Three-dimensional culture method for organoids

Abstract

There was a demand for a culture method that would allow for the large-scale culture of cells requiring three-dimensional culture, such as organoids and gastroids, using simple equipment. [Solution] An initial culture step in which target cells and culture medium are placed in a cylindrical culture vessel, and culture is performed using a horizontal rotation culture method in which the culture vessel is rotated with its side facing downwards in the direction of gravity and the length of the cylinder as the central axis, An induction culture step in which the culture medium is replaced with a culture medium containing an organoid inducer and cultured using the horizontal rotation culture method, The three-dimensional culture method for organoids, characterized by having a culture step in which the culture medium is replaced with a culture medium containing polysaccharides but not containing the organoid inducer, and cultured using the horizontal rotation culture method, allows for the large-scale culture of organoids without the cell aggregate adhering to any wall of the culture vessel, even when it grows large.

Description

Technical Field

[0006]

[0001] The present invention relates to a three-dimensional culture method capable of mass-producing organoids and gastroliths.

Background Art

[0002] For drug creation and substance screening, an evaluation target that substitutes for a living body is required. Conventionally, cells have been preferably used from the viewpoints of ease of handling, cost, and ethics. However, although living tissues are composed of cells, cells do not necessarily reflect the comprehensive characteristics as tissues such as the cooperation between cells in a living body. Therefore, even when evaluating cells alone, there are many cases where the expected effects are not exhibited when used in a living body such as a mouse.

[0003] Organoids, which are artificial organs, are considered to better reflect the characteristics of organs than individual cells, although they are small, and are thus expected in fields such as drug discovery. However, organoids need to be cultured one by one, for example, by inserting cell aggregates positive for a target organ marker into gel-like Matrigel (registered trademark) particles and performing suspension culture, and have been considered unsuitable for mass production.

[0004] Therefore, Patent Document 1 provides a method for producing brain organoids including a step of culturing in a state of being dispersed in a first medium containing an extracellular matrix at a concentration exceeding 10% by volume without inserting into Matrigel (registered trademark).

[0005] Further, Patent Document 2 discloses a method for producing organoids, a step of dissociating a living tissue, <​​​​​​Furthermore, (i) to (iv) are as follows: (i) Extracellular matrix of 0.1–10 volume percent. (ii) Insulin-like growth factor 1 (IGF-1). (iii) Fibroblast growth factor 2 (FGF-2). (iv) At least one selected from the group consisting of Wnt agonists, bone morphogenetic protein (BMP) inhibitors, transforming growth factor-β (TGF-β) inhibitors, and epidermal growth factor (EGF).

[0007] Furthermore, as a unique method, Patent Document 3 provides a method for producing cell aggregates, which includes the step of filling a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells into a microfiber and performing suspension culture to form cell aggregates. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2020 / 100481 [Patent Document 2] International Publication No. 2023 / 171702 [Patent Document 3] International Publication No. 2024 / 034559 [Overview of the project] [Problems that the invention aims to solve]

[0009] While all prior art methods utilize suspension culture, all except Patent Document 3 employ an orbital shaker. However, an orbital shaker rotates within a plane, and it is extremely rare for cell clumps that have settled at the bottom of the shaker to be stirred up and become suspended. [Means for solving the problem]

[0010] Therefore, the present invention provides a simpler, more reliable, and reliable three-dimensional culture method for mass-producing organoids without adhesion to the wall surface.

[0011] More specifically, the three-dimensional culture method of organoids according to the present invention is: The initial culture step involves placing the target cells and culture medium in a cylindrical culture vessel and culturing them using a horizontal rotation culture method, in which the culture vessel's side faces downwards in the direction of gravity and the vessel is rotated around the length of the cylinder as its central axis. An induction culture step in which the culture medium is replaced with a culture medium containing an organoid inducer and cultured using the horizontal rotation culture method, This culture step is characterized by replacing the culture medium with a culture medium containing polysaccharides but not containing the organoid inducer, and culturing using the horizontal rotation culture method. [Effects of the Invention]

[0012] In the three-dimensional culture method for organoids according to the present invention, the cells and culture medium are placed in a cylindrical culture vessel with its side facing downwards in the direction of gravity, and the culture is performed while rotating the cylinder around its central axis as the axis of rotation. As a result, the cells are constantly subjected to a force that moves them away from the inner side of the culture vessel. Therefore, the cells do not adhere to the inner surface of the culture vessel.

[0013] Furthermore, in this culture process, the culture medium is subjected to stirring forces, which makes it easy for cells to clump together and form aggregates.

[0014] Furthermore, adding polysaccharides after cell clumps have formed increases the viscosity of the culture medium, inhibiting contact between cell clumps, thus facilitating their growth as organoids.

