WO2010084943A1 - Method for evaluating cultured cells - Google Patents

Abstract

Disclosed is a method for evaluating the type or function of cultured cells, such as a method for evaluating a bone formation ability, which has been difficult to achieve based on a certain criterion because of the fluctuations among individuals. In the method for evaluating cultured cells, for the purpose of identifying a cell of interest among the cultured cells, multiple test values or indicators for the cultured cells are measured, and the function of the cell of interest is predicted or determined with high accuracy based on a combination of the multiple test values or indicators. The method is characterized in that a cell of interest can be identified not only by designating a certain adaptation range for each of the test values, but also referring other test value or indicator for a sample that goes out of the application range.

Description

Method for evaluating cultured cells Related applications

This application claims the priority of Japanese Patent Application No. 2009-013385 filed on January 23, 2009, and is incorporated herein.

The present invention relates to a method for evaluating cultured cells, and more particularly to a new method for evaluating cultured osteogenic cells.

Realization of regenerative medicine for regenerating deficient living tissue is required. Regenerative medicine is a medical technique that uses a cell, biomaterial, and cell growth factor to create a form and function similar to that of the original tissue from a living tissue that cannot be recovered by the healing ability of the living body.
In regenerative medicine, mesenchymal stem cells are generally used. These mesenchymal stem cells are known to be able to induce differentiation into various cells such as adipocytes and osteoblasts, but in advance determine whether the obtained cells have the desired function It was difficult.

In bone regenerative medicine, bone marrow-derived mesenchymal stem cells are mainly used, and there are reports of adipose-derived stem cells, periosteum-derived stem cells, synovial-derived stem cells, and the like. Among them, bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are relatively easy to collect and have an extremely high bone forming ability, and are currently used for almost all clinical applications of bone regeneration.
However, previous reports by the present inventors have revealed that human-derived osteogenic cells have large individual differences. It was also clarified that the bone forming ability was quickly lost by culture. Therefore, it is important to examine whether the cells to be transplanted actually have the ability to form bone, but it has been found that the methods reported so far are not sufficient. In particular, cell alkaline phosphatase activity (ALP activity) (Patent Document 1), gene expression of bone markers (Non-Patent Document 1), and the like have been used. Although ALP activity is a useful assessment for osteogenic cells, it alone is difficult to define osteogenic cells. Moreover, although gene expression of bone system markers is a useful evaluation, the results of the study by the present inventors have shown that there are large individual differences in expression, and cases where it is difficult to use as an evaluation. In particular, for the evaluation of human-derived cells with large individual differences, one standard is not sufficient, and it is considered important to improve reliability by combining multiple evaluation methods. Has not been reported so far.
Osteogenic cells are defined as cells essential for bone formation, such as osteoblasts. In particular, cells capable of ectopic bone formation are defined as osteogenic cells. Ectopicity is the ability to form bone in parts that are essentially free of bone. Ectopic bone forming ability is verified, for example, by transplanting subcutaneously in immunodeficient animals with a carrier holding human cells. Although bone regeneration is actually expected in the vicinity of bone, it is necessary to regenerate bone toward the bone-free part, so the ability originally required for bone regeneration is this ectopic bone formation. Noh is considered the most appropriate evaluation method. However, there is no method for evaluating cells produced from such a viewpoint.

JP 2005-58225 A

That is, in order to perform bone formation by transplantation, a process as shown in FIG. 1 is performed. As shown in FIG. 1, osteogenic cells are allowed to coexist with a transplant material such as pseudo bone granules, and the pseudo bone granules are decomposed by osteoclasts at the transplant site, while the true bone is regenerated by osteoblasts and the like. At this time, the number of osteogenic cells in the transplant material is of course an important factor, but on the other hand, it is important that proper bone forming ability is maintained. If the number of these osteogenic cells is small or cells with poor bone forming ability are used, proper bone formation is not performed, but it takes a considerable amount of time to confirm actual bone formation. Necessary. If bone formation is not performed properly, the mesenchymal stem cells must be collected again, cultured, transplanted, and reconfirmed, resulting in wasted months. However, treatments that burden the patient, such as transplant surgery, are repeated. Therefore, it is important to appropriately evaluate the function of cultured cells.
On the other hand, even when determining the bone forming ability during cell culture, the time required for the test during differentiation induction is several weeks, so it cannot be easily repeated.

