WO2020013345A1 - Spheroid production method utilizing difference in hydrophilicity - Google Patents

Abstract

A method for forming spheroids characterized by comprising culturing stem cells with the use of a culture container, the surface of which is processed or treated so as to inhibit cell adhesion, to thereby form spheroids.

Description

Method for producing spheroids utilizing difference in hydrophilicity

The present invention relates to a method for producing spheroids utilizing a difference in hydrophilicity.

、 In recent years, stem cell-based cell therapy (cell therapy) and regenerative medicine have become increasingly attractive because they are widely used in various medical and dental fields (1-5). However, our understanding of somatic stem cells is still limited. One reason that somatic stem cells are difficult to understand is that they have characteristic changes between the in vivo and in vitro environments. Traditionally, it has been believed that niches exist that support the maintenance of somatic stem cells in vivo, but are lost in the cell extraction and culture environment (in vitro). Most cell cultures utilize two-dimensional cultures on culture plates, which not only cannot mimic the complex interactions between stem cells and the environment (6), but also the natural phenotype of stem cells. Is also changed (7-9).

Spheroid culture is one of three-dimensional (3D) culture methods, and was known as early as the 1940s (10). Compared to conventional two-dimensional (2D) cultures, spheroid cultures are expected to grow more closely in vivo (10, 11). More recently, spheroid culture has received much attention as a novel selective culture method for neural stem cells (12). This culture technique has since proven to be effective in the selective culture of many other types of somatic stem cells, including skin (13), salivary glands (14), and mesenchymal stem cells (15). Most of the studies have shown that spheroid cultures are a potent selective culture of somatic stem cells, which can enhance the therapeutic potential of stem cells (11).

Although spheroids are widely used, there is no consensus on how to make spheroids. In addition, the basic mechanisms that affect spheroid formation are not well understood.
Broadly, there are three main methods for producing somatic stem cell spheroids.
The most traditional approach is to combine single cells dissociated from tissue in a serum-free environment with several key factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and B27 (16). This is a method of culturing. This method is for selecting cells that can survive without adhering to the dish and that can proliferate and form cell aggregates. Although this approach seems reasonable for selecting stem cell populations, it has usually been very inefficient since most of the cells attach to the dish or do not grow without attaching to the dish.

The second method is based on a dynamic cell culture method, in which cells are cultured in a rotating flask or the medium is agitated to avoid cell attachment to the flask using a mechanical method. (17). The third method is a culture method in which spheroids are formed in a hanging drop culture using gravity using a non-adhesive inverted conical culture well or a gel coat dish (18). Compared to the first method of selectively culturing spontaneously formed spheroids, the second and third culturing methods theoretically simply form cell aggregates without selection. Thus, these two different types of spheroids are defined as spontaneously formed spheroids and mechanically formed spheroids.
By the way, with respect to the invention relating to spheroid formation, for example, a substrate for adhesive cell culture having an albumin adsorption amount on a cell culture surface or a static water contact angle within a predetermined amount or within a range is known (JP-A-2017-077240). Japanese Unexamined Patent Application Publication No. 2007-077241 (Patent Document 2) and the like.
However, the base material has been subjected to surface coating, and it is unknown whether spheroids are formed using the base material.

JP 2017-077240 A JP 2017-077241 A

An object of the present invention is to provide a high-performance method for forming spheroids of stem cells.

The present inventors have conducted intensive studies to solve the above problems, and as a result, found that spheroid formation occurs efficiently and spontaneously by keeping the hydrophobicity of the culture plate constant, thereby completing the present invention. Reached.

That is, the present invention is as follows.
(1) A method for forming spheroids, wherein spheroids are formed by culturing stem cells using a culture vessel having a surface processed or treated so that cell adhesion is inhibited.
(2) The method according to (1), wherein the culture vessel whose surface is processed or treated so as to inhibit cell adhesion has hydrophobicity.
(3) The method according to (2), wherein the culture vessel having hydrophobicity has a roughness of a culture bottom so as to satisfy a condition that a contact angle is in a range of 85 ° to 95 °.
(4) The method according to (3), wherein the contact angle is from 89 ° to 91 °.
(5) The culture vessel whose surface is processed or treated so as to inhibit cell adhesion is made of polystyrene or glass, and the bottom surface is not coated with a fluorine-containing polymer. (1) to (4) The method according to any one of claims 1 to 4.
(6) The culture vessel whose surface has been processed or treated so as to inhibit cell adhesion has a root-mean-square surface roughness of 2.400 nm to 2.500 nm, (1) to (5). A method according to any one of the preceding claims.
(7) The method according to any one of (1) to (6), wherein the stem cells are somatic stem cells, embryonic stem cells, mesenchymal stem cells, or cancer stem cells or tumor-derived cells.
(8) A method for producing spheroids, comprising recovering spheroids formed by the method according to any one of (1) to (7).
(9) A culture vessel having a bottom surface of a culture so as to satisfy a condition that a contact angle is in a range of 85 ° to 95 °.
(10) The culture container according to (9), wherein the contact angle is 89 ° to 91 °.
(11) The culture vessel according to (9) or (10), wherein the culture vessel is made of polystyrene or glass, and the bottom surface is not coated with a fluorine-containing polymer.
(12) The culture vessel according to any one of (9) to (11), having a root-mean-square surface roughness of 2.400 nm to 2.500 nm.
(13) A spheroid produced by the method according to (8).
(14) The spheroid according to (13), wherein the spheroid has a higher ability to differentiate into a tissue than a spheroid formed without using the method according to any one of (1) to (7).
(15) The spheroid according to (14), wherein the tissue is a nerve or a bone.
(16) A regenerative medical material comprising the spheroid according to any one of (13) to (15).
(17) The material according to (15), wherein the regenerative medicine is a regenerative medicine for treating a nerve disease or a bone disease.
(18) A regenerative medicine kit comprising the spheroid according to any one of (13) to (15).
(19) The kit according to (18), wherein the regenerative medicine is a regenerative medicine for treating a nerve disease or a bone disease.

According to the present invention, a method for forming a spheroid and a method for producing the same are provided. According to the present invention, it has become possible to selectively produce high-performance spheroids of stem cells without requiring a special medium as well as a special medium.

