CN113812398B - Cryogenic freezing method of scaffolds for tissue repair - Google Patents
Cryogenic freezing method of scaffolds for tissue repair Download PDFInfo
- Publication number
- CN113812398B CN113812398B CN202111182771.3A CN202111182771A CN113812398B CN 113812398 B CN113812398 B CN 113812398B CN 202111182771 A CN202111182771 A CN 202111182771A CN 113812398 B CN113812398 B CN 113812398B
- Authority
- CN
- China
- Prior art keywords
- trehalose
- tissue engineering
- stem cells
- mesenchymal stem
- cryogenic freezing
- 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.)
- Active
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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- 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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/72—Chitin, chitosan
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Rheumatology (AREA)
- Physiology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A low-temperature freezing method of a scaffold for tissue repair, the scaffold for tissue repair is cultured with mesenchymal stem cells in a 24-well culture plate; changing the liquid every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure; immersing the prepared tissue engineering structure in a cryoprotectant, and incubating for 12 hours in an incubator; the scaffold for tissue repair is made of gallic acid grafted chitosan. The low-temperature freezing method is suitable for low-temperature freezing preservation of tissue engineering structures (such as scaffolds) containing living cells, maintains the survival rate and recovery rate of the cells at a higher level, and can enable the frozen stem cells to continue to maintain the dryness and differentiation potential after recovery.
Description
Technical Field
The present invention relates to a method for preserving biological products, in particular to a method for freezing scaffolds for tissue repair at low temperature, and the biological activity of the biological products (such as cells) in the scaffolds is maintained.
Background
Tissue engineering and regenerative medicine are considered to be a promising approach to repair damaged tissues and maintain biological function. Among the therapeutic methods of tissue engineering and regenerative medicine, cell-based therapeutic methods are one of the most recent hot spots. These therapies have been focused on the ability to regenerate damaged tissues and provide a number of beneficial cytokines. In particular, stem cells can be maintained dry by implanting them in a scaffold. In previous studies, we established tissue engineering structures based on gallic acid grafted chitosan scaffolds and mesenchymal stem cells. In addition, the difficulty in scaffold fabrication is the encapsulation of cells into the scaffold to produce an active composite biomaterial; however, the process of procurement, growth and packaging is very cumbersome and time consuming and difficult to apply to acute diseases. Therefore, the development of an effective cryopreservation strategy to maintain the biological activity and function of the tissue engineering structure is one of the most important research fields of modern tissue engineering and regenerative medicine. The integrity tissue engineering structure capable of being preserved not only can save the time of donors and recipients, but also can establish a global high-quality tissue engineering structure cell biological information base. In preparing engineered tissues for clinical applications, cryopreservation strategies have the potential to enable the off-the-shelf supply of tissue engineered structures.
Disclosure of Invention
It is an object of the present invention to provide a cryogenic freezing method for cryo-preservation of scaffolds for tissue repair.
It is another object of the present invention to provide a cryogenic freezing method for cryo-preservation of scaffolds for tissue repair containing bioactive substances.
A cryogenic freezing method comprising:
scaffolds and mesenchymal stem cells (e.g., 2X 10) for tissue repair 4 Individual cells) were cultured in 24-well culture plates. Changing the culture solution every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure;
the prepared tissue engineering structure was immersed in cryoprotectant and incubated in an incubator for 12 hours.
According to the method, the scaffold for tissue repair is prepared by grafting gallic acid on chitosan. The scaffolds were soaked in 70% v/v ethanol overnight before co-culturing with mesenchymal stem cells, and then irradiated under ultraviolet rays for 12 hours.
The cryoprotectant adopted by the method comprises trehalose derivatives, fetal calf serum and DMEM medium.
The other cryoprotectant comprises 25 mg/mL-100 mg/mL of trehalose derivatives, fetal calf serum and a DMEM medium.
Another cryoprotectant comprises 25 mg/mL-100 mg/mL trehalose derivatives, 10% v/v fetal calf serum and DMEM medium.
Another cryoprotectant comprises 25 mg/mL-100 mg/mL of trehalose derivatives, 10% v/v fetal bovine serum and 90% v/v DMEM medium.