However, the conventional evaluation of target cells, for example, osteogenic cells, is performed by a single determination method as described above, and if the evaluation criteria are raised, regeneration after transplantation is performed. Although the rate increases to some extent, it is considered that the cultured cells that were originally usable can be judged as inappropriate. On the other hand, if the evaluation standard is lowered, cultured cells that cannot form bone can be used for transplantation. Become. Furthermore, according to the research of the present inventors, in human cells, there are large variations among individuals in almost all test values, and it is difficult to make an accurate determination only with a certain reference value or only with a single criterion. It has become clear that even realistic judgment is difficult.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a reliable evaluation method capable of determining a target cell. Although the evaluation method according to the present invention can be suitably used for osteogenic cells, the problem of variation of human cells is common to many cells that are widely used for regenerative medicine and cell therapy in addition to osteogenic cells. This is considered a problem. Therefore, the effect of the present invention is not only for osteogenic cells but also widely for cell therapy using human cells, and whether the obtained cells are the target cells and whether they have functions necessary for the treatment. It provides an effective evaluation method.

As a result of intensive studies conducted by the present inventors in order to solve the above problems, in order to determine a target cell from the cultured cell, a plurality of test values or indices of the cultured cell are measured and combined. It has been found that a reliable evaluation method can be created. For bone-forming cells (bone-forming (+) cells), we developed a bone-forming ability test method as shown in Fig. 2 to predict and evaluate whether bone formation is performed properly after transplantation. It has been found that a method for evaluating a useful osteogenic cell can be created by carrying out the process appropriately and accurately, and the present invention has been completed.

That is, in the method for evaluating cultured cells according to the present invention, in order to determine a target cell from the cultured cells, a plurality of test values or indices of the cultured cells are measured, and the function of the cells is accurately predicted by combining them. An evaluation method for judging,
For each test value, not only can you specify a certain compatible range, but even if the sample is outside the applicable range, you can determine the target cell by referring to another test value or index. It is characterized by being.

Further, the method for evaluating cultured osteogenic cells of the present invention is a method for evaluating cultured cells having a primary determination criterion, a secondary determination criterion, and a tertiary determination criterion.
The cells that have undergone the primary determination are further determined as bone forming (+) cells by performing a secondary determination.
It is characterized in that a bone forming (+) cell is determined by performing a third determination on a cell that is difficult to determine whether it is a bone forming (+) cell.
In the primary determination, the determination criteria are ALP activity (alkaline phosphatase activity), ALP index (ALP activity in differentiation induction group / ALP activity in non-differentiation induction group), cell proliferation ability (number of cells after differentiation induction / initial seeded cells) It is preferable that any one of (number) satisfies the reference value of the primary determination or more.
In the secondary determination, the determination criterion is whether or not all of ALP activity, ALP index, and cell proliferation ability satisfy the reference value of the secondary determination,
A cell that satisfies both the primary determination and the secondary determination is determined as a bone-forming (+) cell,
For cells that did not meet the criteria in the primary judgment but met the criteria in the secondary judgment, the cells moved to the tertiary judgment criteria,
It is preferable to determine a cell that does not meet the criteria in the secondary determination as a bone-forming (−) cell.
In the tertiary determination, the determination criterion is whether any of surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining, or alizarin red staining satisfies the criterion,
It is preferable to determine cells that satisfy this criterion as osteogenic (+) cells, and cells that do not satisfy these criteria as osteogenic (−) cells.
In the primary determination, the reference value is preferably each (average value + 2SD) of bone-forming (−) cells.
In the secondary determination, it is preferable that the reference value is a lower limit value of each measurement value of bone forming (+) cells (average value −2SD) or each value of bone forming (+) cells.
In the third determination, the standard for surface antigen (HLA-DR) analysis is preferably about 6% or more of measured HLA-DR positive cells.
In the primary determination, the reference value for ALP activity is about 166 units (μmol).
p-nitrophenol produced / min / μg protein) is preferred.
In the primary determination, the reference value of the ALP index is about 3.95, The method for evaluating cultured osteogenic cells.
In the primary determination, it is preferable that the reference value of the cell proliferation ability is about 9.7.
In the secondary determination, the reference value for ALP activity is about 67 units (μmol).
p-nitrophenol produced / min / μg protein) is preferred.
In the secondary determination, it is preferable that the reference value of the ALP index is about 1.01.
In the secondary determination, it is preferable that the reference value of the cell growth ability is about 5.6.

