JP2023051354A - Method for producing cell aggregates - Google Patents

Abstract

To form a cell aggregate 1 in which three-dimensionally cultured cells 7 are evenly dispersed and to easily collect the cells from a culture vessel 6.SOLUTION: The method comprises mixing steps (A)-(C) for preparing a collagen-containing cell mixture 5 by diluting uncrosslinked solubilized collagen 4 with a culture medium 3 to a prescribed concentration and mixing in cells 7, and a culturing step (D) of storing the collagen-containing cell mixture 5 in a culture vessel 6 and culturing the same at a prescribed temperature environment. The culturing step (D) involves thermally crosslinking the solubilized collagen 4 and obtaining the cell aggregate 1 by culturing the cells 7 dispersed three-dimensionally in the crosslinked collagen 4A.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to a method for producing cell aggregates, and more particularly to a method for producing cell aggregates by three-dimensionally culturing cells.

BACKGROUND ART Conventionally, cell aggregates obtained by three-dimensionally culturing cells have been used in fields such as regenerative medicine.
As a method for producing such cell aggregates, a method of embedding cells in high-concentration collagen and culturing them is known (Patent Document 1).

Japanese Patent No. 4936892

However, in the method of Patent Document 1, due to the high viscosity of collagen, many times of pipetting are required in order to uniformly mix the cells with the collagen, which may damage the cells. In addition, since air bubbles tend to remain during mixing, there is a fear that hollows may form in the collagen due to thermal cross-linking.
In addition, since the concentration of collagen is high, the fibers become dense, and cells cannot migrate and disperse within the collagen after thermal cross-linking. rice field.
Furthermore, since the cell aggregate adheres to the bottom surface of the culture vessel, there is a risk that the cell aggregate may be damaged when the cell aggregate is peeled off from the culture vessel for recovery.
In view of such problems, the present invention provides cells that can be uniformly dispersed and three-dimensionally cultured, and that cell aggregates can be easily recovered from a culture vessel without damaging the cells. A method for making an aggregate is provided.

That is, the method for producing a cell aggregate according to the invention of claim 1 is a method for producing a cell aggregate by three-dimensionally culturing cells,
A mixing step of diluting uncrosslinked solubilized collagen with a culture medium to a predetermined concentration and mixing cells to prepare a collagen-containing cell mixture; performing a culturing step of culturing the cells in
The method is characterized in that the collagen is thermally crosslinked in the culturing step, and the three-dimensionally dispersed cells are cultured in the crosslinked collagen to produce a cell aggregate.

According to the invention of claim 1, by using the uncrosslinked solubilized collagen diluted to a predetermined concentration, the cells migrate in the crosslinked collagen and three-dimensionally disperse, resulting in homogeneous cells. Aggregates can be made.
In addition, the cultured cell aggregates are spontaneously detached from the bottom surface of the culture vessel, so that the cell aggregates can be easily recovered, and damage to the cell aggregates during recovery can be eliminated.

FIG. 4 is a diagram for explaining a method for producing a cell aggregate according to the present embodiment;

The illustrated embodiments will be described below. FIG. 1 is a diagram for explaining a method for producing a cell aggregate 1 according to the present invention.
The cell aggregate 1 produced in this embodiment can be used for regenerative medicine, etc. Cells collected from a patient are cultured to produce the cell aggregate 1, and this is applied to a patient's defective part for treatment. used to Moreover, the production method according to the present invention is suitable for culturing adhesive cells such as mesenchymal stem cells (MSC), fibroblasts, endothelial cells, and the like.
In addition, the cell aggregate 1 produced in this embodiment is an elastic solid in which cells are three-dimensionally dispersed in crosslinked and gelled collagen. It is possible to obtain a substantially disk-shaped cell structure with a thickness of about 0.1 to several mm.
On the other hand, a so-called cell sheet is obtained by culturing cells two-dimensionally and has a thickness of about 0.01 to 0.04 mm.

