JP7205817B2 - Method for preparing stem cell-like cells - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law Published in the lecture program and proceedings on the website of the 57th Annual Meeting of the Japanese Society of Histocytochemistry published on August 18, 2016

Application of Article 30, Paragraph 2 of the Patent Act Published in the lecture program and proceedings of the 57th Annual Meeting of the Japanese Society of Histocytochemistry, published on September 3, 2016

Application of Article 30, Paragraph 2 of the Patent Law Presented at the 57th Annual Meeting of the Japanese Society of Histocytochemistry held at Kyorin University on September 4, 2016

Application of Article 30, Paragraph 2 of the Patent Law Published in the abstract of the 6th International Conference on Plasma Medicine (ICPM-6) issued on September 4, 2016

Application of Article 30, Paragraph 2 of the Patent Act Presented at the 6th International Conference on Plasma Medicine (ICPM-6) held at Komensky University on September 9, 2016

The present invention relates to a method for preparing stem cell-like cells.

There is a need for new methods of treatment of damaged areas of the brain, protection and regeneration of the cerebral cortex, and new prevention and treatment of psychiatric disorders such as depression. It was believed that once a mature brain is damaged, nerve cells cannot be regenerated, and the brain must live with the aftereffects for the rest of its life. Recently, however, it has become clear that even in the mature brain, new neurons can be generated depending on the location and conditions.

Therefore, development of a method of using neural stem cells and progenitor cells existing in the subventricular zone of the lateral ventricle and other regions to proliferate and differentiate them in vitro and then transplanting them into the damaged brain region again has been studied. there is In addition, differentiating induced pluripotent stem cells (iPS cells) into nerve cells and transplanting them is also under study.

As a method for regenerating nerves in the damaged cerebral cortex, for example, cells (SB623 ) is transplanted (Non-Patent Document 1).

There is also a method of preparing neurospheres and obtaining differentiated nerve cells from the neurospheres. For example, a method of producing neurospheres from tissue obtained from the perilateral ventricles, which is known to contain many neural stem cells, has been reported (Non-Patent Document 2).

It has also been reported that a pathological model such as cerebral ischemia can be prepared by surgical treatment, and neurospheres can be prepared from the cerebral cortex of the pathological model (Non-Patent Document 3).

As various methods for inducing differentiation into cells of the central nervous system, for example, Patent Document 1 discloses culturing pluripotent stem cells or neural progenitor cells in the presence of N-acetyl-D-mannosamine to differentiate orexin neurons. A method of inducing is disclosed.

Patent Document 2 discloses a novel monoclonal antibody capable of efficiently separating neural stem cells and progenitor cells by selectively recognizing neural stem cells and progenitor cells. It is also described that neural stem cells and progenitor cells can be efficiently isolated from human brain tissue using this monoclonal antibody.

US Pat. No. 5,300,001 discloses substantially pure human retinal progenitor cell cultures, forebrain progenitor cell cultures, retinal pigment epithelial cell cultures, and methods for their production.

Patent Document 4 discloses a method for inducing the differentiation of pluripotent stem cells into neural progenitor cells, including culturing the pluripotent stem cells in the presence of a low-molecular-weight BMP inhibitor.

In Patent Document 5, aggregates of pluripotent stem cells are subjected to suspension culture in the presence of a Wnt signal inhibitor and a TGFβ signal inhibitor to obtain a telencephalic marker-positive aggregate, and further the telencephalic marker-positive aggregates are obtained. A method is disclosed for inducing more mature telencephalon or its progenitor tissue in vitro from mammalian pluripotent stem cells by further culturing aggregates in suspension under conditions of high partial pressure of oxygen.

Non-Patent Document 4 discloses that neurons are formed from neural progenitor cells into which HMGA (high mobility group A) genes have been introduced.

In addition, US Pat. No. 6,200,001 discloses a composition suitable for therapeutic use, comprising cryopreserved neural stem cells.

International Publication No. 2013/047773 (published on April 4, 2013) Japanese Patent Application Laid-Open No. 2004-2350 (published on January 8, 2004) Special table 2013-502234 (published on January 24, 2013) Special table 2013-501502 (published on January 17, 2013) International Publication No. 2015/076388 (published on May 28, 2015) Special table 2012-510986 (published on May 17, 2012)

Aleksandra Glavaski-Joksimovic, Tamas Virag, Thomas A. Mangatu, Michael McGrogan, Xue Song Wang, and Martha C. Bohn, “Glial Cell Line-Derived Neurotrophic Factor-Secreting Genetically Modified Human Bone Marrow-Derived Mesenchymal Stem Cells Promote Recovery in a Rat Model of Parkinson's Disease”, Journal of Neuroscience Research 2010, 88:2669-2681. Gil-Perotin S, et al. “Adult Neural Stem Cells From the Subventricular Zone: A Review of the Neurosphere Assay”, The Anatomical Record. 2013, 296:1435-1452 Nakagomi T, et al. “Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice”, European Journal of Neuroscience. 2009, 29: 1842-1852 Kishi Y et. al., "HMGA regulates the global chromatin state and neurogenic potential in neocortical precursor cells", Nature Neuroscience.15: 1127-1133, 2012

However, conventional methods require gene transfer into cells and preparation of a cerebral ischemia model in order to prepare stem cells. The method described in Non-Patent Document 2 does not require the introduction of genes into cells or the preparation of pathological models, but only a small region of the brain where stem cells are densely present ( confined to the dentate gyrus of the hippocampus and the periventricular region).

Attempts have also been made to induce the differentiation of induced pluripotent stem cells (iPS cells) into nerve cells, but gene transfer into cells is necessary to produce iPS cells. In addition, it is not easy to differentiate iPS cells into target nerve cells alone, and there are problems such as canceration due to transplantation of proliferating cells.

The present invention has been made in view of the above problems, and an object thereof is to realize a novel method for preparing stem cell-like cells.

In order to solve the above problems, the method for preparing stem cell-like cells according to the present invention is a method including a plasma irradiation step of irradiating animal tissue with non-equilibrium plasma.

INDUSTRIAL APPLICABILITY The present invention has the effect of providing a novel method for preparing stem cell-like cells from tissue.