[0015] Furthermore, the cylindrical culture vessel rotates around its central axis, and the inclusion of polysaccharides in the culture medium allows for the growth of pluripotent stem cells into gastroids once they are initially introduced.

[0016] In addition, in the three-dimensional culture method of the organoid of the present invention, although it is a simple method, the cell mass to be cultured can grow while maintaining a three-dimensional structure for a long period of time, so that it is possible to extract further target cells from the gastroid.

Brief Description of Drawings

[0017] [Figure 1] It is a diagram showing the main configuration of the culture apparatus of the present invention. [Figure 2] Photographs of cells when polysaccharide (carboxymethyl cellulose) is not added (a), and when added (b) and (c) in the polysaccharide-containing culture step. The starting cells are mouse ES cells. [Figure 3] Cell photographs when the starting cells are human iPS cells are shown (Fig. 3(a)). Fig. 3(a) is a photograph stained for each gene. [Figure 4] It is a photograph showing the staining state of each gene in the photograph of Fig. 3(b). Fig. 4(a) shows the T gene, Fig. 4(b) shows the SOX17 gene, Fig. 4(c) shows the SOX1 gene, and Fig. 4(d) shows staining with DAPI (targeting DNA). [Figure 5] Photographs of mesenchymal stem cells generated in a mouse gastroid ((Fig. 5(a)) and photographs of parathyroid cells generated in a mouse gastroid (Fig. 5(b)). [Figure 6] It is a photograph of the experimental results examining the effect of polysaccharides. Fig. 6(a) is the control where no polysaccharide is added in the polysaccharide-containing culture step. Fig. 6(b) is the case where 0.1% of polysaccharide (carboxymethyl cellulose) is added, and Fig. 6(c) is a photograph when 0.3% of polysaccharide (carboxymethyl cellulose) is added. [Figure 7]Continuing from Figure 6, these are photographs of experimental results investigating the effects of polysaccharides. Figure 7(a) shows the case where 0.6% of polysaccharide (carboxymethylcellulose) was added to the polysaccharide-containing culture process. Figure 7(b) shows the case where 0.9% of polysaccharide (carboxymethylcellulose) was added, and Figure 7(c) shows the case where 1.2% of polysaccharide (carboxymethylcellulose) was added. Figure 7(d) shows the case where 1.5% of polysaccharide (carboxymethylcellulose) was added. [Modes for carrying out the invention]

[0018] The three-dimensional culture method for organoids according to the present invention will be described below with reference to drawings and examples. The following description illustrates one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description may be modified without departing from the spirit of the present invention.

[0019] Figure 1 shows a schematic diagram of the manufacturing apparatus for realizing the three-dimensional culture method of organoids according to the present invention. As shown in Figure 1(a), the cylindrical culture vessel 10 contains culture medium M and target cells C. The target cells C may be individual cells or cell aggregates.

[0020] The culture vessel 10 is covered with a lid 12 and placed so that its sides face gravity. Culture is then performed while rotating the cylindrical container around its axis of rotation (Figure 1(b)). Typically, a rotor is used to rotate the culture vessel 10, with a pair of parallel rolls 20 spaced apart, so that each roll rotates in the same direction at the same speed. Note that in Figure 1, the drive mechanism for rotating the pair of rolls 20 is omitted. This method of culturing while rotating the culture vessel 10 around an axis of rotation perpendicular to the direction of gravity is called the horizontal axis rotation culture method.

[0021] Furthermore, the horizontal rotation culture method may also involve vibration in the pitch direction of the rolls 20 (Figure 1(c)). This shows a pair of rolls 20 on which the culture vessel 10 is placed and rotated being vibrated in the pitch direction P of the rolls 20. This prevents the cultured cell aggregates from adhering to the underside of the lid 12 of the culture vessel 10 or to the bottom of the culture vessel 10.

[0022] The three-dimensional culture method for organoids of the present invention is manufactured using a horizontal rotation culture method by the following steps. First, cells to be cultured (target cells) are obtained. If the target cells are single cells, they can be obtained as follows. First, culture medium is placed in a flat container such as a petri dish, and the cells are cultured in it until they reach a confluent state. Then, the cell sheet formed in the petri dish is separated into single cells by enzymatic treatment or the like. Target cells C in Figure 1 illustrate the case of single cells obtained in this way. Tube cells, such as pluripotent stem cells like ES cells and iPS cells, or tissue stem cells positive for organ markers, can be suitably used as target cells.

