KR101767195B1 - A Composition for Cryopreservation of Cell and a Method for Cryopreservation of Cell Using the Same - Google Patents
A Composition for Cryopreservation of Cell and a Method for Cryopreservation of Cell Using the Same Download PDFInfo
- Publication number
- KR101767195B1 KR101767195B1 KR1020150054751A KR20150054751A KR101767195B1 KR 101767195 B1 KR101767195 B1 KR 101767195B1 KR 1020150054751 A KR1020150054751 A KR 1020150054751A KR 20150054751 A KR20150054751 A KR 20150054751A KR 101767195 B1 KR101767195 B1 KR 101767195B1
- Authority
- KR
- South Korea
- Prior art keywords
- cells
- cell
- cryopreservation
- composition
- present
- Prior art date
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
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The present invention relates to a composition for cryopreservation and a method for cryopreservation using the same. By using the composition of the present invention, cells can be preserved at a high survival rate for a long period even at a temperature of about -70 ° C.
Description
The present invention relates to a composition for cryopreservation and a method for cryopreservation using the same.
When cultured to preserve cells, it is known that their characteristics are likely to be changed by genetic variation or generational variation, and unwanted pollution may be caused by environmental factors. Conventional ultra freezing preservation methods have been used to minimize these genetic or generational variations and preserve cells while preventing contamination by environmental factors. Cryogenic cryopreservation for cells requires a low temperature of about -195 ° C and requires cryopreservation to prevent cell freezing / thawing damage. Conventionally 15% glycerol has been used as a general cryopreservative. However, there are many limitations to maintain a low temperature of -195 ℃. In addition, the effect of preventing freezing / thawing damage of glycerol which has been used as a cryopreservation agent is insufficient, and there is a need for a cryopreservation agent capable of minimizing cell freezing / thawing damage while maintaining cell preservation effect even at a relatively high temperature .
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have sought to develop a novel cryopreservative agent capable of minimizing freezing / thawing damage in cryopreservation for long-term storage of cells. As a result, the present inventors have completed the present invention by confirming that, when a composition for cryopreservation containing bovine serum albumin is used as a cryoprotectant, the cells can be preserved at a high survival rate for a long period even at a temperature of about -70 ° C.
Accordingly, an object of the present invention is to provide a composition for cryopreservation.
Another object of the present invention is to provide a cell freezing preservation method.
It is another object of the present invention to provide a method for preserving cell freezing and obtaining viable cells.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a composition for cryopreservation comprising bovine serum albumin as a cryoprotectant.
The present inventors have sought to develop a novel cryopreservative agent capable of minimizing freezing / thawing damage in cryopreservation for long-term storage of cells. As a result, it was found that when a composition for cryopreservation containing bovine serum albumin is used as a cryoprotectant, the cells can be preserved at a high survival rate over a long period of time at a temperature of about -70 ° C.
The present invention is to improve the cryopreservation method that has been used for preserving cells. The present invention provides a cryopreservation method for efficiently preserving cells in a relatively high temperature range of -70 ° C To a cryopreservation composition capable of preserving cryopreservation. There is no successful cryopreservation of cells at -70 ° C. Cryopreservation at -70 ° C allows cryopreservation without the use of liquid nitrogen, which is difficult to purchase and store. Since the temperature can be maintained only by using a generally used freezer (deep freezer), it is versatile and economical.
According to one embodiment of the present invention, the cells of the present invention are eukaryotic cells or prokaryotic cells derived from microalgae. The term "prokaryotic cell" of the present invention means a cell derived from a prokaryote, and is specifically a cell derived from a bacterium such as Escherichia coli, Staphylococcus aureus or Cholera, or a cyanophyta. "Eukaryotic cell" of the present invention means a cell derived from microalgae belonging to a eukaryote. The composition for cryopreservation of the present invention can be used for lyophilization of the cells described above.