1 is a diagram showing a schematic configuration of an atmospheric non-equilibrium plasma apparatus used in an example of the present invention; FIG. FIG. 2 is a diagram illustrating a method of irradiating atmospheric pressure plasma to brain tissue of rats performed in an example of the present invention. It is a figure which shows the measuring method and measurement result of the temperature change of the water by plasma irradiation used in the Example of this invention. FIG. 2 shows the results of Example 1 of the present invention, showing the results of fluorescent immunohistochemical staining of brain tissue sections after plasma irradiation. It is a figure explaining the formation method of the sphere performed by the Example of this invention. FIG. 2 shows the results of Example 1 of the present invention, showing the results of fluorescence immunohistochemical staining of cells after adhesion culture. The results of Example 1 of the present invention are shown, and the results of observing cells under an optical microscope after performing adhesion culture for 5 to 7 days on the cerebral cortex 3 days after plasma irradiation and then floating culture for 3 days or 7 days are shown. FIG. 10 shows. Shown are the results of Example 1 of the present invention. Fluorescence of spheres obtained by subjecting the cerebral cortex 3 days after plasma irradiation to adhesion culture for 5 to 7 days, suspension culture for 1 week, and a neuron differentiation induction test. It is a figure which shows the result of immunohistological staining. FIG. 2 shows the results of Example 1 of the present invention, and shows the results of fluorescent immunohistochemical staining of spheres after 2-week suspension culture of cerebral cortex 3 days after plasma irradiation. FIG. 3 shows the results of Example 1 of the present invention, and shows the results of fluorescent immunohistochemical staining of skin 3 days after plasma irradiation. The results of Example 1 of the present invention are shown, and the cerebral cortex 3 days after plasma irradiation was subjected to adhesion culture for 5 to 7 days, followed by suspension culture for 1 week, and then a differentiation induction test into astrocytes or oligodendrocytes. FIG. 3 shows the results of fluorescence immunohistochemical staining of spheres. The results of Example 2 of the present invention are shown, and after 3 days of plasma irradiation, the mouse cerebral cortex was subjected to adhesion culture for 5 to 7 days, and then suspension culture was performed for 3 days, 5 days, or 7 days. It is a figure which shows the result observed by. FIG. 3 shows the results of Example 3 of the present invention, and shows the results of observation under a phase-contrast microscope of cells after adhesion culture, suspension culture, and differentiation induction in rat cerebral cortex three days after plasma irradiation.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail. In this specification, unless otherwise specified, "A to B" representing a numerical range means "A or more and B or less".

The method for preparing stem cell-like cells according to the present invention comprises a plasma irradiation step of irradiating animal tissue with non-equilibrium plasma.

The method for preparing stem cell-like cells according to one aspect of the present invention may further include a culturing step of ex vivo culturing part or all of the tissue irradiated with plasma in the plasma irradiation step.

Here, in the present specification, the above-mentioned "stem cell-like cells" are intended to be cells having self-renewal ability and differentiation ability. The "stem cell-like cells" have pluripotency capable of differentiating into multiple cell types like embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Cells may be used, and cells capable of differentiating only into a specific cell type such as somatic stem cells may be used.

In one embodiment of the present invention, the "stem cell-like cells" may be cells positive for at least one of Sox2, Oct4 and NG2.

NG2 (neuron-glial antigen 2) is also called cspg4 (chondroitin sulfate proteoglycan 4). NG2 is known as a glial progenitor cell marker, pericyte marker, or immature glial cell marker (Reference 1: Trotter J, Karram K, Nishiyama A. NG2 cells: Properties, progeny and origin. Brain Research Reviews 63:72-82, 2010).

Sox2 is a transcription factor belonging to the SRY-related HMG-box (SOX) family. Sox2 is known as a marker for ES cells, TS cells or neural stem cells (Reference 2: Maucksch C, Jones KS, Connor B. Concise review: the involvement of SOX2 in direct reprogramming of induced neural stem/precursor cells. Stem Cells Transl Med: 579-583, 2013).

Oct4 is a transcription factor belonging to the POU family and is also called Oct3/4. Oct4 is known as an ES cell and undifferentiated marker (Reference 3: Shi G, Jin Y. Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem Cell Res Ther 1:39, 2010).

In one embodiment, the "stem cell-like cell" may be a cell positive for both Sox2 and Oct4. In another embodiment, the "stem cell-like cell" may be a cell positive for all of Sox2, Oct4 and NG2.

The subject cells are cells positive for at least one of Sox2, Oct4 and NG2 by a known immunological method (e.g., immunohistochemical staining) using an antibody that specifically binds to Sox2, Oct4 or NG2. etc.). Also, the expression of the Sox2, Oct4 or NG2 gene in the subject cells may be confirmed by known genetic engineering techniques (eg, RT-PCR, etc.).

(1. Plasma irradiation step)
The plasma irradiation step is a step of irradiating animal tissues with non-equilibrium plasma. The above-mentioned "non-equilibrium plasma" refers to plasma in which the temperature of electrons is higher than that of ions and neutral particles. Specifically, it refers to plasma in which the electron temperature is 5000 to 20000K (0.5 to 2.0 eV) and the temperature of ions and neutral particles is 300 to 1000K. Incidentally, 1 eV corresponds to ~11600K as a heat statistical distribution.

The above "non-equilibrium plasma" may be atmospheric pressure non-equilibrium plasma, low pressure non-equilibrium plasma, or vacuum non-equilibrium plasma. “Atmospheric non-equilibrium plasma” is non-equilibrium plasma generated under atmospheric pressure (~1.0 atm). “Low pressure non-equilibrium plasma” is non-equilibrium plasma generated under low pressure (10 ⁻⁵ to 10 ⁻¹ atm). “Vacuum non-equilibrium plasma” is non-equilibrium plasma generated under vacuum (10 ⁻⁷ to 10 ⁻⁵ atmospheres). Atmospheric pressure non-equilibrium plasma is preferable because plasma irradiation can be performed under atmospheric pressure and thus irradiation is easy. Since the atmospheric pressure plasma is emitted into the atmosphere, the plasma of the discharge gas reacts with oxygen and nitrogen in the atmosphere to generate oxygen radicals and nitrogen radicals.

In the plasma irradiation step, the method of irradiating animal tissue with non-equilibrium plasma is not particularly limited, but it is preferable to irradiate plasma so as not to increase the temperature of the tissue of the animal to be irradiated. Here, "irradiate the plasma so as not to increase the temperature of the tissue of the animal to be irradiated" means that the surface temperature of the tissue of the animal irradiated with the plasma is increased from 0 to 0 compared to before the plasma irradiation. It refers to irradiating plasma so that the temperature is within the range of 10° C. (preferably 0 to 5° C.). Plasma irradiation conditions that "do not involve an increase in the temperature of the tissue of the animal to be irradiated" can be determined, for example, by measuring the temperature of plasma-irradiated water, as shown in Examples below. It can be said that the plasma irradiation conditions under which the water temperature rise phenomenon cannot be detected by such a method are the irradiation conditions that do not cause tissue temperature rise.

In the plasma irradiation step, the surface temperature of the tissue of the animal to be irradiated is preferably kept at room temperature (27°C) to 42°C, more preferably 36 to 39°C.

