CN110055208B - Non-pancreatin digestion cell harvesting culture method based on temperature-sensitive hydrogel material - Google Patents
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Abstract
The invention discloses a non-pancreatin digestion harvested cell culture method based on a temperature-sensitive hydrogel material. Firstly, a hydrogel solution is prepared by an emulsion polymerization method, the hydrogel can keep a solution state at a low temperature, and can quickly form hydrogel after the physiological temperature of a human body is close to. Based on the temperature-sensitive hydrogel material, the invention realizes non-enzymatic harvesting of cells by the following two schemes: 1) mixing the cell suspension and the hydrogel solution, placing the mixture in a 37 ℃ cell culture box, adding a 37 ℃ culture medium to culture after the mixed solution rapidly forms gel, and then releasing cells by cooling; 2) the hydrogel solution is firstly coated on the surface of a substrate, the substrate is placed in a 37 ℃ cell culture box, after gel is formed, cell suspension and a culture medium at 37 ℃ are added for culture, and then the cells are released by cooling. The cell culture method disclosed by the invention does not need pancreatin digestion and a cell scraper, can better preserve cell surface protein and markers, ensures high cell activity, and is a lossless cell culture method.
Description
Technical Field
The invention relates to a cell harvesting method without pancreatin digestion and scraper, in particular to a cell culture method without pancreatin digestion and cell harvesting based on a temperature-sensitive hydrogel material.
Background
Chitosan is amino polysaccharide from nature, the macromolecular chain of the chitosan is composed of acetylglucosamine and deacetylated glucoseamine through random or block distribution, the chitosan has good biocompatibility, blood compatibility, hemostatic performance, antibacterial performance and natural pharmaceutical activity, has a promoting effect on wound healing, can be degraded by lysozyme in a living body, and the final degradation product is glucosamine which is alkalescent and can be absorbed by the body after matching with the pH value of body fluid, thereby having huge application potential in the fields of biology and medicine.
In the development and utilization of chitosan resources, hydrogel is an important form. Hydrogel materials have a variety of advantageous properties, and their use extends to various areas of daily life and advanced industry. Smart hydrogels have received much attention because they respond to subtle changes in the external environment, as compared to conventional hydrogels. Among them, the hydrogel responding to the external temperature stimulus is called temperature sensitive hydrogel, and also called temperature sensitive hydrogel. The temperature-sensitive hydrogel contains a certain proportion of hydrophilic groups and hydrophobic groups, and the interaction between the hydrophobic groups can be influenced by the change of temperature, so that the network structure of the gel is changed, and the hydrogel is subjected to phase transition. The temperature-sensitive chitosan hydrogel has high water absorption, water retention, good biocompatibility and temperature sensitivity, and has wide application prospect in the fields of biological materials and medical materials such as biochemical separation, cell culture, drug controlled release systems and the like.
The in vitro culture of the seed cells has important significance in the regenerative medicine fields of tissue engineering, cell therapy and the like. Currently, among conventional cell culture methods such as 2D planar and 3D scaffolds, pancreatin digestion and cell scraper are widely used cell harvesting methods. However, such methods can cause damage to cell membrane proteins, which in turn can cause damage to cell membranes, loss of cell surface markers, or partial loss of cell function, which can impair the utility of seed cells in regenerative medicine. The development of the temperature-sensitive chitosan hydrogel material provides a new idea for solving the problems.
Disclosure of Invention
The invention aims to provide a non-pancreatin digestion harvested cell culture method based on a temperature-sensitive hydrogel material aiming at the defects of the prior art.
The preparation method of the chitosan hydrogel with the temperature sensitivity characteristic comprises the following steps:
1) adding 1-20 parts by weight of chitosan, 0.1-2 parts by weight of acid, 0.01-8 parts by weight of initiator, 1-20 parts by weight of N-isopropylacrylamide monomer and 50-98 parts by weight of deionized water into a reactor, stirring and dissolving uniformly, reacting for 1-12 hours at 40-90 ℃, dialyzing the reaction product to be neutral by using the deionized water, and performing vacuum freeze-drying to obtain the poly-N-isopropylacrylamide modified chitosan powder.
