CN116478441B - An assemblable and dissolvable three-dimensional cell culture carrier and its preparation method - Google Patents

Abstract

The invention discloses an assemblable and dissolvable three-dimensional cell culture carrier and a preparation method thereof, and belongs to the field of cell culture technology. The three-dimensional cell culture carrier of the present invention uses chitosan as raw material, and first prepares chitosan microspheres, and then It is prepared by adding an appropriate amount of acid and alkali in sequence; the present invention makes full use of the characteristics of chitosan and can obtain an assemblable and soluble three-dimensional cell culture carrier through simple acid-base action. The obtained carrier has a porous structure and is conducive to The penetration of nutrients and metabolites can be used for in vitro cell culture; the preparation steps are simpler, easy to operate, low cost, and the resulting product has high purity.

Description

An assemblable and dissolvable three-dimensional cell culture carrier and its preparation method

Technical field

The present invention relates to the field of cell culture technology, and in particular to an assemblable and dissolvable three-dimensional cell culture carrier and a preparation method thereof.

Background technique

3D cell culture is the co-culture of material carriers with three-dimensional structures and various cells in vitro, which can allow cells to grow and migrate in three-dimensional space to form a three-dimensional cell-carrier complex, which is more conducive to supporting the state of cells in simulated in vivo environments. and trends. 3D cell culture technology has made considerable contributions to the development of medicine. Researchers are constantly developing and designing more 3D cell culture systems to improve the cell culture environment and help them grow in environmental conditions closer to those in the body. The most important type of 3D culture technology is hydrogels.

Among biodegradable natural macromolecular materials, chitosan is a type of amino polymer polysaccharide extracted from chitin in shrimps, crabs and other crustaceans through chemical deacetylation. It has abundant sources and low cost. It is The second largest category of natural macromolecular materials after cellulose. It has good biocompatibility and biodegradability and is currently the only natural macromolecule with electropositive characteristics. It has been widely used in medicine, food, agriculture, environmental protection, daily chemicals and other fields. Polymer microspheres are an excellent carrier material due to their high dispersion and large specific surface area.

Chitosan is a natural polyglucosamine with a loose network structure. It is a derivative obtained by deacetylating chitin to a certain extent. The microspheres formed by its gel have a network structure, good mechanical properties, stable chemical properties, and good heat resistance.

There have been reports of using chitosan and its derivatives to prepare three-dimensional cell culture carriers. For example, Chinese patent application CN102517211A discloses a method of preparing three-dimensional cell culture carriers using carboxymethyl-modified chitosan as raw material. This method is to dissolve carboxymethyl-modified chitosan in phosphate or 2-(N-morpholino)ethanesulfonic acid buffer with pH=5-8, and then add carbodiimide-activated disulfide Using a substituted dibasic acid cross-linking agent, a three-dimensional chemically cross-linked hydrogel is formed through the amidation reaction between the amino groups in carboxymethyl chitosan and the carboxyl groups in the cross-linking agent, and finally a three-dimensional porous carrier is obtained through freeze-drying; this method Disadvantages include: it requires carboxymethylation modification of chitosan, and a cross-linking agent needs to be added for amidation reaction. The preparation steps are complicated and costly, and the resulting product may contain many impurities and requires purification. .

Another example is the Chinese patent application CN111704739A, which discloses a method in which chitosan and/or gelatin are dissolved as a precursor liquid, and a microfluidic atomizer is used to spray the liquid droplets; the liquid droplets are spray-frozen to obtain ice ball particles; and the ice ball particles are obtained. Carry out vacuum freeze-drying to obtain porous microsphere particles; although this method uses chitosan as the material, it requires the use of special equipment - a microfluidic atomizer, which has high equipment requirements and high cost; and, existing These methods only make chitosan gel microspheres, but cannot assemble the microspheres.

Contents of the invention

The purpose of the present invention is to provide an assemblable and dissolvable three-dimensional cell culture carrier to solve the above problems.

