JP2018033320A - Determination system and determination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a determination system and a determination method that can simply and reliably determine that a culture solution used for culturing cells is "a culture medium not including serum" or "a culture medium including serum".SOLUTION: The determination system comprises: a storage device (1) for storing a culture solution for which it is to be determined whether serum is included or not; an interferon γ supply device (2); a blending device (3); a cell supply device (4) which supplies cells to be cultured with the culture solution; a culture vessel (5) for storing the culture solution provided with the cells so as to be left standing in a constant environment; an analyzer (6) for analyzing fluorescence intensity of the cultured cells; and a determination device (7) for analyzing analysis results. The determination device (7) has a mechanism for determining whether or not human type 2 antigen presenting molecule is expressed, a mechanism for determining that serum is not included in the culture solution if not expressed, and a mechanism for determining that serum is included in the culture solution if expressed.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a culture solution (or medium) used for culturing cells (eg, from fat). More specifically, the present invention relates to a technique for determining whether serum is included in a culture solution.

In recent years, it has been widely known that treatments for administering stem cells to various diseases are effective. At that time, it is necessary to culture the stem cells safely and efficiently. And in fields other than stem cell administration, there is a need to culture cells safely and efficiently.
When culturing cells (for example, adipose-derived mesenchymal stem cells), the cells to be cultured are put into a culture solution and stored in a predetermined environment (for example, 37 ° C., 5% CO ₂ environment). Generally done.

Here, when the above-mentioned culture solution contains serum (so-called “serum medium”), the heterologous antigen is taken up by the cells to be cultured in the culture solution, and the immune reaction occurs when administered. There is a risk of waking up.
There is also the risk of infection from pathogens in the serum. Furthermore, serum is known to have different components depending on the lot, and when culturing with serum, the problem is that the culture process is heterogeneous.
In addition, since the characteristics of cells cultured in a culture medium containing serum depend on the serum mixed in the culture medium, there is a problem that the range of characteristics of the cultured cells is widened.

In order to eliminate the above-mentioned disadvantages, it is preferable to use a culture solution that does not contain serum (so-called “serum-free medium”).
However, among the currently marketed culture solutions, there are cases where a relatively low serum content is sold as “low serum medium” or the like. Therefore, for those who purchase cell culture media for culturing cells, such as medical institutions and research institutions (users), it is a safe and stable culture solution (a culture solution that does not include serum) In many cases, it is difficult to determine whether the culture medium is low in stability or stability (culture medium including serum).
In particular, when cells are cultured in the medical field (eg, administration of stem cells), there is a strong demand to use a “culture medium that does not include serum”, which is highly safe and stable. Nevertheless, no technology has been proposed at present to easily and reliably determine whether a culture solution does not contain serum.

As another conventional technique, a technique for detecting the amount of nucleic acid in a sample using polymerase chain reaction (PCR) has been proposed (for example, see Patent Document 1).
However, the related art (Patent Document 1) does not aim to determine whether or not the culture solution contains serum.

International Publication No. 2006/10269

  The present invention has been proposed in view of the above-mentioned problems of the prior art, and the culture solution (medium) used for cell culture is a “culture solution not containing serum” or “culture containing serum”. It is an object of the present invention to provide a determination system and a determination method that can easily and reliably determine whether the liquid is “liquid”.

The determination system (100) of the present invention includes:
A storage device (1: container) for storing a culture solution to be judged whether or not it contains serum;
An interferon γ supply device (2: an interferon γ dropping unit) that supplies interferon γ to the culture solution stored in the storage device (1);
A mixing device (3) for uniformly mixing the culture solution in the storage device (1) and interferon γ,
A cell supply device (4: cell supply unit or pipette, etc.) for supplying cells to be cultured in a culture solution (culture solution mixed with interferon γ in a mixing device);
A culture container (5: constant temperature unit) for storing a storage device (1) for storing a culture solution supplied with cells to be cultured in a constant environment (for example, in a 37 ° C., 5% CO ₂ environment). )When,
An analyzer (6: for example, a flow cytometer or an imaging cytometer) for analyzing the fluorescence intensity in the cultured cells;
A determination device (7: determination unit: for example, an information processing device having a calculation function such as a PC) that analyzes the result of the analysis device;
The judging device (7) has a function of determining whether or not a human type 2 antigen-presenting molecule (for example, HLA-DR) is expressed, and if the human type 2 antigen-presenting molecule is not expressed, It is characterized by having a function of determining that serum is not included and a function of determining that serum is included in the culture medium when a human type 2 antigen-presenting molecule is expressed.

  In the determination system (100) of the present invention, a part of the cultured cell is recognized between the culture vessel (5) and the analyzer (6) to recognize a fluorescently labeled human type 2 antigen-presenting molecule. An antibody processing unit (11) having a function of treating with a negative control antibody (negative control antibody) which treats a part of cultured cells with an antibody (for example, anti-HLA-DR antibody) and does not recognize any molecule Is preferably arranged.