Another cryoprotectant comprises 50mg/mL trehalose derivatives, fetal bovine serum and DMEM medium.
Another cryoprotectant, comprises 50mg/mL trehalose derivatives, 10% v/v fetal bovine serum and DMEM medium.
Another cryoprotectant, comprising 50mg/mL trehalose derivatives, 10% v/v fetal bovine serum, and 90% v/vDMEM medium.
According to the method, the trehalose derivatives comprise trehalose and epichlorohydrin, and the trehalose and the epichlorohydrin are covalently bonded.
The trehalose derivative comprises the following components in parts by weight:
4-12 trehalose and 3-10 epichlorohydrin, wherein the trehalose and the epichlorohydrin are covalently bonded.
The trehalose derivative comprises the following components in parts by weight:
4-12 parts of trehalose and 3-10 parts of epoxy chloropropane, and is prepared by the following method:
dissolving 4-12 parts by weight of trehalose and NaOH in deionized water, reacting and stirring at 65-75 ℃ for 15-45 minutes at a pH of 11-12 to obtain a transparent viscous liquid. Then adding 3-10 parts by weight of epoxy chloropropane, standing for 36-72 hours, adjusting the pH value of the solution to be neutral (such as the pH value of 7 +/-0.2) by using hydrochloric acid, dialyzing in deionized water for 3-5 days, and freeze-drying to obtain the trehalose derivative.
The trehalose derivative can enter cells and is used as a cryoprotectant for the cells, and the dosage is as follows: but not limited to 25 mg/mL-100 mg/mL, has the effect of protecting frozen cells, improves the survival rate and recovery rate of the cells, and can keep the dryness and differentiation potential of stem cells.
The low-temperature freezing method can maintain the survival rate and the recovery rate of the cells at a higher level by the low-temperature freezing preservation of the tissue engineering structure (such as a scaffold) containing the living cells, and can ensure that the frozen stem cells continue to maintain the dryness and differentiation potential after recovery.
Drawings
FIG. 1 shows the survival rate of mesenchymal stem cells in a tissue engineering structure frozen at low temperature;
FIG. 2 is the recovery rate of mesenchymal stem cells in a tissue engineering structure frozen at low temperature;
FIG. 3 shows the proliferation capacity of mesenchymal stem cells in a tissue engineering structure frozen at low temperature;
FIG. 4 is a graph of the colony forming ability of mesenchymal stem cells in a cryogenically frozen tissue-engineered structure;
FIG. 5 is a data statistics chart showing the differentiation capacity of adipose-forming, osteogenic and chondrogenic mesenchymal stem cells in a tissue engineering structure frozen at a low temperature.
Detailed Description
The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
The tissue engineering structure containing the mesenchymal stem cells adopted by the following embodiments of the invention is prepared by the following method:
culturing scaffold (made of gallic acid grafted chitosan) for tissue repair and mesenchymal stem cells in 24-well culture plate; changing the culture solution every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure;
the prepared tissue engineering structure was immersed in cryoprotectant and incubated in an incubator for 12 hours. Specifically, such as: soaking gallic acid grafted chitosan scaffold in 70% v/v ethanol overnight, and then under ultravioletThe irradiation was carried out for 12 hours. Then, gallic acid grafted chitosan scaffold and mesenchymal stem cells (2 × 10) 4 Individual cells) were cultured in 24-well culture plates. Changing the culture solution every 2 days, and culturing for 7 days to form a tissue engineering structure.
The preparation method of the gallic acid grafted chitosan scaffold comprises the following steps:
first 28mmol of gallic acid and 2.8mmol of EDC were dissolved in 40mL 70% ethanol and 2.8mmol of NHS was added to the solution. The resulting solution was stirred in an ice bath, then 1.5g of chitosan dispersed in 110mL of 70% ethanol was added after 1 hour. The solution was further stirred in an ice bath for 30 minutes and finally at room temperature for 24 hours. The precipitate was collected by filtration and washed with ethanol. After 3 days of dialysis against deionized water to remove possible residual reagents, gallic acid-grafted chitosan was obtained by vacuum freeze-drying. The porous gallic acid grafted chitosan scaffold is prepared by utilizing the physical property that gallic acid grafted chitosan is dissolved in an acid solution and is not dissolved under an alkaline condition. First, 1g of gallic acid-grafted chitosan was dissolved in 50ml of 0.1 v/v% glacial acetic acid. Then 250 mu L of gallic acid grafted chitosan solution is added into each hole of the polytetrafluoroethylene plate, and the mixture is kept in a refrigerator at the temperature of 20 ℃ below zero for 24 hours. The samples were then lyophilized in a freeze dryer. The samples were deacidified with 2 w/v% NaOH solution for 4 hours and lyophilized in a freeze dryer. Finally obtaining the gallic acid grafted chitosan scaffold.
