TWI432577B - Method for controlling attachment of cells - Google Patents

Description

Method for controlling cell adhesion

The present invention relates to a method for controlling cell adhesion, and more particularly to a method for controlling cell adhesion by adjusting the pH of a medium.

Cell culture technology is a technique in which cells are taken out from an organism and cultured under the growth bar of a mimetic organism to propagate, survive, and grow. With this technique, drug efficacy, tissue engineering, and cells can be performed. Screening and other aspects of research. Therefore, with the development of biomedical technology, the importance of cell culture technology is increasing.

In general, animal cells can be divided into three types according to the growth pattern of animal cells, such as suspension cells (such as blood cells, lymphoid tissue cells, hematopoietic stem cells), adherent cells (such as mesenchymal stem cells, epithelial cells) and In the cell between the two types, the attached cell can grow because it needs to be attached to the object, and therefore, if it is to be separated from the surface of the object, special treatment is required. However, common procedures are often complicated, or in the process of separation, the structure of the cell is destroyed, which in turn causes death and affects subsequent research.

In response, Yamada et al. have developed a method for allowing cells to be desorbed smoothly by using a surface grafted with a temperature-responsive polymeric poly(N-isopropene acrylamide) at 37 ° C. Hydrophobic allows cells to attach to them, but when the culture temperature drops to the lowest criticality of the temperature-responsive polymer material, it turns into hydrophilicity, allowing the cells to smoothly separate to the surface. However, in this method, the attachment or detachment of cells is mainly controlled by the effects of different temperatures, and therefore, there is a disadvantage that cells are easily affected by the effects of temperature turbulence during culture. Therefore, it is necessary to develop another method for effectively desorbing cells from the attached surface.

In view of the above, the object of the present invention is to develop a method for effectively desorbing cells from a self-adhesive surface, which is mainly accomplished by applying different characteristics of surface electrical properties of acid-base responsive polymers in different acid-base environments. .

In order to achieve the above object, the present invention provides a method for controlling cell adhesion, comprising: providing a substrate, wherein the substrate has an acid-base responsive polymer on a surface thereof; The cells are cultured on the surface; and the pH of the medium for culturing the cells is adjusted to control the adhesion of the cells.

In a preferred embodiment, the substrate is a cell culture dish, a cell culture dish or a cell culture flask.

In a preferred embodiment, the acid-base response type polymer has an acid separation coefficient (pKa) of 6.0 to 8.0. More preferably, the acid-base response type polymer has an acid separation coefficient of 6.4 to 7.2.

In a preferred embodiment, the acid-base responsive polymer is a polymer having an amine group. More preferably, the amine-based polymer is chitosan, polylysine, polyamine, or polyacrylamide. (due to

In a preferred embodiment, the method of providing an acid-base responsive polymer on the surface of the substrate comprises the step of dissolving the acid-base responsive polymer in a weak acid aqueous solution to obtain a high a molecular solution; coating the polymer solution onto a substrate to obtain a coated substrate; drying the coated substrate to obtain a dried substrate; neutralizing the dried with an alkaline aqueous solution a substrate, a neutralized substrate; and the neutralized substrate is washed with water and dried.

In a preferred embodiment, the pH of the aqueous weak acid solution is between 3.5 and 6.5. More preferably, the pH of the aqueous weak acid solution is between 4 and 6.

In a preferred embodiment, the pH of the medium is between 6.5 and 8.5.

In a preferred embodiment, the adjustment of the pH is accomplished by replacing the medium.

In a preferred embodiment, the step of culturing is carried out in an incubator.

In a preferred embodiment, the adjustment of the pH is accomplished by varying the partial pressure of carbon dioxide in the incubator.

In a preferred embodiment, the adjustment of the pH is accomplished by balancing the carbon dioxide in the incubator with sodium bicarbonate in the medium.

In a preferred embodiment, the attachment system comprises attachment or detachment.

It can be seen from the above that the present invention mainly utilizes the characteristics of the surface electrical properties of the acid-base responsive polymer in different acid-base environments to regulate the adhesion of the cells, and thus can be easily changed by changing the pH of the medium. The cells are desorbed from the acid-base responsive polymer.

The present invention provides a method for effectively desorbing cells from a surface to be attached, which is mainly accomplished by applying characteristics of different surface electrical properties of an acid-base responsive polymer in different acid-base environments.

Specifically, the method for controlling cell adhesion provided by the present invention comprises: providing a substrate, wherein the surface of the substrate has an acid-base responsive polymer; on the surface of the substrate The cells are cultured; and the pH of the medium for culturing the cells is adjusted to control the adhesion of the cells.

In a preferred embodiment, the pH of the medium is from 6.5 to 8.5, more preferably from 6.8 to 7.7.