Moreover, the determination method of the present invention includes:
Storing a culture solution to be determined whether or not it contains serum in a storage device (1: container);
Supplying interferon γ to the culture solution stored in the storage device (1) by the interferon γ supply device (2: interferon γ dropping unit);
A step of uniformly mixing the culture solution in the storage device (1) and the interferon γ by the mixing device (3);
Supplying cells to be cultured in a culture solution (culture solution mixed with interferon γ in a mixing device) by a cell supply device (4: cell supply unit: pipette, etc.);
In the culture vessel (5: constant environment unit), the storage device (1) that stores the culture solution supplied with the cells to be cultured is left standing in a constant environment (for example, in a 37 ° C., 5% CO ₂ environment). And culturing the cells,
Determining whether a human type 2 antigen-presenting molecule (eg, HLA-DR) is expressed in the cultured cells;
Determining that serum is not included in the culture medium when the human type 2 antigen-presenting molecule is not expressed;
It is characterized by having a step of determining that serum is included in the culture medium when a human type 2 antigen-presenting molecule is expressed.

In the determination system (100) and the determination method of the present invention, after interferon γ is added, the time for culturing the cells (the time for culturing the cells in the culture vessel 5) is 12 hours or more (in the cell culturing step, the cells Is preferably 12 hours or longer), and particularly preferably 48 hours or longer.
In the determination system (100) and determination method of the present invention, the concentration of interferon γ is 100 units / ml or more, and preferably 400 units / ml or more.

In the determination method of the present invention, in the step of determining whether or not a human type 2 antigen-presenting molecule (for example, HLA-DR) is expressed in the cultured cells, a part of the cells cultured in the culturing step Is treated with an antibody that recognizes a fluorescently labeled human type 2 antigen-presenting molecule (for example, an anti-HLA-DR antibody), and a part thereof is treated with a negative control antibody (a negative control antibody) that does not recognize any molecule. It is preferable to have a process (antibody treatment process).
The cells of the present invention are cultured using a culture solution that does not include serum, and human type 2 antigen-presenting molecules (eg, HLA-DR) are not expressed even when interferon γ is added.

According to the present invention having the above-described configuration, after interferon γ is added and, for example, fat-derived mesenchymal stem cells are cultured, human type 2 antigen presentation in the stem cells (for example, fat-derived mesenchymal stem cells) Whether or not serum is contained in the culture solution can be determined by measuring whether or not the molecule HLA-DR is expressed.
In other words, in the present invention, when serum is included in the culture solution, when interferon γ is added to the culture solution and cells (for example, fat-derived mesenchymal stem cells) are cultured, Since HLA-DR, which is a type of antigen-presenting molecule, is expressed, as a result of culturing the cell, if a type 2 antigen-presenting molecule (for example, HLA-DR) is expressed, it is determined that serum is included in the culture solution If the type 2 antigen-presenting molecule (for example, HLA-DR) is not expressed, it is determined that serum is not included in the culture medium.
Therefore, according to the present invention, it is possible to easily and reliably determine whether or not a culture solution for culturing cells includes serum as a component.

As described above, according to the present invention, whether or not serum is contained in the culture medium can be easily and reliably determined. Therefore, by determining the culture medium according to the present invention, the culture medium is cultured in the culture medium. It is possible to discriminate whether the cells (for example, adipose-derived mesenchymal stem cells) are cells cultured in the presence of serum.
In stem cell administration therapy, cells cultured in the presence of serum are at high risk of being contaminated with infections, etc., so patients treated with such stem cells are more likely to suffer from infections, etc. It is. By discriminating whether or not the cells are cultured in the presence of serum according to the present invention, it is possible to administer stem cells with a low risk of developing an infectious disease, etc., to further improve the safety of stem cell administration I can do it.

Furthermore, since the above-mentioned culture broth does not contain serum (because it is a so-called “serum-free medium”), the cells to be cultured in the culture broth will not be taken up with foreign antigens and administered to patients. In some cases there is no risk of an immune response.
Furthermore, since the cells are cultured in a culture solution that does not contain serum, the characteristics of the cultured cells do not depend on the serum, and the range of cell characteristics is wider, unlike in the case of a culture solution that includes serum. Is prevented.

In addition, according to the present invention, since it is possible to determine whether human serum and / or animal (eg, bovine) serum is mixed with the culture medium, the culture medium using bovine serum is identified. Unlike the prior art (technique for measuring Neu5Gc), it can also be determined whether or not the cells are cultured using human serum.
Furthermore, according to the present invention, when interferon γ is added in a culture solution containing serum to culture stem cells (for example, adipose-derived mesenchymal stem cells), type 2 antigen-presenting molecules may be expressed in the cultured cells. It was revealed.
In addition, since the cells of the present invention are cultured in a culture solution that does not contain serum, foreign antigens are not taken up and there is no possibility of causing an immune reaction when administered. In addition, there is no infection caused by pathogens mixed in serum. Furthermore, the culture process is homogeneous and the range of characteristics of the cultured cells is not widened.

