CN114836364A - Temperature-sensitive cell culture microcarrier and preparation method thereof - Google Patents
Temperature-sensitive cell culture microcarrier and preparation method thereof Download PDFInfo
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- CN114836364A CN114836364A CN202210623623.9A CN202210623623A CN114836364A CN 114836364 A CN114836364 A CN 114836364A CN 202210623623 A CN202210623623 A CN 202210623623A CN 114836364 A CN114836364 A CN 114836364A
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- 238000004113 cell culture Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims description 8
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 claims abstract description 29
- -1 hydroxypropyl diethylaminoethyl Chemical group 0.000 claims abstract description 29
- 239000004005 microsphere Substances 0.000 claims abstract description 15
- 229920002678 cellulose Polymers 0.000 claims abstract description 12
- 239000001913 cellulose Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 34
- 238000003756 stirring Methods 0.000 claims description 19
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 15
- 239000004202 carbamide Substances 0.000 claims description 15
- 239000005662 Paraffin oil Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 5
- 239000003995 emulsifying agent Substances 0.000 claims description 5
- 230000001804 emulsifying effect Effects 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 5
- RTRZIWZZYGDMFE-UHFFFAOYSA-N 1-chloro-n,n-diethylethanamine;hydron;chloride Chemical compound Cl.CCN(CC)C(C)Cl RTRZIWZZYGDMFE-UHFFFAOYSA-N 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 37
- 238000003306 harvesting Methods 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
- 102000018697 Membrane Proteins Human genes 0.000 abstract description 2
- 108010052285 Membrane Proteins Proteins 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000029087 digestion Effects 0.000 abstract description 2
- 239000008363 phosphate buffer Substances 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- RAGSWDIQBBZLLL-UHFFFAOYSA-N 2-chloroethyl(diethyl)azanium;chloride Chemical compound Cl.CCN(CC)CCCl RAGSWDIQBBZLLL-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000001066 destructive effect Effects 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- 238000011072 cell harvest Methods 0.000 description 2
- 125000001309 chloro group Chemical group Cl* 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- JEQDSBVHLKBEIZ-REOHCLBHSA-N (2s)-2-chloropropanoyl chloride Chemical compound C[C@H](Cl)C(Cl)=O JEQDSBVHLKBEIZ-REOHCLBHSA-N 0.000 description 1
- 101710141544 Allatotropin-related peptide Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- DILRJUIACXKSQE-UHFFFAOYSA-N n',n'-dimethylethane-1,2-diamine Chemical compound CN(C)CCN DILRJUIACXKSQE-UHFFFAOYSA-N 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
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- 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
- C12N5/0068—General culture methods using substrates
- C12N5/0075—General culture methods using substrates using microcarriers
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- 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
- C12N2533/78—Cellulose
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- 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
- C12N2535/00—Supports or coatings for cell culture characterised by topography
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- 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
- C12N2539/00—Supports and/or coatings for cell culture characterised by properties
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
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- Microbiology (AREA)
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Abstract
The invention discloses a temperature-sensitive cell culture microcarrier, which is a microsphere with a porous structure prepared by taking modified cellulose, namely hydroxypropyl diethylaminoethyl cellulose as a material. The invention has the beneficial effects that in the process of harvesting after cell culture, the temperature is reduced to 0-5 ℃, and the microcarrier is converted from a gel state to a solution state, so that cells growing in and on the microcarrier automatically fall off and are separated from the microcarrier. Different from the traditional separation method of trypsase digestion cells, the novel method can better preserve cell surface protein and markers, ensure high cell activity and is a lossless cell harvesting method. And aiming at the defect improvement of the existing temperature-sensitive microcarrier, the cell harvesting rate is greatly improved. The novel temperature-sensitive microcarrier has good cell attaching and detaching capacity, and can be a good material for large-scale culture of attached cells.
