JPH089961A - Culture medium and its production - Google Patents

Abstract

PURPOSE:To prepare a culture medium useful for tissue culture, etc., capable of preventing a collagen gel layer from liquefying by vibration during transportation by forming the collagen gel layer on the bottom of a container, adding a specific colloidal solution and treating it under a prescribed condition. CONSTITUTION:A collagen gel layer such as type 1 collagen from which telopeptide is removed is formed on the bottom of a container, a colloidal solution which is made into a sol state by heating and into a gel state by cooling, comprising a gelatin as a main component and having 1-20% gelatin concentration at pH7-9 and 4-39 deg.C conversion temperature from the sol state to the gel state is added to the collagen gel layer. The colloidal solution is incubated at a temperature between the conversion temperature from the colloidal solution to the gel state and 40 deg.C, water in the colloidal solution is replaced with the colloidal solution and the colloidal solution is gelatinized to give objective culture medium.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is mainly used in the field of cell culture and tissue culture, and is a soft collagen gel capable of culturing while maintaining the function of cells for a long period of time. The present invention relates to an incubator having a collagen gel layer used for the above culture.

[0002]

2. Description of the Related Art Functional cells having functions of secretion and metabolism such as hepatocytes and mammary gland cells, when cultured on a collagen gel, form a cell mass and have a three-dimensional structure close to that of a living body. It functions at a level close to that in the living body, and depending on the composition of the medium, it is possible to maintain the function for a long period of time.

This collagen gel consists of 99% or more of water and is very soft. When the gel is formed, the gel liquefies due to vibration during transportation, and once liquefied, the collagen gel cannot form a gel again. Absent. There is also a method of freeze-drying the gel and transporting it, but when freeze-drying, it is necessary to soak it in water for a long period of several days to several weeks to return it to the gel. Even after returning to, the reformed gel is very stiff,
Does not function as a collagen gel for cells,
Even if cells are cultured on this reformed gel, the above-mentioned cell clusters are not formed and the function of the cells cannot be maintained. Therefore, when the collagen gel is used for culturing cells, the collagen gel must be adjusted each time the culture is performed.

Generally, a collagen gel is formed by neutralizing an acid-solubilized collagen solution and crosslinking it. At that time, in order to adjust the concentration of salts in the gel to the optimum condition for culturing, a salt preparation solution is added in addition to the neutralization solution. As described above, the experimenter has the troublesomeness of preparing a neutralizing solution and a salt adjusting solution each time the collagen gel is prepared. In order to eliminate such troublesomeness, a kit for forming a collagen gel in which a necessary amount of an acidic acid-solubilized collagen solution, a neutralizing solution, and a salt adjusting solution are preset is commercially available from a reagent manufacturer. If such a kit is used, it is not necessary to prepare the neutralizing solution and the salt adjusting solution, but the three kinds of solutions must be preparatively mixed in correct proportions. If the mixing ratio is wrong, no gel will be formed or the hardness of the gel will be different.

In addition, due to the heat generated during neutralization, a gel may be partially formed during neutralization, and a uniform collagen gel may not be obtained. It is necessary to carry out the Japanese operation little by little under ice cooling. The neutralized collagen solution must be dispensed into a gel-forming container, and then heated at about 37 ° C in an incubator to form a uniform gel.

Since it is difficult to transport collagen gel and it is not possible to market preformed gel as a gel,
When culturing on collagen gel, it is said that the experimenter requires a great deal of labor in addition to the original operation of collecting primary cells and culturing cells in order to prepare the collagen gel culture bed by itself as described above. There's a problem.

[0007]

DISCLOSURE OF THE INVENTION The present invention prevents the liquefaction of a soft collagen gel capable of forming a cell mass maintaining a function during transport in the primary culture of functional cells, thereby culturing the collagen gel. It is to provide a culture experimenter with a culture vessel in which a collagen gel is formed in advance, which can be transported without deteriorating the characteristics and without requiring a special device or the like.

[0008]

As a result of earnest research to achieve such an object, the inventors of the present invention have found that the collagen gel once formed does not undergo liquefaction unless it is vibrated, and under the culture conditions. Does not dissolve, and the colloidal solution such as gelatin does not become a sol at a temperature of 4 to 35 ° C depending on the concentration of solute, maintains the gel strength, does not cause liquefaction due to vibration, and the normal culture temperature. 37 ° C
Then, they found that they could be rapidly dissolved and easily removed, and the present invention was completed.

That is, the present invention is a colloidal solution in which a collagen gel layer is formed on the bottom surface of a container, and the collagen gel layer is heated to a sol state and cooled to a gel state. Is embedded in a colloidal solution in the range of 4 to 39 ° C., and the colloidal solution contains gelatin as a main component, and the concentration of gelatin is 1 to 20% (W / V), and further, the collagen gel is a type 1 collagen gel from which telopeptides have been removed, or a collagen gel obtained by gelling type 1 collagen treated with a reducing agent. Is characterized by.