The method for producing the cell aggregate 1 in this example includes a suspension preparation step (A) of mixing the cells 7 to be cultured and the culture medium 3 to prepare the cell suspension 2, and A solution preparation step (B) for preparing a collagen solution 8 by mixing the solubilized collagen 4 from the above, and a collagen-containing cell mixture 5 by mixing the prepared cell suspension 2 and the collagen solution 8. and a culturing step (D) of placing the prepared collagen-containing cell mixture 5 in a culture vessel 6 and culturing the cells 7 in a predetermined temperature environment.
Then, through the suspension preparation step (A), the solution preparation step (B), and the mixture preparation step (C), the uncrosslinked solubilized collagen 4 is diluted to a predetermined concentration with the culture medium 3, and the cells 7 are mixed. A mixing step of preparing the collagen-containing cell mixed solution 5 by allowing the mixture to flow is constituted.
The suspension preparation step (A), the solution preparation step (B), and the mixture preparation step (C) can be performed in a clean space in a clean bench or an isolator, and the culture step (D) is performed in a predetermined incubator. can be performed in a space maintained at a temperature and humidity environment of
Since the clean bench, isolator, and incubator are conventionally known, detailed description thereof will be omitted.

Each step will be described in detail below. The suspension preparation step (A) can be carried out by a very general conventionally known method.
First, mesenchymal stem cells are plate-cultured inside the dish 9, and when they are propagated to a certain amount by repeating passage a plurality of times, they are moved together with the dish 9 from the incubator to the isolator.
Subsequently, after removing the medium from the dish 9 and washing with PBS (phosphate-buffered saline), the PBS is removed, a peeling enzyme agent is added, the mixture is placed in the incubator again, and incubated at 37° C. for a predetermined time. Cells 7 are detached.
After detachment, a culture solution 3 made of DMEM (Dulbecco's Modified Eagle's Medium) is added to a dish 9 in an isolator, placed in a centrifuge tube 10 and centrifuged, and the supernatant is removed from the centrifuge tube 10 after centrifugation. Then, the culture solution 3 heated to 37° C. is added to obtain a suspension of cells 7 .
A small amount of this suspension is sampled, the number of cells is counted using a hemocytometer, and the cell suspension 2 with a predetermined concentration is prepared by diluting with the culture medium 3 so as to obtain the desired cell number concentration.
Here, the cell suspension 2 was prepared so that the cell number concentration was 5.0×10 ⁵ to 60.0×10 ⁵ cells/mL.

Next, the solution preparation step (B) will be described. Solubilized collagen 4 (3% type I atelocollagen) and culture medium 3 (DMEM (Dulbecco's Modified Eagle Medium)) that had been refrigerated at 4 to 10° C. Then, the collagen solution 8 is prepared by diluting the solubilized collagen 4 with the culture solution 3 to a predetermined concentration in the isolator.
The solubilized collagen 4 is obtained by enzymatically decomposing collagen, which is composed of poorly soluble proteins, solubilized while maintaining the fibrous structure.
Collagen solution 8 was prepared to have a collagen concentration of 1.0 to 8.0 mg/mL.
Here, it is important that the solubilized collagen 4 is uncrosslinked, and gelation proceeds due to the crosslinking reaction at room temperature. Uncrosslinked solubilized collagen 4 and culture solution 3 that have been refrigerated at about 10° C. are used.
After that, the work is performed quickly so as not to raise the temperature during the work in the room temperature environment (20 to 25° C.) in the isolator, and the prepared collagen solution 8 is stored again in the refrigerator.