It is a block diagram of an embodiment of the present invention. It is a flowchart which shows the procedure of the determination in embodiment of this invention. It is a characteristic view which shows the fluorescence intensity characteristic of the cell cultured with the culture solution which does not contain serum, (A) shows the fluorescence intensity characteristic of the cell cultured with the culture solution which does not add interferon gamma, (B) Shows the fluorescence intensity characteristics of cells cultured in a culture medium supplemented with interferon γ. It is a characteristic view which shows the fluorescence intensity characteristic of the cell cultured with the culture solution which mixed serum, (A) shows the fluorescence intensity characteristic of the cell cultured with the culture solution which does not add interferon gamma, (B) Shows the fluorescence intensity characteristics of cells cultured in a culture medium supplemented with interferon γ. It is a characteristic view which shows the fluorescence intensity characteristic of the cell which added fetal bovine serum to the culture solution which does not contain serum, and was cultured by adding human serum, (A) added fetal bovine serum (B) shows the fluorescence intensity characteristics of the cells cultured in the culture solution added with fetal bovine serum and interferon γ. ) Shows fluorescence intensity characteristics of cells cultured in a culture solution to which human serum is added but interferon γ is not added, and (D) is fluorescence of cells cultured in a culture solution to which human serum and interferon γ are added. Shows strength characteristics. It is a characteristic view which shows the fluorescence intensity characteristic of the cell culture | cultivated for 12 hours by the culture solution which added interferon (gamma) to the culture solution which mixed serum. 6 is a characteristic diagram showing fluorescence intensity characteristics of cells cultured for 12 hours in a culture solution in which interferon γ is added to a culture solution mixed with serum, as in FIG. 6, in the case where the amount of interferon γ added is larger than that in FIG. 6. FIG. It is a characteristic view which shows the fluorescence intensity characteristic of the cell cultured for 48 hours by the culture solution which added interferon (gamma) to the culture solution which mixed serum. FIG. 9 is a characteristic diagram showing fluorescence intensity characteristics of cells cultured for 48 hours in a culture solution in which interferon γ is added to a culture solution mixed with serum in the same manner as in FIG. 8, when the amount of interferon γ added is larger than that in FIG. 8. FIG. It is a figure which shows the experimental result which measured whether the human type 2 antigen presentation molecule | numerator HLA-DR was expressed to the cell cultured with the culture solution which mixed serum using PCR method.

Embodiments of the present invention will be described below with reference to the accompanying drawings.
First, a determination system and a determination method according to an embodiment of the present invention will be described with reference to FIGS.
In FIG. 1, in the determination system 100 indicated as a whole by the reference numeral 100, the movement of cells (or the order of determination steps) is indicated by arrows. The determination system 100 shown in FIG. 1 includes a storage device 1 (container), an interferon γ supply device 2 (interferon γ dropping unit), a mixing device 3 (including manual pipetting), and a cell supply device 4 (cell supply unit: pipette). Etc.), a culture vessel 5 (constant environment unit), an analysis device 6 (fluorescence intensity analysis unit), and a determination device 7 (determination unit: an information processing device having a calculation function such as a PC).
In FIG. 1, symbol CF represents a culture solution, symbol SC represents a cell to be cultured (for example, adipose-derived mesenchymal stem cell), and symbol CC represents a cultured cell (for example, a fat-derived mesenchymal stem cell).

In the determination system 100, the storage device 1 (container) stores the culture medium CF (culture medium to be determined whether to include serum) supplied from the culture medium supply unit 8. In the process until the cells are cultured (until a predetermined time elapses in step S2 in FIG. 2), the culture solution CF to be determined is stored in the storage device 1 (container).
The interferon γ supply device 2 (interferon γ dripping unit) has a function of supplying interferon γ to the culture solution CF (culture medium to be determined) stored in the storage device 1.
In the illustrated embodiment, after interferon γ is added to the culture medium CF and the mesenchymal stem cells derived from fat are cultured, whether or not HLA-DR that is a human type 2 antigen-presenting molecule is expressed in the stem cells is determined. By measuring, it can be determined whether serum is contained in the culture solution.

The mixing device 3 has a function of uniformly mixing the culture solution CF in the storage device 1 and the interferon γ supplied to the culture solution CF. The mixing device 3 is a conventionally known device, and includes a placement portion 3A for placing the accommodation device 1 and a motion portion 3B for applying a horizontal reciprocating motion to the placement portion 3A. 1 has a function of uniformly mixing the culture solution CF and interferon γ in the storage device 1.
In the present specification, the term “mixing device” is used not only for the electric device as described above but also for manual pipetting by an operator.
The cell supply device 4 (cell supply unit) supplies the cells SC to be cultured in the culture solution CF (culture medium to be determined) mixed with interferon γ by the mixing device 3. In the embodiment, fat-derived mesenchymal stem cells are used as the cells SC to be cultured. The cell supply device 4 also includes not only an automatic device but also a case where an operator uses a pipette or the like.

