 ℃.
The invention has the beneficial effects that:
according to the invention, the non-embedded tissue frozen at low temperature is taken out from the storage place and subjected to programmed rewarming, and then frozen embedding and slicing are carried out, so that the problems of tissue fragmentation and damage when the non-embedded tissue frozen at low temperature is applied to a frozen tissue slice experiment can be effectively solved, and the frozen tissue slice subjected to rewarming treatment has good integrity, good shape and uniformity;
the improved method is simple to operate, and the frozen tissue section of the non-embedded tissue frozen at low temperature is well dyed, and the tissue structure is clearly displayed;
the method is used for the materials which are directly wrapped by a freezing tube or tinfoil paper and then placed in liquid nitrogen or an ultra-low temperature refrigerator of minus 80 ℃ for storage because the frozen section is not considered before the experiment, or some tissues are not frozen and embedded in time because of urgent time or overlarge material taking amount and the like, avoids the conditions of tearing and damage of the tissue sections, solves the problem of material distortion for pathological diagnosis or scientific research, and improves the accuracy of judgment.
Drawings
FIG. 1 is a photomicrograph of a cryosection stained at 100 times microscope in example 1 of the present invention;
FIG. 2 is a photomicrograph of cryo-section staining at 100 times microscope in example 2 of the present invention;
FIG. 3 is a photomicrograph of a cryosection staining at 100 times microscope in comparative example 1.
Detailed Description
The experimental procedures in the following examples are all conventional ones unless otherwise specified. The raw materials in the following examples are all commercially available products and are commercially available, unless otherwise specified.
In the following examples, the non-embedded tissues were all mouse livers.
The present invention is described in further detail below with reference to examples:
example 1
A method for frozen sectioning of non-embedded tissues stored in liquid nitrogen, comprising the following specific steps:
(1) Procedural rewarming:
a. preparing a cell transfer box in advance, and precooling the cell transfer box in a refrigerator at minus 80 ℃ for one night for later use;
b. the cell transfer box is pretreated in liquid nitrogen for 2 hours before use, and the low-temperature frozen tissue is taken out and put into the pretreated cell transfer box;
c. placing the cell transfer box in a refrigerator at-80 ℃ for 0.5h;
d. placing the cell transfer box in a refrigerator at the temperature of-40 ℃ for 0.5h;
(2) Embedding: taking out the tissue subjected to programmed rewarming, and embedding the tissue on dry ice by using an embedding agent OCT;
(3) Slicing: the embedded tissue was placed in an ice-cutting machine and equilibrated for 0.5h before sectioning.
The obtained section was stained by a conventional method, observed under a 100-fold microscope and photographed, and the photograph thereof is shown in FIG. 1. As can be seen from FIG. 1, the stained section has high integrity, good and uniform shape, and clear tissue structure display.
Example 2
A method for frozen sectioning of non-embedded tissues stored in a freezer at-80 ℃ comprising the following specific steps:
(1) Procedural rewarming:
a. preparing a magnetic bead transfer box in advance, and precooling the magnetic bead transfer box in a refrigerator at minus 80 ℃ for one night for later use;
b. taking out the low-temperature frozen tissues and then putting the low-temperature frozen tissues into a pretreated magnetic bead transfer box;
c. placing the magnetic bead transfer box in a refrigerator with the temperature of-80 ℃ for 1h;
d. placing the magnetic bead transfer box in a refrigerator at the temperature of-40 ℃ for 1h;
(2) Embedding: taking out the tissue subjected to programmed rewarming, and embedding the tissue on dry ice by using an embedding agent OCT;
(3) Slicing: the embedded tissue is placed in an ice cutting machine and is balanced for 0.5 to 1 hour and then sliced.
The obtained section was stained by a conventional method, observed under a 100-fold microscope and photographed, and the photograph thereof is shown in FIG. 2. As can be seen from FIG. 2, the stained section has high integrity, good and uniform shape, and clear tissue structure display.
Comparative example 1
A method for frozen sectioning of non-embedded tissues stored in a freezer at-80 ℃ comprising the following specific steps:
(1) Embedding: taking out the non-embedded tissues stored in a refrigerator at the temperature of-80 ℃ and embedding the tissues on dry ice by using an embedding agent OCT;
(2) Slicing: the embedded tissue was placed in an ice-cutting machine and equilibrated for 1h before sectioning.
The obtained section was stained by a conventional method, observed under a 100-fold microscope and photographed, and a photograph thereof is shown in FIG. 3. As can be seen from FIG. 3, the stained sections have poor integrity, and are torn and damaged, uneven in shape, and overlapped or missing in tissue structure.
