CN110973119A - Cryopreservation method applicable to umbilical cord mesenchymal stem cell cryopreservation preparation - Google Patents
Cryopreservation method applicable to umbilical cord mesenchymal stem cell cryopreservation preparation Download PDFInfo
- Publication number
- CN110973119A CN110973119A CN201911223206.XA CN201911223206A CN110973119A CN 110973119 A CN110973119 A CN 110973119A CN 201911223206 A CN201911223206 A CN 201911223206A CN 110973119 A CN110973119 A CN 110973119A
- Authority
- CN
- China
- Prior art keywords
- temperature
- cryopreservation
- cooling
- cells
- program
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a cryopreservation method for umbilical cord mesenchymal stem cell preparations, and particularly, a large number of experiments show that in the process of cryopreservation of cells, the cryopreservation of cells is very favorable after the cells are rapidly cooled and maintained for a certain time, and then the cells are continuously cooled, so that the survival rate of the cells and the quality of the cells after recovery are almost close to the quality of the cells before cryopreservation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cryopreservation method of a preparation suitable for cryopreservation of umbilical cord mesenchymal stem cells.
Background
The stem cell cryopreservation preparation is used as an important preparation form of a new stem cell medicine, the curative effect safety of the cell preparation is directly influenced by the quality of the recovered frozen cells, the cell cryopreservation is one of main methods for preserving the cell preparation, and the cells are preserved at low temperature of liquid nitrogen at the temperature of-196 ℃ by using the cryopreservation technology, so that the cells can be temporarily separated from the growth state to preserve the cell characteristics, and the effect of preserving the seeds of the cells is achieved. The damage of cells during cryopreservation comes mainly from two aspects: ice crystals and osmotic pressure. In the process of cooling the cells, the cells are damaged by too fast or too slow cooling rate so as to influence the resuscitation activity: the temperature is reduced too fast, and water in the cells is rapidly crystallized to cause ice crystal damage to the cells; the temperature is reduced too slowly, and osmotic pressure is generated inside and outside cell walls, so that cells are excessively dehydrated; proper cooling rate control is critical.
There are generally two methods for cryopreserving cells: the traditional method is that a freezing tube is placed in liquid nitrogen at 4 ℃, 20 ℃ below zero, 80 ℃ below zero, and the program cooling is divided into two types: the temperature control program of the isopropanol controls the temperature of a temperature box and the temperature of liquid nitrogen of a program cooling instrument, the program cooling box utilizes the characteristics of the isopropanol of a preset program to enable cells to be cooled to minus 80 ℃ at a constant speed, but the method needs to add the isopropanol to realize program cooling, the isopropanol is toxic to human bodies and can pollute air, the method can not absorb a large amount of heat released by a preparation when the preparation is frozen to a latent heat point, extracellular moisture can not be crystallized continuously, cells can not be dehydrated continuously, ice crystals can cause a large amount of damage to the cells, the program cooling instrument achieves the effect of cooling through volatilization of a large amount of heat absorbed by the liquid nitrogen, and the cooling speed and the cooling process can be controlled through pumping the amount of the liquid nitrogen. However, different temperature reduction procedures have great influence on the survival rate and the cell state of the frozen cells.
Therefore, those skilled in the art have been devoted to developing cell cryopreservation methods with higher cell viability, less impact on cell status, and lower cost.
Disclosure of Invention
The invention aims to provide a method suitable for cryopreservation of a cryopreservation preparation of umbilical cord mesenchymal stem cells.
In a first aspect of the present invention, there is provided a cell cryopreservation method, in which a cell sample is placed in a programmed cooling device for programmed cooling, and the cooling step includes:
(1) first stage cooling
Reducing the temperature of the program temperature reducer to a first temperature at a first temperature reduction speed, wherein the first temperature reduction speed is 0.5-2.5 ℃/min, and the first temperature is-8 to-20 ℃;
(2) second stage cooling
Reducing the temperature of the program temperature reducer to a second temperature at a second temperature reduction speed, wherein the second temperature reduction speed is 5-25 ℃/min, and the second temperature is-30 to-50 ℃;
(3) maintenance of low temperature
Maintaining the temperature of the program cooling instrument at the second temperature + -5 deg.C for 3-10 min;
(4) third stage of temperature reduction
And reducing the temperature of the program temperature reducer to a third temperature at a third temperature reduction speed, wherein the third temperature reduction speed is 5-20 ℃/min, and the third temperature is-60 to-120 ℃.
In another preferred example, the first cooling rate is 0.8 to 1.2 ℃/min; and/or the first temperature is-10 to-15 ℃.
In another preferred example, the first cooling rate is 1.0 ℃/min.