The culture vessel 5 (constant environment unit) is suitable for culturing cells when the storage device 1 that stores the culture solution CF (determination target) supplied with the cells SC to be cultured is stored and left standing. Provide and maintain a safe environment. In the illustrated embodiment, a culture environment at a temperature of 37 ° C. and 5% CO ₂ is maintained in the culture vessel 5.
In the culture vessel 5, the culture solution CF (culture solution to be determined) mixed with interferon γ and supplied with the cells SC to be cultured is accommodated and left in a culture environment at a temperature of 37 ° C. and 5% CO _2. Then, the cells SC are cultured.

An antibody processing unit 11 is disposed between the culture vessel 5 and the analyzer 6 (fluorescence intensity analysis unit), and cells cultured in the culture vessel 5 are supplied to the antibody processing unit 11. In the antibody processing unit 11, a part of the cells supplied from the culture vessel 5 is processed with an antibody that recognizes a fluorescently labeled anti-human type 2 antigen-presenting molecule (for example, an anti-HLA-DR antibody), and one of the cells is processed. It has a function of treating the part with a negative control antibody (negative control antibody) that does not recognize any molecule.
As will be described later, cells cultured in a culture medium containing serum are treated with, for example, an anti-HLA-DR antibody and treated with a negative control antibody. When analyzed with a flow cytometer or an imaging cytometer, the cells treated with the anti-HLA-DR antibody show fluorescence intensity characteristics as indicated by the characteristic B2 in FIG. 4 (B), and the cells treated with the negative control antibody FIG. 4B shows a fluorescence intensity characteristic as indicated by characteristic B1 in FIG.
In other words, by providing the antibody processing unit 11, when determining whether or not serum is included in the culture solution using the fluorescence intensity characteristics described in FIG. The characteristics are displayed simultaneously with the characteristics when the expression of HLA-DR was not induced.

The analyzer 6 (fluorescence intensity analysis unit) is a conventionally known apparatus (for example, a flow cytometer or an imaging cytometer) having a function of measuring and analyzing the fluorescence intensity in a cell, and is a cell cultured in the culture vessel 5. A certain cell CC is analyzed by the analyzer 6. As a result of the analysis by the analyzer 6, the fluorescence intensity characteristic of the cell CC can be grasped.
The determination device 7 (determination unit) analyzes the analysis result of the analysis device 6, determines the fluorescence intensity characteristic of the cell CC, and a human type 2 antigen-presenting molecule (for example, HLA-DR) is expressed in the cultured cell CC. It has a function of determining whether or not serum is contained in the culture solution. The flow cytometer corresponds to the analysis device 6 in FIG. 1, while the imaging cytometer corresponds to a combination of the analysis device 6 and the determination device 7 in FIG.

The determination device 7 is an information processing device having a calculation function such as a PC, and analyzes the analysis result of the analysis device 6 to determine the fluorescence intensity characteristic of the cultured cell CC. A function for determining whether or not a type 2 antigen-presenting molecule (for example, HLA-DR) is expressed, and when the human type 2 antigen-presenting molecule is not expressed in cultured cells CC, A function for determining that serum is not included, and a function for determining that serum is included in the culture medium CF when human type 2 antigen-presenting molecule is expressed in cultured cells CC. Yes.
The determination device 7 includes a main body portion 7A and a display portion 7B, and has a function of displaying the determined fluorescence intensity characteristics and other analysis results and determination results on the display portion 7B. Although not shown, it is possible for an operator to determine “whether serum is included in the culture medium CF” instead of the information processing device, and the term “determination device” in this specification is It is used to include such cases.
A method for determining whether or not a human type 2 antigen-presenting molecule (for example, HLA-DR) has been expressed from the fluorescence intensity characteristic as an analysis result will be described later with reference to FIGS.

Next, in the illustrated embodiment, a procedure (step) for determining whether or not the culture solution contains serum will be described mainly with reference to the flowchart of FIG.
In step S1, a culture solution to be determined whether or not it contains serum is supplied from the culture solution supply unit 8 to the storage device 1, and interferon γ is supplied to the culture solution stored in the storage device 1 by the interferon γ supply device 2. Supply.
And the accommodation apparatus 1 which accommodated the culture solution which supplied interferon (gamma) is mounted in the mounting part 3A of the mixing apparatus 3, a reciprocating motion is provided to the accommodation apparatus 1, and a culture solution and interferon (gamma) are mixed uniformly. As described above, the mixing in step S1 may be manual pipetting.

In step S1, the cell supply device 4 supplies fat-derived mesenchymal stem cells, which are cells to be cultured, to the culture solution mixed with interferon γ.
Furthermore, in step S <b> 1, the storage device 1 that stores the culture solution (culture solution to be determined) supplied with the cells to be cultured is stored in the culture vessel 5. Then, in a state where the storage device 1 is housed and left in the culture container 5, the culture container 5 is maintained in a constant environment (for example, in a 37 ° C., 5% CO ₂ environment) to start cell culture.