FIG. 3 shows the effect of surface hydrophobicity on spheroid formation from mouse skin-derived cells. A) Photographic image of water drops pipetted on the surface of the dish. B) Average contact angle of a 1 μL water droplet on the dish. Data are shown as mean ± standard deviation (n = 5-10), ^*** p <0.0001. C) A 3D image of the surface roughness of Dish1, Dish2, and Dish3 was observed with a scanning probe microscope. D) The morphological changes of the mouse skin-derived cells on the culture dish showed different hydrophobicities on days 1, 3, and 5. Cells were able to attach to Dish1 and Dish2, but showed spheroid formation only on Dish3. Scale bars represent 100 μm (black) and 500 μm (red). It is a figure which shows the expression of a stem cell marker in spheroids from mouse skin-derived cells. A) Immunofluorescent staining for SSEA1, Nanog, Oct4 and Sox2. The scale bar is 50 μm. The time course of expression of SSEA1 (B), nanog (C), Oct4 (D), and Sox2 (E) was compared with that of monolayer culture. Mean ± standard deviation ( ^* P <0.05, ^** P <0.01, ^*** P <0.001, n = 5). FIG. 2 is a diagram showing spheroid-forming ability in different types of cells.ス Spheroid formation of skin-derived cells (A), oral mucosa-derived cells (D), cortical bone-derived cells (E), and NIH3T3 cells (F). Immunofluorescent staining of SSEA1 (B) and Sox2 (C) on spheroids of skin-derived cells. Scale bars represent 500 μm (white) and 100 μm (black). It is a figure which shows the spontaneous spheroid formation process by the skin origin cell (taken every 3 hours). It is a figure which shows the result of alkaline phosphatase staining at the time of spontaneous spheroid culture and planar culture of cells derived from oral mucosa. Characterization of spontaneous spheroids from cells derived from oral mucosa. (A) Morphological changes of oral mucosa-derived cells on special culture dishes. No spheroid formation was observed in serum-free and serum-free media containing bFGF, EGF and B27 supplement. Only cells in the medium containing serum spontaneously formed spheroids and then maintained them. Scale bar = 100 μm (B) Immunofluorescence analysis of Sox2, SSEA1, Oct4 and Nanog expression in spheroids from cells derived from oral mucosa. Positive reactions were observed in almost all spheroid-forming cells. (C) Expression of nestin in spheroids derived from oral mucosa-derived cells. Scale bar = 50 μm. DAPI: 4 ', 6-diamidino-2phenylindole. Spontaneous spheroid formation from oral mucosal and skin-derived cells. (A) Phase difference image of spheroid. In a medium containing serum, oral mucosa-derived cells and skin-derived cells spontaneously aggregate within 24 hours after cell seeding to form dense multicellular spheroids. (B) Size of spheroids derived from oral mucosa and skin-derived cells. The difference was significant at 120 hours; n = 10. (C) Number of spheroids derived from oral mucosa and skin-derived cells. Significant differences were observed only at 120 hours; n = 10. (D) Effect of additives on spheroid size. Spheroid size was significantly larger in media containing bFGF, EGF and B27 supplements than in media without these additives. n = 20. (E) Effect of additives on the number of spheroids. More spheroids were observed in media containing bFGF, EGF and B27 supplements than in media without these additives. n = 6. Scale bar = 100 μm. Data are shown as mean ± SEM; *, p <0.05; ***, p <0.001. Cell proliferation and apoptosis in spheroids of oral mucosal and skin-derived cells. (A) Immunofluorescence of Ki67 and caspase 7 in spheroids from oral mucosal cells and skin cells. Regardless of the origin, the number of Ki67-positive cells was higher in spheroids with additives than in spheroids without additives. Results from caspase 7 immunofluorescence showed positive cells in oral mucosal spheroids without additives, but only a few weakly positive cells were observed in spheroids with additives. Skin-derived spheroids showed only faint staining with spheroids without additives, whereas spheroids with additives did not show positive cells. (B) Expression of Ki67 and caspase 7 in spheroids derived from oral mucosa and skin-derived cells was analyzed using qRT-PCR, and the levels in spheroids were compared with or without additives. Higher expression of Ki67 was observed in both oral mucosal spheroids and skin-derived spheroids with additives than without additives. On the other hand, the expression of caspase 7 was significantly higher in spheroids without additives than in spheroids with additives. (C) The expression levels of caspase 7 in spheroids of oral mucosa-derived and skin-derived cells were compared. Regardless of the additive, caspase 7 expression was significantly higher in spheroids of oral mucosa-derived cells than in spheroids of skin-derived cells. Scale bar = 50 μm. DAPI: 4 ', 6-diamidino-2phenylindole. Data are shown as mean ± SEM. n = 3. *, P <0.05; **, p <0.01; ***, p <0.001. Effect of bFGF, EGF and B27 supplements on stem cell marker expression in spheroids of oral mucosal and skin-derived cells. (A) Expression of Sox2, Fut4 (SSEA1), Oct4 and nestin in spheroids of cells derived from oral mucosa was analyzed using qRT-PCR, and the level in spheroids cultured in the presence and absence of additives was analyzed. Compared. (B) Expression of Sox2, Fut4 (SSEA1), Oct4 and nestin in spheroids of skin-derived cells was analyzed using qRT-PCR, and the levels in spheroids cultured in the presence and absence of additives were compared. did. Data are shown as mean ± SEM. n = 3. *, P <0.05; **, p <0.01; ***, p <0.001. Direct comparison of stem cell marker expression between oral mucosa-derived cells and skin-derived cells spheroids. (A) Expression levels of Sox2, Fut4 (SSEA1), Oct4 and nestin in spheroids of oral mucosa and skin derived cells without bFGF, EGF and B27 supplements were analyzed using qRT-PCR and compared. (B) Expression levels of Sox2, Fut4 (SSEA1), Oct4 and nestin in spheroids of oral mucosal and skin-derived cells cultured with bFGF, EGF and B27 supplements were analyzed using qRT-PCR and compared. Data are shown as mean ± SEM. n = 3. *, P <0.05; **, p <0.01; ***, p <0.001. Neurogenic induction of spheroid-derived cells from oral mucosa and skin. (A) Immunofluorescence image of spheroids. βIII-tubulin- and MAP2-positive cells were not observed, but nestin-, NeuN-, Sox2- and S100β-positive cells were found in oral mucosal cells (two-dimensional culture). Nestin-, βIII-tubulin-, MAP2-, NeuN-, Sox2- and S100β-positive cells were observed in spheroid-derived cells from both oral mucosa and skin. (B) The expression level of the neuronal marker was analyzed using qRT-PCR. The expression levels of MAP2, MBP, nestin and Nurr1 in spheroid-forming cells from oral mucosa were significantly higher than in spheroid-forming cells from skin. (C) Expression of MAP2, MBP and nestin in spheroid-forming cells from oral mucosa and CBDC. The expression levels of MAP2, MBP and nestin in spheroid-forming cells from oral mucosa were significantly higher than in spheroid-forming cells from CBDC. Data are shown as mean ± SEM. n = 3. *, P <0.05; **, p <0.01; ***, p <0.001. Scale bar = 50 μm. DAPI: 4 ', 6-diamidino-2-phenylindole. Phase contrast image of spontaneous spheroids from skin-derived cells. Note the presence of spontaneously formed spheroids, even from passaged cells. (A) Spontaneous spheroid formation from skin-derived cells at the second passage. (B) Spontaneous spheroid formation from skin-derived cells at the third passage. (C) Spontaneous spheroid formation from skin-derived cells at passage 4. (D) Spontaneous spheroid formation from skin-derived cells at the fifth passage. Bone forming ability of spheroids. Monolayer cultured CBDC and spheroid-derived cells were incubated for 7 days in an osteogenic induction medium. ALP assay data showed that the induced spheroid-derived cells had significantly increased ALP activity compared to the induced monolayer cells (a). The qRT-PCR data showed that the derived spheroid-derived cells expressed bone formation-related genes, such as Osterix, BSP, and DMP1, at statistically significant levels (bd). Data are expressed as mean ± SEM. ALP assay, N = 3; qRT-PCR, N = 3. * P <0.05, ** P <0.01, *** P <0.001. Histology 4 weeks after transplantation.

Hereinafter, the present invention will be described in detail.
1. Outline The present invention relates to a method for forming spheroids, which comprises forming spheroids by culturing stem cells using a culture vessel having a surface processed or treated so that cell adhesion is inhibited.
Further, the present invention is a culture container having a roughness of a culture bottom surface so as to satisfy a condition that a contact angle is in a range of 85 ° to 95 °, wherein stem cells are cultured using the culture container. , A method of forming spheroids.

1. Overview After the discovery of selective culture of neural stem cells, spheroid culture has been recognized as a method for selective culture of somatic stem cells. Since then, it has been reported that spheroids are produced by various methods. However, it is not clear what are the fundamental factors for forming spheroids that can enrich a strong stem cell population.

The present inventors paid attention to the adhesiveness (adhesiveness) of cells to a culture vessel during spheroid formation. It is well known that hydrophobicity affects cell adhesion. However, factors that affect cell adhesion include various factors such as surface shape in addition to hydrophobicity.
Therefore, the present invention is characterized in that spheroids are formed by controlling cell adhesion using conditions that affect cell adhesion, not limited to hydrophobic conditions.

Conditions that affect cell adhesion can be set by processing or treating the surface of the culture vessel so that the adhesion of the cells to the culture vessel is inhibited. “Adhesion is inhibited” means that all or a part of the cells is prevented from attaching to the culture vessel, and includes both physical treatment and chemical treatment.
For example, focusing on the contact angle of water (water repellency or hydrophobicity), the degree of cell adhesion obtained by water repellency or hydrophobicity is the most important factor for spontaneous spheroid formation. That is, if the surface of the culture vessel is hydrophobic, it becomes non-adhesive, and the degree of cell adhesion changes depending on the degree. Therefore, the degree of cell adhesion determines good formation conditions for spontaneous spheroid formation. By culturing stem cells using a culture vessel whose surface has been processed or treated so as to inhibit cell adhesion, spheroids can be formed.

Furthermore, when spheroids are formed using the above culture vessel, it is shown that the obtained spontaneous spheroids have high differentiation ability. In particular, in the present invention, (i) bone-derived mesenchymal stem cells have excellent bone and nerve differentiation ability, and (ii) skin and oral mucosa-derived stem cells have excellent nerve differentiation ability. It has been shown. The skin and oral mucosal stem cells produced by the method of the present invention are better than existing cells (spheroids) formed without using the method of the present invention, for example, off-the-shelf bone marrow-derived mesenchymal stem cells (eg, stemilac). It has much higher tissue differentiation ability (eg, bone differentiation ability and nerve regeneration ability). Therefore, the method of the present invention is an excellent method for preparing stem cells for regenerative medicine, and the spontaneous spheroid of the present invention is a cell source useful for regenerative medicine, particularly nerve regeneration and bone regeneration.

2. Surface Properties of Culture Vessel In the present invention, examples of a culture vessel (culture plate) whose surface is processed or treated so as to inhibit cell adhesion include those having hydrophobicity.
Thus, one aspect of the present invention focuses on the surface properties of the culture vessel, especially on hydrophobicity. Primary culture cells were prepared from mouse skin by conventional two-dimensional culture, and the cells were transferred to culture plates with different hydrophobicity, which were confirmed at different contact angles. The size of the contact angle defines the hydrophilicity and hydrophobicity of the culture surface. Yes, in the range of 85 ° to 95 °. Further, the contact angle is preferably 89 ° to 91 °, more preferably 90 °. Only cell culture plates with the above contact angles were able to successfully achieve spheroid formation.