In the plasma irradiation step, the plasma is preferably irradiated with a predetermined gap between the plasma irradiation head and the tissue of the animal. By providing a predetermined distance between the plasma irradiation head and the tissue of the animal, it is possible to irradiate the plasma without increasing the temperature of the tissue of the animal to be irradiated (the tissue to be irradiated). The distance between the plasma irradiation head and the animal's tissue is determined by the length of the plasma jet stream formed from the tip of the plasma head, the plasma irradiation time, and the like. It is preferable to irradiate with a gap between the plasma irradiation head and the tissue of the animal so that the tip of the plasma jet does not come into contact with the tissue to be irradiated. As a result, it is possible to suppress an increase in the temperature of the tissue to be irradiated during plasma irradiation.

The plasma irradiation conditions can be appropriately set according to the type of the plasma generator and the like. For example, in the embodiments described later, a micro hollow discharge type helium atmospheric pressure non-equilibrium plasma generator is used. Then, the tip of the plasma head was fixed at a position 2.5 cm above the upper surface of the target tissue to be irradiated, and a high voltage helium gas discharge of AC 60 Hz and a peak voltage of 7 kV was performed inside the head at a gas flow rate of 2.5 L/min. Plasma irradiation is performed for seconds. In this case, it is estimated that an electron density of 2.2×10 ¹⁵ cm ⁻³ is generated inside the head from an analysis of the Stark broadening width of the hydrogen atom β-ray seen in the emission spectrum (Stark method).

In addition to micro hollow discharge, for example, corona discharge, dielectric barrier discharge, RF discharge, microwave discharge, arc discharge, etc., can be employed as plasma discharge forms in the plasma generator.

As the micro hollow discharge type atmospheric pressure non-equilibrium plasma generator, for example, the atmospheric pressure plasma generator disclosed in JP-A-2013-153995, JP-A-2013-214377, etc. can be used.

As plasma discharge gas species, in addition to helium gas (He), for example, rare gases such as argon gas (Ar), neon gas (Ne), krypton gas (Kr), xenon gas (Xe), and nitrogen gas ( N ₂ ), synthetic atmosphere (N ₂ /O ₂ ) can be used.

When the discharge form of plasma changes, the density of electrons generated inside the plasma head of the plasma generator changes. For example, it is estimated that an electron density of 2.2×10 ¹⁵ cm ⁻³ is generated inside the head in a micro hollow discharge type helium atmospheric non-equilibrium plasma generator under the irradiation conditions described above. On the other hand, it is about 10 ⁹ cm ⁻³ in a known corona discharge or the like, and about 10 ¹³ cm ⁻³ in a dielectric barrier discharge (DBD). Therefore, the micro hollow discharge type is preferred because it can provide higher plasma densities compared to other discharge types.

When using a discharge type plasma generator other than a micro hollow discharge helium atmospheric pressure non-equilibrium plasma generator, (i) the types and amounts of radical molecules and ions generated or brought into the biological tissue to be irradiated Also, the irradiation time, gas flow rate, etc. may be appropriately adjusted in consideration of the concentration gradient, (ii) temperature conditions, and (iii) the current flowing through the tissue. More specifically, for example, the voltage at the irradiation position on the plasma irradiation target tissue is in the range of 3 kV or less (preferably 2 kV or less) on a time average of 20 ms, and the current at the irradiation position on the plasma irradiation target tissue However, the irradiation position may be determined so that the time average is in the range of 0.1 mA (preferably 0.5 mA or less) over 20 ms, and the irradiation time, gas flow rate, etc. may be appropriately adjusted. The voltage and current at the irradiation position on the plasma irradiation target tissue can be measured using a conductive electrode installed at the irradiation position on the plasma irradiation target tissue.

In one embodiment, for example, helium gas at a flow rate of 2.0-2.5 L/min (2.0-2.5 slm) can be irradiated for 60 seconds. The unit "slm" means standard (1 atm 0°C) liter (L) per minute.

In the present invention, the type, origin, etc. of the "tissue" to be irradiated with plasma are not particularly limited, and any tissue can be used. Types of tissue may include, for example, nerve tissue, epithelial tissue, connective tissue, muscle tissue, kidney tissue, liver tissue, hair root tissue, and the like. The nerve tissue may be, for example, central nerve tissue (eg, cerebral cortex, cerebellum, etc.), peripheral nerve tissue, or the like. The epithelial tissue may be, for example, epidermis, gastrointestinal mucosal tissue, or the like. The connective tissue may be, for example, dermis, adipose tissue, and the like. Examples of the muscle tissue include cardiac muscle tissue, skeletal muscle tissue, and smooth muscle tissue.

The animal species from which the tissue is derived is also not particularly limited, but tissues derived from mammals (mammals) are preferred in consideration of industrial applications such as cell therapy/tissue therapy. In addition, although the type of mammal is not particularly limited, it can be a non-human mammal or a human. Examples of non-human mammals include experimental animals such as mice, rats, rabbits, guinea pigs, and primates other than humans; companion animals such as dogs and cats; domestic animals such as cows and horses; Human-derived tissues are particularly preferred for clinical applications.

The tissue may be normal tissue, or may be tissue exposed to stress loading conditions such as ischemia, trauma, and the like.

Moreover, the tissue may be a tissue existing in the animal's body, or a tissue collected from the animal's body. As used herein, the term “tissue” refers to a structural unit in which one type or multiple types of cells are aggregated in a certain pattern. Myocardial sheets, cartilage, etc. prepared by inducing differentiation in vitro are also included in the above "tissue" category.

The above tissue is not limited to those derived from mature individuals, and may be derived from fetal (fetal) individuals, for example.

In addition, the tissue may be a tissue transplanted from a heterologous animal or a tissue transplanted from an allogeneic animal. Transplantation into allogeneic animals also includes autologous transplantation. For example, human tissue implanted into non-human animal tissue is also included in the above tissue category.

In one embodiment of the present invention, the step of transplanting a part or all of the tissue irradiated with plasma in the plasma irradiation step to a tissue different from the tissue irradiated with plasma is performed after the plasma irradiation step, as described below. may be included prior to the culture step.

The tissue irradiated with plasma may be transplanted to the same tissue or xenogeneic tissue from the same animal, or may be the same tissue or xenogeneic tissue from the different animal. Transplantation into allogeneic animals also includes autologous transplantation. For example, after irradiating human tissue with plasma, the plasma-irradiated tissue may be transplanted into the corresponding identical tissue of a xenogenic animal (eg, mouse).

(2. Culture process)
The culturing step is a step of culturing, in vitro, part or all of the tissue irradiated with plasma in the plasma irradiation step.

In the culturing step, it is preferable to culture, in vitro, part or all of the tissues that have undergone vigorous tissue regeneration after the plasma irradiation, among the tissues that have been irradiated with the plasma. The “tissue in which tissue regeneration was active after plasma irradiation” can be confirmed by evaluating an increase in cell proliferation activity. For example, increased cell proliferation activity can be assessed by immunohistochemical staining using, for example, the Ki67 antigen.

Culture conditions in the culture step are not particularly limited as long as the tissue irradiated with plasma in the plasma irradiation step can survive and proliferate, and appropriate culture conditions may be selected depending on the cells to be cultured. For example, when cerebral cortex as a tissue is irradiated with plasma, the tissue can be cultured under known culture conditions suitable for culturing neural stem cells.

The culturing step may be a step of obtaining neurospheres containing stem cell-like cells. Culture conditions for forming neurospheres are known (for example, references: Gil-Perotin S, Duran-Moreno M, Cebrian-Silla A, et al. Adult neural stem cells from the subventricular zone: a review of the neurosphere assay Anat Rec (Hoboken) 296:1435-1452, 2013).

It is preferable to obtain the "neurosphere" having a diameter of 200 micrometers or more. According to the method for preparing stem cell-like cells according to one embodiment of the present invention, neurospheres can be obtained in a shorter period of time than conventional methods. More specifically, in the conventional method of preparing neurospheres from tissue obtained from the perilateral ventricles, a culture period of about 3 weeks is required to obtain neurospheres with a diameter of 100 to 150 micrometers. rice field. In contrast, according to the method for preparing stem cell-like cells according to one embodiment of the present invention, it is possible to obtain neurospheres in 12 to 14 days. Plasma irradiation of tissue was thought to increase the growth rate of neurospheres. In other words, according to the method for preparing stem cell-like cells according to one embodiment of the present invention, neurospheres having a diameter of 150 micrometers or more can be obtained after one week from the start of suspension culture. Neurospheres with a diameter of 200 micrometers or more can be obtained two weeks after the start of suspension culture. It should be noted that "a diameter of 150 micrometers or more at the time when one week has passed since the start of suspension culture" or "a diameter of 200 micrometers or more at the time when two weeks have passed since the start of suspension culture" means any time during the suspension culture period The expression used to define the size of the neurosphere in the method for preparing stem cell-like cells according to the present invention, and limits the culture period to that period (specifically, 1 week or 2 weeks) not a thing Therefore, for example, the suspension culture of neurospheres having a diameter of 150 micrometers or more after one week from the start of the suspension culture is not limited.

The tissue culture period may be appropriately adjusted depending on the tissue to be cultured. After that, suspension culture is preferably carried out for 5 days or longer, and more preferably suspension culture is carried out for 7 to 10 days. Neurospheres containing stem cell-like cells can be obtained by suspension culture of cells collected from the cerebral cortex for the period described above. In addition, neurospheres obtained by floating culture of cells collected from the cerebral cortex for the period described above can be suitably induced to differentiate into nerve cells. According to the method for preparing stem cell-like cells according to one embodiment of the present invention, cells collected from the cerebral cortex are cultured in suspension for one week to obtain neurospheres with a diameter of 170±52 micrometers (SEM). After 2 weeks of suspension culture, neurospheres with a diameter of 279±142 micrometers (SEM) can be obtained.

In one embodiment, the culturing step may further include a step of inducing differentiation of stem cell-like cells. The method of inducing differentiation of stem cell-like cells to obtain desired differentiated cells is not particularly limited. Depending on the purpose, a known differentiation induction method (for example, references: Gil-Perotin S, Duran-Moreno M, Cebrian-Silla A, et al. Adult neural stem cells from the subventricular zone: a review of the neurosphere assay. Anat Rec (Hoboken) 296:1435-1452, 2013) can be adopted.

According to the method for preparing stem cell-like cells according to the present invention, it is possible to easily prepare stem cell-like cells from tissues. Therefore, according to the method for preparing stem cell-like cells according to the present invention, stem cell-like cells that can be used for preparation of cell preparations used in regenerative medicine, preparation of transplant materials such as myocardial sheets, regenerated cartilage, etc. can be easily prepared. It becomes possible to

In addition, according to the method for preparing stem cell-like cells according to one embodiment of the present invention, stem cell-like cells can be prepared from surgically accessible cerebral cortex, and in vitro, the stem cell-like cells Neurospheres comprising The neurospheres thus produced can be used for nerve regeneration and drug discovery medicine.

The present invention is not limited to the above-described embodiments, but can be modified in various ways within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. is also included in the technical scope of the present invention.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited by the examples.

[Example 1]
(experimental animal)
In this example, 8- to 10-week-old male SD rats were used.

(Atmospheric non-equilibrium plasma generator)
FIG. 1 is a diagram showing a schematic configuration of an atmospheric non-equilibrium plasma generator used in an embodiment of the present invention. The non-equilibrium atmospheric pressure plasma generator 1 includes a micro hollow electrode 11 , a cylindrical electrode 12 , a ceramic tube 13 as a cylindrical insulating member, a gas supply path 14 and an AC power source 15 .

A micro hollow in which minute recesses are repeatedly formed is formed at the tip of the micro hollow electrode 11 . The material of the micro hollow electrode 11 is stainless steel (SUS). The material of the cylindrical electrode 12 is copper.

The micro hollow electrode 11 is housed inside the ceramic tube 13 and arranged near the gas inlet of the ceramic tube 13 . On the other hand, the cylindrical electrode 12 is arranged outside the ceramic tube 13 and in the vicinity of the gas ejection port. Helium gas is introduced from the gas inlet of the ceramic tube 13 through the gas supply path 14, and a voltage is applied between the micro hollow electrode 11 and the cylindrical electrode 12 by the AC power supply 15. Plasma is generated inside the The thickness of the plasma jet stream irradiated from the tip of the ceramic tube 13 (that is, the tip of the head) is about 1 mm in diameter.

[Example 1-1]
(Atmospheric non-equilibrium plasma irradiation to the brain)
FIG. 2 is a diagram illustrating a method for irradiating rat brain tissue with atmospheric non-equilibrium plasma. Under isoflurane anesthesia, an incision was made in the scalp of an SD rat, and a hole with a diameter of about 1 mm was made with a dental drill at a position 2 mm posterior and ±2 mm lateral from the bregma of the skull (Fig. 2). Holes indicated by arrows A and B in (a)) were made to expose a portion of the surface of the cerebral cortex.

Plasma irradiation of brain tissue was performed by helium atmospheric non-equilibrium plasma. A plasma jet was generated from helium (He) gas using an atmospheric pressure plasma power supply control unit (manufactured by NU Global), and the tip of the plasma head was fixed at a position 2.5 cm above the exposed cerebral cortex surface. Plasma irradiation was performed for 60 seconds under the irradiation conditions (FIG. 2(b)). The helium gas flow rate was 2.5 L/min (2.5 slm).

(Irradiation conditions)
Discharge gas: Helium Gas flow rate: 2.5 L/min Electrode applied voltage (AC): 7 kV (60 Hz)
Discharge format: micro hollow discharge Plasma was irradiated to the holes indicated by arrow A in arrows A and B of FIG. 2(a). On the other hand, the hole indicated by the arrow B was used as a control without being irradiated with plasma. After plasma irradiation, the scalp was sutured, the affected area was disinfected, and then the rat was allowed to recover from anesthesia.

Under these irradiation conditions, a high-voltage helium gas discharge with an AC 60 Hz peak voltage of 7 kV is generated inside the head to form a plasma jet flow approximately 2.0 cm from the tip of the head while the gas temperature remains below 60°C. be done. Therefore, the light-emitting portion of the plasma jet does not come into contact with the irradiated affected area. Therefore, the atmosphere is involved in the transport diffusion process of the plasma jet over a distance of _2.5 cm from the tip of the head ^{, and} electrons in the plasma collide with oxygen molecules. is irradiated. Since the gas temperature rises to nearly 60° C. due to the temperature rise of the head depending on the irradiation time, the irradiation was limited to 60 seconds and the temperature of the affected area was kept at 37° C. or less. At this time, the voltage at the irradiation position on the plasma irradiation target tissue is 3 kV averaged over 20 ms, and the current at the irradiation position on the plasma irradiation target tissue is 0.1 mA averaged over 20 ms. Met.

In this plasma, it was estimated by the Stark method that an electron density of 2.2×10 ¹⁵ cm ⁻³ was generated inside the head. The oxygen atom density at an irradiation distance of 2.5 cm from the head to the affected area was estimated to reach 3.0×10 ¹³ cm ⁻³ by vacuum ultraviolet absorption spectroscopy (VUVAS). In addition, the Stark method uses a high-resolution (~ 40 pm) spectroscope to fit the Stark broadening width of the hydrogen atom β-ray, which can be seen at a wavelength of about 486 nm on the emission spectrum, with a Voigt function type, and the Lorentzian type was obtained from the analysis of the plasma density per cubic cm by 10 ¹⁶ (ΔS/0.946) ^1.5 as the expansion width ΔS. The Gaussian width is the linewidth determined by Doppler broadening and the like. Vacuum ultraviolet absorption spectroscopy (VUVAS) is a method for measuring the absorption of oxygen atoms at a wavelength of 130 nm in the vacuum ultraviolet region. An emission line of oxygen atoms emitted from a micro hollow cathode light source was passed through a magnesium fluoride window, and the absorption of oxygen atoms present in the plasma jet was measured with an absorption length of 2 mm in this experiment.

A water film covers the surface of the cerebral cortex exposed by incising the scalp of SD rats. Therefore, simulating this, pure water (MillQ water) is irradiated with plasma under the same conditions as described above, and OH radicals generated in water are measured by the spin trap method using the electron spin resonance method (ESR). bottom. Since .OH radicals generated in water have a short life, the .OH radicals were captured by a spin trapping agent and measured as long-lived nitrone (NO). As a result, it was confirmed that .OH radicals were generated. Therefore, plasma irradiation was considered to exert chemical exogenous stress on cells. The spin trapping agent is 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).

Using the apparatus shown in FIG. 3, the temperature change of water due to plasma irradiation was measured. While changing the irradiation distance, plasma was irradiated under the same conditions as described above to water that was shallowly put in a petri dish (to the extent that it was immersed), and the temperature change of the water was measured with a thermocouple (Fig. 3 (a) and ( b)). The irradiation distance is the distance from the tip of the head of the plasma generator to the water surface. The irradiation distance was 15 mm, 20 mm, 25 mm or 45 mm. As shown in FIG. 3(c), at 60 seconds after plasma irradiation, the temperature of water is highest at an irradiation distance of 2.0 cm (20 mm), followed by an irradiation distance of 1.5 cm (15 mm). And if the water temperature was high. On the other hand, as shown in (c) of FIG. 3, the irradiation distance of 2.5 cm (25 mm) (the second lowest water temperature graph at 60 seconds after plasma irradiation) and 4.5 cm (45 mm) (graph showing the lowest water temperature at 60 seconds after plasma irradiation), almost no change in water temperature was observed during plasma irradiation for 60 seconds. If the plasma irradiation raises the temperature of tissue, it is thought that the temperature rise phenomenon of water can also be captured in the irradiation of thermocouples immersed in water. At an irradiation distance of 2.5 cm (25 mm), almost no change in water temperature was observed during plasma irradiation for 60 seconds. It was thought that it would not bring about a temperature rise of

(Preparation of brain tissue section)
3 hours after plasma irradiation, and 1 day, 3 days, and 7 days after plasma irradiation, the plasma-irradiated rats were fixed by whole-body perfusion of 4% formaldehyde solution from the heart under pentobarbital anesthesia, respectively. did

After extracting the brain from the rat, the extracted brain was immersed in a 4% formaldehyde solution and post-fixed overnight at 4°C. After replacing the 4% formaldehyde solution with a 30% sucrose solution and submerging the brain, 50 μm-thick frozen tissue sections were prepared using a cryostat and subjected to immunostaining. The upper diagram of FIG. 4(a) shows the excised brain, and the lower diagram shows a cross section centered on the irradiation site. A region surrounded by a square in FIG. 4(a) represents an affected area irradiated with plasma.

(Staining of brain tissue piece)
Fluorescent immunohistochemical staining of brain tissue sections used Sox2 antibody (manufactured by Santacruz, model number sc-17320), Ki67 antibody (manufactured by Novocastra, model number Ki67p), and NG2 antibody (manufactured by millipore, model number MAB5384) as primary antibodies.

The results of fluorescence immunohistochemical staining are shown in FIGS. 4(b) and 4(c). Fluorescent immunohistochemical staining confirmed many Ki67-positive proliferating cells around the irradiated area in the brain tissue 3 days after plasma irradiation (the portion indicated by the arrowhead in FIG. 4(b)). In addition, 3 days after plasma irradiation, NG2, a marker for glial progenitor cells and pericyte, and Sox-2, a stem cell marker (ES cell and TS cell marker), were found in many positive cells around the irradiated area. Some of them were double-positive with NG2 (FIG. 4(c)). This suggests the possibility that these cells (Sox-2 and NG2 double positive cells) are involved in the formation of neurospheres.

(Formation of sphere)
The rat cerebral cortex surface was irradiated with plasma under the same conditions as described above at an irradiation distance of 2.5 cm for 60 seconds. One day, three days, and seven days after plasma irradiation, the rats were quickly decapitated under pentobarbital anesthesia, and the brain was removed. In the upper diagram of FIG. 5(a), the circled area is the plasma irradiation site.

The plasma-irradiated part of the brain was excised with forceps, immersed in DMEM/F12 (1:1) (manufactured by GIBCO), and finely divided into single cells using a syringe and injection needle (18G, 23G, 27G). crushed. Then, FGF-basic (manufactured by PEPROTEC, final concentration 20 ng/ml), hEGF (manufactured by GIBCO), N-2 supplement (manufactured by GIBCO, 100x) and 10% FBS (manufactured by Equitech-Bio, final concentration 10%). Cultivation was performed using DMEM/F12 (1:1) in a 6-well dish (manufactured by Thermofisher Scientific) coated with poly-D-lysine in a CO ₂ incubator at 37°C.

3 to 5 days after the start of the culture, the adhered and sufficiently proliferated cells were detached with trypsin-EDTA (manufactured by Sigma, final concentration 20%), and then FGF-basic (manufactured by PEPROTEC, final concentration 20 ng / ml), hEGF ( Using DMEM/F12 (1:1) (GIBCO) medium containing PEPROTEC, final concentration 20 ng/ml), N-2 supplement (GIBCO, used at 100x), non-coated 12-well multidishes (Thermofisher Scientific), suspension culture was performed for 1 to 4 weeks.

As controls, a similar experiment was performed using tissue collected from around the lateral ventricle (subventricular zone: SVZ) and cerebral cortex not irradiated with plasma. In the lower diagram of FIG. 5(a), the area surrounded by a square is the area containing the lateral ventricle. The hippocampal dentate gyrus and periventricular region are a small part of the brain where stem cells are densely present. be. FIG. 5(b) shows the culture schedule for each condition.

Fluorescent immunohistochemical staining was performed on some of the cells after adherent culture.

Sample 1: Plasma non-irradiated cerebral cortex Sample 2: Plasma irradiated for 1 minute, 3 days later cerebral cortex Sample 3: Plasma non-irradiated subventricular zone (SVZ)
Fluorescent immunohistochemical staining of adherent cells was performed using Sox2 antibody (manufactured by Santacruz, model number sc-17320), GFAP antibody (manufactured by Sigma, model number G9269), Nestin antibody (manufactured by Millipore, model number MAB353), and Ki67 antibody (manufactured by Novocastra) as primary antibodies. , model number Ki67p), Iba1 antibody (manufactured by Wako Pure Chemical Industries, model number 019-19741), and NG2 antibody (manufactured by Millipore, model number MAB5384) were used.

Sox2 is a marker for ES cells and TS cells, GFAP is a marker for astrocytes, Nestin is a marker for neural stem cells and neural progenitor cells, Ki67 is a marker for cell proliferation, Iba1 is a marker for microglia, and NG2 is a marker for immature glial cells and pericytes.

The results are shown in FIGS. 6 and 7. FIG. FIG. 6 shows the results of fluorescent immunohistochemical staining of cells after adhesion culture. Among the photographs shown in FIG. 6, the photograph at the top of each row represents the results of double staining with Sox2 and GFAP. Among the photographs shown in FIG. 6, the photograph at the bottom of each row represents the result of double staining with Nestin and Ki67. In addition, among the photographs shown in FIG. 6, the photographs in the middle of each row show the results of double staining with Iba1 and NG2. FIG. 7 is a diagram showing the results of observing cells under an optical microscope after 3 days after plasma irradiation, the cerebral cortex was adherently cultured for 5 to 7 days, and then suspension cultured for 3 days or 7 days (one week). be.

In the non-plasma-irradiated cerebral cortex (Sample 1), cells did not adhere and could not be cultured (Fig. 6). Similarly, cells did not adhere to the cerebral cortex one day after plasma irradiation, and culture could not be performed (data not shown). In the cerebral cortex 3 days after plasma irradiation (Sample 2), double-positive cells for Sox2 and GFAP were observed, and Nestin-positive cells and proliferating cells were also observed (Fig. 6). In addition, although cell adhesion occurred in the cerebral cortex 7 days after plasma irradiation, the number of adhering cells was very small compared to the cerebral cortex 3 days after plasma irradiation (data not shown). In the SVZ sample (sample 3), double-positive cells for Sox2 and GFAP were similarly observed, and many Nestin-positive cells and Ki67-positive cells were also observed (Fig. 6).

From this result, plasma treatment revealed cells that had acquired traits similar to those of neural stem cells, but unlike the SVZ sample, the proliferating cells did not overlap with Nestin-positive cells so much that they could not be regarded as neural stem cells. It was considered that different cells may also have appeared.

Further, as shown in FIG. 7(a), after three days of plasma irradiation, the cerebral cortex was cultured in a medium containing FBS for 5 to 7 days, and then suspended in a medium not containing FBS (3 days or 7 days). , the formation of spheres was confirmed. In the cerebral cortex 7 days after plasma irradiation, sphere formation took a long time during the suspension culture process (data not shown). From this result, it was concluded that the 3rd day of plasma irradiation is the number of days for obtaining spheres most efficiently.

In addition, neurospheres prepared from tissue collected from the periphery of the lateral ventricle had a diameter of 100 to 150 μm, whereas spheres prepared from plasma-irradiated tissue (referred to as “plasmaspheres”) were large ( 150 to 500 μm) (FIG. 7(b)). In addition, in conventional methods, only a small area of the brain where stem cells are densely present (dentate gyrus and periventricular region of the hippocampus) can be used to prepare neurospheres. According to the method of the invention, it is possible to prepare spheres from a wide cerebral cortex region, so it can be said that there is an advantage that a large amount of spheres can be obtained.

In addition, as shown in FIG. 5(b), the plasma irradiation significantly shortened the adhesion culture period (14 days in the SVZ was shortened to 5 days at the shortest). From this, it can be said that the plasma irradiation has the advantage that neurospheres can be obtained in a shorter period of time than in the past. This is considered to be one of the major features of the present invention.

(Preparation of sphere section)
A portion of the spheres cultured for the above period was collected with a pipette and fixed with a 4% formaldehyde solution. After replacing the 4% formaldehyde solution with 30% sucrose, 16 μm-thick frozen tissue sections were prepared using a cryostat and subjected to immunostaining.

Immunostaining of the sphere section was performed using the following primary antibodies: Sox2 antibody (manufactured by Santacruz, model number sc-17320), GFAP antibody (manufactured by Sigma, model number G9269), Nestin antibody (manufactured by Millipore, model number MAB353), Ki67 antibody (manufactured by Novocastra, model number Ki67p), Iba1 antibody (manufactured by Wako Pure Chemical Industries, model number 019-19741), NG2 antibody (manufactured by Millipore, model number MAB5384), Oct4 antibody (manufactured by Cell Signaling, model number 2840S), Nanog antibody (manufactured by R & D Systems, model number AF2729), CD11b An antibody (manufactured by Serotec, Model No. MCA275R) was used.

Oct4 is an ES cell and undifferentiated marker, Nanog is an ES cell and pluripotent stem cell marker, and CD11b is a macrophage marker.

The results are shown in FIG. Among the photographs shown in FIG. 9, the first photograph from the left in the upper row represents the results of double staining with Nestin and Ki67, and the second photograph from the left in the upper row shows the results of double staining with Sox2 and Ki67. The results are shown, and the third photograph from the left in the upper row shows the results of double staining with Sox2 and Nestin. In addition, among the photographs shown in FIG. 9 , the first photograph from the left in the lower row shows the results of double staining with Sox2 and Oct4, and the second photograph from the left in the lower row shows double staining with Nanog and Ki67. The results of staining are shown, the third photograph from the left in the lower row shows the results of double staining with Iba1 and NG2, and the fourth photograph from the left in the lower row shows the results of double staining with CD11b and Ki67. there is Nestin, Sox2, GFAP, and Oct4 positive cells were observed in the formed spheres, and among them, Oct4 and Sox2 double positive cells were present. Nanog-positive cells were not clearly visible in this staining. In addition, the center of the sphere was positive for NG2, and Iba1-positive cells were observed around the sphere. The presence of Oct4 and Sox2 double-positive cells was similar to their localization in brain tissue sections after plasma irradiation.

Cells with sphere-forming ability appeared by plasma treatment. In particular, the existence of Oct4-positive cells revealed that highly undifferentiated cells appeared due to plasma irradiation.

(Differentiation induction into neurons)
After one week of suspension culture in an FBS-free medium, differentiation induction test into neurons was performed. In a clean bench, the spheres in the wells were collected with the naked eye with a pipette and collected in a sample tube. A cover glass coated with poly-L-lysine was placed in the well of a non-coated 12-well dish (manufactured by Thermofisher Scientific), B-27 supplement (manufactured by Thermofisher Scientific, 50x), All-trans-retinoic acid (Wako Pure Chemical Industries, Ltd.) Neurobasal medium (registered trademark) supplemented with 0.2 μM (final concentration) was added, and 10-15 spheres were submerged in each well. After 7 days of culture in a CO ₂ incubator at 37° C., the culture medium was gently aspirated, and the spheres adhered to the cover glass were fixed with a 4% formaldehyde solution, followed by immunostaining.

Immunostaining of spheres after induction of differentiation into neurons was performed using a Pan Neuronal Marker (MAB2300 manufactured by Millipore).

The results are shown in FIG. FIG. 8 shows the results of immunostaining of spheres of the cerebral cortex 3 days after plasma irradiation, subjected to adhesion culture for 5 to 7 days, followed by suspension culture for 1 week, and subjected to a neuronal differentiation induction test. When the spheres cultured in suspension for one week were induced to differentiate into neurons, differentiation into neurons positive for Pan-Neuronal Markers was confirmed.

Based on the above results, we were able to prepare neurospheres from the cerebral cortex, which is a terminally differentiated tissue (which contains almost no undifferentiated stem cells). It was considered that stem cells or stem cell-like cells that acquired properties of progenitor cells were induced.

(Induction of differentiation into astrocytes and oligodendrocytes)
The sphere-forming cells obtained by the above-described plasma treatment were cultured in suspension in an FBS-free medium for one week, and then treated under the same conditions as in the test for inducing differentiation into neurons. Spheres differentiated into sites and spheres differentiated into oligodendrocytes were confirmed. These spheres were fixed with a 4% formaldehyde solution and immunostained in the same manner as in the differentiation induction test into neurons.

Immunostaining of spheres after induction of differentiation into astrocytes was performed with an anti-GFAP antibody (G9269 manufactured by SIGMA).

Immunostaining of spheres after induction of differentiation into oligodendrocytes was performed with an anti-O4 antibody (MAB345 manufactured by Millipore).

The results are shown in FIGS. 11(a) and (b). Figures 11 (a) and (b) show differentiation of the cerebral cortex 3 days after plasma irradiation into (a) astrocytes and (b) oligodendrocytes after suspension culture for 5 to 7 days followed by suspension culture for 1 week. FIG. 4 shows the results of immunostaining of spheres subjected to an induction test.

When the spheres suspended in suspension culture for one week were induced to differentiate into astrocytes, differentiation into GFAP-positive astrocytes was confirmed. Regarding the induction of differentiation into oligodendrocytes, differentiation into O4-positive oligodendrocytes could also be confirmed.

[Example 1-2]
(Plasma irradiation to skin)
Under isoflurane anesthesia, the hair on the back of the rat was shaved off with a hair clipper, and the skin surface was irradiated with plasma. As for the plasma irradiation conditions, the tip of the plasma head was fixed at a position 2.5 cm above the skin surface, and the irradiation time was 60 seconds, as in the plasma irradiation to the brain. The flow rate of helium gas was also set to 2.5 L/min as in the plasma irradiation to the brain. After plasma exposure, rats were allowed to recover from anesthesia.

(Preparation of skin tissue section)
Three days after the plasma irradiation, the rats were anesthetized with pentobarbital and fixed by whole-body perfusion of 4% formaldehyde solution from the heart. After removing the skin, it was immersed in a 4% formaldehyde solution and post-fixed overnight at 4°C. After substituting the 4% formaldehyde solution with a 30% sucrose solution and submerging the tissue, a 50 μm-thick floating tissue section was prepared using a microtome and subjected to immunostaining.

(Staining of skin tissue piece)
Fluorescent immunohistochemical staining of skin tissue sections was carried out using, as primary antibodies, Sox2 antibody (manufactured by Santacruz, model number sc-17320), Ki67 antibody (manufactured by Novocastra, model number Ki67p), Iba1 antibody (manufactured by Wako Pure Chemical Industries, model number 019-19741), An NG2 antibody (manufactured by millipore, model number MAB5384) was used. As a control, fluorescent immunohistochemical staining of a piece of skin tissue not irradiated with plasma was performed.

The results are shown in FIG. FIG. 10 shows the results of immunostaining of skin 3 days after plasma irradiation. Each photograph in FIG. 10 shows the results of double staining with NG2 and Ki67. In skin, dermis, and adipose tissue 3 days after plasma irradiation, proliferating cells with many Ki67-positive nuclei were confirmed. In addition, some of them expressed NG2, which is a young cell marker (glial progenitor cell marker or pericyte marker) whose expression was also confirmed after plasma irradiation in central nervous tissue.

[Example 2]
(experimental animal)
Eight-week-old C57BL/6 male mice were used in this example.

(Atmospheric non-equilibrium plasma generator)
An atmospheric non-equilibrium plasma generator similar to that of Example 1 was used.

(Atmospheric non-equilibrium plasma irradiation to the brain)
Under 1-1.5% isoflurane inhalation anesthesia, an incision was made in the scalp of the mouse and a dental drill was used at a position 1.5 mm posterior and 1.5 mm lateral from the bregma of the skull. A hole with a diameter of approximately 1 mm was made to expose a portion of the cerebral cortical surface.

Plasma irradiation of brain tissue was performed by helium atmospheric non-equilibrium plasma. A plasma jet was generated from helium (He) gas using an atmospheric pressure plasma power supply control unit (manufactured by NU Global), and the tip of the plasma head was fixed at a position 2.5 cm above the exposed cerebral cortex surface. Plasma irradiation was performed for 30 seconds under the irradiation conditions. The helium gas flow rate was 2.5 L/min (2.5 slm).

Using the same irradiation conditions as in Example 1, after plasma irradiation, the scalp was sutured, the affected area was disinfected, and then the mice were allowed to recover from anesthesia.

(Formation of sphere)
Three days after plasma irradiation, the mice were quickly decapitated under pentobarbital anesthesia, and the brain was excised.

In the same manner as in Example 1, the plasma-irradiated site of the brain was excised with tweezers, cultured in a CO ₂ incubator at 37° C., and the adhered and sufficiently proliferated cells were transferred to a non-coated 12-well multi-dish (manufactured by Thermofisher Scientific). Suspension culture was performed for 1 to 4 weeks at .

FIG. 12 shows the results of observing cells under a phase-contrast microscope after 3 days after plasma irradiation, the cerebral cortex was adherently cultured for 5 to 7 days, and then suspension cultured for 3, 5, or 7 days (one week). It is a figure which shows.

As shown in FIG. 12, the cerebral cortex 3 days after plasma irradiation was cultured in a medium containing FBS for 5 to 7 days, followed by suspension culture in a medium not containing FBS. . In addition, on day 7 of suspension culture, 15 spheres were randomly selected and the sphere size was measured on a microscopic image.

[Example 3]
(experimental animal)
In this example, 8- to 10-week-old male SD rats were used.

(Atmospheric non-equilibrium plasma generator)
An atmospheric non-equilibrium plasma generator similar to that of Example 1 was used.

(Atmospheric non-equilibrium plasma irradiation from above the skull)
Rat brain tissue was irradiated with plasma in the same manner as in Example 1, except that after incising the scalp of the mouse, the non-equilibrium atmospheric pressure plasma was irradiated from a position 2.5 cm above the skull without providing a through hole in the skull. did

Three days after plasma irradiation, in the same manner as in Example 1, the brain at the plasma-irradiated site was excised, adherent culture, suspension culture, and induction of differentiation into neurons, astrocytes, and oligodendrocytes were performed.

The results are shown in FIG. FIG. 13 shows the results of observation under a phase-contrast microscope of cells after adhesion culture, after suspension culture, and after induction of differentiation in the cerebral cortex three days after plasma irradiation.

As shown in FIG. 13, even when plasma irradiation was performed from above the skull without providing a through hole, formation of large spheres was confirmed as in the case of shaving the skull to expose the cerebral cortex. In addition, when the formed spheres were induced to differentiate into neurons, astrocytes and oligodendrocytes, their ability to differentiate into these cells was confirmed.

〔summary〕
The present invention can also be expressed as follows.

A method for preparing stem cell-like cells according to aspect 1 of the present invention is a method including a plasma irradiation step of irradiating tissue of an animal with non-equilibrium plasma.

The preparation method according to aspect 2 of the present invention, in aspect 1 above, may further include a culturing step of in vitro culturing part or all of the tissue irradiated with plasma in the plasma irradiation step. .

The preparation method according to aspect 3 of the present invention may be a method of obtaining neurospheres containing the stem cell-like cells in the culture step of aspect 2 above.

A preparation method according to aspect 4 of the present invention may be a method of obtaining neurospheres having a diameter of 200 micrometers or more in the above aspect 3.

The preparation method according to aspect 5 of the present invention may be any one of aspects 1 to 4 above, wherein the stem cell-like cells are cells positive for at least one of Sox2, Oct4 and NG2.

A preparation method according to aspect 6 of the present invention may be a method according to any one of aspects 1 to 5, wherein the plasma is atmospheric pressure non-equilibrium plasma.

A preparation method according to aspect 7 of the present invention is the preparation method according to any one of aspects 1 to 6, wherein in the plasma irradiation step, the plasma is applied at a predetermined distance between the plasma irradiation head and the tissue of the animal. A method of irradiating in a state may also be used.

A preparation method according to aspect 8 of the present invention may be a method according to any one of aspects 1 to 7, wherein the tissue of the animal is central nervous tissue.

The preparation method according to aspect 9 of the present invention may be a method according to any one of aspects 1 to 8, wherein the animal tissue is obtained by transplanting human tissue into non-human animal tissue.

The preparation method according to aspect 10 of the present invention may be a method in which, in any one of aspects 1 to 9, the animal is a non-human animal.

The present invention can provide a novel method for preparing stem cell-like cells. Stem cell-like cells prepared according to the present invention can be used for preparation of cell preparations used in regenerative medicine, preparation of transplant materials, and the like. Therefore, the present invention can be used in industries related to regenerative medicine.

1 Atmospheric non-equilibrium plasma generator 11 Micro hollow electrode 12 Electrode 13 Ceramic tube 14 Gas supply path 15 AC power supply

Claims (8)

A method for preparing stem cell-like cells positive for at least one of Sox2, Oct4 and NG2, which comprises a plasma irradiation step of irradiating nonequilibrium plasma to a non-human animal tissue or a tissue collected from the living body of an animal. There is
The preparation method, wherein the tissue is central nervous tissue or skin tissue. 2. The preparation method according to claim 1, further comprising a culturing step of ex vivo culturing part or all of the tissue irradiated with plasma in the plasma irradiation step. 3. The preparation method according to claim 2, wherein the tissue is a central nervous tissue, and neurospheres containing the stem cell-like cells are obtained in the culturing step. 4. The preparation method according to claim 3, wherein the neurospheres having a diameter of 200 micrometers or more are obtained. The preparation method according to any one of claims 1 to 4 , wherein the plasma is an atmospheric non-equilibrium plasma. 6. The preparation method according to any one of claims 1 to 5 , wherein in the plasma irradiation step, the plasma is irradiated with a predetermined gap between the plasma irradiation head and the tissue of the animal. 3. The preparation method according to claim 1 or 2, wherein the animal tissue is central nervous tissue. The preparation method according to any one of claims 1 to 7 , wherein the animal tissue is obtained by transplanting a human tissue into a non-human animal tissue.

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