Description
Technical Field
The invention relates to the technical field of cell culture materials, in particular to a temperature-sensitive cell culture microcarrier and a preparation method thereof.
Background
The commercial temperature-sensitive cell culture surface is available on the market, and is mainly realized by grafting a temperature-sensitive material onto the surface of a culture device. The surface can lead the cells to automatically fall off by changing the temperature after the cell culture is finished, thereby reducing the damage to the cells.
However, various temperature-sensitive cell culture surfaces in the prior art have various defects. For example, chinese patent application "201310250743. X temperature sensitive microcarriers and processes for their preparation and methods of use" provides a microcarrier that does not use pancreatic enzymes or chemical reagents to dissociate cells when they detach from the carrier surface, but only by changing the temperature. When the culture temperature is 37 ℃, cells are combined on the microcarriers and are separated when the temperature is reduced to normal temperature, but the separation temperature and the combination temperature have small difference, so the operation difficulty is high, and the cell yield is only 50-70%.
Disclosure of Invention
Aiming at the problems in the prior art, the application provides a temperature-sensitive cell culture microcarrier which has a temperature-sensitive temperature different from that of the existing temperature-sensitive cell culture microcarrier, can better separate cells and keep the activity of the cells.
In order to achieve the purpose, the temperature-sensitive cell culture microcarrier provided by the invention is prepared into microspheres with a porous structure by taking modified cellulose, namely hydroxypropyl diethylaminoethyl cellulose as a material.
The invention also provides a preparation method of the temperature-sensitive cell culture microcarrier, which comprises the following steps: adding 100mL of 2-4 wt% hydroxypropyl diethylaminoethyl cellulose solution into 300mL of paraffin oil, mixing the hydroxypropyl diethylaminoethyl cellulose solution and the paraffin oil according to a volume ratio of 1: 3-5, adding 10-20 mL of emulsifier span 80, fully stirring and emulsifying, heating to 50 ℃, solidifying the hydroxypropyl diethylaminoethyl cellulose solution into balls, performing solid-liquid separation, fully washing to remove impurities, adding into a freeze dryer, and obtaining the porous hydroxypropyl diethylaminoethyl cellulose solid microspheres under the condition of vacuum freeze drying.
In the above scheme, the preparation method of the hydroxypropyl diethylaminoethyl cellulose solution comprises the following steps: dissolving 2-3 g of cellulose powder in an aqueous solution containing sodium hydroxide and urea, wherein the weight ratio of sodium hydroxide to urea to water is 15:85, the weight ratio of sodium hydroxide to urea is 2-4: 1, fully stirring and dissolving for 24 hours at-18 ℃ to obtain a transparent and clear cellulose solution, adding a mixture of 1-2 g of diethylaminochloroethane hydrochloride and 1-2 g of epoxypropane, and fully stirring for 24 hours at 5-25 ℃ to obtain the cellulose.
The invention has the beneficial effects that: the temperature-sensitive microcarrier has the advantages that after cell culture is finished, the temperature is controlled, the temperature-sensitive microcarrier can be mutually converted in a gel state and a solution state, when the cells are cultured at 5-37 ℃, the microcarrier is in the gel state after swelling, in the harvesting process, the temperature is reduced to 0-5 ℃, and the microcarrier is converted from the gel state to the solution state, so that cells growing in and on the microcarrier automatically fall off and are separated from the microcarrier. Different from the traditional separation method of trypsase digestion cells, the novel method can better preserve cell surface protein and markers, ensure high cell activity and is a lossless cell harvesting method. And aiming at the defect improvement of the existing temperature-sensitive microcarrier, the cell harvesting rate is greatly improved. The novel temperature-sensitive microcarrier has good cell attaching and detaching capacity, and can be a good material for large-scale culture of attached cells.
Drawings
FIG. 1 is a morphological diagram of cells harvested after culture with the microcarriers of example 1.
Detailed Description
The present invention is further illustrated by the following specific examples, which should not be construed as limiting the scope of the invention.