According to one embodiment of the present invention, the bovine serum albumin of the present invention inhibits freezing / thawing damage of cells. During freezing and thawing for long-term preservation of cells, cells undergo damage due to temperature changes. As the freezing progresses, the water outside the cell first freezes, and as a result, the environment outside the cell forms a salt state rather than the inside of the cell. In order to maintain the equilibrium state, water migration occurs inside the cell, (plasmolysis) occurs. To prevent this, cryopreservatives are used. Cryopreservation agents are classified into two categories: cell protective cryoprotectants (colligative cryoprotectants) and cell penetration cryopreservation agents. The cytoprotective genus freezing preservative functions to inhibit the formation of ice crystals outside the cell, and the cell penetration type cryopreservation agent penetrates the cell membrane to substitute water in cells and inhibits the formation of ice crystals during freezing. In particular, cell-protective type fastidious cryopreservation agents are not used for cryopreservation of microalgae cells, and cell permeation-type cryopreservation agents such as DMSO and glycerol have conventionally been used. However, in the case of the cell permeation-type cryopreservation agent, there are fatal disadvantages to the cell when the concentration is high. The present inventors have found that when bovine serum albumin is used as a cryopreservative, the viability of cryopreserved cells, particularly microalgae cells, can be dramatically increased without harmful effects in the cell penetration type cryopreservative.
According to one embodiment of the present invention, the bovine serum albumin of the present invention is contained in an amount of 0.5 w / v% to 80 w / v%. The bovine serum albumin of the present invention preferably contains 1 w / v% to 80 w / v%, more preferably 5 w / v% to 50 w / v%, still more preferably 10 w / v% to 40 w / v%, most preferably from 20 w / v% to 30 w / v%. When the bovine serum albumin of the present invention is contained in an amount less than 0.5 w / v%, the effect as a cryoprotectant can not be exhibited significantly. When the bovine serum albumin is contained in an amount exceeding 80 w / v% Although our best knowledge does not negatively affect the cell itself, there is an inefficiency in reuse after thawing of frozen cells, and the rate of increase in microalga survival rate decreases with increasing content.
In one embodiment of the present invention, the composition of the present invention has a short-term cryopreserved survival rate of microalgae of 50% or more at -70 캜. The term " short-term cryopreserved survival rate "of the present invention means the survival rate after freezing at -70 ° C for 1.5 hours, followed by freezing at 196 ° C for 7 days, followed by rapid thawing at 37 ° C for 5 minutes. In the case of glycerol, which has been conventionally used as a cryopreservative in the past, even at the concentration showing the highest survival rate in the short-term cryopreserved survival rate test according to the concentration used, it did not exceed 40% (see FIG. However, in the case of an embodiment of the present invention using bovine serum albumin, the short-term cryopreserved survival rate was about 50% even with the use of 1 w / v% of the whole medium, and when using 10 w / v% Showing a short-term cryopreserved survival rate (see FIG. 1).
In one embodiment of the present invention, the composition of the present invention has a long-term cryopreserved survival rate of microalgae of 50% or more at -70 캜. The term " long-term cryopreserved survival rate "of the present invention represents the survival rate when stored for 6 months in a frozen state at -70 ° C and rapidly thawed in a 37 ° C water bath for 5 minutes. In the case of the control group without bovine serum albumin, the survival rate of long-term cryopreservation was less than 5%, but when the bovine serum albumin was added, the survival rate of long-term cryopreservation was more than 50% (cf.
In one embodiment of the present invention, the microalgae of the present invention specifically include, for example, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta, Bacillariophyta, Phaeophyta, Rholophyta, and Charophyta, and is not particularly limited.
In one embodiment of the present invention, the microalgae of the present invention are green algae.
In one embodiment of the present invention, the green algae of the present invention belong to the genus Clamidomonas, and specifically, for example, Chlamydomonas reinhardtii CW15- . The clamidomonas of the present invention is a single celled green alga having two flagella with the same length. Clamidomonas is widely used in photosynthesis studies with chlorella and is the optimal photosynthesis study model because it undergoes a simple metamorphosis step compared to chlorella. However, long-term preservation is limited by genetic variation or generational variation, and efficient cryopreservation is necessary to solve such a problem. The present inventors have found that the above problem can be solved by using bovine serum albumin as a cryopreservative.
In one embodiment of the present invention, the microalgae of the present invention are cells of an exponential phase. In the present invention, the term "exponential phase" means a period in which the increase of protoplasm is maximized and the rate is constant during the development of microalgae. The present invention can be used for long-term preservation of microalgae at the time when generation time is shortest and metabolites are most actively used for synthesis of protoplasts.