In another preferred example, the second cooling rate is 10 to 20 ℃/min, and the second temperature is-35 to-45 ℃.
In another preferred example, in the step (3), the temperature of the programmed cooling instrument is maintained at the second temperature ± 3 ℃; preferably, the temperature of the programmed thermometer is maintained at the second temperature.
In another preferred example, in the step (3), the maintaining time is about 5 min.
In another preferred example, the third cooling rate is 8-15 ℃/min, and the third temperature is-80 to-100 ℃.
In another preferred embodiment, the cell sample is a stem cell sample.
In another preferred embodiment, the cell sample is an umbilical cord mesenchymal stem cell sample.
In another preferred embodiment, the cell density in the cell sample is 1 x 106/ml～1*108Per ml; preferably 1 x 107/ml。
In another preferred example, the method further comprises the steps of: the cell sample temperature was maintained at 4 ℃ prior to temperature programming.
In another preferred embodiment, the cell sample is stored in a cryopreservation bag for cryopreservation.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows cryopreservation procedure 1.
Figure 2 shows cryopreservation procedure 2.
Figure 3 shows cryopreservation procedure 3.
Figure 4 shows cryopreservation procedure 4.
Figure 5 shows the cryopreservation procedure 5.
Figure 6 shows the cryopreservation procedure 6.
FIG. 7A, FIG. 7B and FIG. 7C show the cell state of the P8 generation umbilical cord mesenchymal stem cells after cryopreservation and inoculation.
Fig. 8A, 8B, 8C, 8D, 8E, 8F, and 8G show results of the osteogenic ability test, respectively.
Fig. 9A, 9B, 9C, 9D, 9E, 9F, and 9G show results of the measurement of the fat-forming ability, respectively.
Fig. 10A, 10B, 10C, 10D, 10E, 10F, and 10G show the results of chondrogenic potential tests, respectively.
Detailed Description
The inventor finds that different freezing procedures have great influence on the cell survival rate and the cell state through extensive and intensive research, and finds that the cell freezing process is very favorable for freezing the cells by maintaining the low temperature for a certain time after rapid cooling and then continuously cooling, so that the cell survival rate and the cell quality after recovery are almost close to the cell quality before freezing. On the basis of this, the present invention has been completed.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
It should be noted that the terms "first," "second," and "third" in the description herein are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The main advantages of the invention are:
(1) the cell cryopreservation method provided by the invention can greatly reduce the damage to the cells in the cryopreservation process and improve the cell survival rate of the cells after cryopreservation;
(2) the method disclosed by the invention is used for freezing the umbilical cord mesenchymal stem cells, so that the cell survival rate and the cell quality after recovery are almost close to the cell quality before freezing, the differentiation capability of the cells can be effectively maintained, and an unexpected technical effect is shown.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
EXAMPLE 1 preparation of cryopreserved cell samples
Umbilical cord mesenchymal stem cells are separated according to a conventional method, cultured until the passage of P5, and continuously cultured according to the following steps.
(1) Resuscitating P5 generation cells 300 million/vial in T225 (purchased from Corning corporation);
(2) cells were observed every day, with a fusion degree of 80% passaged in T225 at 300 ten thousand/vial, which was P6;
(3) cells were observed every day, with a fusion degree of 80% passaged in T225 at 300 ten thousand/vial, which was P7;
(4) cells were observed every day, with a fusion degree of 80% passaged in T225 at 300 ten thousand/vial, which was P8;
(5) cells were observed daily, cells were harvested at 80% confluence, counted, and viability assayed, and cells were resuspended in frozen stock (cat # 12648010, available from ThermoFisher) to adjust cell concentration 1 x 107/ml。
(6) The 10ml cell suspension was pipetted into a cryopreservation bag using a 10ml pipette gun and sealed with a heat sealer.
(7) And vertically placing the freezing storage bag in a freezing storage iron box.
(8) And sticking the temperature probe to the lower part in the middle of the freezing storage bag.
Example 2 cell cryopreservation
The umbilical cord mesenchymal stem cell sample prepared in example 1 was transferred to a programmed cooling apparatus (model 7453, Thermo corporation) for cryopreservation.
The cryopreservation procedures were as follows.