The experimental method for the survival rate of the mesenchymal stem cells in the tissue engineering structure comprises the following steps:
the prepared tissue engineering structure was washed twice with PBS buffer, and the mesenchymal stem cells were digested with 0.25% trypsin and 1mM EDTA. The cells were then centrifuged and the resulting cell suspension, trypan blue 1:1, was mixed and counted on a hemocytometer. The survival rate of the cells was (viable cells/total cells) × 100.
The experimental method for the Survival Rate (Survival Rate,%) and the resuscitation Rate (Cell Recovery Rate,%) of mesenchymal stem cells in the frozen tissue engineering structure is as follows:
the frozen tissue engineering structures were washed twice with PBS buffer. Mesenchymal stem cells were digested with 0.25% trypsin and 1mM EDTA. The cells were then centrifuged. The cell suspensions, trypan blue 1:1 were then mixed and counted on a hemacytometer. The survival rate of the cells (live cells after cryopreservation/total cells after cryopreservation) × 100, and the recovery rate of the cells (live cells after cryopreservation/live cells before cryopreservation) × 100.
The repopulation capacity (Cell Proliferation) of mesenchymal stem cells in a tissue engineering structure is determined by CCK-8. The specific method comprises the following steps: digesting the mesenchymal stem cells from the tissue engineering structure, then inoculating the mesenchymal stem cells into a 24-well cell culture plate at a density of 10,000/well and 5% CO at 37 ℃ 2 And (4) culturing. The solution was mixed with the solution of CCK-8 in a volume of 9:1 using alpha-MEM to obtain a working solution of CCK-8, 200. mu.L of the working solution of CCK-8 was added and incubated with the cells for 2 hours, and then 100. mu.L of the sample solution was taken out and absorbance was measured at 450 nm.
The experimental method for the dryness and three differentiation capacities of the mesenchymal stem cells in the tissue engineering structure comprises the following steps:
and (3) detecting the formation capacity of the mesenchymal stem cell colony in the tissue engineering structure by using a crystal violet staining method. Digesting the mesenchymal stem cells from the tissue engineering structure. Then, the mesenchymal stem cells were inoculated into 6-well cell culture plates at a density of 200 cells/well and 37 ℃ with 5% CO 2 The culture was carried out for 14 days. Before crystal violet staining, wash 2 times with PBS and fix with 4% paraformaldehyde for 15 min at room temperature. Add crystal violet staining solution and incubate for 30 min.
Three differentiation potentials of the mesenchymal stem cells on the tissue engineering structure are detected through adipogenesis, osteogenesis and cartilage differentiation. According to the manufacturer's instructions. Adopting a adipogenic culture medium, an osteogenic culture medium and a chondrogenic culture medium to respectively mediate MSCgo TM The differentiation capability of the mesenchymal stem cells after the tissue engineering structure is frozen is researched. After 21 days of culture, cells were stained with adipogenic, osteogenic and cartilage staining kit. Washed 2 times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were stained with oil Red o (oil Red o), alizarin Red s (alzarin Red s) and alisin Blue (Alcian Blue) for adipogenesis, osteogenesis and cartilage differentiation. The cells were then washed three times with wash solution to remove excess dye. Then observed with an optical microscope The differentiated cells were examined. Finally, oil red O, alizarin Red S and Alisin blue stained cells were analyzed for adipogenesis, osteogenesis and chondrogenesis at wavelengths of 500nm, 550nm and 600 nm.
Example 1
The survival rate of mesenchymal stem cells in the tissue engineering structure is shown in figure 1.