2) Adding 1-20 parts of poly N-isopropylacrylamide modified chitosan powder in 1) into 80-99 parts of PBS solution, and stirring and dissolving uniformly to obtain a poly N-isopropylacrylamide modified chitosan solution, which is called hydrogel solution for short.
The molecular weight of the chitosan is 3-150 ten thousand, and the deacetylation degree is 50-98%.
The acid is at least one of hydrochloric acid, acrylic acid, citric acid and acetic acid.
The initiator is at least one of azobisisobutyronitrile, azobisisoheptonitrile, dimethyl azobisisobutyrate, azobisisobutylamidine hydrochloride, hydrogen peroxide, ammonium persulfate, potassium persulfate and tert-butyl hydroperoxide.
The poly-N-isopropylacrylamide modified chitosan macromolecule has good water solubility. The temperature-sensitive chitosan hydrogel can quickly form hydrogel (within 5 seconds to 60 minutes) when the temperature is close to the physiological temperature of a human body, and can keep a solution state at room temperature.
The invention relates to a temperature-sensitive hydrogel material-based culture method for collecting cells without trypsinization digestion, which comprises the following two culture schemes:
1) cell culture protocol one of harvest without trypsinization: the cell suspension and the hydrogel solution prepared by the invention are uniformly mixed at a lower temperature and then are added into a culture dish, then the culture dish is placed in a cell culture box at 37 ℃, the mixed solution is added with a culture medium at 37 ℃ for culture after rapidly forming gel, the culture is transferred to room temperature to reduce the culture temperature after a period of culture, and the cells are centrifugally separated and collected after being released.
2) And (2) harvesting cells without trypsinization: the hydrogel solution prepared by the invention is coated on the bottom surface of a culture dish at room temperature, then is placed in a 37 ℃ cell culture box, after gel is formed, a mixed solution of cell suspension and culture medium at 37 ℃ is added into the culture dish for culture, after a period of culture, the culture is transferred to room temperature to reduce the culture temperature, and after the cells are released, the cells are centrifugally separated and collected. The lower temperature is 10-28 ℃.
The cell culture method is realized based on a special hydrogel material, and by utilizing the temperature-sensitive characteristic of the hydrogel material, the cell surface protein and the marker can be better preserved without pancreatin digestion and a cell scraper, so that the high activity of the cell is ensured, and the cell culture method is a lossless cell culture method.
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FIG. 1 is a diagram showing a solution-gel of poly (N-isopropylacrylamide) -modified chitosan having temperature-sensitive characteristics, prepared according to the present invention;
FIG. 2 is a rheological profile of a poly N-isopropylacrylamide-modified chitosan hydrogel prepared according to the present invention.
Detailed Description
The invention is further illustrated by the following figures and specific examples.
Example 1:
1) weighing 1.5g of chitosan powder with the viscosity-average molecular weight of 65w and the deacetylation degree of 90%, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate, dispersing the chitosan powder, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate in 95 mL of deionized water, then adding 0.2mL of hydrochloric acid, stirring and dissolving uniformly, reacting for 8 hours at 60 ℃, then dialyzing the reaction product to be neutral by using the deionized water, and performing vacuum freeze-drying to obtain poly-N-isopropyl acrylamide modified chitosan powder;
2) weighing 1.0g of poly N-isopropylacrylamide modified chitosan powder obtained in the step 1), dispersing in 25ml of PBS solution, and stirring and dissolving uniformly to obtain poly N-isopropylacrylamide modified chitosan aqueous solution with the mass concentration of 4%;
3) uniformly mixing 0.75mL of the poly-N-isopropylacrylamide modified chitosan solution prepared in the step 2) with 0.25mL of cell suspension at room temperature, adding the mixture into a culture dish, then placing the culture dish into a 37 ℃ cell culture box, adding 2mL of 37 ℃ culture medium into the mixture after the mixture rapidly forms gel to culture, transferring the culture to room temperature after a period of culture, reducing the culture temperature, releasing the cells, and centrifugally separating and collecting the cells.