In step S2, the elapsed time from the start of the culture is measured, and it is determined whether or not a predetermined time has elapsed. When the predetermined time has elapsed (step S2 is “Yes”), the process proceeds to step S3. On the other hand, when the predetermined time has not elapsed (step S2 is “No”), the process returns to step S2, and the loop of “No” in step S2 is repeated.
In the next step S3, it is determined whether a human type 2 antigen-presenting molecule (HLA-DR) is expressed in cells cultured after a predetermined time (adipose-derived mesenchymal stem cells). The “determination” in step S3 is performed by the determination device 7 based on the result of the fluorescence intensity analysis of the analysis device 6.

In determining whether or not the human type 2 antigen-presenting molecule (HLA-DR) is expressed in step S3, first, a part of the cells cultured in step S2 is fluorescently labeled with the human type 2 antigen presenting. It is treated with an antibody that recognizes a molecule (for example, an anti-HLA-DR antibody), and a part thereof is treated with a negative control antibody (negative control antibody) that does not recognize any molecule. Referring also to FIG. 1, cells cultured in the culture vessel 5 are supplied to the antibody processing unit 11, and a part of the cells supplied from the culture vessel 5 is fluorescently labeled in the antibody processing unit 11. The cells are treated with an antibody that recognizes an anti-human type 2 antigen-presenting molecule (for example, an anti-HLA-DR antibody), and the rest of the cells are treated with a negative control antibody (negative control antibody) that does not recognize any molecule.
If the cells treated by the antibody treatment unit 11 are cells cultured in a culture solution containing serum, the treated cells are analyzed by flow cytometry or imaging cytometry.
As a result, the cells treated with the anti-HLA-DR antibody showed a fluorescence intensity characteristic as indicated by characteristic B2 in FIG. 4B (step S3 is “Yes”), and the cells treated with the negative control antibody were FIG. 4B shows a fluorescence intensity characteristic as indicated by characteristic B1 (step S3 is “No”).

If it is determined in step S3 that human type 2 antigen-presenting molecule (HLA-DR) is expressed in the cultured cells (step S3 is “Yes”), the process proceeds to step S4.
On the other hand, when it is determined that the human type 2 antigen-presenting molecule (HLA-DR) is not expressed in the cultured cells (step S3 is “No”), the process proceeds to step S5.

In step S4, based on the determination result (step S3 is “Yes”) that “a human type 2 antigen-presenting molecule (HLA-DR) is expressed in the cultured cells”, the culture solution (the culture solution to be determined) ) Is determined to contain serum.
On the other hand, in step S5, based on the determination result (step S3 is “No”) that “the human type 2 antigen-presenting molecule (HLA-DR) is not expressed in the cultured cells” in step S3, It is determined that serum is not included in the (culture medium to be determined).

In the illustrated embodiment, the time for culturing cells in the culture vessel 5 (predetermined time in step S2) after adding interferon γ to the culture medium (culture medium to be determined) is described with reference to FIGS. As will be described later, it is 12 hours or longer (the time for culturing the cells in the step of culturing the cells is 12 hours or longer), and it is particularly preferable to culture for 48 hours or longer.
The concentration of interferon γ is 100 units / ml or more, particularly preferably about 400 units / ml, as will be described later with reference to FIGS.

In the illustrated embodiment, the adipose-derived mesenchymal stem cells are cultured at a temperature of 37 using a culture solution sold by the applicant and not containing serum (trade name “sf-DOT”). ° C., and cultured under 5% CO _2, the stem cells the culture (derived mesenchymal stem cells fat), after staining with anti-HLA-DR antibody is fluorescently labeled, the fluorescence intensity by flow cytometry When measured, the characteristics as shown in FIG. 3 to 9, the horizontal axis indicates the fluorescence intensity, and the vertical axis indicates the parameter relating to the number of the corresponding cells.
In FIG. 3A, interferon γ is not added, and HLA-DR is not expressed.

On the other hand, interferon γ (Prospec) was added to the same culture medium used in the experiment for obtaining the characteristics of FIG. 3A (culture medium without serum: trade name “sf-DOT”: sold by the applicant). The product name “IFN-γ” sold by the company is used in the illustrated embodiment), and the adipose-derived mesenchymal stem cells are cultured at 37 ° C. in a 5% CO ₂ environment using the culture solution to which the interferon γ is added. After culturing and staining the cultured stem cells (adipose-derived mesenchymal stem cells) with a fluorescently labeled anti-HLA-DR antibody, the fluorescence intensity was measured by flow cytometry. As a result, the characteristics as shown in FIG.
As shown in FIG. 3B, even when interferon γ is added to a culture solution not containing serum, the expression of HLA-DR, which is a human type 2 antigen-presenting molecule, is not induced.