The comprehensive results show that the non-embedded tissue frozen at low temperature is taken out from the storage place, is subjected to programmed rewarming, and is then subjected to frozen embedding and slicing, so that the problems of tissue fragmentation and damage when the non-embedded tissue frozen at low temperature is applied to a frozen tissue slice experiment can be effectively solved, and the frozen tissue slice subjected to rewarming treatment has good integrity, good form and uniformity; the improved method can be applied to non-embedded tissues frozen at low temperature for frozen tissue sections, has good effect, and can be used as a rescue method for subsequent frozen tissue morphology.
Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, rather than limitations, and that many variations and modifications of the invention are possible to those skilled in the art, without departing from the spirit and scope of the invention.
Claims (7)
1. A method for freezing and storing tissues at low temperature for frozen sectioning is characterized by comprising the following steps of procedural rewarming, embedding and sectioning:
(1) Procedural rewarming:
a. preparing a transfer box in advance, and precooling the transfer box in a refrigerator at minus 80 ℃ for one night for later use;
b. taking out the low-temperature frozen tissues and putting the tissues into a pretreated transfer box;
c. placing the transfer box in a refrigerator at-80 ℃ for 0.5-1h;
d. placing the transfer box in a refrigerator at the temperature of-40 ℃ for 0.5-1h;
(2) Embedding: taking out the tissue subjected to programmed rewarming, and embedding the tissue on dry ice by using an embedding agent;
(3) Slicing: the embedded tissue is placed in an ice cutting machine and is balanced for 0.5-1h, and then is sliced.
2. The method for cryosectioning of a cryo-cryopreserved tissue according to claim 1, wherein said cryo-cryopreserved tissue is non-embedded tissue stored in liquid nitrogen or non-embedded tissue stored at-80 ℃.
3. The method for cryosectioning of cryo-preserved tissue according to claim 2, wherein when the cryo-preserved tissue is non-embedded tissue preserved in liquid nitrogen, the pre-treated transport cassette is placed in liquid nitrogen for 1.5-2.5h before the tissue is transported.
4. The method for cryopreserving tissue for cryosectioning according to claim 1, wherein the temperature change rate of the transport cassette is less than or equal to 1 ℃/min, and the transport cassette is a cell transport cassette or a magnetic bead transport cassette.
5. The method of claim 1, wherein the embedding agent is OCT.
6. The method of claim 1, wherein said section is 6-10 μm thick.
7. Use of the method of any one of claims 1-6 in cryogenically frozen non-embedded tissue for freezing tissue sections.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211073313.0A CN115226707B (en) | 2022-09-02 | 2022-09-02 | Method for frozen section of low-temperature frozen tissue and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211073313.0A CN115226707B (en) | 2022-09-02 | 2022-09-02 | Method for frozen section of low-temperature frozen tissue and application thereof |
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| CN115226707A true CN115226707A (en) | 2022-10-25 |
| CN115226707B CN115226707B (en) | 2024-02-02 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60149525A (en) * | 1984-01-17 | 1985-08-07 | Hoxan Corp | Preparation of small piece of frozen liver for artificial liver |
| US5452584A (en) * | 1994-07-13 | 1995-09-26 | Duke University | Method of freezing biological tissue specimens with opti-cryo-fluid |
| WO2008047154A2 (en) * | 2006-10-21 | 2008-04-24 | Elizabeth Acton | Controlled rate freezing |
| CN205087344U (en) * | 2015-10-22 | 2016-03-16 | 复旦大学附属肿瘤医院 | Biological tissue freezes and deposits container |
| CN113340699A (en) * | 2021-06-02 | 2021-09-03 | 中国人民解放军陆军特色医学中心 | Organoid tissue frozen section embedding kit and embedding method |
| CN215422473U (en) * | 2021-05-26 | 2022-01-07 | 宁夏医科大学总医院 | Liquid nitrogen quick freezing device for frozen section tissue |
-
2022
- 2022-09-02 CN CN202211073313.0A patent/CN115226707B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60149525A (en) * | 1984-01-17 | 1985-08-07 | Hoxan Corp | Preparation of small piece of frozen liver for artificial liver |
| US5452584A (en) * | 1994-07-13 | 1995-09-26 | Duke University | Method of freezing biological tissue specimens with opti-cryo-fluid |
| WO2008047154A2 (en) * | 2006-10-21 | 2008-04-24 | Elizabeth Acton | Controlled rate freezing |
| CN205087344U (en) * | 2015-10-22 | 2016-03-16 | 复旦大学附属肿瘤医院 | Biological tissue freezes and deposits container |
| CN215422473U (en) * | 2021-05-26 | 2022-01-07 | 宁夏医科大学总医院 | Liquid nitrogen quick freezing device for frozen section tissue |
| CN113340699A (en) * | 2021-06-02 | 2021-09-03 | 中国人民解放军陆军特色医学中心 | Organoid tissue frozen section embedding kit and embedding method |
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| CN115226707B (en) | 2024-02-02 |
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