[0023] Next, as shown in Figure 1(a), culture medium M and target cells C are placed in the culture vessel 10, and the culture is performed while rotating the culture vessel 10 on its horizontal axis, as shown in Figure 1(b). The rotation speed of the culture vessel 10 is preferably between 10 rpm and 60 rpm. Since the cells themselves are light, their settling rate in the medium is slow. Therefore, if the vessel is rotated too quickly, the cells will not be agitated and their position will not change. On the other hand, if the vessel is rotated too slowly, the cells will settle and adhere to the sides of the culture vessel 10.

[0024] The size of the culture vessel 10 is not particularly limited as long as it can be rotated horizontally, but culture vessels 10 of sizes such as 25 ml or 50 ml are suitably used on a laboratory scale.

[0025] Furthermore, an air pocket 14 is provided between the culture vessel 10 and the culture medium M. That is, the culture vessel 10 is not filled with culture medium. The culture medium M should occupy 30% to 70%, more preferably 40% to 60%, of the volume of the culture vessel 10. That is, the air pocket 14 should occupy 70% to 30%, more preferably 60% to 40% of the volume of the culture vessel 10. The presence of the air pocket 14 increases the efficiency of stirring and reduces suspension and temperature changes within the culture vessel 10. It also improves the efficiency of gas exchange. This is called the initial culture process.

[0026] When culturing organoids, an inducer is used to induce the target cell C into organoids. Suitable inducers include known inhibitors or growth promoters. For example, a GSK3b inhibitor is suitable for inducing Wnt / betacatein activation. Suitable GSK3b inhibitors include Chiron and BIO. The timing of inducer addition may vary depending on the cells being cultured.

[0027] After adding the inducing agent to culture medium M, culture is continued for a certain period of time. Alternatively, the culture medium may be replaced with one containing the inducing agent. The horizontal rotation culture method is also used during this stage. Culture in the state containing the inducing agent is called the induction culture step. The length of the induction culture step may vary depending on the cells. During the induction culture step, the target cells C aggregate while being induced into organoids.

[0028] Next, the culture medium containing the inducing agent is replaced with a medium that does not contain the inducing agent but contains polysaccharides. After cell aggregates are formed, the medium is replaced again with a medium that does not contain the inducing agent but contains polysaccharides, and horizontal rotation culture is continued. This is called the main culture process. In this main culture process, the cell aggregates grow independently. Conventionally, organoids that have grown and become large sometimes adhere to other organoids, but in the three-dimensional culture method of the present invention, the cells are lifted while falling in the direction of gravity within the viscous culture medium M, so as a result, individual cells are cultured without changing their relative positions much. Consequently, contact with other organoids is avoided.

[0029] While there are no particular limitations on the polysaccharides that can be used in this culture process, carboxymethylcellulose was used in the examples described later. A concentration of carboxymethylcellulose of 0.1% to 0.9% is preferable. Too little will cause cell aggregates to stick together. Too much will conversely hinder the growth of cell aggregates.

[0030] In the three-dimensional culture method for organoids according to the present invention, contact between cell aggregates is avoided, allowing for longer-term culture (currently confirmed to be 10 days) compared to the conventional method which could only culture for about 4 days. As a result, it was also possible to differentiate other cells from gastroids (pseudoembryos). [Examples]

[0031] <Culture equipment> A rotator was used consisting of a pair of rods, each 20 mm in diameter and 200 mm long, with a resin coating on the surface. These rods were placed parallel to each other at a distance of a few millimeters and rotated in the same direction at the same speed. The culture vessel was a 25 ml container with a lid (a filter-equipped tube used for centrifugation). The rotator's rod rotation speed could be variably adjusted.

[0032] (Comparative Example 1: Sample without carboxymethylcellulose) Mouse ES cells were cultured to confluence. The culture conditions were 37°C and 5% CO2. These cells were enzymatically treated with a 0.1% trypsin and 0.25% EDTA (ethylenediaminetetraacetic acid) solution to separate into single cells, which were then harvested.

[0033] 5-10 ml of Ndiff® 227 culture medium was placed in a culture vessel, the number of cells was measured, and 1 x 10⁴ to 1 x 10⁵ cells were seeded per ml.

[0034] The tube was placed in a rotator and cultured at a rotation speed of 25 rpm (initial culture step). The culture environment was 37°C. After culturing in this state for 48 hours, 3 μM Chiron was added, and the culture was continued in the rotator for another 24 hours (induction culture step). Subsequently, the culture medium was changed to Ndiff® 227, and cultivation was continued for one week. In this comparative example, since no polysaccharides were used, this culture step was omitted.

[0035] (Example 1: Sample with added carboxymethylcellulose) Mouse ES cells were cultured in the same manner as in Comparative Example 1. However, 0.1% carboxymethylcellulose (polysaccharide) was added during the culture in Ndiff® 227 after the Chiron treatment (induction culture step) (main culture step).