In order to achieve the above object, the technical solution adopted by the present invention is as follows: an assemblable and soluble three-dimensional cell culture carrier. The three-dimensional cell culture carrier uses chitosan as raw material. Chitosan microspheres are first prepared, and then It is prepared by adding appropriate amounts of acid and alkali in sequence.

The above-mentioned preparation method of the assemblable and dissolvable three-dimensional cell culture carrier includes the following steps:

(1) Prepare chitosan solution by adding chitosan powder solvent in acid solution;

(2) Drop the chitosan solution prepared in step (1) into the alkaline solution using an injection needle, and then stir to prepare chitosan microspheres;

(3) Wash the chitosan microspheres prepared in step (2) with water to remove the alkali solution on the surface;

(4) Put the cleaned chitosan microspheres into a container, absorb the water, add it to the acid solution, and continue to dissolve for 10-60 seconds. When the microspheres are in a semi-dissolved state, stack them together into the required shape, and add The alkali solution is terminated to obtain fused microspheres, thereby preparing the assemblable and dissolvable three-dimensional cell culture carrier.

The present invention makes full use of the characteristics of chitosan: the large number of amino groups present in its molecule enables it to dissolve in acidic solutions with pH below 6.5 and precipitate in alkaline solutions. The basic principle of preparation is: control and production of chitosan. The acidity and alkalinity and reaction time of the sugar microsphere process are controlled by using a small amount of medium and weak acid. The chitosan is dissolved by the acid, so that the microspheres are slowly partially dissolved. When the microspheres are in a semi-dissolved state, they are stacked into a certain shape, and then an alkaline solution is used. It is allowed to precipitate out, achieving the effect of assembling the partially dissolved microspheres together, and obtaining a three-dimensional cell culture carrier that can be assembled and dissolved, and can be used for in vitro cell culture.

As a preferred technical solution: the acid solution in step (1) is an acid solution with a pH of 0-7. For example, it can be an organic acid with a volume percentage concentration of 2-3%, or an inorganic acid, as long as the pH value meets the requirements.

As a preferred technical solution: the diameter of the chitosan microspheres prepared in step (2) is 0.1-5mm.

As a preferred technical solution: the alkali solution in step (2) is an alkali solution with a pH of 7-14, such as 0.5 mol/L sodium hydroxide, potassium hydroxide, etc.

As a preferred technical solution: in step (3), the water used is reverse osmosis water.

As a preferred technical solution: in step (4), the shape of the stack includes but is not limited to cylinder, sphere, and cube.

As a preferred technical solution: in step (4), the acid is an acid with a pH of 1-7, including but not limited to hydrochloric acid and acetic acid.

As a preferred technical solution: in step (4), the alkali is an alkali solution with a pH of 7-14, including but not limited to sodium hydroxide.

Compared with the existing technology, the advantage of the present invention is that the present invention makes full use of the characteristics of chitosan and can obtain an assemblable and soluble three-dimensional cell culture carrier through simple acid-base interaction. The obtained carrier has a porous structure and has It is beneficial to the penetration of nutrients and metabolites, and can be used for in vitro cell culture; the preparation steps are simpler, easy to operate, low cost, and the obtained product has a single component and does not require other additives.

Description of the drawings

Figure 1 is a photo of chitosan microspheres without acid treatment produced by dropping a chitosan solution of a certain mass concentration into a sodium hydroxide solution;

Figure 2 is a photo of slightly adherent chitosan microspheres obtained after a certain amount of acid treatment and then adding sodium hydroxide to terminate the reaction;

Figure 3 is a photo of the surface of chitosan microspheres prepared in Example 1 under a scanning electron microscope;

Figure 4 is a photo of cell adhesion on the surface of a three-dimensional cell carrier under a fluorescence microscope after staining and labeling;

Figure 5 is a comparison chart of pellets with different amounts of acid and different treatment times.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings.