The poly-N-isopropylacrylamide modified chitosan powder prepared by the invention has good water solubility at room temperature, and the solution is in a clear and transparent state as shown in figure 1; after the temperature is raised to 37 ℃, the chitosan solution is quickly changed into white hydrogel; cooling to room temperature, and converting into solution state; it can be seen that the poly-N-isopropylacrylamide modified chitosan can present reversible changes from 'solution' to 'gel' at room temperature and at human physiological temperature.
The rheological property of the poly-N-isopropylacrylamide modified chitosan hydrogel prepared by the invention is shown in figure 2, and when the temperature is lower than 30 ℃, the loss modulus is greater than the storage modulus, and the hydrogel is in a solution state; when the temperature is increased to 31 ℃, the loss modulus is rapidly reduced, and the storage modulus is rapidly increased, so that sol-gel conversion is realized; when the temperature is raised to 37 ℃ which is the physiological temperature of a human body, the storage modulus is larger than the loss modulus, and a gel state is presented; it can be seen that the gelation temperature of the poly-N-isopropylacrylamide modified chitosan hydrogel is 32 ℃, and the temperature response speed is very high.
Example 2:
1) weighing 1.5g of chitosan powder with the viscosity-average molecular weight of 65w and the deacetylation degree of 90%, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate, dispersing the chitosan powder, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate in 95 mL of deionized water, then adding 0.2mL of hydrochloric acid, stirring and dissolving uniformly, reacting for 8 hours at 60 ℃, then dialyzing the reaction product to be neutral by using the deionized water, and performing vacuum freeze-drying to obtain poly-N-isopropyl acrylamide modified chitosan powder;
2) weighing 1.0g of poly N-isopropylacrylamide modified chitosan powder obtained in the step 1), dispersing in 25ml of PBS solution, and stirring and dissolving uniformly to obtain poly N-isopropylacrylamide modified chitosan aqueous solution with the mass concentration of 4%;
3) coating 0.75mL of the poly-N-isopropylacrylamide modified chitosan solution prepared in the step 2) on the bottom surface of a culture dish at room temperature, then placing the culture dish in a 37 ℃ cell culture box, adding 0.25mL of cell suspension at 37 ℃ and 2mL of culture medium mixed solution into the culture dish for culture after gel formation, transferring the culture to the room temperature after a period of culture, reducing the culture temperature, releasing the cells, and centrifugally separating and collecting the cells.
Example 3:
1) weighing 1.5g of chitosan powder with the viscosity-average molecular weight of 65w and the deacetylation degree of 90%, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate, dispersing the chitosan powder, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate in 95 mL of deionized water, then adding 0.2mL of hydrochloric acid, stirring and dissolving uniformly, reacting for 8 hours at 60 ℃, then dialyzing the reaction product to be neutral by using the deionized water, and performing vacuum freeze-drying to obtain poly-N-isopropyl acrylamide modified chitosan powder;
2) weighing 1.5g of poly N-isopropylacrylamide modified chitosan powder obtained in the step 1), dispersing in 30 ml of PBS solution, and stirring and dissolving uniformly to obtain poly N-isopropylacrylamide modified chitosan aqueous solution with the mass concentration of 5%;
3) uniformly mixing 0.75mL of the poly-N-isopropylacrylamide modified chitosan solution prepared in the step 2) with 0.25mL of cell suspension at a lower temperature, adding the mixture into a culture dish, then placing the culture dish in a 37 ℃ cell culture box, adding 2mL of 37 ℃ culture medium into the mixture after the mixture rapidly forms gel for culture, transferring the culture to the room temperature after a period of culture, reducing the culture temperature, releasing the cells, and centrifugally separating and collecting the cells.