In addition, spheroid formation was spontaneous, efficient, and stable. Since spheroid formation was achieved in a medium (αMEM supplemented with 10% FBS) containing no additives (factors) such as EGF, bFGF and B27, it can be said that the spheroid production process does not depend on the above factors.

Furthermore, as a result of immunofluorescence and quantitative real-time PCR, the expression of ES cell markers such as SSEA-1, SOX-2, OCT4, and nanog was shown, and the selective properties of a strong stem cell population according to the method of the present invention were confirmed. confirmed. This phenomenon was reproducible and applicable not only to skin-derived stem cells, but also to oral mucosa-derived stem cells, mesenchymal stem cells and 3T3 cells. Therefore, the method of the present invention shows the robustness of the above phenomenon.

3. Contact Angle at the Bottom of the Culture Vessel The culture vessel (sometimes referred to as a culture plate or dish) used in the present invention has a contact angle in the range of 85 ° to 95 °, for example, 85 °, 86 °. , 87 °, 88 °, 89 °, 90 °, 91 °, 92 °, 93 °, 94 °, or 95 °, preferably 89 ° to 91 ° (eg, 89 °, 90 ° or 91 °), More preferably, it is 90 °.
Contact angle refers to the angle between the liquid surface and the solid surface where the free surface of the stationary liquid contacts the solid wall. The contact angle is used as an index indicating wettability and can be measured by various methods. For example, in the present invention, a θ / 2 method, a tangent method, a curve fitting method and the like can be mentioned. Methods for measuring these contact angles are well known.

The form of the culture vessel used in the present invention is not particularly limited, and may be any form such as a flask, a multiwell plate, and a petri dish (dish).
In addition, the material of the culture vessel used in the present invention is not limited, and is made of, for example, polystyrene or glass.
Various coatings are applied to ordinary culture vessels in order to impart cell adhesion and the like, but the culture vessels used in the present invention do not require such coatings. For example, a culture vessel in which the bottom surface (culture surface) is not coated with a fluorine-containing polymer is used. Furthermore, it is sufficient that the culture surface has a surface roughness (line roughness) such that the contact angle satisfies the above range.

In the present invention, the surface roughness can be a roughness represented by JIS standards such as JIS B 0601 and JIS B 0651 as parameters, and the arithmetic average roughness (Ra), the maximum height (Rz), There is a root mean square surface roughness (Rms), but is not limited thereto.
In the present invention, these parameters can be used alone or in an appropriate combination.
For example, when Rms is used as a parameter, Rms on the culture surface of the culture vessel used in the present invention is 2.400 nm to 2.500 nm.

4. Culture method In the present invention, stem cells are cultured using the culture plate.
First, the method for obtaining stem cells is not particularly limited, and can be collected from any tissue. For example, it can be collected from adipose tissue, bone marrow, skin, oral mucosa, salivary gland, periodontal ligament, dental pulp, cartilage, umbilical cord, placenta, and the like. Examples of stem cells include somatic stem cells, embryonic stem cells, mesenchymal stem cells, cancer stem cells or tumor-derived cells, and the like. The stem cells used in the present invention may be derived from any animal species including human.

The stem cells are cultured in animal cell culture medium containing fetal bovine serum (eg, MEM medium, DMEM medium, RPMI-1640 medium, etc.) under conditions used for normal animal cell culture, for example, at 37 ° C., 5% CO ₂ , and 95% air. Cultivate underneath to form spheroids. When the cells are cultured using the culture container of the present invention, the cells start to aggregate before they become confluent and spontaneously form clumps.
Thereafter, the formed spheroids may be collected. The spheroid thus produced is referred to herein as “spontaneous spheroid”.

The spheroid of the present invention does not involve cells other than stem cells when forming spheroids. That is, the spheroids of the present invention are characterized by being substantially constituted only by stem cells and containing almost no cells having no spheroid-forming ability. For example, the proportion of stem cells in spheroids obtained by the method of the present invention is, for example, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5 or more. % Or 99.9% or more. Spheroids produced by the conventional hanging drop method and the like are, in addition to stem cells, a mixture containing differentiated cells, etc., the spheroids of the present invention have a high purity of the cells constituting them and have high functions as stem cells. Become.
In order to identify the properties of spheroids formed from stem cells, a gene or gene product that is uniquely expressed in spheroids can be used as a marker. For example, SSEA1 (also known as Fut4), SOX2, OCT4, Nanog, Nestin, etc. are used alone or in an appropriate combination.

In order to confirm the expression of each marker, an RT-PCR method, a Western blot method, or the like is used. Primers can be designed from each genetic information and can be obtained by ordinary chemical synthesis.
The spheroid of the present invention has, for example, the following properties.
The ability to differentiate into tissues (eg, nerves and bones) is higher than spheroids formed without using the method of the present invention.
It expresses stem cell markers such as Sox2, SSEA1, Oct4, Nanog and Nestin.
It has the ability to differentiate into nerve and Schwann cells after inducing differentiation into neural cells.
It has the ability to differentiate into dopamine-producing cells after induction of neural differentiation.

5. Materials for Regenerative Medicine As described above, the spontaneous spheroids of the present invention have an excellent ability to differentiate into tissues. Accordingly, the present invention provides a regenerative medicine material or composition comprising spontaneous spheroids.
The tissue to be subjected to regenerative medicine is not particularly limited, and may be any tissue, such as nerves and bones.

In order to use the spontaneous spheroid of the present invention for treating a neurological disease, the spheroid can be transplanted to a site where the spheroid is needed (for example, a central nerve such as brain or spinal cord, or a peripheral nerve). Alternatively, the cells can be cultured in the presence of a nerve growth factor, a neurotrophic factor, or the like, and differentiated into nerves for use.
In addition, in order to use the spontaneous spheroid of the present invention for the treatment of bone disease, the spheroid is required to be used in a site that requires it (for example, atrophy of alveolar bone, tumor, removal of cyst, defective site after resection, intractable disease). It can be transplanted to bone fractures, bone defects after tumor resection, bone and cartilage defects in osteoarthritis, and fracture sites in osteoporosis (including joints). Local injection or intravenous administration to osteoporosis patients improves bone density and lowers the risk of fractures. Alternatively, the cells can be cultured in the presence of a bone differentiation inducing factor (dexamethasone, β-glycerophosphate, ascorbic acid, BMP, etc.) and differentiated into osteoblasts before use.
Other tissues other than nerves and bones can be treated in the same manner as described above.

6. Kit The present invention provides a regenerative medicine kit containing the spontaneous spheroid. The amount of the spontaneous spheroids per unit kit may be arbitrary and can be appropriately prepared. The target of regenerative medicine is the same as described above.
The kit of the present invention can include a culture container, a differentiation factor, a buffer, a culture solution, instructions for use, and the like.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.
[Example 1]

To focus on the adhesive effects of cell culture plates to identify various factors that could affect spheroid formation. It is well known that hydrophobicity affects cell attachment (19). Thus, the effect of the hydrophobicity of the culture plate on spheroid formation was examined.

Materials and Methods Selection of Cell Culture Plates and Their Characteristics In this example, various commercially available cell culture plates were tested to evaluate hydrophobicity as a water contact angle. Finally, three different dishes, namely, adhesion-dependent cell culture dishes (hereinafter referred to as Dish1) (Falcon, REF 353001 and 353002, Corning Inc.) and suspension culture dishes (hereinafter referred to as Dish2) (Nunc) ^TM , Lot # 171099 and 150340, Thermo Fisher Scientific Inc. and a low attachment surface (LAS) cell culture plate (hereinafter referred to as Dish3) were selected for further analysis.
The contact angle of a 1 μl ultrapure water droplet on the culture surface was determined as hydrophilicity (the surface wettability of the culture plate) by a surface contact angle measurement device (portable contact angle meter PCA-1, Kyowa Interface Science Co., Ltd.). It was measured. Since the hydrophobicity mainly depends on the surface roughness, the surface roughness was examined with a scanning probe microscope (SPM) (SPM-9500J3, Shimadzu) having a diameter of 2.5 × 2.5 μm.

Animals The experimental animals used were according to the guidelines of the National Institutes of Health (NIH), care and use of laboratory animals for the experimental procedures at Matsumoto Dental University, and the “Matsumoto Dental University Animal Experimental Committee” (approval number 269 and 319).
C57BL / 6J mice (female, 3 weeks old) were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and maintained at a laboratory animal facility at Matsumoto Dental University High Tech Center. The mice were maintained under controlled temperature (25 ± 2 ° C.) and lighting (12 hour light / dark cycle). After the animals had been acclimated in the new environment for about one week, they were used in the following experiments.

Preparation of cells Skin-derived cells (SDC) and oral mucosa-derived cells (OMDC)
Mice were killed by overdose of anesthesia and hair was removed with a hair clipper. After sterilization with 70% alcohol, dorsal skin and buccal and palatal mucosa were collected. Primary cultures were obtained using conventional explant culture techniques. Briefly, tissue sections were washed twice with phosphate buffered saline (PBS, Cat No: 166-23555, Wako, Osaka, Japan) and cut into fragments approximately 4 mm in size. The tissues were dispensed onto the well surface in a 6-well tissue culture plate (Falcon, Product No. 353046) and placed in a humidified incubator at 37 ° C., 5% CO ₂ .