Example 1

Take 0.1g chitosan (Aladdin, degree of deacetylation ≥95%) and add it to 10mL of acetic acid solution with a volume percentage concentration of 2%. Use an injection needle with a diameter of 0.4mm to drop the solution into 500mL of 0.5mol/L. Sodium hydroxide aqueous solution in the precipitation bath, and then stirred with a magnetic stirrer; after waiting for half an hour, the chitosan microspheres gradually solidified and formed. The photo is shown in Figure 1. It can be seen in Figure 1: The obtained chitosan microspheres Uniform size;

Then collect the chitosan microspheres and wash them in RO water to wash away the sodium hydroxide; put the microspheres into a container (well plate), absorb the RO water, add a 20% acetic acid solution by volume, and continue to dissolve for 30 seconds. , stack the microspheres into a certain shape in a semi-dissolved state, add sodium hydroxide to terminate, and obtain fused microspheres. The photo is shown in Figure 2. From Figure 2, it can be seen that the chitosan microspheres are slightly adherent. It can achieve the effect of being assembled, and there are sufficient pores between the microspheres, which can provide good conditions for cell growth.

Example 2

Take 0.2g of chitosan (Aladdin, degree of deacetylation ≥95%) and add it to 10mL of acetic acid solution with a volume percentage concentration of 2%. Use an injection needle with a diameter of 0.4mm to drop the solution into 500mL of 0.5mol/L. Put the sodium hydroxide aqueous solution in the precipitation bath, and then stir it with a magnetic stirrer; wait for half an hour, and the chitosan microspheres will gradually solidify and form;

Collect the chitosan microspheres and wash them in RO water to remove the sodium hydroxide. Put the microspheres into a container (EP tube), absorb the RO water, add a 20% acetic acid solution by volume, and continue to dissolve for 23 seconds. The microspheres are stacked into a certain shape in a semi-dissolved state, and then sodium hydroxide is added to terminate. Fusion microspheres.

Example 3

Take 0.3g of chitosan (Aladdin, degree of deacetylation ≥95%) and add it to 10mL of acetic acid solution with a volume percentage concentration of 2%. Use an injection needle with a diameter of 0.4mm to drop the solution into 500mL of 0.5mol/L. Put the sodium hydroxide aqueous solution in the precipitation bath, and then stir it with a magnetic stirrer; wait for half an hour, and the chitosan microspheres will gradually solidify and form;

Collect the chitosan microspheres and wash them in RO water to remove the sodium hydroxide. Put the microspheres into a container (orifice plate), absorb the RO water, add a 20% acetic acid solution by volume, continue to dissolve for 25 seconds, stack the microspheres into a certain shape in a semi-dissolved state, and add sodium hydroxide to terminate, to obtain Fusion microspheres.

Example 4

Take 0.3g of chitosan (Aladdin, degree of deacetylation ≥95%) and add it to 10mL of acetic acid solution with a volume percentage concentration of 3%. Use an injection needle with a diameter of 0.4mm to drop the solution into 500mL of 0.5mol/L. Put the sodium hydroxide aqueous solution in the precipitation bath, and then stir it with a magnetic stirrer; wait for half an hour, and the chitosan microspheres will gradually solidify and form;

Collect the chitosan microspheres and wash them in RO water to remove the sodium hydroxide. Put the microspheres into a container (EP tube), absorb the RO water, add a 20% acetic acid solution by volume, continue to dissolve for 20 seconds, stack the microspheres into a certain shape in a semi-dissolved state, and add sodium hydroxide to terminate, to obtain Fusion microspheres.

Test example 1

Use a scanning electron microscope to observe the surface morphology of chitosan microspheres and observe their porous structure, as shown in Figure 3. It can be seen from Figure 3 that each microsphere gel has a porous structure, which is conducive to the penetration of nutrients and metabolites. .