Further, a collagen gel layer is formed on the bottom surface of the container, and a colloidal solution is formed on the collagen gel layer by heating to a sol state and ice-cooling to a gel state.
A colloidal solution whose conversion temperature between the sol state and the gel state is in the range of 4 to 39 ° C. is added, and the mixture is incubated at a temperature between the temperature at which the colloidal solution is converted to the sol state and 40 ° C. Is replaced with the colloidal solution, and then the colloidal solution is gelled, which is a method for producing an incubator.

The incubator according to the present invention is a incubator in which the collagen gel formed on the bottom of the container is embedded in a colloidal solution and gelled to reinforce the soft collagen gel and to transport the collagen gel without liquefying it. It is a feature.

[0012] There are examples of embedding cells with agar, alginic acid, etc. as gel-like substances having no cytotoxicity. However, these do not dissolve in water or require high temperature for dissolution. When the collagen gel is embedded in the collagen gel during the embedding process, it is hard to be solidified in the collagen gel and difficult to remove from the collagen gel. In this case, the structure of the collagen gel changes and the collagen gel becomes hard, and when cells are cultured on such a collagen gel, a single layer does not form a cell mass, and it becomes difficult to maintain the function of the cells for a long time. It becomes impossible to culture on collagen gel.

The colloidal solution has a normal transportation temperature (normal temperature).
Is in a gel state, which is a general cell culture temperature. 3
There is no particular problem as long as it dissolves easily at 7 ° C., is easy to remove, and is not toxic to cells. As such a colloidal solution, a gelatin solution can be mentioned. In particular, gelatin does not change the structure or hardness of the collagen gel even when the formation of cell mass is performed on the collagen gel.
Since gelatin is originally derived from collagen, the composition of protein is close to that of collagen, and even if gelatin remains, it does not change the properties of the collagen gel surface.
The colloidal solution used in the present invention is most suitable.

Next, the method for producing the main incubator will be described by taking the case of using a gelatin colloidal solution as an example. First,
Dispense a mixture of acidic collagen solution, salt concentration adjusting solution, and alkaline solution for neutralization into the well of a dish or a plate with multiple wells used for normal tissue culture, and heat at about 37 ° C. Then, the mixture is solidified, and the collagen gel is embedded with a colloidal solution of gelatin.

The origin of the gelatin used is not particularly specified, and those derived from the dermis and bones of pigs and cows which are generally commercially available and easily available can be used. The concentration of gelatin in the solution to be adjusted is appropriately 1 to 20%. If the concentration is lower than this, the strength of the gelatin gel formed is weak and the gelatin gel may collapse during transportation. Further, in a solution that is thicker than this, the viscosity of the gelatin solution becomes high, making it difficult to dispense the solution at the time of embedding and to remove the gelatin solution at the start of culture.

The water used for preparing the gelatin colloidal solution is physiological saline, physiological phosphate buffer, or various serum-free media for the purpose of adjusting the salt concentration in order to prevent changes in the salt concentration of the medium during culturing. It is better to use Further, the pH of the colloidal solution is preferably adjusted to 7 to 9 because it is necessary to prevent the collagen gel from being dissolved.

After the gelatin colloidal solution is dispensed, it may be solidified as it is to form a gelatine gel, but in order to reinforce the strength of the collagen gel itself, the gelatine solution is dispensed into a container and then 37 It is preferable to incubate at about 0 ° C. for several hours, replace the water content in the collagen gel with a gelatin colloid solution, and then solidify to form a gelatin gel layer.

In the collagen gel layer embedded with the gelatin gel, the gelatin gel acts as a reinforcing and shock absorbing material at a temperature of 0 to 30 ° C., and the collagen gel layer is liquid due to vibration and shock during transportation. It doesn't break up or collapse.

As the container used in the present invention, an incubator generally used for tissue culture or cell culture such as a petri dish, a plate having a plurality of wells, and a flask is used. As a special one, there is one with a fine mesh or membrane on the bottom of the cylinder.In this case,
The formation of the collagen gel layer and the embedding with gelatin gel can be performed in a container, and the container used in this way is not particularly limited. Also, the material of the container is not particularly limited as long as it is not cytotoxic.

The thickness of the collagen gel layer is not particularly limited as long as a gelatin gel layer having a sufficient thickness can be formed in the container, and the gelatin gel layer has a thickness equal to or greater than that of the collagen gel layer. It is possible to reinforce the strength of the collagen gel layer.

Next, the method of using the main culture vessel will be described. First, before seeding the cells, the main incubator is incubated at 37 ° C. The gelatin gel layer dissolves immediately, so pipette off the gelatin colloid solution,
The medium used for culturing the target cells is added, and the mixture is incubated for several hours to allow the medium to adapt to the collagen gel layer.
All that is required is to seed the cells, and no preparation of reagents for preparing the collagen gel is required.