In the method for evaluating osteogenic cells according to the present invention, the ALP activity used as a criterion is high in osteogenic cells, such as osteoblasts. If the ALP activity after differentiation induction is low, it is possible that the cells do not contain osteogenic cells or have no bone forming ability. When the ALP index, which is the ratio of ALP activity in the induction group, is increased, it is indicated that the cells are osteogenic cells and may have osteogenic ability. On the other hand, if the ALP activity is sufficiently high, the differentiation induction of osteogenic cells is very likely to be good. However, even if ALP activity is high, sufficient bone formation cannot be expected after bone transplantation if cell proliferation ability is lost.
Therefore, the evaluation method according to the present invention also evaluates cell proliferation ability.

Even if the above three evaluations (ALP activity, ALP index, cell proliferation ability) are combined, a more careful evaluation is required for a group in which it is difficult to determine the presence or absence of osteogenic cells. For these groups, the present inventors further analyzed the surface antigen (HLA-DR) by flow cytometry, TRAP staining, and alizarin red staining, and either positive (HLA by flow cytometry). -DR fraction positive, TRAP positive, alizarin red positive), and it is judged that there is an osteogenic cell as an auxiliary, provides a reasonable basis for samples having almost all individual differences.

Although the direct relationship between this HLA-DR positive cell and bone forming ability is unknown, the present inventors have confirmed that a clear correlation was found between HLA-DR activity and bone forming ability. TRAP staining (tartaric acid-resistant acid phosphatase staining) is used as an osteoclast marker, but it is necessary to interact with osteoblasts (osteogenic cells) to mature from osteoclast precursor cells to osteoclasts Therefore, it seems that it can be used as an indirect indicator of the process by which stem cells differentiate into osteogenic cells. Also, alizarin red staining represents calcification ability in vitro and stains calcium deposits. As a result of confirmation by the present inventors, it has been shown that cells showing osteogenic potential are positive for alizarin red staining, and staining is negative for cells that have been passaged and have lost osteogenic potential.

As described above, in the present invention, a comparatively simple and clear method of ALP activity evaluation, ALP index, and cell proliferation ability determination are used to select those clearly having bone forming ability and those not having bone forming ability. For groups that were not clear in the evaluation of HLA-DR, HLA-DR analysis, TRAP staining, and alizarin red staining would be discarded as “no osteogenic ability” even though sufficient osteogenic ability could be expected. It is preventing.

According to the present invention, it is possible to provide a method that can evaluate the type and function of a target cell that has been difficult to judge by a certain standard due to variations among individuals. Further, when evaluating osteogenic cells, it is possible to almost certainly evaluate the bone forming ability of cultured cells, and a method relating to evaluation essential for treatment using cells can be provided.

Bone-forming ability test schedule. The flowchart which shows the evaluation method of the osteogenic cell concerning this invention. Explanatory drawing of the culture | cultivation process and differentiation induction process of the osteogenic cell to which this invention is applied suitably. The figure which showed distribution of ALP activity of a human cultured osteoblast-like cell. The figure which showed distribution of the ALP index of a human cultured osteoblast-like cell. The figure which showed distribution of the proliferation ability of a human cultured osteoblast-like cell. ALP activity measurement results. Cell proliferation ability measurement results. Surface antigen analysis results by flow cytometry. TRAP staining measurement results. Results of alizarin red staining.

DESCRIPTION OF SYMBOLS 10 ... Bone marrow fluid 12 ... Cell culture flask 14 ... Porous pseudo bone granule 16 ... Deep bottom container

Hereinafter, preferred embodiments of the present invention will be described. Hereinafter, the case where the target cell is an osteogenic cell will be described as an example. First, a method for producing granular cultured bone that can be effectively applied to treatment by the method for evaluating osteogenic cells of the present invention will be described with reference to FIG. Granule-type cultured bone can be produced by collecting bone marrow fluid, culturing bone marrow-derived mesenchymal stem cells, seeding mesenchymal stem cells, and inducing differentiation into cultured osteoblast-like cells.
The following steps show a form using bone marrow-derived cells. However, the evaluation method of the present invention can be cultured separately from periosteum, fat, peripheral blood, etc. in addition to bone marrow, and cultured bones in the same manner. It can also be suitably used in granulated cultured bone produced by inducing differentiation into blast-like cells.