Test example 2

Cells were cultured on the assemblable and dissolvable three-dimensional cell culture carrier prepared in Example 1, stained with DAPI: 4',6-diamidino-2-phenylindole, and labeled with cell nuclei, and observed with an immunofluorescence microscope after 24 hours. Cell morphology and adhesion spreading. The results are shown in Figure 4. It can be seen from Figure 4 that chitosan gel beads can support cell adhesion, which is beneficial to in vitro cell culture and the cells grow well.

Test example 3

Cells were cultured on the assemblable and dissolvable three-dimensional cell culture carrier prepared in Example 2. Philloidin:actin dye was used to mark the cell microfilament skeleton. After 24 hours, immunofluorescence microscopy was used to observe cell morphology and adhesion spreading. The results are shown in Figure 4. It can be seen from Figure 4 that chitosan gel beads can support cell adhesion, which is beneficial to in vitro cell culture and the cells grow well.

The most important invention of this invention is that chitosan gel microspheres stick together to make 3D scaffolds of different shapes. This application is realized through the above-mentioned process control. For example, in the above-mentioned process, there is too much acid and the time is too long. It will cause the ball to dissolve, destroy the structure of the ball, and thereby destroy the support of the ball, as shown in Figure 5: From left to right in Figure 5, add a hydrochloric acid solution of pH 1 to dissolve for 30 seconds, and a hydrochloric acid solution of pH 4 to dissolve 30s, pH 6 hydrochloric acid solution for 30s, and 1M HAC solution for 30s. It can be seen from Figure 5 that too much acid for too long will destroy the support of the pellet.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Based on the embodiments of the present invention, those skilled in the art can obtain all the results without any creative work. Other embodiments fall within the protection scope of the present invention.

Claims (7)

1. A three-dimensional cell culture carrier capable of being assembled and dissolved, which is characterized in that: the three-dimensional cell culture carrier is prepared by taking chitosan as a raw material, firstly preparing chitosan microspheres, and then sequentially adding a proper amount of acid and alkali, wherein the specific preparation method comprises the following steps of:

(1) Preparing chitosan powder solvent into chitosan solution in an acid solution, wherein the pH value of the acid solution is 0-7;

(2) Dripping the chitosan solution prepared in the step (1) into an alkali solution by using an injection needle, and stirring to prepare chitosan microspheres;

(3) Washing the chitosan microspheres prepared in the step (2) with water to remove alkali liquor on the surface;

(4) And (3) filling the cleaned chitosan microspheres into a container, absorbing water, adding the water into an acid solution with the pH of 1-7, continuously dissolving for 10-60s, stacking the microspheres together in a semi-dissolved state to form a required shape, and adding an alkali solution to terminate, thereby obtaining the fused microspheres, and preparing the spliced and soluble three-dimensional cell culture carrier.

2. The three-dimensional cell culture support of claim 1, wherein the three-dimensional cell culture support is a three-dimensional cell culture support comprising: the diameter of the chitosan microsphere prepared in the step (2) is 0.1-5mm.

3. The three-dimensional cell culture support of claim 1, wherein the three-dimensional cell culture support is a three-dimensional cell culture support comprising: the alkali solution in the step (2) is an alkali solution with the pH value of 7-14.

4. The three-dimensional cell culture support of claim 1, wherein the three-dimensional cell culture support is a three-dimensional cell culture support comprising: in the step (3), the water is selected from one of distilled water and reverse osmosis water.

5. The three-dimensional cell culture support of claim 1, wherein the three-dimensional cell culture support is a three-dimensional cell culture support comprising: in the step (4), the stacking shape is one selected from a cylinder, a sphere and a cube.

6. The three-dimensional cell culture support of claim 1, wherein the three-dimensional cell culture support is a three-dimensional cell culture support comprising: in the step (4), the acid is selected from one of hydrochloric acid and acetic acid.

7. The three-dimensional cell culture support of claim 1, wherein the three-dimensional cell culture support is a three-dimensional cell culture support comprising: in the step (4), the alkali is alkali solution with pH of 7-14.