In one embodiment of the present invention, the composition further comprises DMSO (dimethyl sulfoxide) as a cryopreservation agent. DMSO has been known as a conventional cell penetration type cryopreservation agent and has been used as a cryopreservative under liquid nitrogen at -196 ° C. However, as can be seen from the results of FIG. 9, when DMSO alone is used at -70 ° C., It is not possible to observe the effect of increasing. However, when DMSO and bovine serum albumin are used together as a cryopreserving agent, a synergistic effect which is unpredictable as compared with the case of using DMSO or bovine serum albumin alone is exerted, and excellent cell survival effect is exerted.
In one embodiment of the invention, the DMSO of the present invention is comprised between 0.5 v / v% and 50 v / v%. More specifically, the DMSO of the present invention may be present in an amount ranging from 1 v / v% to 40 v / v%, more specifically from 3 v / v% to 35 v / v%, still more specifically from 5 v / . DMSO has the property of conventional cell penetration that, when used in large amounts, may have a negative effect on the cells.
According to another aspect of the present invention, the present invention provides a cell freezing preservation method comprising the steps of:
(a) adding cells to a preservative solution containing the composition for cryopreservation as described above;
(b) cryopreserving the preservation solution to which the cell has been added at -60 캜 to -80 캜.
The term "preservative solution" of the present invention means various media generally used for culturing cells. Specifically, for example, TAP (Tris-Acetate-Phosphate) medium, Allen's medium, BG11, Waris-H, Bold's Basal Medium, CM (Modified Closterium Medium, Watanabe et al. of Strains, Sixth Edition, 2000. Microalgae and Protozoa, Diatom Medium, Modified DM, HR-v1 or AW-v1, preferably TAP medium, There is no limitation. In step (b) of the present invention, the freezing temperature of the cells may be from -60 캜 to -80 캜, and appropriate adjustment is possible if necessary. The freezing temperature is preferably -65 째 C to -80 째 C, more preferably -70 째 C to -80 째 C.
In one embodiment of the invention, the cells of the invention are eukaryotic or prokaryotic cells derived from microalgae. The term "prokaryotic cell" of the present invention means a cell derived from a prokaryote, and is specifically a cell derived from a bacterium such as Escherichia coli, Staphylococcus aureus or Cholera, or a cyanophyta. "Eukaryotic cell" of the present invention means a cell derived from microalgae belonging to a eukaryote.
The cell cryopreservation method of the present invention corresponds to the method of using the composition for cryopreservation of cells, which is another embodiment of the present invention, so that redundant contents are omitted in order to avoid the excessive complexity described in the present specification.
According to another aspect of the present invention, the present invention provides a method of cryopreserving cells and obtaining viable cells comprising the steps of:
(a) adding cells to a preservative solution containing the composition for cryopreservation as described above;
(b) cryopreserving the preservation solution to which the cell has been added at -60 캜 to -80 캜; And
(c) thawing the frozen-assisted cells to obtain live cells.
In the present invention, a viable cell refers to an individual showing a physiological function inherent to a cell, and specifically refers to a cell that is not stained blue by Evans Blue staining and shows green color. &Quot; Obtaining viable cells "of the present invention includes not only obtaining viable cells separately from non-viable cells, but also obtaining viable cells and non-viable cells in a mixed state .
In one embodiment of the invention, the cells of the invention are eukaryotic or prokaryotic cells derived from microalgae. The term "prokaryotic cell" of the present invention means a cell derived from a prokaryote, and is specifically a cell derived from a bacterium such as Escherichia coli, Staphylococcus aureus or Cholera, or a cyanophyta. "Eukaryotic cell" of the present invention means a cell derived from microalgae belonging to a eukaryote.
The method of cryopreservation and the method of obtaining viable cells according to the present invention corresponds to a method of using a composition for microcavity cryopreservation according to another aspect of the present invention, so that redundant contents are omitted in order to avoid excessive complexity described in the present specification.
The features and advantages of the present invention are summarized as follows:
(a) The present invention provides a composition for cryopreserving cells.
(b) The present invention provides a cell freezing preservation method.
(c) The present invention provides a method of cryopreserving cells and obtaining viable cells.
(d) When the present invention is used, genetic variation or generational variation can be minimized in long-term preservation of cells.