• Bone marrow fluid collection Bone marrow fluid should be collected approximately one month before surgery. First, local anesthesia is performed on the bone marrow fluid collecting part, and aseptically sucked and collected from the posterior superior iliac crest.

-Cultivation of bone marrow-derived mesenchymal stem cells Bone marrow fluid 10 diluted 4-fold with cell culture medium is seeded in cell culture flask 12, culture is started as shown in Fig. 3 (A), and 4 days after the start of culture. Change the whole medium. As the cell culture medium, either serum-containing αMEM or serum-free medium can be used for both primary culture and differentiation culture.
Confirmation that there is no infection with general bacteria and fungi during the culturing process is made by observing the medium every time the medium is exchanged (if the medium is infected, the medium becomes cloudy), and at the start of culture, before differentiation induction, and cultured osteoblasts Confirm the sterility test (whether it meets the standards of the Japanese Pharmacopoeia) at the time of recovery of the cells.
Thereafter, the whole medium is changed twice a week. The subculture timing is determined based on the state of the cells, but it is about 21 to 28 days after the start of culture. Viable cells are counted at the time of passage.

・ Seeding of mesenchymal stem cells Passage is performed as follows. After evacuating the culture medium in the flask, washing with darbecolate buffer (D-PBS), and then evacuating D-PBS, adding a cell dissociating agent, and culturing at 37 ° C. for 10 minutes. After confirming that the cells are detached, add D-PBS or a medium to collect the cells, and then centrifuge. Resuspend in medium and count cells. After the measurement, the cultured bone marrow fluid 10 is put into a deep bottom container 16 in which porous pseudo-bone granules 14 as shown in FIG. When the bone marrow fluid 10 is put into the deep bottom container 16 containing the pseudo bone granules 12, the bone marrow fluid 10 and the pseudo bone granules 12 float as shown in FIG.

-Differentiation induction into cultured osteoblast-like cells Cells are seeded on the porous pseudo-bone granule 14 and then allowed to stand overnight to recover the function of the cells. After one day, as shown in FIG. 3D, cells such as cells involved in bone formation and the porous pseudo bone granule 14 are submerged in the lower part of the deep container 16. After confirming this, the medium is replaced with a differentiation-inducing medium. The differentiation induction period is 1 to 3 weeks, and the medium is changed about twice a week. As differentiation induction proceeds, as shown in FIG. 3 (E), the morphology of cultured osteoblast-like cells attached around the pseudo-bone granule 14 changes, and the bone matrix protein is secreted into a lump.
Meanwhile, in parallel with this process, cells are seeded in a 12-well culture dish for evaluation. Cells are 2 × 10 ⁴ per well.

Hereinafter, the method for evaluating osteogenic cells according to the present invention will be described with reference to the flowchart of FIG. As shown in FIG. 2, osteogenic cells are determined based on a primary determination criterion, a secondary determination criterion, and a tertiary determination criterion. In addition, the ratio in the parenthesis in FIG. 2 indicates the ratio to the initial cell. However, these ratios are merely examples and are not particularly limited, and vary depending on the selection of cells to be used.

Primary determination criteria The primary determination is performed based on ALP activity, ALP index, and cell proliferation ability.
In the primary determination, if the reference value of any of the three evaluation methods is exceeded, the cell is determined to be the primary determination standard (O), and if not exceeding all the reference values, the cell is primary It is determined that it is a criterion (×).

Secondary determination criteria The secondary determination is also performed based on the ALP activity, the ALP index, and the cell proliferation ability as in the primary determination. However, as shown below, these evaluation reference values are different from the primary determination.
When the primary determination criterion (◯) cell exceeds the reference value in all of the three evaluation methods in the secondary determination, the cell is determined to be a bone-forming (+) cell, and any reference value is determined. If not, the cell is determined to be an osteogenic (−) cell.
When the primary determination criterion (×) cell satisfies all of the above three evaluation methods in the secondary determination, the cell is transferred to the tertiary determination criterion. If any of the reference values is not exceeded, the cell is Determined to be osteogenic (−) cells.

Criteria for tertiary determination The tertiary determination is performed based on surface antigen analysis by flow cytometry, TRAP staining, and alizarin red staining.
If any of the tertiary criteria is positive, the cell is determined to be a bone-forming (+) cell, and if all are negative, the cell is determined to be a bone-forming (-) cell. .

In the bone-forming cell evaluation method according to the present invention, it is determined whether or not it is a bone-forming (+) cell by combining a plurality of criteria. The importance of combining these methods is as follows.