Adipose-derived mesenchymal stem cells cultured in a culture medium in which 10% (all in this specification, volume%) fetal bovine serum is added to an αMEM culture medium that is classically used for culturing mesenchymal stem cells. FIG. 4 shows the result of fluorescence intensity measurement by flow cytometry after staining with a fluorescently labeled anti-HLA-DR antibody.
FIG. 4A shows the fluorescence intensity characteristics when no interferon γ is added to the culture medium. The characteristics shown in FIG. 4 (A) are similar to the characteristics shown in FIGS. 3 (A) and 3 (B), and expression of HLA-DR, which is a human type 2 antigen-presenting molecule, is not induced.

On the other hand, FIG. 4 (B) shows the case where the stem cells are cultured by adding interferon γ to a culture solution (indicated by “αMEM + 10% FCS”) in which 10% fetal bovine serum is added to the αMEM culture solution. The characteristics of fluorescence intensity are shown.
In the characteristics shown in FIG. 4B, the characteristic curve B1 shows the fluorescence characteristics of the cells treated with the negative control antibody, and the characteristic curve B2 shows the fluorescence characteristics of the cells treated with the anti-HLA-DR antibody. Since the characteristic curves B1 and B2 can be clearly distinguished,
It can be judged from FIG. 4 (B) that HLA-DR was expressed.

FIG. 5 shows a culture solution sold by the applicant, which contains a serum-free mesenchymal stem cell at a temperature of 37 ° C. using a culture solution that does not contain serum (trade name “sf-DOT”). After culturing in a 5% CO ₂ environment, the cultured stem cells (adipose-derived mesenchymal stem cells) were stained with a fluorescently labeled anti-HLA-DR antibody, and then the fluorescence intensity was measured by flow cytometry. Experimental results are shown.
FIG. 5 (A) shows fluorescence labeling of cells cultured in a culture solution containing 10% fetal bovine serum but not interferon γ added to a serum-free culture solution (trade name “sf-DOT”). It is the experimental result which measured the fluorescence intensity by flow cytometry after dyeing | staining with the anti-HLA-DR antibody and negative control antibody which were made. In FIG. 5A, expression of HLA-DR, which is a human type 2 antigen-presenting molecule, is not induced. On the other hand, FIG. 5 (B) shows the fluorescence-labeled anti-HLA− for the cells cultured in a culture solution (serum-free culture solution: trade name “sf-DOT”) supplemented with fetal bovine serum and interferon γ. It is the experimental result which measured the fluorescence intensity by flow cytometry after dyeing with DR antibody and a negative control antibody. As is apparent from FIG. 5B, HLA-DR, which is a human type 2 antigen-presenting molecule, is expressed.
From the experimental results of FIG. 5 (A) and FIG. 5 (B), the expression of HLA-DR is suppressed by the components of the culture solution (trade name “sf-DOT”) that does not include the serum used in this experiment. However, it was revealed that the expression of HLA-DR is induced when serum is added to the culture solution.

FIG. 5C shows fluorescence labeling of cells cultured in a culture solution containing 10% human serum but not interferon γ added to a serum-free culture solution (trade name “sf-DOT”). It is the experimental result which measured the fluorescence intensity by flow cytometry after dyeing | staining with the anti-HLA-DR antibody and negative control antibody which were made. In FIG. 5C, the expression of HLA-DR, which is a human type 2 antigen-presenting molecule, is not induced.
On the other hand, FIG. 5 (D) shows the anti-HLA-DR antibody labeled with fluorescence for cells cultured by adding human serum and interferon γ to a serum-free culture solution (trade name “sf-DOT”). It is the experimental result which measured the fluorescence intensity by flow cytometry after dyeing | staining with a negative control antibody. As is clear from FIG. 5D, HLA-DR, which is a human type 2 antigen-presenting molecule, is expressed.
From the experimental results of FIGS. 5 (C) and 5 (D), it was revealed that HLA-DR is expressed by addition of interferon γ in human serum as well as bovine serum (fetal bovine serum). . This shows that cells cultured in a culture medium supplemented with serum express HLA-DR, a human type 2 antigen-presenting molecule, if interferon γ is added regardless of the origin of the serum. It was.

The inventor conducted experiments on the culture time after addition of interferon γ and the concentration of interferon γ. As interferon γ, the trade name “IFN-γ” sold by Prospec was used in the experiment. The “unit”, which is a parameter for the concentration of interferon γ, is also the “unit” in the interferon γ (trade name “IFN-γ” sold by Prospec).
FIG. 6 shows the fluorescence intensity characteristics of cells cultured for 12 hours in a culture solution in which interferon γ is added to a culture solution (αMEM + 10% FCS, 10% fetal bovine serum mixed) mixed with serum.
FIG. 6 shows a characteristic 6-1 that is a fluorescence intensity characteristic when treated with a negative control antibody and a fluorescence intensity characteristic 6-2 that is stained with a fluorescently labeled anti-HLA-DR antibody. . The fluorescence intensity in the characteristic 6-1 when stained with the fluorescently labeled anti-HLA-DR antibody is slightly smaller than the fluorescence intensity in the fluorescent intensity characteristic 6-1 when treated with the negative control antibody. It was strong. This is a characteristic indicating that HLA-DR, which is a human type 2 antigen-presenting molecule, is expressed, as in the data of FIG. 4 (B).