[0022]

EXAMPLES Next, the present invention will be described more specifically by way of examples. Example 1 0.3% acidic collagen type 1 solution, 10-fold concentrated PBS
(Physiological phosphate buffer solution) and 0.01N sodium hydroxide aqueous solution were aseptically mixed at a ratio of 8: 1: 1 under ice cooling, and 1 ml was dispensed into an injection-molded polystyrene dish having a diameter of 35 mm. Then, the mixture was heated at 37 ° C. to form a collagen gel layer.

On the other hand, 10% while heating with pure water
(W / V) gelatin colloidal solution was prepared, and MEM medium powder was added at a rate of 9.4 g / l for autoclave sterilization, and after cooling to 37 ° C., glutamine was added,
Adjust the pH to 7.4 by adding sodium bicarbonate and
A gelatin colloidal solution containing M medium was prepared. 2 ml of this gelatin colloid solution was poured into a petri dish on which a collagen gel layer was previously formed, and the mixture was heated at 37 ° C. for 10 hours to replace the water in the collagen gel with the gelatin colloid solution and solidify at room temperature. The collagen gel was embedded in a gelatin gel, a transport test was performed, and then a culture test was performed.

Comparative Example 1 A petri dish having a collagen gel layer formed thereon in the same manner as in Example 1 was subjected to a transport test without being embedded in a gelatin colloid solution, and then subjected to a culture test.

Comparative Example 2 The collagen gel formed in the same manner as in Example 1 was not embedded in the gelatin colloid solution, and was not subjected to the transport test, and was used as it was for the culture test.

Transport test The temperature of the petri dish of Example 1 and Comparative Example 1 was not particularly adjusted, 1300 kg was transported by truck, and then the state of the collagen gel was examined. In Example 1, after transportation, the jelly layer was dissolved by heating to 37 ° C., the gelatin solution was removed, L-15 medium was added, and the mixture was incubated at 37 ° C. for 12 hours. For Comparative Example 1, after transportation, L
-15 medium was added and incubated at 37 ° C for 12 hours. For Comparative Example 2, no transportation was performed and L-15
The medium was added and incubated at 37 ° C. for 12 hours. After removing the medium, the weight and thickness of the collagen gel layer were measured, and the ratio was calculated for Example 1 and Comparative Example 1 with Comparative Example 2 as 100.

In addition, a cell morphology test and a cell function test were performed on each sample in the following manner. Cell morphology test Rats (Wistar strain,
Liver parenchymal cells were collected from males, 5 weeks old, and 10% FBS
Using L-15 medium supplemented with (fetal bovine serum), 1 × 10 ⁶ cells were seeded per petri dish, the medium was exchanged 2 hours after seeding, and then the medium was exchanged every 2 days.
The morphology of cells was observed on the day. Cell Function Test Simultaneously with the above cell morphology test, the amount of albumin in the medium was measured 3 and 7 days after seeding, and Comparative Example 2
The albumin amount was shown as 100.

The results of each observation and measurement are shown in Table 1. The incubator in which the collagen gel layer of the present invention is formed can be transported without damaging the collagen gel layer, and the collagen gel layer can be transported after the transportation. It is clear that the culture characteristics of the are maintained unchanged.

[0029]

[Table 1]

[0030]

EFFECTS OF THE INVENTION According to the present invention, the collagen gel layer can be prevented from being liquefied due to vibration during transportation, and the culture characteristics are not impaired. Therefore, three-dimensional culture of functional cells such as hepatocytes. It is possible to directly provide the experimenter with an incubator in which a soft collagen gel is formed, and the experimenter is not required to prepare the collagen gel, and is suitable as an incubator that can easily perform the collagen gel culture.

Claims (5)

[Claims] 1. A collagen gel layer is formed on the bottom surface of a container,
The collagen gel layer was embedded in a colloidal solution that was in a sol state when heated and turned into a gel state when cooled, in which the conversion temperature between the sol state and the gel state was in the range of 4 to 39 ° C. An incubator characterized by that. 2. A colloidal solution containing gelatin as a main component,
The incubator according to claim 1, wherein the gelatin concentration is in the range of 1 to 20%. 3. The incubator according to claim 1, wherein the pH of the colloidal solution is in the range of 7-9. 4. The incubator according to claim 1, wherein the collagen gel is gel of type 1 collagen from which telopeptide has been removed or type 1 collagen treated with a reducing agent. . 5. A collagen gel layer is formed on the bottom of the container,
Onto the collagen gel layer, a colloidal solution which is in a sol state when heated and is in a gel state when cooled, in which the conversion temperature between the sol state and the gel state is in the range of 4 to 39 ° C., is added. After incubating at a temperature between the temperature at which the colloidal solution is converted to the sol state and 40 ° C. to replace water in the collagen gel with the colloidal solution,
A method for producing an incubator, which comprises gelling the colloidal solution.