After the tissue adhered to the plate, it was supplemented with 10% fetal bovine serum (FBS, BioWest, Nuail, France) and 1% penicillin-streptomycin-amphotericin B (Biological Industries USA, Inc., Cromwell, CT, USA). 0.5 mL of αMEM (Wako, Osaka, Japan) was added and the plates were placed at 37 ° C. in a humidified incubator with 95% air and 5% CO ₂ . The medium was changed every three days.
On the 10th day from the start of the culture, the cells were dissociated with 0.25% Trypsin-EDTA (Life Technologies Japan Ltd., Cat No: 2520056, Tokyo, Japan) at 37 ° C for 2 to 3 minutes. The dissociated cells were filtered with a 40 μm cell strainer (Corning, Inc., NY, USA), washed twice with αMEM containing 10% FBS, and centrifuged at 300 g for 5 minutes at 4 ° C. Finally, 2 × 10 ⁵ cells were plated in 60 mm diameter cell culture dishes (Falcon, product number 353002) containing αMEM supplemented with 10% FBS, 1% penicillin-streptomycin-amphotericin B, and 5% CO 2. _2. Cultured in a humidified incubator with 95% air. The medium was changed every two days.
When the cells reached 80-90% confluence, the cells were detached. Cells at passages 2-3 were used in this example.

Compact bone-derived cells (CBDC)
Cultivation of CBDC was based on the protocol reported in a previous study by the inventor (20). The method is as follows.
First, the femur and tibia were carefully dissected to remove other tissue. The epiphysis of the femur and tibia were cut and the bone marrow was flushed using a syringe with a 28G needle. Bone was minced into small pieces in PBS containing 0.25% collagenase (Wako) and 20% FBS. The minced bone chips were transferred to a 50 ml centrifuge tube containing the above collagenase solution (13 ml) and incubated at 37 ° C. for 45 minutes in a bioshaker at a speed of 90 rpm. The cells were then collected through a 70 μm cell strainer and centrifuged at 4 ° C., 300 × g for 5 minutes.

The cell pellet was collected and gently resuspended in αMEM supplemented with 10% FBS, 1% penicillin-streptomycin-amphotericin B, and 10 ng / ml bFGF (PeproTech, Rocky Hill, USA). The cell suspension was seeded on a 35 mm cell culture dish (Falcon, # 353001) and cultured at 37 ° C. in a 5% CO ₂ , 95% humidified incubator. The medium was changed every three days. On day 7, the cells were subcultured at a density of 2.5 × 10 ⁵ cells / cm ^{2 in} a new culture dish. Cells from the third passage were used for subsequent experiments.

NIH 3T3 cells
NIH3T3 (cell number JCRB0615; Health Science Research Resources Bank, Osaka, Japan) cells were cultured in αMEM supplemented with 10% FBS and 1% penicillin-streptomycin-amphotericin B. Cultures were maintained at 37 ° C. in a water-saturated atmosphere of 95% air and 5% CO 2. Cells were routinely passaged every 3-4 days, and cells at passages 8-10 were used for experiments.

Spheroid-forming ability test The above SDC, OMDC, CBDC and NIH3T3 were seeded on the three different plates, and αMEM supplemented with the same concentration of 10% FBS, 1% penicillin-streptomycin-amphotericin B (1. 5 × 10 ⁴ cells / cm ² ). According to other research reports (21, 22), when a cell aggregate having a diameter of 50 μm or more was formed, it was regarded as a spheroid in this example. After 3 days of culture, fresh medium was added (however, aspiration of the medium was avoided). Spheroid formation was observed and counted on days 1, 3, 5, and 7 in each plate. Primary spheroids formed between 3-5 days were used for gene and protein expression studies.

Detection of Alkaline Phosphatase (AP) Activity AP activity was tested on day 3 after plating on culture plates. Cells were fixed with 4% paraformaldehyde phosphate buffer (Wako, 163-12014) at 4 ° C for 15 minutes. Fixed cells were washed twice in distilled water and stained with Blue-Color AP Staining Kit (SBI, https://www.systembio.com/, CA, USA) according to the manufacturer's instructions. The stained image was taken with an Olympus IX70 inverted microscope (Olympus, Toyo, Japan).

On day 3 after seeding the culture plate, the cells were fixed with 4% paraformaldehyde phosphate buffer (Wako, 163-12014) for 15 minutes at room temperature, washed four times with PBS (-), 30% at room temperature with 0.5% Triton X-100, 5% bovine serum albumin (BSA, Nacalai Tesque, Inc., Kyoto, Japan) and 5% normal goat serum (abcam, ab7481, Cambridge, UK) in PBS. Permeabilized for 5 minutes and blocked. The cells were then incubated overnight at 4 ° C. with the primary antibody in PBS containing 1% BSA. After washing three times with PBS, cells were incubated with the appropriate species-specific secondary antibody containing a fluorescent dye for 2 hours at room temperature. The cells were washed three times with PBS and then counterstained with DAPI (fluoroshield mounting medium containing ab104139, DAPI, Abcam, Cambridge, UK) for 30 minutes.

Images were captured by fluorescence microscopy (Keyence, model BZ-X710, Osaka, Japan).
The primary antibodies are as follows:
Mouse anti-SSEA1 [MC-480] (1:50; ab16285, Abcam, Cambridge, UK),
Rabbit anti-Oct4-ChIP grade (1: 250; ab19857, Abcam, Cambridge, UK),
Rabbit anti-Nanog (1: 100; ab80892, Abcam, Cambridge, UK), and rabbit anti-SOX2 (1: 250, ab97959, Abcam, Cambridge, UK).

The secondary antibodies are as follows:
Goat anti-mouse IgG H & L (Alexa Fluor 488) (ab150113, Abcam, Cambridge, UK),
Anti-mouse IgM (Alexa Fluor 488) (ab150121, Abcam, Cambridge, UK), and anti-rabbit IgG H & L (Alexa Fluor 647) (ab150079, Abcam, Cambridge, UK).

Quantitative real-time PCR (qRT-PCR)
Twelve hours, one, three, and five days after cell seeding, total RNA was extracted from cells with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) and PrimeScript ^™ RT Master Mix (Perfect Real Time) according to the manufacturer's protocol. (TaKaRa, Cat # RR036A) to synthesize cDNA. qRT-PCR was performed three times using SYBR (registered trademark) Premix Ex TaqTM II (TaKaRa, RR820A) using a Thermal Cycler Dice Real Time System fluorimeter (TaKaRa TP900). RT-PCR has a volume of 25 μl and contains 2 μl (2 μg) cDNA, 12.5 μL SYBR Premix Ex Taq II (TaKaRa, RR820A), 1 μl each of specific forward and reverse primers, and 8.5 μl sterile water.

qRT-PCR consists of 40 cycles of 95 ° C. for 30 seconds, followed by 95 ° C. for 5 seconds, 60 ° C. for 30 seconds, followed by a final cycle of 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 95 ° C. for 15 seconds. Separation was performed. Data was analyzed by 2-ΔΔCt ratio quantification. Data were normalized to β-actin levels and compared to normalized control values.
The expected amplicon primers are as follows:
SSEA1 (also known as Fut4)

Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA). Tukey's post-test was performed for significant F ratios. Significance levels were P <0.05 and are shown as mean ± SD unless otherwise indicated. All data were obtained from at least three independent experiments unless otherwise indicated.

Results Selection of Culture Dishes with Different Hydrophobicities To investigate the effect of various surface hydrophobicities, the water contact angles of various culture plates were examined, which varied between the three culture plates. The water contact angles of the three culture plates are shown in FIG. 1A. The water contact angle of the most common culture plate (Dish 1) was around 60 °. The water contact angle of the non-adhesive plate (Dish 2) was approximately 75.12 °. However, only one of the culture plates (Dish 3) showed a very large contact angle (90.83 °).

The water contact angle of each culture plate was relatively stable between the same lot from the same manufacturer and there was a significant difference between the three culture plates (FIG. 1B). In order to understand the difference in water contact angle, the surface roughness of the dish was evaluated using SPM. The images showed differences in surface roughness and correlated with the observed water contact angle. Dish 1 with the smallest water contact angle showed the highest roughness, Dish 3 with the largest water contact angle showed the lowest surface roughness, and Dish 2 with the middle water contact angle showed a medium surface roughness. (FIG. 1C).
In FIG. 1C, Table 1 shows the measurement results of the surface roughness.

When skin-derived cells are seeded on these dishes, the cells proliferate well on each dish. However, only the cells of dish # 3 showed spheroid production (FIG. 1D). Cells growing on the dish gradually formed cell aggregates by day 1, but most cell aggregates remained attached to the dish. Spheroid formation was evident by day 3 and a number of spheroids were found to be floating in the medium. The formation of new spheroids was not evident after day 6. On the other hand, in Dish # 1 and Dish # 2, spheroid formation was not observed at the same seeding density.