Although not clearly shown, in the inventor's experiment, the characteristic curve indicating that HLA-DR is expressed was not clearly expressed when the culture time was 11 hours or less. From this, it was found that the cells should be cultured for 12 hours or more after the addition of interferon γ when determining whether or not the culture solution contains serum.
Further, in the experiment of FIG. 6, the concentration of interferon γ is 100 units / ml, but if the concentration of interferon γ is less than that (95 units / ml or less in the inventors' experiment), HLA-DR is expressed. The characteristic curve indicating that this was not clearly expressed. Therefore, it was found that the concentration of interferon γ should be 100 units / ml or more.

FIG. 7 also shows the fluorescence intensity characteristics of cells cultured for 12 hours in a culture solution in which interferon γ is added to a culture solution (αMEM + 10% FCS mixed with fetal calf serum) mixed with serum, as in FIG.
In FIG. 7, the concentration of added interferon γ is 400 units / ml. When FIG. 6 is compared with FIG. 7, the characteristic which shows that the direction of FIG. 7 is expressing HLA-DR is clear.
From FIG. 7, it was found that the concentration of interferon γ is preferably 400 units / ml or more.

FIG. 8 also shows the fluorescence intensity characteristics of cells cultured in a culture solution in which interferon γ is added to a culture solution (αMEM + 10% FCS, 10% fetal bovine serum mixed) mixed with serum, as in FIG. 6.
In FIG. 8, the culture time is 48 hours, and the concentration of added interferon γ is 100 units / ml. When FIG. 6 and FIG. 8 are compared, the characteristics indicating that HLA-DR is expressed in FIG. 8 are clearer.
From comparison between FIG. 6 and FIG. 8, it was found that the culture time is preferably 48 hours or more.

Similarly to FIG. 8, FIG. 9 shows the fluorescence intensity characteristics of cells cultured in a culture solution in which interferon γ is added to a culture solution (αMEM + 10% FCS, 10% fetal bovine serum mixed) mixed with serum.
In FIG. 9, the culture time is 48 hours, and the concentration of added interferon γ is 400 units / ml. Comparing FIGS. 6 to 9, the expression of HLA-DR is most obvious in FIG.
6 to 9, it was found that the culture time is preferably 48 hours or more, and the concentration of interferon γ is preferably 400 units / ml or more.

According to experiments conducted by the inventors, it was possible to show the expression of HLA-DR, a human type 2 antigen-presenting molecule, by PCR.
In experiments conducted by the inventors, RNA was extracted from cells and then reverse transcription was performed to prepare cDNA. The reverse transcription reaction is based on 1 μg of RNA, and the 5 ′ end is phosphorylated with a ^6- mer or ^9- mer deoxyribonucleotide mixture having a random sequence (deoxyribonucleotide mixture: 4 ⁶ or 4 ⁹ sequences are possible). As a primer, reverse transcriptase (trade name “PrimeScript ™ II Reverse Transcriptase” sold by Takara Bio Inc.) was used. A PCR reaction was performed using the obtained cDNA to determine whether HLA-DR, which is a human type 2 antigen-presenting molecule, was expressed.
In order to express HLA-DR by PCR, the following were mixed.
0.1 μl (final concentration: 0.025 unit / μl) of 5 units / μl of enzyme (trade name “TaKaRa Taq” sold by Takara Bio Inc.),
2 μl of a conditioner (PCR Buffer: for mixing 1/10 of the reaction solution)
1 μl of nucleic acid mixture (trade name “dNTP Mixture” sold by Takara Bio Inc.)
0.4 μl of forward primer (5 μM) (final concentration: 0.1 μM),
0.4 μl of reverse primer (5 μM) (final concentration: 0.1 μM),
CDNA derived from 25 ng RNA.

Here, the primer sequences are as follows.
In FIG. 10, in the gene used as an internal standard (gene expressed in all: symbol “GAPDH” in FIG. 10),
The forward primer is aggcacatcgctcagacac
Reverse primer is gcccaatacgaccacaatcc
It is.
On the other hand, in the gene HLA-DR,
The forward primer is gggaaaagccaccactggaaaa
The reverse primer is aaaacagatgaggacgtgtgg
It is.
Using a general thermal cycler,
95 cycles of 15 seconds, 60 ° C for 1 minute, and 25 cycles of reaction.

10 μl of the PCR reaction solution was mixed with 10 μl of a staining reagent (trade name “GRGreen Loading buffer” manufactured by Biocraft Co., Ltd.) and then subjected to agarose gel electrophoresis.
After agarose gel electrophoresis, a band as shown in FIG. 10 was detected as a result of using an analyzer (trade name “ImageQuant LAS4000” manufactured by GE Healthcare).