Cryopreservation procedure 1
S1: 4.0 ℃ wait
S2: the temperature of the program cooling instrument is reduced to-4.0 ℃ at the cooling speed of 1.0 ℃/min
S3: the temperature of the program cooling instrument is reduced to-40.0 ℃ at the cooling speed of 25 ℃/min
S4: the temperature of the program cooling instrument is reduced to-12.0 ℃ at the cooling speed of 10 ℃/min
S5, cooling the temperature of the program cooling instrument to-40.0 ℃ at a cooling speed of 1.0 ℃/min
S6 end
Cryopreservation procedure 2
S1: 4.0 ℃ wait
S2: the temperature of the program cooling instrument is reduced to-15.0 ℃ at the cooling speed of 1.0 ℃/min
S3: the temperature of the program cooling instrument is reduced to-40.0 ℃ at the cooling speed of 15 ℃/min
S4: the temperature of the program cooling instrument is reduced to-22.0 ℃ at the cooling speed of 10 ℃/min
S5, cooling the temperature of the program cooling instrument to-80.0 ℃ at a cooling speed of 1.0 ℃/min
S6 end
S1: 4.0 ℃ wait
S2: the temperature of the program cooling instrument is reduced to-15.0 ℃ at the cooling speed of 1.0 ℃/min
S3: the temperature of the program cooling instrument is reduced to-40.0 ℃ at the cooling speed of 10 ℃/min
S4: the temperature of the program cooling instrument is reduced to-30.0 ℃ at the cooling speed of 1 ℃/min
S5, cooling the temperature of the program cooling instrument to-81.0 ℃ at a cooling speed of 1.0 ℃/min
S6 end
S1: 4.0 ℃ wait
S2: the temperature of the program cooling instrument is reduced to-15.0 ℃ at the cooling speed of 1.0 ℃/min
S3: the temperature of the program cooling instrument is reduced to-40.0 ℃ at the cooling speed of 10 ℃/min
S4: maintaining at-40.0 deg.C for 10min
S5, cooling the temperature of the program cooling instrument to-81.0 ℃ at a cooling speed of 1.0 ℃/min
S6 end
S1: 4.0 ℃ wait
S2: the temperature of the program cooling instrument is reduced to-15.0 ℃ at the cooling speed of 1.0 ℃/min
S3: the temperature of the program cooling instrument is reduced to-40.0 ℃ at the cooling speed of 10 ℃/min
S4: maintaining at-40.0 deg.C for 5min
S5, cooling the temperature of the programmed cooling instrument to-100.0 ℃ at a cooling speed of 1.0 ℃/min
S6 end
S1: 4.0 ℃ wait
S2: the temperature of the program cooling instrument is reduced to-15.0 ℃ at the cooling speed of 1.0 ℃/min
S3: the temperature of the program cooling instrument is reduced to-40.0 ℃ at the cooling speed of 10 ℃/min
S4: maintaining at-40.0 deg.C for 5min
S5, cooling the temperature of the program cooling instrument to-100.0 ℃ at a cooling speed of 10 ℃/min
S6 end
Control (isopropanol temperature programmed box):
(1) the cell suspension adjusted by the freezing solution is subpackaged into 2ml freezing tubes, and 1ml cell suspension is added into each tube.
(2) And (3) placing the freezing tube in an isopropanol program cooling box placed at normal temperature, transferring the freezing tube to a refrigerator at the temperature of minus 80 ℃ for 4 hours, and then transferring the freezing tube to a liquid nitrogen tank.
The human cells before and after the cryopreservation recovery of each cryopreservation procedure are subjected to cell or rate detection by adopting a conventional method, and the calculation formula of the cell viability rate (trypan blue staining principle: normal viable cells have complete cell membrane structure and can exclude trypan blue so that the trypan blue cannot enter cells, cells with lost activity or incomplete cell membranes have increased cell membrane permeability and can be stained into blue by the trypan blue) is as follows: (number of viable cells/total number of cells) 100%
The smaller the difference between the cell survival rates before and after cryopreservation recovery indicates that the degree of damage to the cells during the freezing process is lower.
The results are shown in table 1:
TABLE 1
The P8 umbilical cord mesenchymal stem cells were cryopreserved for resuscitation by each cryopreservation procedure, then the same number of viable cells were seeded on T75 (purchased from Corning corporation) and the cell status was observed after 8 hours of fluid exchange, and some typical cell statuses are shown in fig. 7A (procedure 6), fig. 7B (procedure 1) and fig. 7C (isopropyl alcohol temperature programmed box procedure). As can be seen from the figure, procedure 6 frozen cells were better conditioned and not aged; procedure 1 the frozen cells after recovery exhibited aging with enlarged inclusion, whereas the frozen cells after recovery using isopropanol procedure exhibited complete aging with very poor state and rag-like appearance. Procedures 2, 3, 4, 5 cryopreserved cells with better cell status after recovery.
After recovery, the cell phenotype and the three-line differentiation capability are detected.