As described above, ALP activity has also been used for confirmation of bone formation ability so far. However, as shown in FIG. 4, the distribution of ALP activity is close to cells with and without bone forming ability, and it was difficult to set a clear boundary. If it is set low, many cells having no bone forming ability are mixed, and if it is set high, cells having bone forming ability are excluded.

When a criterion is actually considered using ALP activity, for example, it is conceivable to set a boundary with an average value −2SD of ALP activity of cells showing bone formation. Assuming that the value of ALP activity has a normal distribution, 95.45% of cells that actually have osteogenic potential should be able to be recovered, but on the other hand, looking at FIG. 4, it does not have osteogenic potential. It is possible that most cells will also be recovered.
It is also conceivable to set the lower limit value of bone-forming (+) cells. However, when FIG. 4 is seen, there are many cells that do not have the ability to form bone even when the bone formation (+) cell is below the lower limit.

As described above, the usefulness of the ALP index has been reported by the present inventors for the determination of bone forming ability. However, referring to FIG. 5, the distribution of the ALP index is also close between cells with and without bone forming ability, and it was difficult to set a clear boundary.

Although not used for the determination of bone forming ability so far, the present inventors have clarified that, among cell proliferating ability, the proliferating ability particularly during differentiation induction is related to the osteogenic ability. . However, referring to FIG. 6, the distribution of cell proliferating capacity is also close between cells having bone forming ability and cells not having bone forming ability, and it was difficult to set a clear boundary.

From the above, it can be seen that it is difficult to appropriately determine bone-forming (+) cells using conventionally used indices and simple combinations thereof.
Therefore, the present inventors have many overlapping portions of data of bone forming (+) cells and bone forming (−) cells in the above-described determination method, and therefore, the strict criteria (1) that only bone forming (+) cells can be collected. The following criteria are provided first.

However, since many bone-forming (+) cells are included in a portion not selected by the primary determination criterion, the relief is performed using the secondary determination criterion and the tertiary determination criterion.
In addition, cells that satisfy the primary determination criteria may contain a small amount of osteogenic (−) cells. For this reason, the secondary determination standard is performed also about these cells.
As described above, in the osteogenic cell evaluation method according to the present invention, the primary determination to the tertiary determination are performed. As a result, the present inventors have determined that the osteogenic (+) cells are almost 100%. % Bone forming (+) cells have been revealed.

Next, the criteria used in the primary determination and the secondary determination will be described. First, reference values and evaluation methods (ALP activity, ALP index, cell proliferation ability) will be described in detail.

In the present invention, the reference value for primary determination was set as each (average value + 2SD) of bone-forming (−) cells. At this time, assuming that the sample data has a normal distribution, the probability that a cell having no bone forming ability is erroneously included is (100−95.45) /2=2.275%.
The reference value for secondary determination was set as the lower limit of each measured value of bone forming (+) cells (average value −2SD) or each value of osteogenic (+) cells.

The reference value may vary depending on the population of patients (gender, age, race, etc.) from which cells are collected. That is, it is important to select an appropriate value that matches the patient as the reference value. Therefore, the reference values shown below are determined by the patient's cells collected by the present inventors, and are not limited to these. With more data, it is possible to set this value more precisely.
In addition, the specific reference value of the secondary determination shown below is exemplified by the lower limit value of each measurement value of bone-forming (+) cells, but each (average value −2SD) of bone-forming (+) cells. ) Can be used to obtain reasonable results.

ALP activity For the measurement of ALP activity, for example, p-nitrophenyl phosphate tablet set (Sigma-Aldrich) and cell counting kit WST-8 (Dojindo Laboratories) can be used.
Hereinafter, the measurement method will be described by taking ALP activity measurement with the above product as an example. First, 100 μl of WST-8 solution is added to each well. After a color reaction for 1 to 4 hours in a carbon dioxide incubator, the absorbance is measured using a microplate reader. After WST-8 analysis, cell membrane protein was dissolved and extracted, p-nitrophenyl phosphate solution was added to the lysate, allowed to stand at room temperature for 10 minutes, the reaction was stopped with NaOH, and p-nitrophenol (p-nitro The absorbance of the degradation product of phenyl phosphate by ALP is measured. ALP activity is expressed in moles of p-nitrophenol per hour / mass of protein (μmol p-nitrophenol produced / min / μg protein). Alternatively, it can also be expressed as p-nitrophenol absorbance (OD; 405 nm) / WST-8 absorbance (OD; 450 nm).