Characterization of spheroids derived from skin-derived cells Immunofluorescence analysis was performed to characterize spheroid-forming cells. Spheroids were positive for SSEA1, nanog, oct-4, and sox2 (FIG. 2A), which was confirmed by qRT-PCR (FIGS. 2B, C, D, E). SSEA1 expression was higher in spheroids even 12 hours after cell seeding (FIG. 2B). Oct4 and Sox2 expression was also high in spheroids and was relatively stable by 72 hours (FIGS. 2C, D). Nanog expression in spheroids was also higher than monolayer expression, with expression increasing for 72 hours (FIG. 2E).

Possible mechanisms of spheroid formation and its universality To investigate possible mechanisms of spheroid formation with specific hydrophobicity (90 ° water contact angle is known as the boundary between hydrophobicity and hydrophilicity) Was observed (FIG. 4). FIG. 5 shows an AP-stained image of spheroids and cells when oral mucosa-derived cells were cultured for spheroid formation and planar monolayer culture.

-Initial spheroid formation is observed in certain parts of the culture plate, then proliferates and sometimes aggregates with other spheroid-forming cells. Over time, these cell aggregates become rounder and larger, and then gradually detach from the dish. Surrounding the spheroid-forming cells are spindle-shaped fibroblasts, which also proliferate but do not form spheroids. In order to confirm the characteristics of the spheroid-forming cells, immunofluorescence of ES cell markers (SSEA-1 and Sox2) was performed from an early stage after seeding the cells on Dish # 3 (FIGS. 3A to 3C). These spheroid-forming cells were positive for both SSEA1 and Sox2, and non-spheroid-forming cells were generally negative for these ES cell markers.

調 べ る To examine the generality of this phenomenon, cells from oral mucosa, cells from compact bone (20) and NIH-3T3 cells were tested. All of these cells were able to form spheroids only on Dish # 3, but not on Dish # 1 and Dish # 2, which is not limited to skin-derived cells. The universality of this phenomenon was confirmed (FIGS. 3D, E, F).

Discussion Although there are many publications on the importance of spheroid cultures for somatic stem cells, there is no single, generally accepted protocol. For example, original methods for neural stem cell culture utilize dissociated cells without traditional two-dimensional cell culture. Spheroid formation was performed under static conditions without mechanical migration, and spheroids formed spontaneously from certain fractions of floating cells. More recently, cell cultures on coated plates have also been utilized to form spheroids. However, there are many studies using physical or mechanical stimuli, including rotation (23), shaking (24) or agitating movement (25). As a method of forming spheroids using gravity, there are a hanging drop method (26) and a method using a specific inverted cone-shaped non-adherent culture dish (27). Overall, these methods can be divided into two main groups.

に お い て In the first reported method using naturally floating cells, this process was considered “spontaneous spheroid formation”. And another method based on spheroid formation using some kind of mechanical force can be considered as “mechanical spheroid formation”. There is diversity not only in the method of forming spheroids, but also in the medium. Initial spheroid-forming cultures utilize a serum-free culture medium (DMEM / F12) containing additives (bFGF, EGF and B27 or other reagents) (28). On the other hand, there are some publications using serum-containing medium (29) without using these additives. Each study showed some evidence demonstrating the superiority of each method and the need for these additives, but it is not yet clear why there are so many variations and they are really essential for spheroid culture . Furthermore, it is not clear whether spheroids produced by these various methods are identical. Therefore, we decided to look at a very basic point: the effect of culture plate adhesion on spheroid formation.

To achieve this, we focused on two things. First, since the hydrophobicity is known to reflect cell adhesion (30 to 32) and can be easily evaluated by the water contact angle, the difference in the hydrophobicity of the culture plate was focused on. Second, we tried to distinguish between spontaneous spheroid formation and mechanical spheroid formation and found the prerequisites for achieving spontaneous spheroid formation. Spontaneous spheroid formation appears to be justified in achieving selective culture of stem cells, and mechanical spheroid formation, by its nature, may involve various types of cells.

Surprisingly, the results of this example showed a direct relationship between hydrophobicity and spheroid formation. Spheroid formation was observed only on the plate with the highest specific water contact angle (FIG. 1, Dish # 3). This means that the culture plate has the lowest adhesion.

Time course images showed that the presence of at least two populations was observed in the cultures on this culture plate (FIG. 4). One type of cell is spindle-shaped and looks like fibroblasts or typical mesenchymal stromal (stem) cells. This type of cell has a relatively high ability to adhere and can remain attached to the dish. The other type of cells appears oval or rectangular in shape, migrates rapidly, and begins to form cell aggregates. When cells of this type contacted cells of the same type, they began to attach, forming larger cell aggregates, proliferating, and forming spheroids. Once the spheroids reached a certain size, they began to spontaneously detach from the dish.

From this stage of immunofluorescence analysis (FIG. 2A), only the aggregated cells express the stem cell marker, indicating that the aggregated cells are somatic stem cells and have a lower adherence capacity than other types of cells. Can result in spontaneous spheroid formation. It is unknown whether spindle cells play any particular role in maintaining niche-like stem cells. However, it is reasonable to speculate that at least some of these spindle cells act as a niche for stem cells over a period of time in vivo or in vitro and support stem cell function. In this case, these cells can be designated as "feeders" of the niche in vivo, and even as virtual niches in vitro, and should be further investigated.

ス Spheroid formation from skin cells has already been reported (so-called SKP) (33) and is a basic protocol based on the original method for neural stem cell culture (34). However, the efficiency was so low that it was almost impossible with cells from adult tissues, so that embryonic skin tissue had to be used. On the other hand, spheroids having stem cells can be efficiently obtained by the method of the present inventors.

主 The main difference between the original method and the method of the present invention was cell passage (only isolated cells were used for SKP without culturing, and cells passaged 2-3 were used in this study). In addition, SKP studies utilize regular culture plates that showed higher adherence. Therefore, even stem cells may be attached to the culture dish. The results of our studies indicate that stem cells can still survive conventional two-dimensional cell culture, and the authors confirmed spheroid formation even after 5 passages, but with reduced efficiency.

Enzymatic separation of cells is not easy and many stem cells may not dissociate completely. Also, the method of producing SKP is too harsh due to the high adhesion of the plate, so that even stem cells cannot float and attach to the dish and lose their spheroid properties.

比較 Comparing this example with other tests using mechanical stimulation is also an interesting issue. In order to confirm this, a time-lapse experiment using the hanging drop method, which is a typical mechanical spheroid formation protocol, was also performed. As a result, during cell aggregation, some of the cells began to grow to form small spheroids, and the size of those spheroids changed. On the other hand, many cells do not form spheroids. It is a dead or non-proliferating differentiated cell. Eventually, all of the different types of cells aggregate and form single or multiple spheroids. This includes mechanically formed spheroids, also stem cell populations with varying levels of stem cells, and differentiated cells, which represent the relatively non-selective nature of mechanical spheroids.

However, theoretically, it can be expected that the ratio of non-dividing cells will decrease over time, and that most of the spheroid-forming cells will be occupied by stem cells or stem cell-derived cells. In this case, the main advantage of spontaneous spheroid formation may be the purity of the stem cells and the rapid selection of the stem cells. To confirm this idea, qRT-PCR results showed significantly higher stem cell marker expression from the very beginning (even after 12 hours) (FIGS. 2B-E).

It is also possible that stem cell aggregation to form spheroids can affect stem cell properties and enhance or alter the expression of stem cell markers, which can be tested under different and more specific study designs. You. Another important question is the need for a serum-free environment and their growth factors. As far as the inventor can confirm, the method of this example does not exclude the possibility that these factors for maintaining stem cells or spheroids may play a role, however, these growth factors and additives do not affect spheroid formation. Is not necessary.
The next issue is the universality of this phenomenon. Therefore, we tested the feasibility of this phenomenon using three other different cell types. Interestingly, not only cells derived from skin (FIGS. 3A to 3C) but also cells derived from oral mucosa (FIG. 3D), cells derived from cortical bone (FIG. 3E), and NIH-3T3 (FIG. 3F) were observed.

Spheroids could be formed with stem cells in a similar manner, although some differences were noted in the efficiency and shape of spheroid formation. This supports that the method of the present invention is very universal.
In conclusion, the novel method of the present invention is easy and reproducible for spheroid formation and is less expensive. Therefore, the method of the present invention contributes to understanding the mechanism of the nature of somatic stem cells from various tissues, and can also be applied to various clinical treatments.

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[Example 2]
SUMMARY The present inventors have developed a new method for spontaneous spheroid formation using specific low-adhesion culture plates with a water contact angle of about 90 degrees. Although the method of the present invention can be applied to various cells, this example describes application to oral mucosa-derived cells and skin-derived cells. First, the feasibility of spontaneous spheroid formation was tested. Next, the properties of spheroids from oral mucosa and skin-derived cells were compared, with a particular focus on stemness and neurogenic potential.