The results of the osteogenic ability test are shown in fig. 8A, 8B, 8C, 8D, 8E, 8F, and 8G, and correspond to the program 1, the program 2, the program 3, the program 4, the program 5, the program 6, and the isopropyl alcohol temperature reduction box, respectively.
The results of the measurement of the fat-forming ability are shown in fig. 9A, 9B, 9C, 9D, 9E, 9F, and 9G, and correspond to the program 1, the program 2, the program 3, the program 4, the program 5, the program 6, and the isopropyl alcohol temperature-lowering cassette, respectively.
The results of the chondrogenic capacity test are shown in fig. 10A, 10B, 10C, 10D, 10E, 10F, and 10G, and correspond to the program 1, the program 2, the program 3, the program 4, the program 5, the program 6, and the isopropyl alcohol temperature reduction cassette, respectively.
The pictures show that the frozen cells of the isopropanol temperature-reducing program box can not be osteogenic, and the capacity of adipogenesis and chondrogenesis is obviously poor. Cryopreservation procedure 1 the cryopreserved cells have poor osteogenic capacity and adipogenic capacity and good chondrogenic capacity; cryopreservation procedure 2 the cryopreserved cells have poor osteogenesis capacity and adipogenesis capacity and good chondrogenesis capacity; cryopreservation procedure 3 frozen cells had poor adipogenic ability, and adequate osteogenic and chondrogenic ability. Cryopreservation procedures 4, 5 and 6 were performed to obtain cells excellent in osteogenic ability, adipogenic ability and chondrogenic ability. Therefore, the cryopreservation method provided by the invention can effectively maintain the differentiation capacity of the umbilical cord mesenchymal stem cells.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A cell freezing method is characterized in that a cell sample is placed in a programmed cooling instrument for programmed cooling, and the cooling step comprises the following steps:
(1) first stage cooling
Reducing the temperature of the program temperature reducer to a first temperature at a first temperature reduction speed, wherein the first temperature reduction speed is 0.5-2.5 ℃/min, and the first temperature is-8 to-20 ℃;
(2) second stage cooling
Reducing the temperature of the program temperature reducer to a second temperature at a second temperature reduction speed, wherein the second temperature reduction speed is 5-25 ℃/min, and the second temperature is-30 to-50 ℃;
(3) maintenance of low temperature
Maintaining the temperature of the program cooling instrument at the second temperature + -5 deg.C for 3-10 min;
(4) third stage of temperature reduction
And reducing the temperature of the program temperature reducer to a third temperature at a third temperature reduction speed, wherein the third temperature reduction speed is 5-20 ℃/min, and the third temperature is-60 to-120 ℃.
2. The cell cryopreservation method of claim 1, wherein the first cooling rate is 0.8-1.2 ℃/min; and/or the first temperature is-10 to-15 ℃.
3. The cell cryopreservation method according to claim 1, wherein the second cooling rate is 10-20 ℃/min, and the second temperature is-35 to-45 ℃.
4. The method of claim 1, wherein in step (3), the temperature of the programmed cooling device is maintained at a second temperature ± 3 ℃; preferably, the temperature of the programmed thermometer is maintained at the second temperature.
5. The method for cryopreserving cells according to claim 1, wherein the maintaining time in the step (3) is about 5 min.
6. The cell cryopreservation method of claim 1, wherein the third cooling rate is 8-15 ℃/min, and the third temperature is-80 to-100 ℃.
7. The method of claim 1, wherein the cell sample is a stem cell sample.
8. The method of claim 1, wherein the cell density in the cell sample is 1 x 106/ml～1*108Per ml; preferably 1 x 107/ml。
9. The method of claim 1, further comprising the step of: the cell sample temperature was maintained at 4 ℃ prior to temperature programming.
10. The method of claim 1, wherein the cell sample is stored in a cryopreservation bag for cryopreservation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911223206.XA CN110973119B (en) | 2019-12-03 | 2019-12-03 | Cryopreservation method applicable to umbilical cord mesenchymal stem cell cryopreservation preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911223206.XA CN110973119B (en) | 2019-12-03 | 2019-12-03 | Cryopreservation method applicable to umbilical cord mesenchymal stem cell cryopreservation preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110973119A true CN110973119A (en) | 2020-04-10 |
| CN110973119B CN110973119B (en) | 2022-03-08 |
Family
ID=70089702
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911223206.XA Active CN110973119B (en) | 2019-12-03 | 2019-12-03 | Cryopreservation method applicable to umbilical cord mesenchymal stem cell cryopreservation preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110973119B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN109122665A (en) * | 2018-09-12 | 2019-01-04 | 银丰生物工程集团有限公司 | With the method for a variety of stem cells of freezing and storing umbilical tissue preserration, method for resuscitation and application |
| CN109497041A (en) * | 2018-11-23 | 2019-03-22 | 西安九州医学中心有限公司 | A kind of cryopreservation methods of adipose tissue |
| CN109864063A (en) * | 2018-06-29 | 2019-06-11 | 江苏艾尔康生物医药科技有限公司 | A kind of method and application that the Programmed cryopreservation of source of people retinal pigment epithelium freezes |
-
2019
- 2019-12-03 CN CN201911223206.XA patent/CN110973119B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN109864063A (en) * | 2018-06-29 | 2019-06-11 | 江苏艾尔康生物医药科技有限公司 | A kind of method and application that the Programmed cryopreservation of source of people retinal pigment epithelium freezes |
| CN109122665A (en) * | 2018-09-12 | 2019-01-04 | 银丰生物工程集团有限公司 | With the method for a variety of stem cells of freezing and storing umbilical tissue preserration, method for resuscitation and application |
| CN109497041A (en) * | 2018-11-23 | 2019-03-22 | 西安九州医学中心有限公司 | A kind of cryopreservation methods of adipose tissue |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110973119B (en) | 2022-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mazur et al. | A two-factor hypothesis of freezing injury: evidence from Chinese hamster tissue-culture cells | |
| CN108207930B (en) | Cocktail type cryoprotectant and application thereof | |
| Chen et al. | Freezing characteristics of cultured Catharanthus roseus (L). G. Don cells treated with dimethylsulfoxide and sorbitol in relation to cryopreservation | |
| Mazur | MANIFESTATIONS OF INJURY IN YEAST CELLS EXPOSED TO SUBZERO TEMPERATURES I: Morphological Changes in Freeze-Substituted and in “Frozen-Thawed” Cells | |
| CN103667061A (en) | Strain preservation method | |
| CN104839144A (en) | Vitrification freezing fluid of oocytes | |
| CN112913833B (en) | Programmed cooling method for human umbilical cord mesenchymal stem cell working cell bank | |
| Mocé et al. | Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols | |
| CN112772637A (en) | DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution | |
| CN110973119B (en) | Cryopreservation method applicable to umbilical cord mesenchymal stem cell cryopreservation preparation | |
| US9078430B2 (en) | Cell preparation method | |
| US9005886B2 (en) | Method for cryopreservation of human spermatozoa free from seminal plasma using a fast and simple aseptic vitrification-devitrification process; portable kit for carrying out the method; and use of the same for treatment of disorders related to reproductive failures | |
| Schrcnemakers et al. | A two-step or equilibrium freezing procedure for the cryopreservation of plant cell suspensions | |
| CN103004751A (en) | Hematopoietic stem cell cryopreserving method and protective agent | |
| Harrison et al. | A comparative study in methods of cryoprotection for human semen | |
| US20150024487A1 (en) | Cryopreservation storage device for cell collection bag, and using method thereof | |
| Hoch | Preservation of cell ultrastructure by freeze-substitution | |
| CN105284789B (en) | A kind of acipenser dabryanus spermary cell freezen protective liquid and spermary cell freezing and storing method and application | |
| Withers et al. | A fine-structural study of the freeze-preservation of plant tissue cultures: I. The frozen state | |
| CN112674080A (en) | Programmed cooling method for human neural stem cell working cell bank | |
| Liu et al. | Response of murine osteoblasts and porous hydroxyapatite scaffolds to two-step, slow freezing and vitrification processes | |
| CN117814212A (en) | Cryopreservation method of neural stem cells | |
| Towill | Cryopreservation by vitrification | |
| CN111406736A (en) | Cell cryopreservation liquid | |
| JP2014217356A (en) | Method for producing container having automatic ice-forming ability for freeze-preservation of reproductive cell |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 2066 Lane 2, 4 Floors, Wangyuan Road, Fengxian District, Shanghai, 20149 Patentee after: Zhejiang Quansheng Biotechnology Co.,Ltd. Address before: 2066 Lane 2, 4 Floors, Wangyuan Road, Fengxian District, Shanghai, 20149 Patentee before: Shanghai Quansheng Biotechnology Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 313301 Building 6, Zhejiang North Life Health Microenterprise Park, Yuedu Middle Road, Dipu Street, Anji County, Huzhou City, Zhejiang Province (self declared) Patentee after: Zhejiang Quansheng Biotechnology Co.,Ltd. Address before: 2066 Lane 2, 4 Floors, Wangyuan Road, Fengxian District, Shanghai, 20149 Patentee before: Zhejiang Quansheng Biotechnology Co.,Ltd. |
|
| CP02 | Change in the address of a patent holder |