FIG. 7 shows an example of ALP activity measurement results.
The average value of ALP activity + 2SD of osteogenic (−) cells was 2.31 {p-nitrophenol absorbance (OD; 405 nm) / WST-8 absorbance (OD; 450 nm)}. This value corresponds to 166 units (μmol p-nitrophenol produced / min / μg protein). For this reason, the present inventors set the reference value for primary determination of ALP activity as about 166 units (μmol p-nitrophenol produced / min / μg protein). That is, the reference value for primary determination of ALP activity is preferably 166 ± 5 units (μmol p-nitrophenol produced / min / μg protein).
The conversion formula for the unit of ALP activity per cell (μmol p-nitrophenol produced / min / μg protein) was determined by the present inventors depending on the cells used in this study.

The lower limit of the measured ALP activity of the bone-forming (+) cells was 0.93 {p-nitrophenol absorbance (OD; 405 nm) / WST-8 absorbance (OD; 450 nm)}. This value corresponds to 67 units (μmol p-nitrophenol produced / min / μg protein). For this reason, the present inventors set the reference value for secondary determination of ALP activity as about 67 units (μmol p-nitrophenol produced / min / μg protein). That is, the reference value for secondary determination of ALP activity is preferably 67 ± 5 units (μmol p-nitrophenol produced / min / μg protein).

ALP index The ALP index was measured by measuring ALP activity for cells to which a differentiation-inducing medium was added (differentiation-inducing group) and cells in which normal medium was added as a control (non-differentiation-inducing group). (ALP activity of non-differentiation induction group).

The average value of ALP index + 2SD of osteogenic (−) cells was 3.95. For this reason, the inventors set the reference value for the primary determination of the ALP index as about 3.95. That is, it is preferable that the reference value for primary determination of the ALP index is 3.95 ± 0.15.
Further, the lower limit value of the measured ALP index value of the bone-forming (+) cells was 1.01. For this reason, the present inventors set the reference value for secondary determination of the ALP index to about 1.01. That is, the reference value for secondary determination of the ALP index is preferably 1.01 ± 0.15.

-Cell proliferation ability Cell proliferation ability can be calculated | required by the ratio of the number of cells before and behind culture | cultivation, ie, (number of cells after differentiation induction / number of initial seed | inoculated cells). Actually, it can be obtained by measuring the number of cells after the differentiation induction period, but it can be calculated as (number of recovered cells / number of seeded cells) by keeping the seeded cells constant. . Here, the cell proliferation rate during the differentiation induction period is defined as the cell proliferation ability. For example, the determination can be made with the ratio of the OD values of the cell counting kit (WST-8). That is, after performing the above-mentioned color reaction, the absorbance (OD value) at 450 nm is measured, and divided by the OD value of WST-8 relative to the number of cells before differentiation induction, the cell proliferation rate during differentiation induction is obtained. Can be sought. The cell proliferation ability determination according to the present invention can be applied from passage 1 to passage 4.

FIG. 8 shows an example of measurement results of cell proliferation ability.
The average proliferation ability + 2SD of osteogenic (−) cells was 9.7. For this reason, the present inventors set the reference value for the primary determination of cell proliferation ability to be about 9.7. That is, it is preferable that the reference value for the primary determination of cell proliferation ability is 9.7 ± 0.3.
Moreover, the lower limit of the proliferative ability measurement value of the bone forming (+) cells was 5.6. For this reason, the present inventors set the reference value for secondary determination of cell proliferation ability to be about 5.6. That is, it is preferable that the reference value for primary determination of cell proliferation ability is 5.6 ± 0.3.

Next, the criteria and evaluation methods (surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining, alizarin red staining) used in the tertiary determination will be described in detail.
In the present invention, the criterion for tertiary determination was set to 6% or more of surface antigen (HLA-DR) positive cells, TRAP positive, and alizarin red positive.

-Surface antigen analysis by flow cytometry Although the existing flow cytometer can be used for 6-color flow cytometry analysis, the FACS Aria flow cytometer (BDIS) was used here.

