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.
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.