Next, the mixture preparation step (C) will be described. The collagen solution 8 prepared in the solution preparation step (B) and the cell suspension 2 prepared in the suspension preparation step (A) are are mixed at a ratio of 1:1 to prepare a collagen-containing cell mixture 5 in which the solubilized collagen 4 is diluted to a predetermined concentration.
Regarding the mixing step in this example, the cell suspension 2 is prepared in the suspension preparation step (A), the collagen solution 8 is prepared in the solution preparation step (B), and the mixed solution is prepared. Although the cell suspension 2 and the collagen solution 8 are mixed in the step (C), the present invention is not limited to this, and the solubilized collagen 4 is added to the cell suspension 2 prepared in the suspension preparation step (A). The collagen-containing cell mixed solution 5 may be prepared by mixing, or the cells 7 may be mixed with the collagen solution 8 prepared in the solution preparation step (B). The solubilized collagen 4 and the cells 7 may be mixed at the same time. The point is that the collagen-containing cell mixed solution 5 in which the solubilized collagen 4 is diluted to a predetermined concentration and the cells 7 are mixed can be prepared.
In addition, since the cells 7 are damaged when the temperature drops from 37 ° C., after preparing the cell suspension 2 in the suspension preparation step (A), the mixture is prepared in the mixture preparation step (C) as soon as possible. Since it is better to mix with the collagen solution 8 and move to the next culture step (D), the solution preparation step (B), the suspension preparation step (A), and the mixed solution preparation step (C) Before preparing the cell suspension 2, the collagen solution 8 is prepared in advance and stored in a refrigerator.

By mixing the cell suspension 2 and the collagen solution 8 at a ratio of 1:1, the collagen concentration in the resulting collagen-containing cell mixture 5 was 0.5 to 4.0 mg/mL. The number concentration is 2.5×10 ⁵ to 30.0×10 ⁵ cells/mL.
Here, if the collagen concentration in the collagen-containing cell mixture 5 is less than 0.5 mg/mL, the cell aggregate 1 to be produced becomes soft, and it becomes difficult to handle with tweezers or the like when removing from the culture vessel 6. end up
On the other hand, when the collagen concentration exceeds 4.0 mg/mL, the viscosity becomes high, and many times of pipetting are required to uniformly mix the cells in the collagen, which may damage the cells. In addition, since air bubbles tend to remain during mixing, there is a risk that cavities may form in the collagen due to thermal cross-linking.
In addition, when the collagen concentration is high, the fibers become dense, and the cells cannot migrate and disperse within the collagen after cross-linking. can no longer be produced.
Next, when the cell number concentration in the collagen-containing cell mixture 5 is less than 2.5×10 ⁵ cells/mL, the contraction action of the cells is not sufficiently exhibited, and the cell aggregates are separated from the bottom surface of the culture vessel 6. It becomes difficult to exfoliate spontaneously.
Conversely, when the cell number concentration exceeds 30.0×10 ⁵ cells/mL, the contraction action of the cells becomes strong, and the cell aggregates after detachment may be deformed and warped. Since the rate of adhesion increases, a spheroid structure is likely to be formed, resulting in non-uniform tissue strength and cracking of cell aggregates.
In addition, since cells are damaged in a low temperature state, after mixing the cell suspension 2 with the collagen solution 8 that has been cold-stored in the mixture preparation step (C), the culture step (D) is started immediately. There is a need.

Finally, in the culture step (D), the collagen-containing cell mixture 5 is contained in the culture vessel 6 using a pipette or other instrument in the isolator, and the cells 7 are seeded. Transfer to an incubator in which the humidity is maintained in an environment suitable for culture.
The storage portion for storing the collagen-containing cell mixture 5 in the culture vessel 6 generally has a flat and circular bottom, but there is no problem even if the bottom is curved, and a shape other than a circular shape may be used.
Also, in this embodiment, the culture container 6 having one accommodating portion is used, but a plurality of accommodating portions may be formed like a so-called well plate.