Example 1
A temperature-sensitive cell culture microcarrier is prepared by the following specific steps:
1) 2g of cellulose powder was dissolved in an aqueous solution containing sodium hydroxide and urea in a weight ratio of sodium hydroxide/urea/water of 11/4/85, and the solution was dissolved at-18 ℃ for 24 hours with stirring to obtain a transparent and clear cellulose solution, and a mixture of 1g of diethylaminoethyl chloride hydrochloride and 1g of propylene oxide was added thereto and stirred at 25 ℃ for 24 hours to obtain a hydroxypropyl diethylaminoethyl cellulose solution.
2) Adding 100mL of 2 wt% hydroxypropyl diethylaminoethyl cellulose solution into 300mL of paraffin oil, mixing the hydroxypropyl diethylaminoethyl cellulose solution and the paraffin oil according to a volume ratio of 1/3, adding 10mL of emulsifier span 80, fully stirring and emulsifying, heating to 50 ℃, solidifying the hydroxypropyl diethylaminoethyl cellulose solution into spheres, after solid-liquid separation, fully washing with 0.1M hydrochloric acid and absolute ethyl alcohol, adding into a freeze dryer, and obtaining the porous hydroxypropyl diethylaminoethyl cellulose solid microspheres under a vacuum freeze drying condition. The modified microsphere has both porous structure and temperature sensitive characteristic, and may be used in cell culture and non-destructive harvesting.
Example 2
1) Dissolving 3g of cellulose powder in 100g of an aqueous solution containing sodium hydroxide and urea in a weight ratio of sodium hydroxide/urea/water of 10/5/85, dissolving the mixture at-18 ℃ for 24 hours under stirring to obtain a transparent and clear cellulose solution, adding a mixture of 2g of diethylaminoethyl chloride hydrochloride and 2g of propylene oxide, and stirring the mixture at 5 ℃ for 24 hours to obtain a hydroxypropyl diethylaminoethyl cellulose solution.
2) Adding 100mL of 3 wt% hydroxypropyl diethylaminoethyl cellulose solution into 300mL of paraffin oil, mixing the hydroxypropyl diethylaminoethyl cellulose solution and the paraffin oil according to a volume ratio of 1/5, adding 20mL of emulsifier span 80, fully stirring and emulsifying, heating to 50 ℃, solidifying the hydroxypropyl diethylaminoethyl cellulose solution into balls, performing solid-liquid separation, fully washing with 0.1M hydrochloric acid and absolute ethyl alcohol, adding into a freeze dryer, and obtaining the porous hydroxypropyl diethylaminoethyl cellulose solid microspheres under vacuum freeze drying conditions. The modified microsphere has both porous structure and temperature sensitive characteristic, and may be used in cell culture and non-destructive harvesting.
Example 3
A temperature-sensitive cell culture microcarrier is prepared by the following specific steps:
1) dissolving 2g of cellulose powder in an aqueous solution containing sodium hydroxide and urea in a weight ratio of sodium hydroxide/urea/water of 12/3/85, dissolving the solution at-18 ℃ for 24 hours under stirring to obtain a transparent and clear cellulose solution, adding a mixture of 2g of diethylaminoethyl chloride hydrochloride and 2g of propylene oxide, and stirring the mixture at 20 ℃ for 24 hours to obtain a hydroxypropyl diethylaminoethyl cellulose solution.
2) Adding 100mL of 4 wt% hydroxypropyl diethylaminoethyl cellulose solution into 300mL of paraffin oil, mixing the hydroxypropyl diethylaminoethyl cellulose solution and the paraffin oil according to a volume ratio of 1/4, adding 15mL of emulsifier span 80, fully stirring and emulsifying, heating to 50 ℃, solidifying the hydroxypropyl diethylaminoethyl cellulose solution into balls, performing solid-liquid separation, fully washing with 0.1M hydrochloric acid and absolute ethyl alcohol, adding into a freeze dryer, and obtaining the porous hydroxypropyl diethylaminoethyl cellulose solid microspheres under vacuum freeze drying conditions. The modified microsphere has both porous structure and temperature sensitive characteristic, and may be used in cell culture and non-destructive harvesting.