[0036] Photographs of cells cultured in Comparative Example 1 and Example 1 are shown in Figure 2. Figure 2(a) is a sample from Comparative Example 1 without carboxymethylcellulose, and Figures 2(b) and 2(c) are samples from Example 1 with carboxymethylcellulose added. Figure 2(c) is a magnified portion of Figure 2(b). In the absence of carboxymethylcellulose, nearly spherical cell aggregates (organoids) with uniform density were obtained.

[0037] On the other hand, cells cultured in a medium supplemented with carboxymethylcellulose were nearly elliptical, exhibiting a bias in color intensity, and showing an axis aligned in the direction of color intensity. Furthermore, expression of genes such as Sox1 and T was observed in these cell aggregates, and their expression was concentrated. Thus, rotator culture with supplemented carboxymethylcellulose was able to culture not only organoids but also gastroids.

[0038] (Example 2) The same experiment as in Example 1 was performed using human iPS cells. Specifically, the target cells C and culture medium M (Ndiff® 227) were packed into culture vessel 10 to 50% of its volume, and the cells were rotated horizontally for 24 hours. After that, the medium was replaced with Ndiff® 227 with Chiron added, and the cells were rotated horizontally for another 24 hours. After that, the medium was replaced with Ndiff® 227 containing carboxymethylcellulose, and the main culture was performed.

[0039] The results are shown in Figures 3 and 4. Figure 3(a) is a normal micrograph. The entire cell has grown elongated, with a clear contrast in density along the axial direction, being darker on the right and lighter on the left. Figure 3(b) is a fluorescence image stained with SOX1, SOX17, T, and DAPI. Figure 4 shows images of each gene individually. Figures 4(a) to (d) show the cells stained with T, SOX17, SOX1, and DAPI, respectively.

[0040] The cultured cell aggregates were found to be gastroyotic, with SOX17, T, and SOX1 present in different cell populations.

[0041] As described above, it has been found that even when using human iPS cells, gastroid culture is possible by using the culture method according to the present invention.

[0042] (Example 3) Gastroids exhibit differentiation that mimics that of an early embryo. Therefore, gastroids contain a variety of cells that correspond to the stages of early embryonic development. By using gastroids, it is possible to acquire target cells, even those for which differentiation induction methods are not yet known, without requiring special induction methods and under more physiological conditions.

[0043] As a result of culturing gastroids, we were able to find the cells shown in Figure 5(a). These were mesenchymal stem cells. Furthermore, parathyroid cells (CaSR-positive cells), which are considered difficult to differentiate in vitro due to their very complex developmental process, could also be obtained by culturing them for 10 days in a medium of Ndiff® 227 and 0.3% carboxymethylcellulose (see Figure 5(b)).

[0044] (Example 4) In Example 1, using mouse ES cells, experiments were conducted in which the concentration of carboxymethylcellulose was varied in Ndiff® 227 culture medium after Chiron treatment. In other words, this experiment examined the changes in polysaccharide concentration during the culture process. The final stage of culture was 4 days. The results are shown in Figures 6 and 7.

[0045] In the case of 0% carboxymethylcellulose (Figure 6(a)), some cells were aggregated. In the following photographs, carboxymethylcellulose is abbreviated as "MC". On the other hand, up to 0.1% (Figure 6(b)), 0.3% (Figure 6(c)), 0.6% (Figure 7(a)), and 0.9% (Figure 7(b)), the size of the cells remained unchanged or even increased, showing an elongated shape. However, at 1.2% (Figure 7(c)) and 1.5% (Figure 7(d)) ​​of carboxymethylcellulose, the cell aggregates did not grow much.

[0046] From the above, it was found that a carboxymethylcellulose concentration of 0.1% to 0.9% is preferable. [Industrial applicability]

[0047] This invention is suitable for mass production of organoids (gastroids). [Explanation of Symbols]

[0048] 10 (cylindrical) culture vessels 12. Lid (of the culture vessel) 14 Air section 20 rolls M medium C. Target cells for culture P (of the roll) pitch direction oscillation

Claims

1. The initial culture step involves placing the target cells and culture medium in a cylindrical culture vessel and culturing them using a horizontal rotation culture method, in which the culture vessel's side faces downwards in the direction of gravity and the vessel is rotated around the length of the cylinder as its central axis. An induction culture step in which the culture medium is replaced with a culture medium containing an organoid inducer and cultured using the horizontal rotation culture method, A method for three-dimensional culture of organoids, characterized by comprising the culture step of replacing the culture medium with a culture medium containing polysaccharides but not containing the organoid inducer, and culturing using the horizontal rotation culture method.

2. A method for three-dimensional culture of an organoid according to claim 1, wherein the polysaccharide is carboxymethylcellulose.

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