Example 4:
1) weighing 1.5g of chitosan powder with the viscosity-average molecular weight of 65w and the deacetylation degree of 90%, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate, dispersing the chitosan powder, 1.75g N-isopropyl acrylamide and 0.075g of ammonium persulfate in 95 mL of deionized water, then adding 0.2mL of hydrochloric acid, stirring and dissolving uniformly, reacting for 8 hours at 60 ℃, then dialyzing the reaction product to be neutral by using the deionized water, and performing vacuum freeze-drying to obtain poly-N-isopropyl acrylamide modified chitosan powder;
2) weighing 1.5g of poly N-isopropylacrylamide modified chitosan powder obtained in the step 1), dispersing in 30 ml of PBS solution, and stirring and dissolving uniformly to obtain poly N-isopropylacrylamide modified chitosan aqueous solution with the mass concentration of 5%;
3) coating 0.75mL of the poly-N-isopropylacrylamide modified chitosan solution prepared in the step 2) on the bottom surface of a culture dish at room temperature, then placing the culture dish in a 37 ℃ cell culture box, adding 0.25mL of cell suspension at 37 ℃ and 2mL of culture medium mixed solution into the culture dish for culture after gel formation, transferring the culture to the room temperature after a period of culture, reducing the culture temperature, releasing the cells, and centrifugally separating and collecting the cells.
Claims (4)
1. A temperature-sensitive hydrogel material-based culture method for non-pancreatin digestion harvested cells is characterized by comprising the following steps:
1) preparing a poly N-isopropylacrylamide modified chitosan macromolecule: adding 1-20 parts by weight of chitosan, 0.1-2 parts by weight of acid, 0.01-8 parts by weight of initiator, 1-20 parts by weight of N-isopropyl acrylamide monomer and 50-98 parts by weight of deionized water into a reactor, stirring and dissolving uniformly, and then adding the mixture into the reactor at 60 parts by weightoC, reacting for 1-12 hours, then dialyzing the reaction product to be neutral by using deionized water, and carrying out vacuum freeze-drying to obtain poly-N-isopropylacrylamide modified chitosan powder; the acid is hydrochloric acid;
2) preparing a poly N-isopropylacrylamide modified chitosan aqueous solution: adding 1-20 parts of poly N-isopropylacrylamide modified chitosan powder in 1) into 80-99 parts of PBS solution, stirring at room temperature, and uniformly dissolving to obtain poly N-isopropylacrylamide modified chitosan solution, which is called hydrogel solution for short;
3) non-pancreatin digestion harvest cell culture: mixing the cell suspension and the hydrogel solution prepared in step 2) at a lower temperature, adding the mixture into a culture dish, and placing the culture dish in a place 37oIn the C cell incubator, the mixed solution is added into 37 after the mixed solution forms gel rapidlyoC, culturing in culture medium, transferring the culture to room temperature, reducing culture temperature, releasing cells, and centrifugingSeparating; the lower temperature is 10-28%oC；
Alternatively, the hydrogel solution prepared in step 2) is applied to the bottom surface of a petri dish at a relatively low temperature and then placed in a bath 37oC in the cell incubator, after gel is formed, 37 is addedoC, adding the cell suspension and culture medium mixed solution into a culture dish for culture, transferring the culture to room temperature after culture to reduce the culture temperature, and centrifugally separating after the cells are released; the lower temperature is 10-28%oC。
2. The method of claim 1, wherein the cell culture is obtained by non-pancreatin digestion and harvest of temperature-sensitive hydrogel materials, and the method comprises the following steps: the molecular weight of the chitosan is 3-150 ten thousand, and the deacetylation degree is 50-95%.
3. The method for culturing cells harvested by non-pancreatin digestion based on temperature-sensitive hydrogel material according to claim 1, wherein the initiator is ammonium persulfate or potassium persulfate.
4. The method for culturing harvested cells based on temperature-sensitive hydrogel material by non-pancreatin digestion according to claim 1, wherein the cells are adherent cells.
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