Comparative example 1
The existing temperature-sensitive cell culture microcarrier is prepared by the following specific steps:
1) adding 1.0G of glucan microsphere (G50) and 10mL of dimethyl sulfoxide (DMSO) into a 25mL round-bottom flask, adding 1.0G of 2-chloropropionyl chloride, adding magnetons, stirring for 1.5h, washing for 3-5 times by using methanol to obtain chemically modified glucan microspheres with a large number of chlorine atoms on the surfaces;
2) n-isopropylacrylamide (NiPAAm) was grafted by free radical ATRP polymerization as follows: adding dextran microsphere with chloromethylated surface and containing a large amount of chlorine atoms into a 50mL round-bottom flask containing magnetons, sequentially adding 2.0g of N-isopropylacrylamide (NIPAAm) and copper chloride (CuCl) 2 )0.34g of tris- (N' N dimethylamino ethylamine) (Me6Tren)0.46g and 25mL of deionized water, adding 0.13g of ascorbic acid in an oxygen-free environment, reacting at room temperature for 24h, washing with water for 3-5 times, and drying to obtain the temperature-sensitive dextran microspheres grafted with the N isopropylacrylamide polymer.
Examples of the experiments
The temperature-sensitive cell culture microcarriers prepared in the above examples and comparative examples were used for cell culture.
The culture method comprises the following steps:
1) taking 3g of the prepared temperature-sensitive microcarrier, putting the temperature-sensitive microcarrier into a 100mL rotary bottle, adding 80mL of PBS (phosphate buffer), soaking overnight, pouring off the redundant PBS, adding new PBS, standing for about 20min, pouring off the redundant PBS after the microcarrier is settled, and repeating the steps for three times, and finally, leaving about 50mL of PBS for sterilization pretreatment;
2) PBS was discarded in the clean bench, 20mL of complete medium (10% FBS + MEM) was added and soaked for 6 h;
3) 10ml of the suspension was added to the cells at a cell density of 1X 10 6 cells/mL HepG2 cells (from China center for type culture Collection) in spinner flasksAdding a complete culture medium to a volume of 100mL, inoculating cells at a stirring speed of 50rpm, reducing the speed to 20rmp/min after the cells are added, culturing at a stirring speed of 40rpm after 8 hours at a culture temperature of 37 ℃, and replacing half volume of culture solution every 2 days;
4) culturing until the day eight, the cells are full of microcarriers, discarding the culture solution, adding 100mL of precooled PBS into a roller bottle, changing the temperature of the culture solution from 37 ℃ to 4 ℃, standing for 10min, converting the microcarriers into a liquid state, and centrifuging to collect the cells. Calculated, cell harvest.
The detection shows that the cell has two-dimensional culture morphology under a microscope with the magnification of 40 times, and under the microscope, the HepG2 cell grows in a cylindrical or polygonal attached manner, the cells are tightly connected, the cell morphology is clear, and the state is good, as shown in figure 1.
As a result, the results of the evaluation of the results of the microcarrier culture of cells and the morphological health of the cells in the comparative examples are shown in the following table.
| Evaluation index | Cell harvest rate | Cellular activity |
| Example 1 | 98％ | 95％ |
| Example 2 | 99％ | 97％ |
| Example 3 | 92％ | 94％ |
| Comparative example | 68％ | 90％ |
The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the protection of the invention and the scope of the claims are not limited to the examples provided. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (4)
1. A temperature-sensitive cell culture microcarrier is characterized in that: modified cellulose, namely hydroxypropyl diethylaminoethyl cellulose is used as a material to prepare the microsphere with a porous structure.