In the present invention, the substrate may be a vessel for culturing cells well known to those skilled in the art, including, but not limited to, cell culture dishes, cell culture dishes or cell culture flasks.

The "acid-base response type polymer" as used in the present invention means a polymer which is positively charged in a weak acid environment and is not charged in a weak alkali environment. In general, the acid-base response type polymer preferably has an acid separation coefficient of 6.0 to 8.0, more preferably 6.4 to 7.2. In a preferred embodiment, the acid-base responsive polymer comprises an amine group-containing polymer (such as chitosan, polylysine, polyacrylamide, or polyacrylamide), but not Limited to this.

In the present invention, the method of providing an acid-base responsive polymer on the surface of the substrate comprises the steps of: dissolving the acid-base responsive polymer in a weak acid aqueous solution to obtain a polymer solution; The polymer solution is coated on a substrate to obtain a coated substrate; the coated substrate is dried to obtain a dried substrate; and the dried substrate is neutralized with an alkaline aqueous solution to obtain The neutralized substrate; and the neutralized substrate is washed with water and dried. The aforementioned weak acid aqueous solution and alkaline aqueous solution are well known to those skilled in the art. For example, the weak acid aqueous solution may be an aqueous acetic acid solution, an aqueous formic acid solution, an aqueous citric acid solution or an aqueous phosphoric acid solution, but is not limited thereto, and the alkaline aqueous solution is not limited thereto. It may be an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, or an aqueous lithium hydroxide solution, but is not limited thereto. The pH of the weak acid aqueous solution is preferably between 3.5 and 6.5, and more preferably between 4 and 6.

The cells used in the present invention may be any kind of mammalian cells, including human lung carcinoma (H1299), mouse embryonic fibroblast (3T3) or bovine corneal interstitial cells (bovine). Keratocyte/fibroblast), but not limited to this.

In a preferred embodiment, the adjustment of the pH is accomplished by replacing the medium.

In another preferred embodiment, the step of culturing is carried out in an incubator. The adjustment of the pH is accomplished by balancing the carbon dioxide in the incubator with sodium bicarbonate in the medium. Preferably, the partial pressure of the carbon dioxide is 0.01 to 30.00%. More preferably, the partial pressure of the carbon dioxide is from 0.10 to 15.00%. Further, it is not difficult to understand that in the present invention, the pH can also be adjusted by changing the partial pressure of carbon dioxide in the incubator.

There is no particular limitation on the selection of the medium, and it can be any commercially available medium, and since it is well known to those skilled in the art, it will not be repeated here. Preferably, the medium contains 0.01 to 50.00% serum. More preferably, the medium contains 0.10 to 20.00% serum.

Moreover, it will be appreciated that the methods provided herein can be used not only to control cell attachment, but also to control cell desorption, which is entirely dependent on the needs of the user.

The technical features of the present invention have been specifically described in the description of the invention, and other materials and formulations are well known in the art, and those skilled in the art can easily implement the present invention. The features and advantages of the present invention are exemplified by the embodiments.

Example 1: Preparation of a substrate having an acid-base responsive polymer on its surface material:

1. Chitosan: purchased from Sigma-aldrich under the trade name Chitosan.

2. Acetic acid: purchased from Sigma-aldrich under the trade name Acetic Acid.

3. 24-well plate: purchased from Corning, the trade name is Multiwell Culture Plate.

4. Sodium hydroxide: purchased from Sigma-aldrich under the trade name Sodium Hydroxide.

Preparation:

0.5 g of chitosan having an acid separation coefficient of 6.5 was dissolved in an aqueous solution of acetic acid having a concentration of 1% and a pH of 4 to obtain a polymer solution. 200 μl of the polymer solution was added to a polystyrene 24-well plate for culturing cells, and at this time, the polymer solution was applied to the 24-well plate. The coated 24-well plate was then placed in an oven at 60 ° C, dried for 24 hours, and the solution was evaporated to dryness. After evaporation to dryness, a 1 M aqueous sodium hydroxide solution was added and neutralized. After the neutralization, the neutralized 24-well disk was washed with water and dried to obtain a substrate having an acid-base responsive polymer on the surface.

Example 2: Preparation of medium material:

1. Sodium bicarbonate: purchased from Sigma-aldrich under the trade name Sodium Bicarbonate.

2. DMEM: purchased from Invitrogen under the trade name Dulbecco's Modified Eagle Medium.

3. Hydrochloric acid: purchased from Sigma-aldrich under the trade name Hydrogen Chloride.

Preparation method:

According to Table 1, after adding 600 mg/L to 3600 mg/L of sodium bicarbonate in DMEM medium containing 10% FBS, the pH of the medium was adjusted to 7.40 by titration with 1 M sodium hydroxide and 1 M hydrochloric acid. Thereafter, a carbon dioxide incubator having a partial pressure of 5% was placed, and after one hour of equilibration, cell culture solutions having a pH of 6.99 to 7.65 were respectively obtained.