(e) The cells can be frozen and stored in a -70 ° C freezer, not in a liquid nitrogen tank (-196 ° C), so that they are more economical and more effective in terms of safety.
Figure 1 shows the number of cells counted and survived when each cryopreservative was used.
Figure 2 shows the cell viability according to each cryopreservative.
Fig. 3 shows the culture conditions of the cells according to each freezing preservative.
Figure 4 shows the number of cells counted and cell status after cryopreservation for one week and one month at -70 ° C using bovine serum albumin as a cryopreservative.
FIG. 5 shows cell viability measured after cryopreservation at -70 ° C for one week and one month using bovine serum albumin as a cryopreservative.
6 shows the results of comparing the number of cells counted after cryopreservation for 6 months at -70 ° C using bovine serum albumin as a cryopreservation agent and immediately after thawing and for one week of culture.
7 shows the state of the cells after cryopreservation for 6 months at -70 ° C using bovine serum albumin as a cryopreservative.
8 shows the results of cell viability in the case of cryopreservation of Cladidomonas reinhardtii cells in TAP medium using 1 w / v% to 50 w / v% of bovine serum albumin as a cryopreservation agent.
9 shows the results of improving the cell survival rate when bovine serum albumin and DMSO are mixed and used as a cryopreservative.
FIG. 10 shows cell viability and colonies of prokaryotic cells (Escherichia coli DH5-Alpha) after cryopreservation at -70 ° C using bovine serum albumin as a cryopreservative.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Example 1: Preparation of cell suspension for cryopreservation and cryopreservation
The cryoprotectants were selected from methanol, which is the most used microalgae, glycerol which is mainly used in microorganisms, and bovine serum albumin which is a new cryoprotectant. For bovine serum albumin, bovine serum albumin (Cat. No. AD0023) from Bio Basic was dissolved in distilled water to prepare 50% bovine serum albumin solution. Various cryopreservants were sterilized using a 0.22 μm membrane filter and mixed at various concentrations ranging from 1 w / v% to 50% in TAP (Tris-Acetate-Phosphate) medium.
The experimental strain, Chlamydomonas reinhardtii CW15- (obtained from the University of British Columbia, Dr. Lee Jae-hyuk), was cultured as a healthy cell of the exponential phase. The cultured strains were counted with a hemocytometer and suspended in a TAP medium supplemented with a cryoprotectant at a concentration of 350 × 10 4 cells / ml.
Example 2: Comparison of the cryopreservation efficiency of existing cryopreservatives and new cryopreservatives
In order to compare the efficiency of existing cryopreservatives with those of new cryopreservatives, cryopreservation experiments were carried out. Cryopreservation and thawing experiments were carried out using the method proposed by the Chlamydomonas resource center (http://chlamycollection.org/cryopreservation/).
First, 1.8 ml of the above-prepared cell suspension for cryopreservation was dispensed into a 2 ml polyethylene freeze storage tube. After this, Mr. Nalgene's' Mr. Frosty 'two-compartment freezing containers (Cat. No. 5100-0001) were placed in a freezing storage tube. At this time, 250 ml of isopropanol was added in advance to the freezing container, and then the temperature was adjusted to a low temperature of 4 ° C. The frozen containers containing the microalgae strains were stored in a -70 ° C freezer for 1 hour and a half and the temperature was lowered. The tubes were then transferred separately to a liquid nitrogen tank (below -196 ° C) and cryopreserved for 7 days.
Cells were cryopreserved in a 37 ° C water bath for 5 minutes and centrifuged at 3200 rpm at 20 ° C. The recovered cell pellet was suspended in fresh TAP medium and then mixed with the same amount of Evans Blue solution. Cells dead by the Evans blue solution were stained blue and living cells appeared green, so cell viability was determined using cell counting methods (see Figure 1).
The survival rate of cells using bovine serum albumin as a cryopreservation agent was higher than that of known methanol or glycerol (Fig. 2).
In addition, the survival rate of bovine serum albumin was further expanded to show cell viability. As a result, the survival rate was about 90% or more at about 25% (w / v) And showed similar survival rates (see FIG. 8).