2. The temperature-sensitive cell culture microcarrier of claim 1, wherein:
the preparation method of the hydroxypropyl diethylaminoethyl cellulose solution comprises the following steps: dissolving 2-3 g of cellulose powder in an aqueous solution containing sodium hydroxide and urea, wherein the weight ratio of sodium hydroxide to urea to water is 15:85, the weight ratio of sodium hydroxide to urea is 2-4: 1, fully stirring and dissolving for 24 hours at-18 ℃ to obtain a transparent and clear cellulose solution, adding a mixture of 1-2 g of diethylaminochloroethane hydrochloride and 1-2 g of epoxypropane, and fully stirring for 24 hours at 5-25 ℃ to obtain the cellulose.
3. A method for preparing the temperature-sensitive cell culture microcarrier according to claim 1, wherein the method comprises the following steps: adding 100mL of 2-4 wt% hydroxypropyl diethylaminoethyl cellulose solution into 300mL of paraffin oil, mixing the hydroxypropyl diethylaminoethyl cellulose solution and the paraffin oil according to a volume ratio of 1: 3-5, adding 10-20 mL of emulsifier span 80, fully stirring and emulsifying, heating to 50 ℃, solidifying the hydroxypropyl diethylaminoethyl cellulose solution into balls, performing solid-liquid separation, fully washing to remove impurities, adding into a freeze dryer, and obtaining the porous hydroxypropyl diethylaminoethyl cellulose solid microspheres under the condition of vacuum freeze drying.
4. The method for preparing a temperature-sensitive cell culture microcarrier according to claim 3, wherein:
the preparation method of the hydroxypropyl diethylaminoethyl cellulose solution comprises the following steps: dissolving 2-3 g of cellulose powder in an aqueous solution containing sodium hydroxide and urea, wherein the weight ratio of sodium hydroxide to urea to water is 15:85, the weight ratio of sodium hydroxide to urea is 2-4: 1, fully stirring and dissolving for 24 hours at-18 ℃ to obtain a transparent and clear cellulose solution, adding a mixture of 1-2 g of diethylaminochloroethane hydrochloride and 1-2 g of epoxypropane, and fully stirring for 24 hours at 5-25 ℃ to obtain the cellulose.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006174826A (en) * | 2004-11-24 | 2006-07-06 | Sanyo Chem Ind Ltd | Temperature-sensitive and swelling resin bead for cell culture |
| CN103409361A (en) * | 2013-06-24 | 2013-11-27 | 上海瀚正生物技术服务有限公司 | Thermosensitive microcarrier as well as preparation technology and application method thereof |
| CN104703580A (en) * | 2012-10-11 | 2015-06-10 | 陶氏环球技术有限公司 | Glyoxal-free cellulose derivatives for personal care compositions |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006174826A (en) * | 2004-11-24 | 2006-07-06 | Sanyo Chem Ind Ltd | Temperature-sensitive and swelling resin bead for cell culture |
| CN104703580A (en) * | 2012-10-11 | 2015-06-10 | 陶氏环球技术有限公司 | Glyoxal-free cellulose derivatives for personal care compositions |
| CN103409361A (en) * | 2013-06-24 | 2013-11-27 | 上海瀚正生物技术服务有限公司 | Thermosensitive microcarrier as well as preparation technology and application method thereof |
Non-Patent Citations (2)
| Title |
|---|
| YIFEN WEN ET AL.: "Intracellular delivery cellulose-based bionanogels with dualtemperature/pH-response for cancer therapy", COLLOIDS AND SURFACES B: BIOINTERFACES, vol. 133, pages 246 - 253, XP029250688, DOI: 10.1016/j.colsurfb.2015.06.017 * |
| 林莹等: "甲基纤维素温敏水凝胶的凝固及体外释药特性", 清华大学学报, vol. 46, no. 6, pages 836 - 838 * |
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