In FIG. 10, the symbol “FBS” indicates a culture solution containing fetal bovine serum in the same manner as the symbol “FCS”, and the symbol “FBS_IFNγ” represents a sample obtained by adding interferon γ to the culture solution containing fetal bovine serum. Show. The symbol “HS” indicates a culture solution containing human serum, and the symbol “HS_IFNγ” indicates a sample obtained by adding interferon γ to a culture solution containing human serum.
In FIG. 10, the white band in the descending “HLA-DR” indicates that HLA-DR, which is a human type 2 antigen-presenting molecule, was expressed. A white band is shown in FBS_IFNγ obtained by adding interferon γ to fetal bovine serum and HS_IFNγ obtained by adding interferon γ to human serum.
In the experiment showing the results in FIG. 10, it was confirmed by PCR that HLA-DR, which is a human type 2 antigen-presenting molecule, was expressed by adding interferon γ in the case of a medium containing serum. .

  In addition to this, according to the experiment by the inventors, a culture solution having a low serum content (for example, in the trade name “MessenPro RS” manufactured by Thermo Fisher Scientific Co., Ltd.), when interferon γ is added, FIG. ), FIG. 5 (B), and FIGS. 7 to 9, it was confirmed that HLA-DR, which is a human type 2 antigen-presenting molecule, is expressed.

It should be noted that the illustrated embodiment is merely an example, and is not a description to limit the technical scope of the present invention.
For example, in the illustrated embodiment, the presence or absence of serum in the culture medium is determined based on whether or not HLA-DR is expressed as a type 2 antigen-presenting molecule. In the present invention, human type 2 antigen-presenting molecules other than HLA-DR are used. This includes the case where it is determined whether or not the culture solution contains serum depending on whether or not HLA-DQ or HLA-DP is expressed.
In the illustrated embodiment, whether or not a type 2 antigen-presenting molecule (HLA-DR) is expressed is measured by flow cytometry, but the present invention is not limited to flow cytometry, and PCR And other measurement methods that can measure the presence or absence of expression of a human type 2 antigen-presenting molecule.
Furthermore, in the illustrated embodiment, the case of culturing fat-derived mesenchymal stem cells is described, but the same applies to umbilical cord-derived, bone marrow-derived, amniotic membrane-derived, dental pulp-derived, placenta-derived mesenchymal stem cells. In the present invention, it is possible to determine whether or not the culture solution used for culturing mesenchymal stem cells derived from the umbilical cord, bone marrow, amniotic membrane, dental pulp, and placenta includes serum. Alternatively, the present invention can also be applied when culturing various cells other than stem cells.

1 ... Storage device (container)
2. Interferon gamma supply device (interferon gamma dropping unit)
3 ... Mixing device 3A ... Placement unit 3B ... Movement unit 4 ... Cell supply device (cell supply unit)
5 ... Culture container (constant environment unit)
6 ... Analyzer (fluorescence intensity analysis unit),
7 ... Judgment device (judgment unit)
7A: Main unit 7B: Display unit 8: Culture solution supply unit 100: Determination system CC: Cultured cell CF ... Culture solution SC ... Cell to be cultured

Claims (5)

A storage device for storing a culture solution to be determined whether or not to contain serum;
An interferon γ supply device for supplying interferon γ to the culture solution stored in the storage device;
A mixing device for uniformly mixing the culture solution in the storage device and interferon γ,
A cell supply device for supplying cells to be cultured in a culture solution;
A culture container that houses a storage device that stores a culture solution supplied with cells to be cultured, in a constant environment; and
An analyzer for analyzing fluorescence intensity in cultured cells;
It has a judgment device that analyzes the results of the analysis device,
The determination device determines whether or not a human type 2 antigen-presenting molecule has been expressed, and determines that serum is not included in the culture medium when the human type 2 antigen-presenting molecule is not expressed. A determination system characterized by having a function and a function of determining that serum is included in a culture medium when a human type 2 antigen-presenting molecule is expressed.   The determination system according to claim 1, wherein the time for culturing the cells in the culture vessel is 12 hours or more. Storing a culture solution to be determined whether or not to contain serum in a storage device;
Supplying interferon γ to the culture solution stored in the storage device by the interferon γ supply device;
A step of uniformly mixing the culture solution in the storage device and interferon γ by a mixing device;
Supplying a cell to be cultured in a culture solution by a cell supply device;
In the culture vessel, the step of culturing the cells by allowing the storage device containing the culture solution supplied with the cells to be cultured to stand in a constant environment; and
Determining whether a human type 2 antigen-presenting molecule is expressed in the cultured cells;
Determining that serum is not included in the culture medium when the human type 2 antigen-presenting molecule is not expressed;
A determination method comprising a step of determining that serum is included in a culture solution when a human type 2 antigen-presenting molecule is expressed.   The determination method according to claim 3, wherein the time for culturing the cells in the step of culturing the cells is 12 hours or more.   A cell that is cultured using a culture solution that does not contain serum, and human type 2 antigen-presenting molecule is not expressed even when interferon γ is added.

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