The interior of the incubator is maintained in an environment similar to that of normal cell culture, for example, an internal chamber temperature of 37° C., a humidity of 100%, and a carbon dioxide concentration of 5%. During the culturing step (D), the culture vessel 6 is transferred to the isolator at an appropriate timing and the medium is exchanged in the same manner as in normal cell culturing.
First, as shown in (a) of FIG. 1(D), immediately after starting the culture in the incubator, the solubilized collagen 4 contained in the collagen-containing cell mixture 5 undergoes a cross-linking reaction due to the internal temperature of the incubator. , and the crosslinked collagen 4A settles on the bottom of the culture vessel 6 as shown in (b).
Since the diluted solubilized collagen 4 has a low viscosity and a low fiber density, the cells 7 migrate in the crosslinked collagen 4A and are three-dimensionally dispersed to every corner. It becomes attached to the fibers that make up 4A.
As the culture of the cells 7 progresses using the fiber as a scaffold, the cells 7 dispersed three-dimensionally within the crosslinked collagen 4A are attracted to each other. The constituent fibers are also attracted and become contracted toward the center of the culture vessel 6 .
After that, when the culture of the cells 7 further progresses in the crosslinked collagen 4A, the cell aggregates 1 are formed and spontaneously detached from the bottom surface of the culture vessel 6 as shown in (c) of (D). there is
After the cell aggregates 1 are formed in this manner, the culture vessel 6 is removed from the incubator, and the cell aggregates 1 are collected from the culture vessel 6 in the isolator. At this time, since the cell aggregates 1 are spontaneously detached from the bottom surface of the culture vessel 6, the cell aggregates 1 can be easily collected.

According to the method for producing the cell aggregate 1 according to the above embodiment, in the mixing step, the cell suspension 2, the culture medium 3, and the solubilized collagen 4 are mixed to dilute the solubilized collagen 4, thereby reducing the collagen concentration. By preparing the concentrated collagen-containing cell mixture 5, in the culture step (D), the cells 7 can freely migrate throughout the crosslinked collagen 4A, and the cells are three-dimensionally evenly dispersed. be able to.
As a result, the dispersed cells 7 are attracted to each other as the culture progresses, and the fibers constituting the crosslinked collagen 4A are contracted to form the cell aggregate 1 . Since the cells in the formed cell aggregate 1 are three-dimensionally evenly distributed, the cells shrink toward the center, so that they spontaneously separate from the bottom surface of the culture vessel 6 and can be easily collected. It's becoming
According to the method for producing the cell aggregate 1 according to the present invention, there is no need to use a detachment enzyme agent for detaching the cell aggregate 1, or to apply a special coating treatment to the culture vessel 6. Does no damage.

Experimental results obtained by varying the collagen concentration and cell number concentration of the collagen-containing cell mixture 5 will be described below.
Cell number concentration was fixed at 7.5×10 ⁵ cells/mL, and culture was performed with different collagen concentrations of 0.5 mg/mL, 1.0 mg/mL, 4.0 mg/mL, and 8.0 mg/mL. gone.
As a result, when the collagen concentration was 0.5 mg/mL, 1.0 mg/mL, and 4.0 mg/mL, the cell aggregate 1 was formed, and the cell aggregate 1 was spontaneously detached from the bottom surface of the culture vessel 6. bottom.
In contrast, when the collagen concentration was 8.0 mg/mL, the cell aggregate 1 did not spontaneously detach. This is probably because at 8.0 mg/mL, the collagen concentration was high and the fiber density was high, so that the elastic force of the crosslinked collagen 4A exceeded the contractile force of the cells 7, and the cell aggregate 1 could not contract. .

Next, the collagen concentration was fixed at 1.0 mg/mL, and the cell number concentrations were 1.5×10 ⁵ cells/mL, 2.5×10 ⁵ cells/mL, 7.5×10 ⁵ cells/mL, Cultivation was performed at different concentrations of 30.0×10 ⁵ cells/mL.
Cell aggregates 1 were formed when the cell number concentration was 2.5×10 ⁵ cells/mL, 7.5×10 ⁵ cells/mL, and 30.0×10 ⁵ cells/mL, and spontaneously effectively peeled off.
In contrast, when the cell number concentration was 1.5×10 ⁵ cells/mL, cell aggregate 1 did not spontaneously detach. This is because the cell concentration is low at 1.5×10 ⁵ cells/mL and the cell density in the cell aggregate 1 is low, so that the contractile force of the cells is lower than the elastic force of the crosslinked collagen 4a, and the cell aggregate 1 could not shrink.
Moreover, when the cell number concentration was 2.5×10 ⁵ cells/mL and 7.5×10 ⁵ cells/mL, it took 4 days to detach the cell aggregate 1, but the cell number concentration was 30. When the concentration was 0×10 ⁵ cells/mL, the cells were peeled off in 2 days. This is thought to be due to the fact that the higher the cell number concentration, the stronger the contractile force of the cells, and the shorter the time required for contraction and detachment of the cell aggregate 1 .