Oral mucosal cells were obtained from the palate and buccal mucosa of C57BL / 6J mice. Similarly, skin cells were obtained from the back of the same strain of mice. Cells from passage 2 to 3 were seeded on specific low-adhesion culture plates to form spontaneous spheroids. The effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and B27 on spheroid formation and maintenance were evaluated. Immunofluorescence and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to examine the expression of pluripotency markers, cell proliferation and apoptosis markers, and neural differentiation markers.

The use of this culture plate enabled spontaneous spheroid formation of both oral mucosa-derived cells and skin oil sticks. This process depended on the presence of serum but was independent of additives such as bFGF, EGF and B27 supplements (although these added factors improved spheroid formation efficiency and were essential for spheroid maintenance). T). Spheroids from oral mucosa-derived cells expressed stem cell markers such as Sox2, SSEA1, Oct4, Nanog and Nestin. The expression of Sox2 in spheroids from oral mucosal cells was higher than in spheroids from skin-derived cells. Both types of spheroid-forming cells were capable of differentiating into neural and Schwann cells after induction of differentiation to nervous system cells, whereas MAP2, MBP, nestin and Nurr1 gene expression was derived from oral mucosal-derived spheroids. Significantly higher in cells.

Here, Nurr1 is a marker for dopamine-producing cells, and it is known that dopamine-producing cells are reduced in Parkinson's disease. The high expression of the Nurr1 gene means that the spheroids of the present invention differentiate into dopamine-producing cells. Therefore, the spheroid of the present invention is useful as a therapeutic agent for Parkinson's disease.

The results showed that spontaneous spheroids from oral mucosa-derived cells contain highly potent stem cells as good as skin-derived stem cells. High expression of certain neuronal marker genes suggests the benefit of these cells for regenerative treatment of neuronal disorders.

(4) Spontaneous spheroids obtained by the method of the present invention have excellent neuronal differentiation ability and have been shown to be useful for treating neurological diseases. In particular, spontaneous spheroids prepared from oral mucosal cells were shown to be excellent neural stem cells. By using the spontaneous spheroid prepared by the method of the present invention, neural stem cells can be prepared. Further, upon spontaneous spheroid formation by the method of the present invention, additional factors such as bFGF and EGF are important for improving the formation efficiency and maintaining the spheroid. Therefore, in the present invention, it is also possible to use these additional factors.

Background Spheroid formation has been used for the selective culture of stem cells from various tissues, including neurons, skin, salivary glands, bone marrow stroma, periodontal ligaments, and pulp tissue [1-5]. Various methods are used to form spheroids. The hanging drop method utilizes droplets of a cell suspension, and the cells begin to aggregate by gravity at the bottom of the droplet, eventually forming spheroids [6]. Media rotation has also been used [7,8]. Because these methods rely on physical forces and the adhesion between cells is achieved by forced contact of cells, we call them "mechanical spheroid formation" methods, and the process of spheroid formation is It is not selective for stem cells [9].

他 Other methods, including the method of the present invention, do not use physical forces and spheroid formation occurs under static conditions. Therefore, we have named these approaches the "spontaneous spheroid formation" method [9]. Since spontaneous spheroid formation starts only from possible stem cells, it theoretically allows selective culture of stem cells. So far, most of the reported spontaneous spheroid formation has been limited to neural stem cells and skin-derived stem cells [10, 11], and the detailed characteristics of spontaneous spheroids derived from oral mucosal cells have been reported. Not.

口 Among the cell sources for somatic stem cells, oral mucosa is considered to be very unique. Cell lineage studies have shown that even adult tissues contain neural crest-like stem cells [12,13]. Oral mucosa and dermal fibroblasts have similar morphology and function, but there are some essential differences. For example, wound healing in the oral mucosa is faster and produces less scarring compared to wound healing in the skin [14, 15]. These features of the oral mucosa can be attributed to the presence of highly potent neural crest-derived cells [16]. Therefore, some researchers consider oral mucosa to be a preferred source for somatic stem cells [17]. To support this notion, several recent studies have shown that stem cell populations in the lamina propria of the lips and oral mucosa exhibit neural crest-like characteristics, leading to osteoblasts, chondroblasts, adipocytes, and neural lineages. It has been reported that it has a very broad differentiation potential, including that of [18-21]. However, to the inventors' knowledge, there is no direct comparison of stem cells from skin and oral mucosa.

In this example, the possibility of spontaneous spheroid formation from cells derived from oral mucosa was tested. Next, the role of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and B27 supplements, which have been reported as essential factors for nerve spheroid formation, on spontaneous spheroid formation and maintenance was examined. Finally, the characteristics of spontaneous spheroids from oral mucosal and skin-derived cells were compared, with a special focus on their stem cell and neural differentiation potential.

Materials and Methods Cell Culture Animal experiments were approved by the Animal Experiment Committee of Matsumoto Dental University (no. 289). Primary cultured cells were obtained using a conventional explant culture method. Mice (3-4 weeks old) were killed by overdose of anesthesia, then the buccal and palatal mucosa and the back skin were removed and cultured. The basal medium is 10% fetal bovine serum (FBS; Biowest, Nuail, France), 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin B (Biological Industries USA, Inc., Cromwell, CT, USA). (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with. Cells were passaged at 80% -90% confluence. The medium was changed every 3-4 days. Cells from 2-3 passages were used for the experiments.

培養 The culture of cortical bone-derived cells (CBDC) was modified based on the protocol reported in our previous work [9]. Briefly, femurs and tibias were incised, epiphyses were cut, and bone marrow was perfused. The cortical bone was then cut into small pieces and incubated in a collagenase solution at 37 ° C. for 45 minutes in a bioshaker. Cells were harvested and cultured in αMEM supplemented with 10 ng / ml basic fibroblast growth factor (bFGF) (PeproTech, Rocky @ Hill, USA). Bone chips were seeded with the same culture medium in other wells of the same dish, and further cells were collected.

Spheroid Formation The method used for spheroid formation has been described in our previous publication [9]. Briefly, monolayer cultured cells are trypsinized and the cells are 55 mm dishes (1.5 × 10 ⁴ cells / cm ² ) (Azunol®, # 1-8549-02, AS ONE, Osaka, Japan). And spheroids were produced in a basal culture medium. The medium was changed every three days. In order to evaluate the spheroid formation efficiency of oral mucosa and skin-derived cells, 1 to 5 with 4 parallel phase contrast microscope fields / dish (x4 objective lens (Olympus IX70 inverted microscope, Olympus Optical CO, Ltd, Tokyo, Japan)). During the day, the number and size of spheroids were measured. To assess spheroid maintenance, basic fibroblast growth factor (bFGF; Gibco, Carlsbad, CA, USA), epidermal growth factor (EGF; PeproTech, Rocky Hill, NJ, USA) and B27 supplement (Gibco, Carlsbad). , CA, USA) was added to the basal medium. Cultures were performed with one or several of these additives to determine the need for each factor. Spheroid diameter and number were measured for 5 days.

Neural Differentiation Spheroids from oral mucosal, skin and CBDC derived cells were transferred to new regular culture dishes. After these cultures reached 50% to 60% confluence, the medium was replaced with a neural differentiation induction medium. The medium used for neuronal differentiation was L-glutamine, phenol red (Wako), 50 ng / ml nerve growth factor, 50 ng / ml brain-derived neurotrophic factor, 10 ng / ml NT-3 (all Peprotech), 10% FBS, ΑMEM supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin B (Biological Industries). The medium used for Schwann cell differentiation was L-glutamine, phenol red (Wako), 5 μM forskolin forskolin (Sigma), 50 ng / ml heregulin-1β (Peprotech), 2% v / v N2 supplement (Invitrogen), ΑMEM supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin B (Biological Industries). Cells were differentiated for one or two weeks and 50% of the medium was changed every two days.

Immunofluorescent staining Immunofluorescent staining was performed as previously reported [9]. Primary antibodies targeting the following proteins were used: SSEA1 (1:40, ab16285, Abcam), Sox2 (1: 250, 97959, Abcam), Oct4 (1: 250, ab19857, Abcam), Nanog (1: 100, ab80892, Abcam), βIII-tubulin (1: 250, ab87087, Abcam), nestin (1: 200, ab6142, Abcam), NEUN (1: 100, ab177487, Abcam), MAP2 (1:50, ab32454). , Abcam), and S100β (1: 100, ab52642, Abcam).

The secondary antibodies used were as follows: goat anti-mouse IgM @ Alex @ Fluor @ 488 (1: 200, ab150121, Abcam), goat anti-mouse IgG @ Alex @ Fluor @ 488 (1: 500, ab150113, Abcam), and goat anti-mouse Rabbit IgG Alex Fluor 647 (1: 200-1: 500, ab150079, Abcam), and nuclei were counterstained with 4 ', 6-diamidino-2-phenylindole (DAPI, ab104139, Abcam) for 30 minutes at room temperature. .