The proliferation capacity of mesenchymal stem cells in tissue engineering structures is shown in figure 3.
The colony forming ability of the mesenchymal stem cells in the tissue engineering structure is shown in figure 4, and the three differentiation abilities of the mesenchymal stem cells in the tissue engineering structure are shown in figure 5.
Example 2
The tissue engineering structures were cryopreserved in fetal bovine serum (90% FBS) cryopreserved tubes containing 10% DMSO. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.
The survival rate of the mesenchymal stem cells in the tissue engineering structure after cryopreservation is shown in fig. 1. The survival rate of the mesenchymal stem cells in the tissue engineering structure after cryopreservation is shown in fig. 2.
The proliferation ability of the mesenchymal stem cells in the tissue engineering structure after freezing storage is shown in figure 3, the colony formation ability of the mesenchymal stem cells is shown in figure 4, and the three differentiation abilities of the mesenchymal stem cells are shown in figure 5.
Example 3 electrochemical analysis of Breast cancer Stem cells in the Co-existence of interfering cells
4.00g trehalose and 1.82g NaOH were dissolved in 10mL deionized water, reacted at 75 ℃ and stirred for 30 minutes to give a clear viscous liquid. Then 3.23g of epichlorohydrin were added. The solution was allowed to stand, causing an increase in viscosity. After 48 hours, the pH of the solution was adjusted to 7.00 with hydrochloric acid. Dialyzed against deionized water for 3 days, and then lyophilized in vacuo to give trehalose polymer.
The prepared tissue engineering structure was immersed in a frozen stock solution of the above-synthesized trehalose derivative (25mg/mL) and incubated in an incubator for 12 hours. Each tissue engineering construct was then transferred to a sterile 1.5mL cryovial and 0.75mL trehalose derivative cryovial (25mg/mL) was added. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.
The survival rate of the mesenchymal stem cells in the frozen tissue engineering structure is shown in figure 1, and the recovery rate of the mesenchymal stem cells is shown in figure 2.
Example 4
The prepared tissue engineering structure was immersed in a frozen stock solution of trehalose derivative (50mg/mL) prepared in example 3 and incubated in an incubator for 12 hours. Each tissue engineering construct was then transferred to a sterile 1.5mL cryovial and 0.75mL trehalose derivative cryovial (25mg/mL) was added. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.
The survival rate of the mesenchymal stem cells in the frozen tissue engineering structure is shown in figure 1, and the recovery rate of the mesenchymal stem cells is shown in figure 2.
The proliferation ability of mesenchymal stem cells in the tissue engineering structure after cryopreservation is shown in fig. 3, the colony formation ability of mesenchymal stem cells is shown in fig. 4, and the differentiation ability of adipose, osteogenic and chondrogenic of mesenchymal stem cells is shown in example 4 in fig. 5.
Example 5
The prepared tissue engineering structure was immersed in the frozen stock solution of trehalose derivatives (100mg/mL) of example 3 and incubated in an incubator for 12 hours. Each tissue engineering construct was then manually transferred to a sterile 1.5mL cryovial and 0.75mL trehalose derivative cryovial (25mg/mL) was added. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.
The survival rate of the mesenchymal stem cells in the frozen tissue engineering structure is shown in figure 1, and the recovery rate of the mesenchymal stem cells is shown in figure 2.
Compared with the above examples, the trehalose derivatives provided by the present application have high cell recovery rate (39.56 ± 2.95% -52.02 ± 4.01%) and survival rate (60.75 ± 5.02% -85.38 ± 2.39%) for tissue engineering scaffolds kept frozen, and mesenchymal stem cells in the frozen tissue engineering scaffolds have desiccation and three differentiation capacities, so that the trehalose derivatives can be used as a substitute for DMSO for cryogenically freezing tissue engineering scaffolds containing cells.
The trehalose derivative with the concentration of 50mg/mL is added into the cryopreservation liquid, so that the cell recovery rate and the cell survival rate of the cells can be maintained at high levels, and the economic benefit is better.