Further, the collagen concentration was fixed at 1.0 mg/mL and the cell number concentration was fixed at 7.5×10 ⁵ cells/mL. , 1.56 mm, 2.33 mm, and 3.12 mm.
When the liquid depth was 3.12 mm, the cell aggregates 1 were formed and spontaneously separated from the culture vessel 6, but when the liquid depth was 1.56 mm, the cell aggregates 1 were not formed. Although formed, it did not detach from the culture vessel. When the depth of the liquid was 2.33 mm, the cell aggregates 1 were formed, but sometimes they were peeled off and sometimes they were not peeled off.
The adhesive force between the bottom surface of the culture vessel 6 and the crosslinked collagen 4A is constant regardless of the depth of the liquid because it depends on the collagen concentration and the culture area (contact area of the precipitated crosslinked collagen 4A).
On the other hand, since the contractile force of the cells 7 becomes stronger as the number of cells increases, if the collagen concentration and the culture area are the same, the deeper the liquid and the larger the amount of the collagen-containing cell mixture 5, the greater the number of cells. As a result, the contractile force of the cells 7 exceeds the adhesive force between the bottom surface of the culture vessel 6 and the crosslinked collagen 4A, and the cell aggregate 1 can be easily peeled off from the culture vessel 6.
On the other hand, when the depth of the liquid is shallow, the amount of the collagen-containing cell mixture 5 is small, and the number of cells is small, so that the contractile force of the cells 7 reduces the adhesive force between the bottom surface of the culture vessel 6 and the crosslinked collagen 4A. It is considered that the cell aggregate 1 cannot be detached from the culture vessel 6.

As a result of the above, when the produced cell aggregate 1 was observed and comprehensively judged, the collagen concentration was 0.5 to 4.0 mg/mL, and the cell number concentration was 2.0×10 ⁵ to 30.0×. It can be seen that 10 ⁵ cells/mL is desirable.
Moreover, in order to spontaneously detach the formed cell aggregates 1 from the culture vessel 6, it is desirable to set the depth of the collagen-containing cell mixture 5 in the culture vessel 6 at the time of seeding to 3 mm or more. .

1 Cell Aggregate 2 Cell Suspension 3 Culture Solution 4 Solubilized Collagen 4A Cross-Linked Collagen 5 Collagen-Containing Cell Mixture 6 Culture Vessel 7 Cells 8 Collagen Solution

Claims (4)

A method for producing a cell aggregate by three-dimensionally culturing cells to produce a cell aggregate,
A mixing step of diluting uncrosslinked solubilized collagen with a culture medium to a predetermined concentration and mixing cells to prepare a collagen-containing cell mixture; performing a culturing step of culturing the cells in
A method for producing a cell aggregate, comprising thermally cross-linking collagen in the culture step, and culturing three-dimensionally dispersed cells in the cross-linked collagen to produce the cell aggregate. The mixing step includes a suspension preparation step of mixing cells and a culture medium to prepare a cell suspension, and a solution preparation step of mixing uncrosslinked solubilized collagen and a culture medium to prepare a collagen solution. and a mixture preparation step of mixing the prepared cell suspension and the collagen solution to prepare a collagen-containing cell mixture. 3. The method for producing a cell aggregate according to claim 1, wherein the collagen concentration in the collagen-containing cell mixture is 0.5 to 4.0 mg/mL. 3. The method for producing a cell aggregate according to claim 2, wherein in the solution preparation step, the solubilized collagen cooled to 4 to 10° C. and the culture solution are mixed.

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