In order to examine cell proliferation and apoptosis in spheroids, immunofluorescent staining of Ki67 and caspase 7 was performed. Spheroids were solidified in iPGell (Genostaff, Tokyo, Japan) according to the manufacturer's instructions, fixed with 4% paraformaldehyde in phosphate buffer, embedded in paraffin, and sectioned at 8 μm thickness. . Primary antibodies targeting the following proteins were used for immunostaining of the sections: Ki67 (1: 100, ab15580, Abcam) and Caspase 7 (1: 100, ab69540, Abcam). After overnight incubation at 4 ° C. with the primary antibody, the sections were washed three times with PBS and each incubated with the secondary antibody. Secondary antibodies used were donkey anti-mouse IgG Alex Alex Fluor 488 (1: 250, ab96875, Abcam) and donkey anti-rabbit IgG Alex Alex Fluor 647 (1: 250, ab 150075, Abcam) and nuclei were counterstained with DAPI solution. .

Fluorescence was observed with a phase contrast microscope (KEYENCE 顕 微鏡 BZ-X710, Keyence, Osaka, Japan) and analyzed with BZ-X Analyzer software.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen Corporation, Carlsbad, Calif., USA) and cDNA was cloned from PrimeScript ™ RT Master Mix (Perfect Real Time) (TaKaRa, cat # RR036A, Kats, Japan). Synthesized. qRT-PCR was performed at least three times according to the manufacturer's protocol. The data was quantified using the ΔΔCt method and normalized by the expression level of β-actin. Table 2 shows the primer sequences used for PCR.

Statistical analysis Statistical analysis was performed using Student's t-test. The results are shown as the mean mean standard error (SEM) of at least three experiments.

Results Spontaneous spheroids derived from oral mucosa-derived cells The spheroid-forming ability of oral mucosa-derived cells on a specific culture dish was examined. In the serum-free medium, some of the cells derived from the oral mucosa adhered to the culture dish, exhibited fibroblast morphology after 24 hours, and apoptosis occurred after 72 hours (FIG. 6A). In serum-free medium containing bFGF, EGF and B27 supplement, cells from the oral mucosa adhered to the dish and showed some cell aggregates, but no spheroid formation was observed (FIG. 6A). In the serum-containing medium, oral mucosa-derived cells spontaneously formed spheroids within 24 hours, and spheroids were maintained during the observation period (72 hours) (FIG. 6A).
To examine the expression of the stem cell marker gene, spheroid-forming cells were analyzed by immunofluorescence. Spontaneous spheroids formed from cells derived from oral mucosa expressed Sox2, SSEA1, Oct4, Nanog (FIG. 6B) and Nestin (FIG. 6C).

Effect of additives on spheroid formation and maintenance of oral mucosa and skin-derived cells In media containing serum, oral mucosa-derived cells and skin-derived cells form compact multicellular spheroids within 24 hours without additives. Aggregated spontaneously and maintained for 72 hours (FIG. 7A). The spheroid morphology was not affected by the presence of the additives or the origin of the cells, and the differences between the groups were not significant.

直径 To further investigate the changes in spheroids over time, the diameter and number of spheroids were evaluated up to 120 hours. The size of spheroids derived from oral mucosa-derived cells tended to increase up to 72 hours after cell seeding, and then gradually decreased over time (FIG. 7B). On the other hand, the size of spheroids from skin-derived cells did not change until 120 hours, and the difference between spheroids was significant at 120 hours (p <0.001). The number of spheroids from oral mucosa-derived cells increased at 48 hours, peaked, and then gradually decreased (FIG. 7C). The number of spheroids from skin-derived cells gradually increased until 96 hours and then decreased. Number differences were significant only at 120 hours (p <0.001).

直径 The spheroid diameter and number were then measured in the presence of one or several of the additives. Compared to spheroids without additives, spheroid size was significantly larger in media containing bFGF, EGF and B27 supplement (p <0.001) (FIG. 7D). The closest results were obtained when spheroids were cultured with bFGF and B27 supplement. In addition, the number of spheroids was larger in the group in which three kinds of additives were added and cultured for 24 to 120 hours (p <0.001) than in the group in which no additives were added (FIG. 7E). Similar results were obtained with the group treated with the bFGF and B27 supplements, the number being higher at all time points than the group without additives.

Analysis of cell proliferation and apoptosis in spheroids In both spheroids from oral mucosa and skin-derived cells, more Ki67-positive cells were observed in spheroids with additives than in spheroids without additives (FIG. 8A). Caspase 7 positive cells were observed in oral mucosal spheroids without additives, but only a few weakly positive cells with additives were observed. Skin-derived spheroids showed faint caspase 7-positive cells in spheroids without additives, but no positive cells were observed in spheroids with additives (FIG. 8A).

Proliferation and apoptosis gene expression was analyzed using qRT-PCR. Ki67 expression was higher in spheroids cultured with additives than in spheroids without additives (FIG. 8B). On the other hand, the expression of caspase 7 was significantly higher in spheroids cultured without additives than in spheroids with additives (FIG. 8B). When caspase 7 expression was compared between spheroids of oral mucosa-derived cells and spheroids of skin-derived cells, the expression was significantly higher in spheroids of oral mucosa-derived cells regardless of the presence of additives (FIG. 8C). ).

Comparison of stem cell marker expression in spheroids of oral mucosa-derived and skin-derived cells Expression of pluripotency-related genes was analyzed using qRT-PCR. In spheroids derived from oral mucosa-derived cells, the expression of Sox2, Fut4 (SSEA1) and nestin was higher in spheroids cultured with additives than in spheroids without additives, but the expression of Oct4 was higher in the presence of additives. Unaffected (FIG. 9A). In spheroids derived from skin-derived cells, the expression of Fut4 (SSEA1) and nestin was significantly higher when spheroids were cultured with additives (FIG. 9B). On the other hand, the expression of Sox2 decreased in the presence of the additive. As with the oral mucosal spheroids, the expression of Oct4 showed no significant difference regardless of the presence or absence of additives.

Next, stem cell marker expression was compared between oral mucosa-derived cell spheroids and skin-derived cell spheroids. The expression of Sox2 was higher in spheroids of cells derived from oral mucosa than in spheroids of cells derived from skin, and the tendency was not affected by the presence of additives (FIGS. 10A and B). Nestin expression was higher in spheroids of oral mucosa-derived cells than in spheroids of skin-derived cells without additives (FIG. 10A). However, this difference was not observed when spheroids were cultured with the additives (FIG. 10B). The expression of Fut4 (SSEA1) in spheroids of oral mucosa-derived cells was higher than that of skin-derived cell spheroids at 120 hours without additives and at 24 hours with additives, but no difference was observed under other conditions. Oct4 expression did not show a significant difference between oral mucosa-derived cells and skin-derived cells spheroids, and this trend was not affected by the presence of the additives.

Comparison of neural differentiation potential between spheroid-derived cells from oral mucosa and spheroid-derived cells from skin Two-dimensionally cultured oral mucosal cells contained nestin, NeuN, Sox2 and S100β positive cells, while βIII -Tubulin and MAP2 were negative (Figure 11A). Spheroid-derived cells from both oral mucosa and skin were positive for nestin, βIII-tubulin, MAP2, NeuN, Sox2 and S100β (FIG. 11A). To determine the level of differentiation, MAP2, MBP, NF-M, GFAP and nestin mRNA expression levels were analyzed. In addition, the expression of the specific dopaminergic neuronal marker Nurr1 was also evaluated by qRT-PCR (FIG. 11B). MAP2, MBP, nestin and Nurr1 mRNA expression in oral mucosal spheroid-derived cells was significantly higher than skin spheroid-derived cells. NF-M expression levels did not change between spheroid-derived cells of both origins. GFAP expression in oral mucosal spheroid-derived cells was higher than in skin spheroid-derived cells, but the difference was not significant due to large variability.

Although relatively high expression of several neuronal differentiation markers was observed in spheroids of oral mucosa-derived cells, it was not clear whether their levels were comparable to levels in other somatic stem cells such as mesenchymal stem cells. Did not. Therefore, the present inventors next discovered that spheroids derived from oral mucosa and CBDC-derived spheroids, which are known as an excellent source of mesenchymal stem cells in mice and can differentiate into neurogenic cell lineages, Among them, the expression levels of MAP2, MBP and nestin were compared [22-24]. Following neural induction, the expression of MAP2, MBP, and nestin in oral mucosal spheroid-derived cells was much higher than that of CBDC in spheroid-derived cells (20.08, 3.02, and 16.18, respectively). Times) (FIG. 11C).