.

Example 3: Attachment test

The epithelial lung cancer cell line (H1299), the embryonic fibroblast (3T3), and the keratocyte/fibroblast were prepared as in Example 1 by using the medium sample 1 to the medium sample 6 of the second embodiment. A 24-well plate was used as a control group with a 24-well plate coated with chitosan. The number of cells per well was 2 × 10 ⁴ , and culture was carried out for 24 hours in a culture environment of partial pressure of 5% carbon dioxide at 37 ° C. The cells were observed by an inverted phase contrast microscope, and the results are shown in the first figure. Further remove the culture solution and use Assay (purchased from Invitrogen, trade name Proliferation Assay) Tests the number of cells attached, the results of which are shown in the second figure.

The first panel A to the first panel C show the microscopic observation results of the epithelial lung cancer cell line (H1299), the embryonic fibroblast (3T3), and the keratocyte/fibroblast of Example 3. The results from the first panel A to the first panel C show that in the 24-well plate coated with chitosan, the adhesion of the cells to the well plate decreases with the increase of the pH value of the medium, and the criticality of the attachment is The point is approximately between the medium sample 3 and the medium sample 4, that is, the pH is between 7.36 and 7.48. The second panel A to the second panel C show the quantified results of the cell attachment amount of the epithelial lung cancer cell line, the embryonic fibroblast, and the corneal interstitial cells of Example 3, respectively, and the results are the same as those in the first A map. The observations of the first C chart. Compared to the present invention, the same phenomenon was not observed as a 24-well plate of uncontrolled chitosan as a control group. As described above, it is confirmed that the present invention can control cell adhesion by adjusting the pH of the medium.

Example 4: Desorption experiment-1/replacement medium

Based on the results of Example 3, a suitable pH medium was screened for the desorption experiment. In the present example, the medium sample 2 was used as a patch medium. The epithelial lung cancer cell line, the embryonic fibroblast, and the corneal interstitial cells were cultured in the 24-well plate prepared in Example 1, and the 24-well plate without the chitosan coating was used as a control group. The amount is 2×10 ⁴ , cultured in a 5% carbon dioxide, 37° C culture environment, and cultured for 24 hours. After the attachment medium is removed, the medium sample 6 is added, and then cultured under 5% carbon dioxide and 37° C. Incubate for 4 hours, observe with an inverted microscope and a sputum photographic system, and after 4 hours of culture, Assay tested the amount of cell desorption and the results are shown in the third figure.

The third panel A shows the microscopic observation of corneal stromal cells at different times after the addition of the medium sample 6. From the results of the third panel A, it can be found that the corneal interstitial cells cultured on the chitosan are added to the medium sample 6, and after culturing for 4 hours at 5% carbon dioxide at 37 ° C, the originally attached cells will slowly aggregate. When the pellet is formed, the entire cell mass will desorb from the surface of the well plate by 4 hours.

The third panel B shows the quantified results of the amount of cell desorption of corneal interstitial cells after 4 hours of culture in the culture medium sample 6. The results of the third panel B show that 90% of the cells will desorb from the well plate, while in the control group, no such phenomenon was observed.

Figure 3C shows the observation of fibroblasts at different times after addition of medium sample 6. The third panel D shows the observation of epithelial lung cancer cells at different times after the addition of the medium sample 6. From the results of the third graph C and the third graph D, the phenomenon similar to the third graph B can also be found. From the above, it was confirmed that the present invention can control the adhesion of the cells by adjusting the pH of the medium for culturing the cells.

Example 5: Desorption experiment-2/conversion of carbon dioxide partial pressure

Based on the results of Example 3, a suitable pH medium was screened for the desorption experiment. In this example, the culture medium sample 2 was used as the attachment medium, and the corneal interstitial cells were cultured in the 24-well plate prepared in Example 1, and the 24-well plate without the chitosan coating was used as the control group, and the cells per well were used. The number was 2×10 ⁴ , and after culturing for 24 hours in a culture environment of 5% carbon dioxide and 37° C., the cell disk was moved to a 0% carbon dioxide, 37° C culture environment, and observed with an inverted microscope and a photographic system. After 4 hours of cultivation, Assay tested the amount of cell desorption and performed a LIVE/DEAD cell assay on the desorbed cells (purchased from Invitrogen under the trade name Cell Viability Assays), the results are shown in Figure 5.