Claims (8)
1. A cryogenic freezing method, comprising:
culturing the scaffold for tissue repair and mesenchymal stem cells in a 24-well culture plate; changing the liquid every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure;
immersing the prepared tissue engineering structure in a cryoprotectant, and incubating for 12 hours in an incubator;
the scaffold for tissue repair is made of gallic acid grafted chitosan;
the cryoprotectant contains trehalose derivatives, is obtained by covalently bonding trehalose and epichlorohydrin, and has the dosage of 25 mg/mL-100 mg/mL.
2. The cryogenic freezing method according to claim 1, wherein the scaffold for tissue repair is immersed in 70% v/v ethanol overnight before being co-cultured with mesenchymal stem cells, and then irradiated under ultraviolet rays for 12 hours.
3. A method of cryogenic freezing as claimed in claim 1, wherein the trehalose derivative is present in an amount of 50 mg/mL.
4. A low-temperature freezing method as claimed in claim 1, wherein the trehalose derivative comprises 4-12 parts by weight of trehalose and 3-10 parts by weight of epichlorohydrin.
5. A cryogenic freezing process according to claim 1, wherein the product is obtained by
Dissolving 4-12 parts by weight of trehalose and NaOH in deionized water, reacting and stirring at 65-75 ℃ for 15-45 minutes at a pH of 11-12 to obtain a transparent viscous liquid;
and then adding 3-10 parts by weight of epoxy chloropropane, standing for 36-72 hours, adjusting the pH value of the solution to be neutral by using hydrochloric acid, dialyzing in deionized water for 3-5 days, and freeze-drying to obtain the trehalose derivative.
6. The cryogenic freezing method of claim 1, wherein the cryoprotectant further comprises fetal bovine serum and DMEM medium.
7. A cryogenic freezing method according to claim 1, wherein the cryoprotectant further comprises 10% v/v fetal bovine serum.
8. A cryogenic freezing method according to claim 1, wherein the cryoprotectant further comprises 90% v/v DMEM medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111182771.3A CN113812398B (en) | 2021-10-11 | 2021-10-11 | Cryogenic freezing method of scaffolds for tissue repair |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111182771.3A CN113812398B (en) | 2021-10-11 | 2021-10-11 | Cryogenic freezing method of scaffolds for tissue repair |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113812398A CN113812398A (en) | 2021-12-21 |
| CN113812398B true CN113812398B (en) | 2022-07-29 |
Family
ID=78916333
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111182771.3A Active CN113812398B (en) | 2021-10-11 | 2021-10-11 | Cryogenic freezing method of scaffolds for tissue repair |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113812398B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979104A (en) * | 2010-11-04 | 2011-02-23 | 南通大学 | Porous chitosan scaffold, and neural stem cell porous chitosan scaffold and application thereof |
| CN103965372A (en) * | 2014-04-16 | 2014-08-06 | 南京农业大学 | Preparation method of chitosan-gallate grafted copolymer |
| CN105435310A (en) * | 2015-12-24 | 2016-03-30 | 钟达 | Method for preliminarily constructing tissue-engineered cartilages in vitro from Wharton jelly of umbilical cord |
| CN110072992A (en) * | 2016-12-14 | 2019-07-30 | 株式会社大塚制药工场 | Mammalian cell freezen protective liquid |
| CN110628089A (en) * | 2019-10-11 | 2019-12-31 | 浙江理工大学 | Preparation method of poly-gallic acid/chitosan antibacterial material |
| CN111035611A (en) * | 2019-11-22 | 2020-04-21 | 中冠赛尔生物科技(北京)有限公司 | Mesenchymal stem cell biological scaffold and preparation method and application thereof |
| CN111282020A (en) * | 2020-02-14 | 2020-06-16 | 中国人民解放军陆军军医大学 | Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof |
| CN112870445A (en) * | 2021-01-29 | 2021-06-01 | 周建大 | Preparation method and application of soft tissue repair material |
| CN112961264A (en) * | 2021-03-01 | 2021-06-15 | 华中科技大学 | Chitosan-gallic acid graft copolymer and preparation method and application thereof |
| CN113318276A (en) * | 2021-03-29 | 2021-08-31 | 中山大学附属第一医院 | Preparation method of multiple-crosslinking injectable hydrogel and preparation method and application thereof |
-
2021
- 2021-10-11 CN CN202111182771.3A patent/CN113812398B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979104A (en) * | 2010-11-04 | 2011-02-23 | 南通大学 | Porous chitosan scaffold, and neural stem cell porous chitosan scaffold and application thereof |