DISCUSSION Characteristics of spontaneous spheroids of cells derived from oral mucosa During mechanical formation of spheroids, ideally almost all cells in culture are involved in spheroid formation. Therefore, the process is not selective [22]. On the other hand, spontaneous spheroid formation occurs under static conditions. It starts only with cells that can survive without attachment, and the cells have the ability to proliferate and form spheroids. Thus, spontaneous spheroid formation is selective, but not as efficient as mechanical spheroid formation. Indeed, skin-derived precursors (SKPs) that are well known to form spheroids spontaneously can be obtained from embryonic tissue and difficult to obtain from adult tissue [23]. The present inventor's method for spontaneous spheroid formation can overcome this limitation because spheroid formation is possible even with cells cultured for 4 to 5 passages (FIG. 12). It is also important to ask whether spheroids formed using the method of the invention are identical to spontaneously formed spheroids, such as SKP spheroids. Spheroids spontaneously formed from cells derived from oral mucosa are positive for Sox2, SSEA1, Oct4, Nanog and Nestin, and are capable of neurogenic differentiation, which is a previously reported spontaneous spheroid. [2, 12].

１ One of the fundamental differences between this method and the previously reported spontaneous spheroid formation procedure is the presence or absence of additives. Spontaneous spheroid formation has been reported to require a serum-free environment and the addition of growth factors such as bFGF, EGF and B27 supplements [24, 25]. On the other hand, spontaneous spheroid formation induced by the method of the present invention can be achieved in a serum-containing environment without these additives.

The role of additives for spontaneous spheroid formation and maintenance With additives, the number of spheroids at 24 hours was about twice as high as without additives, which is It shows some roles. Additives promote spheroid formation, but are not required, since spontaneous spheroid formation was feasible without additives. On the other hand, without additives, spheroid size and number decreased with time. This tendency was more apparent in spheroids of cells derived from oral mucosa than in spheroids of cells derived from skin. Therefore, it is also possible to add these additives in spontaneous spheroid formation.

Neuronal differentiation of spontaneous spheroid-forming cells from oral mucosa After neural induction, planar culture of oral mucosal cells failed to express neural markers such as MAP2 and βIII-tubulin. The presence of these differentiated neuronal markers in spheroid-derived cells suggests a high neural differentiation potential of spheroid-forming cells. Since spheroids from both oral mucosal and skin-derived cells contained cells that were positive for nestin, βIII-tubulin, MAP2, NeuN, Sox2 and S100β, qRT-PCR Were compared.

の Among the markers tested, MAP2, MBP and nestin expression levels were significantly higher in spheroids of oral mucosa-derived cells than in spheroids of skin-derived cells. Furthermore, higher expression of the dopaminergic neuronal marker Nurr1 in spheroids of cells derived from oral mucosa was observed. Taken together, these findings suggest the benefits of using these oral mucosal cells for the treatment of neurodegenerative diseases such as Parkinson's disease. More importantly, the expression of MAP2, MBP and nestin was much higher in spheroids of cells from oral mucosa than spheroids from mesenchymal stem cells from CBDC. This can be attributed to the presence of neural crest-derived stem / progenitor cells in oral mucosa-derived spheroids, and can further emphasize the unique advantages of this cell source for application to neurodegenerative diseases.

Conclusions The results of this study showed that spontaneous spheroid formation was feasible in cells from oral mucosa using our method. Although spontaneous spheroid formation was possible without bFGF, EGF, B27 supplements, they improved the efficiency of spheroid formation and, more importantly, were essential for spheroid maintenance. Spontaneous spheroids of oral mucosa-derived cells include powerful stem cells that are as good as skin-derived stem cells. High expression of specific neuronal marker genes suggests the benefit of these cells for regenerative treatment of neuronal disease.

References in Example 2 Reynolds BA, Weiss S.L. Generation of neurons and astrocytes from the isolated cells of the adult mammarian central neural system. Science. 1992; 255 (5052): 1707-10.
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4. Techawattanawial W, Nakahama K, Komaki M, Abe M, Takagi Y, Morita I. Isolation of multipotent stem cells from adult rat rate periodical ligation by neurosphere-forming culture system. Biochem Biophys Res Commun. 2007; 357 (4): 917-23.
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14． Eslami A, Galant-Behem CL, Hart DA, Wiebe C, Hondoust D, Gardner H, Hakkinen L, Larjava HS. Expression of integration alphavbeta6 and TGF-beta in scales vs. scar-forming wound hearing. J Histochem Cytochem. 2009; 57 (6): 543-57.
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21. Ebisawa K, Kato R, Okada M, Sugimura T, Latif MA, Hori Y, Narita Y, Ueda M, Honda H, Kagami H. Gingival and dermal fibroblasts: Their similarities and differences reviewed from gene expression. Journal of Bioscience and Bioengineering. 2001; 113 (3): 255-8.
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[Example 3]
Method The culture method of CBDCs and the method of spontaneously forming spheroids were performed according to Examples 1 and 2.
Induction of bone differentiation of CBCSc Twenty-four hours after spheroid formation, the cells were passaged to another adherent culture dish. The medium was replaced with an osteogenesis induction medium at 50-60% confluence. This medium contains 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 50 μM L-ascorbic acid (Wako Pure Chemical Industries, Ltd.) and 10 mM β-glycerophosphate (Sigma-Aldrich). . The medium was changed every two days.

Measurement of ALP activity ALP activity was measured on the 7th day of bone differentiation induction to confirm bone differentiation. Cells that did not induce differentiation were used as controls. cell counting kit-8 (CCK- 8) (Dojindo Laboratories, Kumamoto, Japan) and p- nitrophenol ^{(SIGMA Fast TM p-Nitrophenyl Phosphate} Tablet;. Sigma-Aldrich Co.LLC) were used according to suppliers of description. Absorbance was measured at 450 nm for formazan and 405 nm for p-nitrophenol using an iMark ^™ Microplate Absorbance Reader (BIO-RAD Laboratories, Hercules, CA, USA).

Analysis of Bone Differentiation Markers by qRT-PCR qRT-PCR was performed in the same manner as in Example 1.
Table 3 shows the primers used.

Bone regeneration by transplantation experiment 1 × 10 ⁶ CBDC cells were seeded on a β-TCP block (Ospherion, Olympus Terumo Biomaterial) having a diameter of 2 mm and a thickness of 2 mm. The next day, the medium was replaced with a bone differentiation induction medium, differentiation was induced for 7 days, and then transplanted subcutaneously to the back of a 6-week-old male SCID mouse. Four weeks after transplantation, the cells were taken out, evaluated by μCT, decalcified, embedded in paraffin, and sectioned. The sections were observed by HE staining.

Result 1. Analysis of Bone Differentiation Markers The analysis results of the bone differentiation markers are shown in FIG.
2. In Vivo Bone Regeneration A histological image 4 weeks after transplantation of CBDC cells is shown in FIG. From the histological image, formation of bone tissue was observed (pink portion), and the ability of the spontaneous spheroid of the present invention to regenerate bone was confirmed.

SEQ ID NOS: 1 to 40: Synthetic DNA

Claims (19)

1. A method for forming a spheroid, comprising forming a spheroid by culturing a stem cell using a culture vessel having a surface processed or treated so that cell adhesion is inhibited.
2. The method according to claim 1, wherein the culture vessel whose surface has been processed or treated so as to inhibit cell adhesion has hydrophobicity.
3. 3. The method according to claim 2, wherein the culture vessel having hydrophobicity has a roughness of a culture bottom so as to satisfy a condition that a contact angle is in a range of 85 ° to 95 °.
4. 4. The method according to claim 3, wherein the contact angle is from 89 ° to 91 °.
5. The culture vessel having a surface processed or treated so as to inhibit cell adhesion is made of polystyrene or glass, and the bottom surface is not coated with a fluorine-containing polymer. The method described in the section.
6. The culture vessel according to any one of claims 1 to 5, wherein the culture vessel having a surface processed or treated so as to inhibit cell adhesion has a root-mean-square surface roughness of 2.400 nm to 2.500 nm. The described method.
7. 7. The method according to claim 1, wherein the stem cells are somatic stem cells, embryonic stem cells, mesenchymal stem cells, or cancer stem cells or tumor-derived cells.
8. A method for producing spheroids, comprising recovering spheroids formed by the method according to any one of claims 1 to 7.
9. A culture vessel having a bottom surface of a culture so as to satisfy a condition that a contact angle is in a range of 85 ° to 95 °.
10. 10. The culture container according to claim 9, having a contact angle of 89 ° to 91 °.
11. The culture vessel according to claim 9 or 10, wherein the culture vessel is made of polystyrene or glass, and the bottom surface is not coated with a fluorine-containing polymer.
12. The culture vessel according to any one of claims 9 to 11, which has a root-mean-square surface roughness of 400 nm to 2.500 nm.
13. A spheroid produced by the method according to claim 8.
14. 14. The spheroid according to claim 13, wherein the spheroid has a higher ability to differentiate into a tissue than a spheroid formed without using the method according to any one of claims 1 to 7.
15. The spheroid according to claim 14, wherein the tissue is a nerve or a bone.
16. A regenerative medicine material comprising the spheroid according to any one of claims 13 to 15.
17. The material according to claim 15, wherein the regenerative medicine is a regenerative medicine for a nerve disease or a bone disease.
18. A regenerative medicine kit comprising the spheroid according to any one of claims 13 to 15.
19. The kit according to claim 18, wherein the regenerative medicine is a regenerative medicine for a nerve disease or a bone disease.

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