In addition, when thawed cells were cultured continuously, cells using previously known methanol or glycerol as cryopreservation agents were denatured as color and died like the cells of negative control, but cells using bovine serum albumin as a cryopreservation agent showed the original strain color (Fig. 3). ≪ tb >< TABLE >
Example 3: Cryopreservation experiment using bovine serum albumin as a cryopreservative at -70 캜
A new cryopreservation method was used to confirm that the excellent cryopreservation efficiency of bovine serum albumin, a newly obtained cryopreservative in Example 2, was maintained even when using a -70 ° C freezer. Concentrations of bovine serum albumin 10 w / v% were set as cryopreservation concentrations. The experimental group in which the temperature was dropped for 1 hour and 30 minutes in the -70 ° C freezer as in Example 2 was kept in the -70 ° C freezer continuously for one week and one month without being transferred to the liquid nitrogen tank, . As a result, high survival rate was confirmed (see FIGS. 4 and 5).
In addition, when DMSO is used instead of bovine serum albumin, bovine serum albumin alone is used, and when 10 v / v% of DMSO is added to bovine serum albumin based on TAP medium, I experimented to see if it affected. As a result, when DMSO alone was used, the effect of increasing the cell survival rate was hardly observed, and when the bovine serum albumin alone was used, the expected increase in cell survival rate was observed as the concentration was increased. Surprisingly, when 10% v / v% of DMSO was mixed with bovine serum albumin, remarkable increase in cell viability was observed. In particular, in the case of using 1% bovine serum albumin and DMSO in combination, remarkable results were observed in which the survival rate of less than 10% was increased to more than 80% when bovine serum albumin or DMSO alone was used (see FIG. 9). In FIG. 9, when the CPA concentration (%) was 1, the test results were obtained using 1 v / v% of DMSO, 1 w / v% of BSA, 1 w / v% of BSA and 10 v / v% of DMSO And 5% of DMSO, 5% of BSA, 5% of BSA, and 10% of DMSO, respectively, in the case of CPA concentration (%) 5.
Example 4: Long-term cryopreservation experiment at -70 캜 for 6 months or longer
In order to confirm that the experiment conducted in Example 3 was effective even for long-term preservation, a long-term cryopreservation experiment was conducted for 6 months. Cells were frozen after the same experiment as in Example 3, and then thawed after 6 months to confirm cell viability. As a result, high survival rate was confirmed (see FIGS. 6 and 7).
The number of cells was counted again one week after the thawing, and it was observed that the cells were maintained in a healthy state for continuous cultivation. As a result, it was confirmed that the cells maintained a healthy state in which they could cultivate after 7 days of thawing (see FIG. 6) .
Example 5: Prokaryotic (< RTI ID = 0.0 > Escherichia coli DH5-Alpha) was used for the cryopreservation experiment
Cell viability was measured after cryopreservation at -70 ° C using 10 v / v% glycerol or 10 w / v% bovine serum albumin as a cryopreservative. The cell culture of Escherichia coli DH5-Alpha, an experimental subject, was suspended using 10 v / v% glycerol or 10 w / v% bovine serum albumin as a cryoprotectant, respectively. Cell suspensions were dispensed in 1.8 mL aliquots into 2 mL polyethylene freeze storage tubes. After this, Mr. Nalgene's' Mr. Frosty 'two-compartment freezing containers (Cat. No. 5100-0001) were placed in a freezing storage tube. At this time, 250 mL of isopropanol was added in advance to the freezing container, and the temperature was adjusted to a low temperature of 4 ° C. The frozen containers containing the microalgae strains were stored in a -70 ° C freezer for one and a half hours and then stored at -70 ° C in a freezer.
To confirm cell viability, the cells were thawed in a 37 ° C water bath for 5 minutes, then diluted to 1/100000 and plated on solid LB medium. The cell viability was then checked by counting the number of colonies grown by overnight incubation in a 37 ° C incubator.
As a result, it was confirmed that 10% v / v% of glycerol and 10% w / v% of bovine serum albumin, which were previously used cryopreservation agents, did not significantly differ in cell survival rate, 10).
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (17)
The bovine serum albumin is contained in an amount of more than 10% (w / v) to less than 40% (w / v) relative to 100% (w / v) / v). < / RTI >
(a) adding cells to a preservative solution comprising the composition of any one of claims 1, 3, 6 to 11; And
(b) cryopreserving the preservation solution to which the cell has been added at -60 캜 to -80 캜.