| CN103965372A (en) * | 2014-04-16 | 2014-08-06 | 南京农业大学 | Preparation method of chitosan-gallate grafted copolymer |
| CN105435310A (en) * | 2015-12-24 | 2016-03-30 | 钟达 | Method for preliminarily constructing tissue-engineered cartilages in vitro from Wharton jelly of umbilical cord |
| CN110072992A (en) * | 2016-12-14 | 2019-07-30 | 株式会社大塚制药工场 | Mammalian cell freezen protective liquid |
| CN110628089A (en) * | 2019-10-11 | 2019-12-31 | 浙江理工大学 | Preparation method of poly-gallic acid/chitosan antibacterial material |
| CN111035611A (en) * | 2019-11-22 | 2020-04-21 | 中冠赛尔生物科技(北京)有限公司 | Mesenchymal stem cell biological scaffold and preparation method and application thereof |
| CN111282020A (en) * | 2020-02-14 | 2020-06-16 | 中国人民解放军陆军军医大学 | Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof |
| CN112870445A (en) * | 2021-01-29 | 2021-06-01 | 周建大 | Preparation method and application of soft tissue repair material |
| CN112961264A (en) * | 2021-03-01 | 2021-06-15 | 华中科技大学 | Chitosan-gallic acid graft copolymer and preparation method and application thereof |
| CN113318276A (en) * | 2021-03-29 | 2021-08-31 | 中山大学附属第一医院 | Preparation method of multiple-crosslinking injectable hydrogel and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113812398A (en) | 2021-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109090100A (en) | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method | |
| KR101679889B1 (en) | Method for the cryopreservation of cells, artificial cell constructs or three-dimensional complex tissues assemblies | |
| Wang et al. | Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability | |
| CN113735989A (en) | Trehalose derivative and cryoprotectant and application thereof | |
| KR102506822B1 (en) | Method for obtaining an enriched population of functional mesenchymal stem cells, cells obtained therefor, and compositions comprising the same | |
| KR101321144B1 (en) | Vitrification medium for stem cells and vitrification method for stem cells using the same | |
| US20200163326A1 (en) | Cryopreservation | |
| CN104800891A (en) | Extracellular matrix biological material for improving biological anti-oxidizing function of mesenchymal stem cells of in-vitro culture, preparation method and application thereof | |
| CN107185047A (en) | Organization engineered cartilage graft and preparation method thereof | |
| Sajed et al. | Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment | |
| CN113812398B (en) | Cryogenic freezing method of scaffolds for tissue repair | |
| CN105713869A (en) | In-vitro three-dimensional isolated culture and storage method for hCDMSC (human chorion-derived mesenchymal stem cell) for clinical adoptive therapy | |
| CN114686421B (en) | Preparation method and application of lung tissue extracellular matrix-free microcarrier | |
| CN107164306B (en) | Solid culture medium for cornea model low-temperature preservation and preparation method and application method thereof | |
| CN107308497B (en) | The building of nucleus pulposus cell source property activity microcarrier | |
| Farooque et al. | Protocol development for vitrification of tissue-engineered cartilage | |
| TWI837281B (en) | Cell cryopreservation solution and gradual freezing method of cells | |
| CN113133445B (en) | Preservation method, transportation method and treatment method of mesenchymal stem cells | |
| CN111374123B (en) | Use method of neutral amino acid as cell cryoprotectant and application thereof | |
| CN114958731A (en) | Nano-pore silk fibroin scaffold-based engineered adipose tissue model, and preparation method and application thereof | |
| CN114946833B (en) | Application of schisandrin B in preparation of in-vitro biological sample preservation solution | |
| CN115053894A (en) | Cryopreservation liquid supporting high-density storage of umbilical cord blood mesenchymal stem cells and application thereof | |
| CN101332312A (en) | Processing method of tissue engineered implant | |
| CN117770230A (en) | Non-freezing type low-temperature transportation method and culture medium for PDLSC membrane | |
| CN116439229A (en) | Umbilical cord mesenchymal stem cell preservation solution and preservation method |
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 |