Figure 4A shows the results of microscopic observation of the corneal interstitial cells of Example 5. The results showed that the corneal interstitial cells cultured on chitosan were transferred to a culture environment of 0% carbon dioxide and 37 ° C, and after 4 hours of culture, the originally attached cells were slowly aggregated into groups for 4 hours. At the time, the entire cell mass will desorb from the well plate. Figure 4B shows the quantitative results of the amount of cell desorption of the corneal interstitial cells of Example 5. As a result, it was found that about 90% of the cells were desorbed from the well plate, and in the control group, the same phenomenon was not observed. In addition, the fifth panel shows the results of the LIVE/DEAD cell assay showing the corneal interstitial cells obtained by desorption. The results showed that the cell pellets that were desorbed were composed of living cells (green fluorescence, please refer to the color scheme attached), and almost no dead cells (red fluorescence) were observed. From the above, it was confirmed that the present invention can control the adhesion of the cells by adjusting the pH of the medium for culturing the cells, and the cells obtained by desorption still have an extremely high survival rate.

In summary, the present invention changes the surface electrical properties of the acid-base responsive polymer by changing the pH of the medium, thereby affecting the adhesion of the cells to the cells. Thereby, a method for easily desorbing cells from the attached surface can be provided, and the cells obtained by desorption still have an extremely high survival rate.

Other implementations

All features disclosed in this disclosure can be combined in any manner. Features disclosed in this specification can be replaced with features of the same, equivalent or similar purpose. Therefore, the features disclosed in this specification are one of a series of equivalent or similar features.

In addition, according to the disclosure of the present specification, those skilled in the art can easily make appropriate changes and modifications to different methods and situations without departing from the spirit and scope of the present invention. The implementation aspect is also included in the scope of the patent application.

The first panel A shows the microscopic observation results of the epithelial lung cancer cell line (H1299) of Example 3.

The first panel B shows the results of microscopic observation of the embryonic fibroblasts (3T3) of Example 3.

The first panel C shows the results of microscopic observation of the corneal interstitial cells (keratocyte/fibroblast) of Example 3.

Fig. 2A shows the results of quantification of the amount of cell attachment of the epithelial lung cancer cell line of Example 3.

Figure B is a graph showing the quantified results of the amount of cell attachment of the embryonic fibroblasts of Example 3.

Fig. 2C shows the results of quantification of the amount of cell attachment of the corneal interstitial cells of Example 3.

The third panel A shows the observation of corneal stromal cells at different times after the addition of the medium sample 6.

The third panel B shows the quantified results of the amount of cell desorption of corneal interstitial cells after 4 hours of culture in the culture medium sample 6.

Figure 3C shows the observation of fibroblasts at different times after addition of medium sample 6.

The third panel D shows the observation of epithelial lung cancer cells at different times after the addition of the medium sample 6.

Figure 4A shows the results of microscopic observation of the corneal interstitial cells of Example 5 at different times.

Figure 4B shows the quantitative results of the amount of cell desorption of the corneal interstitial cells of Example 5.

The fifth panel shows the results of the LIVE/DEAD cell assay of the corneal interstitial cells obtained by desorption.

Claims (14)

A method for controlling cell adhesion, comprising: providing a substrate, wherein the substrate has an acid-base responsive polymer on a surface thereof; culturing cells on a surface of the substrate; and adjusting the culture The pH of the medium used for the cells to control the adhesion of the cells; wherein the pH of the medium is 6.5 to 8.5. The method of claim 1, wherein the substrate is a cell culture dish, a cell culture dish or a cell culture flask. The method of claim 1, wherein the acid-base responsive polymer has an acid separation coefficient (pKa) of 6.0 to 8.0. The method according to claim 3, wherein the acid-base responsive polymer has an acid separation coefficient of 6.4 to 7.2. The method of claim 1, wherein the acid-base responsive polymer is a polymer having an amine group. The method of claim 5, wherein the amine-based polymer is chitosan, poly-lysine, polyacrylamine, or polyacrylamide. The method of claim 1, wherein the method of providing an acid-base responsive polymer on the surface of the substrate comprises the step of dissolving the acid-base responsive polymer in a weak acid aqueous solution, Obtaining a polymer solution; coating the polymer solution onto a substrate to obtain a coated substrate; drying the coated substrate to obtain a dried substrate; neutralizing with an alkaline aqueous solution The dried substrate is subjected to a neutralized substrate; and the neutralized substrate is washed with water and dried. The method of claim 7, wherein the pH of the aqueous weak acid solution is between 3.5 and 6.5. The method of claim 8, wherein the pH of the aqueous weak acid solution is between 4 and 6. The method of claim 1, wherein the adjusting of the pH is accomplished by replacing the medium. The method of claim 1, wherein the step of culturing is carried out in an incubator. The method of claim 11, wherein the adjusting of the pH is accomplished by changing a partial pressure of carbon dioxide in the incubator. The method of claim 11, wherein the adjustment of the pH is accomplished by balancing carbon dioxide in the incubator with sodium bicarbonate in the medium. The method of claim 1, wherein the attachment system comprises attachment or detachment.