(a) adding cells to a preservative solution comprising the composition of any one of claims 1, 3, 6 to 11;
(b) cryopreserving the preservation solution to which the cell has been added at -60 캜 to -80 캜; And
(c) thawing the cryopreserved cells to obtain live cells.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20140046507 | 2014-04-18 | ||
| KR1020140046507 | 2014-04-18 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20150121667A KR20150121667A (en) | 2015-10-29 |
| KR101767195B1 true KR101767195B1 (en) | 2017-08-24 |
Family
ID=54430603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150054751A KR101767195B1 (en) | 2014-04-18 | 2015-04-17 | A Composition for Cryopreservation of Cell and a Method for Cryopreservation of Cell Using the Same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101767195B1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102635317B1 (en) | 2017-01-26 | 2024-02-08 | 중앙대학교 산학협력단 | Composition for cryopreservating spermatogonial stem cells containing necrostatin-1 and method of cryopreservation of spermatogonial stem cells using same |
| KR20180088071A (en) | 2017-01-26 | 2018-08-03 | 중앙대학교 산학협력단 | Composition for cryopreservating spermatogonial stem cells containing melatonin and method of cryopreservation of spermatogonial stem cells using same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011505152A (en) * | 2007-12-04 | 2011-02-24 | プロテオバイオアクティブズ・ピーティーワイ・リミテッド | Protection of progenitor cells and regulation of their differentiation |
-
2015
- 2015-04-17 KR KR1020150054751A patent/KR101767195B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011505152A (en) * | 2007-12-04 | 2011-02-24 | プロテオバイオアクティブズ・ピーティーワイ・リミテッド | Protection of progenitor cells and regulation of their differentiation |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20150121667A (en) | 2015-10-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Taylor et al. | Cryopreservation of eukaryotic algae–a review of methodologies | |
| Seo et al. | Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide | |
| CN104145943A (en) | Cryopreservation protection liquid for Wharton jelly tissues of human umbilical cord and preparation and application of cryopreservation protection liquid | |
| CN102428911A (en) | Cryogenic storing protection liquid for human umbilical cord Wharton's jelly tissue block | |
| Katkov et al. | Low-and high-temperature vitrification as a new approach to biostabilization of reproductive and progenitor cells | |
| CN108094405B (en) | Cell cryopreservation composition and application thereof | |
| Smith et al. | Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi | |
| CN107306940A (en) | The spermary Cryoprotectant and the preparation method of spermatogonium of a kind of seawater fish | |
| JP2019514442A (en) | Antifreezing medium and method for preventing recrystallization | |
| KR101767195B1 (en) | A Composition for Cryopreservation of Cell and a Method for Cryopreservation of Cell Using the Same | |
| Togawa et al. | Cryopreservation of Marchantia polymorpha spermatozoa | |
| Poncet et al. | Cryopreservation of the unicellular marine alga, Nannochloropsis oculata | |
| Chen et al. | Cryopreservation of tissues and organs: present, bottlenecks, and future | |
| CN106754376B (en) | A kind of microorganism low-temperature preservation protective agent | |
| Pandey et al. | In vitro conservation and cryopreservation of vegetatively propagated crop germplasm | |
| Eichlerová et al. | Preservation of basidiomycete strains on perlite using different protocols | |
| KR20180070937A (en) | Composition for cryopreservation of microalgae and method for cryopreservation of microalgae using the same | |
| KR102569522B1 (en) | Composition for cryopreservation of cell and method for cryopreservation of cell using the same | |
| Zhuang et al. | Cryopreservation of filaments of Scytosiphon lomentaria by vitrification | |
| US10611996B2 (en) | Preservation and storage of biological specimens | |
| US20070259327A1 (en) | Cryopreservation medium and cryopreservation method for tissues and cells | |
| Harding et al. | Deployment of the encapsulation/dehydration protocol to cryopreserve microalgae held at The Sammlung von Algenkulturen, Universität Göttingen, Germany | |
| Day | Cryopreservation of macroalgae | |
| Fleck et al. | A brief review of cryobiology with reference to cryo field emission scanning electron microscopy | |
| Hong et al. | A modified cryopreservation method of psychrophilic chlorophyta Pyramimonas sp. from Antarctica |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |