JP6377929B2 - Stem cell culture apparatus and stem cell culture method - Google Patents

Description

  The present invention relates to a stem cell culture apparatus and a stem cell culture method, and more particularly to a stem cell culture apparatus and a stem cell culture method for activating stem cells by irradiating a low-power laser.

Conventionally, regenerative medicine has been known for the purpose of regenerating and restoring the cells, tissues, and organs of living bodies lost due to accidents and diseases.
For example, in bone tissue repair treatment after surgically removing trauma or bone tumor, autologous bones such as the iliac bones and ribs of the patient are collected and transplanted to the affected area. However, this method requires a surgical treatment of a healthy affected part in addition to the treatment of the affected part, which puts a double burden on the patient and increases the burden of medical expenses.

For this reason, recently, regenerative medical techniques that focus on stem cells that can be differentiated into various tissues and organs in a living body have attracted attention.
Regenerative medicine that focuses on stem cells is a revolutionary technique that regenerates lost tissues and organs by transplanting stem cells, and by direct differentiation into cells that make up the target tissue and the paracrine effect from transplanted stem cells. It is a treatment method (for example, Patent Document 1).

  In particular, mesenchymal stem cells are known as cells that can differentiate into various tissues and regenerate the tissues. Mesenchymal stem cells are present in bone marrow and subcutaneous fat, but subcutaneous fat can be collected most safely. In regenerative treatment in the medical and dental fields, a method using subcutaneous fat is considered useful. Subcutaneous fat can be collected by aspiration fat under normal anesthesia.

Japanese Patent No. 4649224

  However, tissue regeneration requires a large amount of stem cells, and it is necessary to culture and proliferate stem cells from the collected subcutaneous fat, but the proliferative ability of stem cells varies greatly depending on the condition of the collected patient, and surgery It was difficult to smoothly and efficiently prepare a necessary amount of stem cells at a necessary time.

In other words, the proliferation rate of stem cells varies greatly depending on the age, sex, presence or absence of the underlying disease, and there is a problem that there is no stable cell culture method.
On the other hand, in the clinical field, it is important to strictly observe the schedule management of the scheduled operation, and it is required that the necessary number of stem cells can be cultured and reliably prepared on the operation day.

  In addition, investing in highly specialized and strictly controlled facilities for stem cell culture, requiring a large amount of equipment and consumables, and investing highly skilled and skilled labor capital Don't be. For this reason, there is a barrier that the labor force and facility maintenance management man-hours to be input are increased, and there is a problem that the treatment cost to be borne by the patient increases.

  On the other hand, in the medical field, optical therapy represented by laser is not limited to incision and transpiration, but is expected to be applied to tissue regeneration treatment of the retina and skin. The outlook is unclear as to whether there is any.

  Therefore, in order to solve the above-mentioned problems, the present invention activates a stem cell existing in a tissue collected from a human or a non-human (animal) or a cell thereof, and can proliferate dramatically, a stem cell culture apparatus, It is another object of the present invention to provide a stem cell culture method.

In order to solve the above-mentioned problems, an invention according to claim 1 is directed to a culture medium for culturing stem cells, an irradiation energy with respect to the stem cells of more than 0 and 10 joules / cm ² or less , and a laser power density of 0.1 W / and a laser irradiation means for irradiating a carbon dioxide laser , which is a low-power laser of cm ² or less , and irradiating the low-power laser to activate the stem cells.

The stem cell culture apparatus according to the present invention activates a stem cell by irradiating the collected tissue or a stem cell existing in the cell with a laser having an irradiation energy of more than 0 and 10 joules / cm ² or less. It is possible to proliferate much more than the case.
In addition, the stem cell culture device according to the present invention can activate stem cells without damaging them by irradiating a low-power laser of, for example, about 1 watt or less.

  For this reason, since the stem cell culture apparatus according to the present invention can activate stem cells and increase the proliferation rate of stem cells, it efficiently operates equipment that has been invested to improve productivity and is required for equipment and consumables. Costs and labor costs can be reduced, and culture costs can be effectively suppressed.

Here, the laser power density is a laser output (W) irradiated per unit area (cm ² ) of the culture medium. By irradiating a low-power laser with a laser power density of 0.1 W / cm ² or less, stem cells can be activated without being damaged by heat.

  Since the carbon dioxide laser can perform patient-friendly treatment, particularly in the field of dentistry, it is highly reliable and widely used in gingival incisions and the like, so it can be easily set to a predetermined low output. It is easy to handle and can reduce capital investment and maintenance costs.

Further, it has been found through experiments that when the irradiation energy is set to 0.5 Joule / cm ² , the carbon dioxide laser has a more advantageous effect than the case where the diode laser is irradiated. It was confirmed that when stem cells were irradiated with a carbon dioxide laser, the stem cells were proliferated much more than when they were left untreated.

  In this way, the stem cell culture apparatus according to the present invention can proliferate stem cells effectively by simple means that is highly reliable and widely spread by irradiating the stem cells with a low-power carbon dioxide laser. Therefore, it is possible to reduce the labor force and capital investment to be input and suppress the treatment cost borne by the patient, thereby favorably contributing to the practical use of the stem cell culture apparatus.

The invention according to claim 2 is the stem cell culture apparatus according to claim 1 , wherein the laser irradiation means sets the irradiation energy of the low-power laser to 0.1 or more and 2.5 joules / cm ² or less. Irradiation, and the stem cells are allowed to proliferate.

According to the second aspect of the invention, the growth rate can be particularly improved by irradiating the low-power laser with the irradiation energy set to 0.1 or more and 2.5 joules / cm ² or less.

In general, the growth rate of stem cells varies greatly depending on the state of the collected patient, etc., but when the irradiation energy is set to 0.1 or more and 2.5 Joules / cm ² or less, it is compared with the case where the laser is not irradiated. Thus, it was confirmed by experiments that the growth rate was suppressed and the growth rate was improved.

  For this reason, the stem cell culturing apparatus according to the present invention can effectively proliferate stem cells in a short period of time to improve productivity, and more effectively suppress culture costs.

The invention according to claim 3 is the stem cell culture apparatus according to claim 1 or 2 , wherein the laser irradiation means defocuses the irradiation light of the low-power laser and irradiates the stem cell. It is characterized by comprising focus irradiation means.

According to the invention which concerns on Claim 3 , it can adjust and manage the irradiation energy of a laser appropriately by providing the defocusing irradiation means.

The invention according to claim 4 is the stem cell culturing apparatus according to claim 3 , wherein the defocus irradiation means includes a defocus diameter adjusting means for changing a diameter of a defocus irradiation region irradiated to the stem cells. It is characterized by that.

According to the invention which concerns on Claim 4 , the irradiation range of a laser can be suitably adapted to the culture medium which proliferates a stem cell by providing the defocus diameter adjustment means.

The invention according to claim 5 is the stem cell culturing device according to any one of claims 1 to 4 , wherein the number of cells for measuring the number of proliferating cells of the stem cells after being proliferated by the activation It has a measuring means.

According to the invention which concerns on Claim 5 , a required number of stem cells can be culture | cultivated and prepared according to a surgery scheduled day by measuring the number of the proliferation cells after proliferation by a cell number measurement means. For example, the degree of proliferation of stem cells can be determined by comparing the number of proliferating cells after irradiation with a low-power laser and the number of proliferating cells when left untreated.

The invention according to claim 6 is the stem cell culturing apparatus according to any one of claims 1 to 5 , wherein the medium containing means for containing the medium and having an opening, and a lid for covering the opening And a lid opening / closing device for opening and closing the lid.

According to the invention of claim 6 , by providing a lid opening / closing device that opens and closes the lid covering the opening, the lid is opened and proliferated only when a stem cell is inserted or a passage operation is performed. During the period, it is possible to prevent contamination so that impurities such as bacteria do not enter by closing the medium storage means (storage device base such as a petri dish or bag-shaped bag). For this reason, it is possible to contribute to the automation of the apparatus by simple means to improve productivity and to reduce costs such as labor costs and equipment maintenance costs.

Lid invention according to claim 7, which covers a stem cell culture device according to any one of claims 1 to 6, a medium containing means having an opening housing the medium, the opening And the lid of the medium containing means is made of a material that transmits the low-power laser.

According to the invention according to claim 7 , because the lid of the culture medium storage means is made of a material that transmits the low-power laser, it can irradiate the low-power laser while keeping the lid on. Contamination can be more effectively suppressed.

The invention according to claim 8 is the stem cell culturing apparatus according to any one of claims 1 to 6 , further comprising medium containing means for containing the medium and having an opening, the opening being downward The low-power laser is irradiated from below in a state of being directed to.

According to the invention of claim 8 , impurities such as bacteria can be obtained by a simple means of directing the opening downward by irradiating the low-power laser from below with the opening directed downward. Contamination can be suppressed by making it difficult to enter. For this reason, it is possible to contribute to automation, improve productivity, and reduce costs such as labor costs and equipment maintenance costs.

The invention according to claim 9 is the stem cell culture apparatus according to any one of claims 1 to 8 , further comprising a movable table for moving the culture medium.

According to the invention which concerns on Claim 9 , since the movable table which moves the said culture medium is provided, many culture media can be arrange | positioned on a movable table and the said low output laser can be irradiated sequentially. In addition, by moving the culture medium from the laser irradiation position by the movable table, it is possible to insert stem cells in a state where the culture medium has been moved from the position where the low-power laser is irradiated, or to perform processing such as passage processing. Therefore, stem cells can be efficiently cultured with improved operability and productivity.

The invention according to claim 10 is a low-power laser in which the irradiation energy exceeds 0 joule / cm ² and the laser power density is 0.1 W / cm ² or less with respect to the stem cells accommodated in the medium. A stem cell culture method comprising activating the stem cells by irradiation with a carbon dioxide laser .

The invention according to claim 11 is characterized in that the low-power laser is irradiated with an irradiation energy set to 0.1 or more and 2.5 joules / cm ² or less to proliferate the stem cells. 10. The stem cell culture method according to 10 .

The invention according to claim 12 is characterized in that the low-power laser irradiation light is defocused and irradiated to the entire culture medium. The stem cell culture according to any one of claims 10 and 11 Is the method.

According to a thirteenth aspect of the present invention, the medium is accommodated in a medium accommodating means having a lid made of a material that transmits the low-power laser, and the stem cell is irradiated with the low-power laser through the lid. The stem cell culturing method according to any one of claims 10 to 12 , wherein the method is a stem cell culture method.

The invention according to claim 14 is characterized in that the medium is accommodated in a medium accommodating means having an opening, and the low-power laser is irradiated from below in a state where the opening is directed downward. The stem cell culture method according to any one of Items 10 to 13 .

The invention according to claim 15 is characterized in that the stem cell is activated by irradiating the low-power laser, and then the stem cell is allowed to proliferate to a target proliferation number by providing a predetermined rest period. The stem cell culturing method according to any one of claims 10 to 14 .

  The present invention provides a stem cell culturing apparatus and a stem cell culturing method capable of activating stem cells present in tissues or cells collected from humans and non-humans (animals) and dramatically expanding them.

It is a figure which shows the whole structure of the stem cell culture apparatus which concerns on embodiment of this invention. It is a graph which shows operation | movement of the proliferation cell control means in the stem cell culture apparatus which concerns on embodiment of this invention. The operation of the stem cell culture device according to the embodiment of the present invention is shown, (a) opening and closing the lid and irradiating the laser, (b) irradiating the laser from the bottom with the petri dish opening, (C) is a figure which shows a mode that a laser is irradiated with the cover of the material which permeate | transmits a laser. It is a graph which shows the result of the animal experiment for confirming the effect of the stem cell culture method which concerns on embodiment of this invention, and is a case where irradiation energy shall be 0.5-10J / cm < ² >. 5 is a comparison table in which the results of FIG. 4 are arranged. It is a graph which shows the result of the animal experiment for confirming the effect of the stem cell culture method which concerns on embodiment of this invention, and is a case where irradiation energy is 0.1-1 J / cm < ² >. 7 is a comparison table in which the results of FIG. 6 are arranged.

A stem cell culture apparatus 1 according to an embodiment of the present invention will be described in detail with reference to the drawings as appropriate.
The stem cell culture apparatus 1 is an apparatus for culturing and proliferating stem cells K collected from a human body or an animal, etc. As shown in FIG. 1, a medium 2 for culturing the stem cells K and a low-power laser for the stem cells K And a laser irradiation means 3 for irradiating a carbon dioxide gas laser L (hereinafter simply referred to as “laser L” unless otherwise specified), and the number of proliferating cells of the stem cell K after being proliferated by activation is measured. The cell number measuring means 4, the movable table 5 for moving the culture medium 2, the temperature adjusting means 11 for keeping the environment of the culture medium 2 in a predetermined temperature range (about 37 degrees C), and the environment of the culture medium 2 in a predetermined gas (5 Gas exchange means 12 for filling with air containing carbon dioxide gas), air purifying means 13 for removing air impurities, air pressure adjusting means 14, and light irradiation for promoting proliferation by irradiating stem cells K with light. Means 15; It includes a humidity adjusting unit 16 to keep the humidity range of the constant, the.

The laser irradiation means 3 is an apparatus that irradiates the stem cells K seeded in the culture medium 2 with the laser L having an appropriate irradiation energy at an appropriate time, and is 10 joules (J) / cm ² or less for the stem cells K. The laser L controlled to an appropriate irradiation energy is irradiated.
The laser L is controlled to a low output of about 1 watt or less so as not to damage the stem cell K.

  The laser irradiation unit 3 defocuses the laser L emitted from the laser oscillator 31, the mirror 31 a that reflects the laser L in a predetermined direction, and the laser L emitted from the laser oscillator 31 and irradiates the medium 2. Irradiation means 32, proliferation cell control means 33 for controlling the irradiation energy, irradiation time, and irradiation timing of laser L, and program input means 34 for inputting an operation program to proliferation cell control means 33 are provided.

The laser oscillator 31 is a device that generates a carbon dioxide laser L.
The carbon dioxide laser L oscillates around wavelengths of 10.6 μm, 9.3 μm, and 9.6 μm, and can be used with high output and high efficiency. In the present invention, the low output of about 0.5 W to 1 W is used. Used in.

In the present embodiment, the carbon dioxide laser L is used. However, the carbon dioxide laser L is not limited to this, and a He—Ne laser, Ar ion can be used as long as it can be controlled to an appropriate irradiation energy of 10 J / cm ² or less. Other types of lasers such as lasers, semiconductor lasers, Nd: YAG lasers, Ho: YAG lasers, Er: YAG lasers can also be used.

The defocusing irradiation means 32 defocuses the oscillated laser L and irradiates the stem cell K with the laser L at an appropriate laser power density (W / cm ² ) and irradiation energy (joule / cm ² ). A defocusing lens 32a and a moving device 32b for moving the defocusing lens 32a in the axial direction.
The laser power density is a laser output (W) irradiated per unit area (cm ² ) of the culture medium.

  With this configuration, when the defocusing lens 32a is moved upward by the moving device 32b, the defocusing irradiation unit 32 expands the irradiation range of the laser L and decreases the laser power density (see the laser L1 displayed by a one-dot difference line). When the defocus lens 32a is moved downward, the irradiation range of the laser L can be reduced and the laser power density can be increased (see the laser L2 indicated by a two-dot difference line).

Specifically, the defocus irradiation unit 32 irradiates the stem cell K with the laser L so that the laser power density is 0.1 W / cm ² or less. For example, when the diameter of the culture medium 2 (the petri dish 21) is 35 mm and the entire culture medium 2 is irradiated with the laser L having a laser output of 1 W, the laser power density is 1 (W) / (π × 1.75 × 1.75). ) ≈0.1 (W / cm ² ).
By irradiating the stem cell K with a low-power laser L having a laser power density of 0.1 W / cm ² or less, the stem cell can be activated without being damaged by heat. The irradiation energy (joule / cm ² ) can be adjusted by the length of the laser L irradiation time.

For example, as shown in FIG. 2, the proliferation cell control means 33 performs the first laser irradiation after seeding the stem cells K on the culture medium 2, for example, 0.1 J The proliferation rate of the stem cell K can be controlled by appropriately executing, for example, a second laser irradiation after irradiating a / cm ² laser and passing a rest period of 10 days.

  The program input means 34 is an input device for inputting a program executed by the proliferation cell control means 33.

  The cell number measuring means 4 is an apparatus for measuring the number of proliferating cells of the stem cell K after being irradiated with the laser L and proliferated by activation. For example, the cell number measuring means 4 is measured using a reagent called trypan blue and a hemocytometer. Can do.

  The movable table 5 is a device that allows the culture medium 2 to be moved in a state where a large number of culture media 2 are placed. The turntable system rotates the table 51 by arranging a large number of culture media 2 on the table 51 in the circumferential direction. A reciprocating table system in which the table 51 is reciprocated while being guided by a linear movement guide, an XY moving table system in which the table 51 is moved in the horizontal front-rear direction, the left-right direction, and the like can be appropriately applied.

  With this configuration, a large number of culture media 2 can be arranged on the movable table 5 and sequentially irradiated with the laser L. Further, by moving the culture medium 2 from the laser irradiation position by the movable table 5, the stem cell K is introduced in a state where the culture medium 2 is moved from the position where the laser L is irradiated, or processing such as subculture operation is performed. Therefore, stem cells K can be efficiently cultured with improved operability and productivity.

As shown in FIG. 3A, the culture medium 2 is stored in a petri dish 21 serving as a culture medium storage means so that impurities such as bacteria do not enter. The petri dish 21 has an opening 22 for introducing the stem cells K and performing passage operations, and includes a lid 23 that covers the opening 22. The lid 23 can be freely opened and closed by a lid opening / closing device 24.
In addition, a culture medium accommodation means is not limited to the petri dish 21, A bag-like (bag) accommodation base material may be sufficient, and various accommodation instrument base materials are employable.

  With this configuration, the lid 23 is opened only when the stem cells K are introduced or passage processing is performed, and the petri dish 21 is closed during the growth period to prevent contamination such as bacteria from entering. can do. For this reason, it is possible to contribute to the automation of the apparatus by simple means to improve productivity and to reduce costs such as labor costs and equipment maintenance costs.

In the present embodiment, the lid opening / closing device 24 is provided and the opening / closing operation is performed. However, the present invention is not limited to this, and another method for irradiating the medium 2 accommodated in the petri dish 21 with the laser L will be described. To do.
FIG. 3B shows a method of irradiating the laser L from below with the opening 22 of the petri dish 21 facing down. FIG. 3C shows a method of irradiating the laser L with the lid 25 being applied by applying the lid 25 made of a material that transmits the laser L.
Examples of the material that transmits the carbon dioxide laser L include zinc selenide (ZnSe), polyethylene PE), and diamond (C).

Then, the effect of the stem cell culture method using the stem cell culture apparatus 1 which concerns on embodiment of this invention is demonstrated, referring FIGS. 4-7. 4 and 5 are a graph and a comparison table showing the results of animal experiments for confirming the effects of the stem cell culture method according to the embodiment of the present invention, and the irradiation energy is 0.5 to 10 J / cm ² . Is the case. 6 and 7 are a graph and a comparison table when the irradiation energy is 0.1 to 1 J / cm ² .

In FIG. 4, stem cells K to be cultured (see FIG. 1) are those obtained by collecting a stromal cell population from the groin adipose tissue of an experimental animal (F344 rat). An experiment was conducted in which the collected stem cells K (rat adipose tissue-derived stem cells) were cultured in a basic medium (DMEM / 10% FBS).
30000 ASCs (Adipose-derived stem cells) after the 3rd passage were seeded in a circular culture dish (dish) having a diameter of 35 mm to start the culture (day: -2).

Two days after the start of the culture (day: 0), the carbon dioxide laser L having a predetermined irradiation energy is irradiated once, and the number of cells is counted 4 days (day: 4) and 7 days (day: 7) after the irradiation date. The cell proliferation number was measured by means 4 (see FIG. 1).
The irradiation energy of the irradiated carbon dioxide laser L was adjusted by the laser oscillator 31 and the defocus irradiation means 32 (see FIG. 1) as follows.

Sample 1 was not irradiated with laser L (indicated as Control). In Sample 2, the output of the laser L was 0.5 W, and the irradiation energy was 0.5 J / cm ² (simplified and displayed as 0.5 J / 0.5 W). Sample 3 had an output of 1 W and an irradiation energy of 0.5 J / cm ² (shown as 0.5 J / 1 W). Sample 4 had an output of 0.5 W and an irradiation energy of 2.5 J / cm ² (displayed as 2.5 J / 0.5 W). Sample 5 had an output of 0.5 W and an irradiation energy of 5 J / cm ² (displayed as 5 J / 0.5 W). Sample 6 had an output of 1 W and an irradiation energy of 10 J / cm ² (displayed as 10 J / 1 W). Sample 7 had an output of 0.5 W and an irradiation energy of 10 J / cm ² (displayed as 10 J / 0.5 W).
When the petri dish has a diameter of 35 mm, the laser power density is about 0.05 (W / cm ² ) when the output is 0.5 W, and about 0.1 (W / cm ² ) when the output is 1 W. ).

  Although the growth rate varies from individual to individual, in this experiment, it increased slightly from 30000 to 34000 (day: 0) in 2 days after seeding of the stem cells K (day: -2). In the sample 1 that was not irradiated with the laser L, up to 200,000 cells grew relatively smoothly in the next 4 days (day: 4), but after that, the growth rate decreased and 24 in the next 3 days. It has grown to 10,000 (day: 7).

As shown in FIG. 4, when the growth rate of the stem cell K is considered after 7 days from the irradiation of the laser L, the growth rate of the sample 3 and the sample 2 is the highest. As shown in FIG. The growth rate is 157% and 152% with respect to the sample 1 which is not irradiated with L. Sample 4 also has a suitable growth rate of 139%.
That is, when the irradiation energy in the range of 0.5~10J / cm ^2, although both can be expected to improve the growth rate, most growth rate is high, especially when the 0.5J / cm ^{2, 2.} Even at 5 J / cm ² , the growth rate can be preferably improved.

As shown in FIG. 4, sample 2 and sample 3 have the same irradiation energy (0.5 J / cm ² ), different outputs, sample 2 is 0.5 W, and sample 3 is 1 W. It can be seen that the difference in output has no significant effect. The same tendency is observed when 0.5 W of sample 6 and 1 W of sample 7 are used.

Next, the case where the irradiation energy is set to 0.1 to 1 J / cm ² will be described with reference to FIG.
In FIG. 6, 100000 ASCs (Adipose-derived stem cells) after the 3rd to 4th passages were seeded in a circular culture dish (dish) having a diameter of 100 mm and the culture was started (day: -2).

  Two days after seeding with the stem cells K (day: -2) (day: 0), the carbon dioxide laser L having a predetermined irradiation energy is irradiated once, 5 days after the irradiation day (day: 5), 10 days later ( Day: 10), and 15 days later (day: 15), the number of cell proliferation was counted by the cell number counting means 4 (see FIG. 1). Moreover, since the output has the same tendency at 0.5 W and 1 W, the output is all 0.5 W in FIG.

The sample 11 was not irradiated with the laser L (displayed as Control). Sample 12 had an irradiation energy of 1 J / cm ² (shown as 0.1 J / 0.5 W). Sample 13 had an irradiation energy of 0.5 J / cm ² (indicated as 0.5 J / 0.5 W). Sample 4 had an irradiation energy of 1 J / cm ² (indicated as 1 J / 0.5 W).
Sample 15 is obtained by irradiating a diode laser with a wavelength of 900 nm at 0.5 J / cm ² for comparison with the carbon dioxide laser L of Samples 12 to 14 (indicated as Diode).
When the petri dish has a diameter of 100 mm, the laser power density is about 0.006 (W / cm ² ) when the output is 0.5 W.

  As shown in FIG. 6, although there are individual differences in the growth rate, the tendency of the overall growth rate in this experiment is that the growth rate is relatively high for 5 days after irradiation with the laser L, and thereafter 10%. The growth rate increased substantially until the day, and after 10 days, the growth rate increased again until the 15th day.

Considering the growth rate of the stem cell K after 5 days from the irradiation of the carbon dioxide laser L, the growth rate of the sample 13 and the sample 12 is the highest. As shown in FIG. The growth rate is 200% and 188%.
Further, even after 15 days from the irradiation of the carbon dioxide laser L, the growth rate of the sample 13 and the sample 12 is the highest, and the growth rates of the sample 11 not irradiated with the laser L are 164% and 143%, respectively. It is.

In the diode laser of sample 15, when a diode laser with a wavelength of 900 nm is irradiated at 0.5 J / cm ² , as shown in FIG. The number decreased to 79%, and the growth rate was not improved.
Further, when the irradiation energy was set to 0.5 Joule / cm ² , it was recognized that the carbon dioxide laser had a more advantageous effect than the case where the diode laser was irradiated. It was confirmed that when stem cells were irradiated with a carbon dioxide laser, the stem cells were proliferated much more than when they were left untreated.

From the above, it employs a carbon dioxide gas laser L, when the irradiation energy in the range of 0.5~10J / cm ^2, the irradiation energy, than 5 J / cm ² and 2.5J / cm ² 0.5J / The growth rate is higher when cm ² . That is, the smaller the irradiation energy, the higher the proliferation rate.
Further, the irradiation energy in the case of the range of 0.1~1J / cm ² is the growth rate at 0.5 J / cm ² is the highest, the 0.5 J / cm ² even 0.1 J / cm ² The improvement of the proliferation rate which is excellent similarly to time is recognized.
Accordingly, in these experiments, data with an irradiation energy of less than 0.1 J / cm ² has not been acquired (see FIG. 6 and FIG. 7), but if the results of the experiments in FIGS. It is presumed that a constant increase in growth rate can be expected even at less than 0.1 J / cm ² .

Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and can be implemented with appropriate modifications.
For example, in the present embodiment, the gas exchange means 12, the air cleaning means 13, the atmospheric pressure adjustment means 14, the light irradiation means 15, and the humidity adjustment means 16 are provided. Depending on the type, etc., they are used in appropriate combinations.

DESCRIPTION OF SYMBOLS 1 Stem cell culture apparatus 2 Medium 3 Laser irradiation means 4 Cell number measurement means 5 Movable table 11 Temperature adjustment means 12 Gas exchange means 13 Air cleaning means 14 Atmospheric pressure adjustment means 15 Light irradiation means 16 Humidity adjustment means 21 Petri dish (medium accommodation means)
DESCRIPTION OF SYMBOLS 22 Opening part 23,25 Lid 24 Lid opening / closing device 31 Laser oscillator 32 Defocusing irradiation means 33 Proliferation cell control means 34 Program input means K Stem cell L Carbon dioxide laser (laser)
L1, L2 laser

Claims (15)

A medium for culturing stem cells;
A laser irradiation means for irradiating the stem cell with a carbon dioxide laser that is a low-power laser having an irradiation energy exceeding 0 and not more than 10 joules / cm ² and a laser power density of not more than 0.1 W / cm ^2. ,
A stem cell culture apparatus, wherein the stem cells are activated by irradiating the low-power laser. The laser irradiation means is configured to irradiate the low-power laser with an irradiation energy set to 0.1 or more and 2.5 joules / cm ² or less, and to proliferate the stem cells;
The stem cell culture apparatus according to claim 1 . The laser irradiation means includes defocus irradiation means for defocusing the irradiation light of the low-power laser to irradiate the stem cells;
The stem cell culture apparatus according to claim 1 or 2 , wherein The defocusing irradiation means includes a defocusing diameter adjusting means for changing a diameter of a defocusing irradiation region irradiated to the stem cells;
The stem cell culture apparatus according to claim 3 . Comprising cell number measuring means for measuring the number of proliferating cells of the stem cells after being proliferated by the activation,
The stem cell culture apparatus according to any one of claims 1 to 4 , wherein: Medium containing means for containing the medium and having an opening;
A lid covering the opening;
A lid opening and closing device for opening and closing the lid;
The stem cell culture device according to any one of claims 1 to 5 , comprising: Medium containing means for containing the medium and having an opening;
A lid that covers the opening,
The lid of the medium containing means is made of a material that transmits the low-power laser,
The stem cell culture apparatus according to any one of claims 1 to 6 , wherein: A medium containing means for containing the medium and having an opening;
Irradiating the low-power laser from below with the opening directed downward;
The stem cell culture apparatus according to any one of claims 1 to 6 , wherein: Having a movable table for moving the medium;
The stem cell culture apparatus according to any one of claims 1 to 8 , wherein: The stem cells are irradiated with a carbon dioxide laser , which is a low-power laser whose irradiation energy exceeds 0 and is 10 joules / cm ² or less and whose laser power density is 0.1 W / cm ² or less. A method for culturing stem cells, which comprises activating the cells. The low-power laser is irradiated with an irradiation energy set to 0.1 or more and 2.5 joules / cm ² or less to proliferate the stem cells,
The stem cell culture method according to claim 10 . Irradiating the entire medium by defocusing the irradiation light of the low-power laser,
The stem cell culturing method according to claim 10 or 11 , wherein: Storing the medium in a medium containing means having a lid made of a material that transmits the low-power laser, irradiating the stem cell with the low-power laser through the lid,
The stem cell culturing method according to any one of claims 10 to 12 , wherein: Storing the medium in a medium storing means having an opening,
Irradiating the low-power laser from below with the opening directed downward;
The stem cell culturing method according to any one of claims 10 to 13 , wherein: Irradiating the stem cell with the low-power laser and activating the stem cell, and then proliferating to the target proliferation number by providing a predetermined rest period for the stem cell,
The stem cell culturing method according to any one of claims 10 to 14 , wherein:

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