The following substances were used for antibodies. Used by fluorescent isothiocyanate complex (FITC-), phycoerythrin complex (PE-), peridinin chlorophyll protein complex (PerCP-Cy5.5-), allophycocyanin complex (APC-), Alexa Fluor 405 complex It was broken.
In addition to the above-mentioned complex, biotinylated antibodies against HLA-ABC, HLA-DR, CD3, CD14, CD19, CD34, CD73, CD90, CD106, CD146, mouse-IgG1k, mouse-IgM (all BD Pharmingen), biotinylated antibodies against CD10 and CD29 (Dako) and biotinylated antibodies against CD45 (Invitrogen) were also used. CD105 antibody (Immunotech) with covalently bound FITC was also used. The biotinylated antibody was detected with Streptavidin Pacific Blue (Invitrogen) or Streptavidin PerCP-Cy5.5 (BD Pharmingen) complex.
STRO-1 antibody (R & D Systems) was detected with PE-conjugated anti-mouse IgM. Propidium iodide (Dojindo Laboratories) was used to detect dead cells.

The surface antigen analysis was performed by flow cytometry using the above tens of types of antibodies. As a result of investigations by the present inventors, it has been clarified that bone marrow-derived mesenchymal stem cells may or may not be able to obtain cells having bone forming ability even if cultured by the same technique. However, in the expression of the above-mentioned antibodies other than the antibody against HLA-DR, there was no difference between cells having the ability to form bone and cells not having them. However, it was revealed that there was only a significant difference in the expression of antibodies against HLA-DR. For this reason, in the surface antigen analysis according to the present invention, a biotinylated antibody against HLA-DR is used as an antibody.

Hereinafter, a surface antigen analysis method using a biotinylated antibody against HLA-DR suitable for the present invention will be described. Cells at passages 0 and 3 were detected by trypsin-EDTA, and 1 × 10 ⁶ cells were suspended in 50 μl of ice-cold phosphate buffered saline (PBS; Nissui Pharmaceutical). The cells were then incubated with a biotinylated antibody against HLA-DR for 20 minutes on ice. The cells were then washed and incubated with streptavidin complex for 20 minutes on ice. Finally, cells were washed, resuspended in 200 μl ice-cold PBS, stained with propidium iodide and analyzed by flow cytometer. Data analysis was performed using FlowJo software (TreeStar).

FIG. 9 shows the results of surface antigen analysis by flow cytometry. The X-axis and Y-axis in FIG. 9 show the responses to different antibodies (HLA-DR and CD14, respectively). The numerical value on the lower right represents the fraction of antibody on the Y axis (CD14) negative and antibody on the X axis (HLA-DR) positive.

As shown in FIG. 9A, when the measured HLA-DR positive cells are 6% or more, it is determined as a cell fraction containing osteogenic (+) cells. However, as shown in FIG. 9B, when the measured HLA-DR positive cells are less than 6%, it is determined as a fraction that has a high possibility of not containing osteogenic (+) cells.
The lower limit of 6% of the HLA-DR positive cells is an empirically determined value found by many inventors in the subject, but the present invention is not limited to this value. That is, it is possible to set this numerical value more strictly by using data of a larger number of samples. In this case, specifically, a value obtained by subtracting 2SD or the like from the average value of cells forming bone, Among the values obtained by adding 2SD or the like to the average value of the cells that did not form, it can be approximated as a higher numerical value.

-TRAP staining For TRAP staining, for example, a TRAP staining kit (Wako Pure Chemical Industries, Ltd.) in which the fixing solution is a buffer solution containing 50 mM tartaric acid (pH 5.0, chromogenic substrate 30 mg / vial) can be used. Below, the TRAP dyeing | evaluation method at the time of using an above described TRAP dyeing | staining kit is shown.
First, D-PBS is warmed in a water bath and cultured in a differentiation induction medium containing 10% serum, 1% penicillin streptomycin, 1% amphotericin B and dexamethasone, β glycerophosphate, and ascorbic acid in α-MEM.
For further rigor, Osteo Clast as a medium other than the above medium
It is preferable to use precursor Basal Medium M-CSF (-), RANKL (-), Osteo Clast precursor Basal Medium M-CSF (+), RANKL (+) and compare the results.
Next, the medium in the 96-well plate during culture is removed, D-PBS is added at 250 μl / well and washed, and D-PBS is removed. Next, 50 μl / well of the TRAP staining kit fixing solution is added and fixed for 5 minutes. After 5 minutes, the fixing solution is removed, and distilled water is added at 250 μl / well for washing to remove the distilled water. This washing with distilled water is repeated three times. Before removing distilled water for the third time, turn off the electricity of the clean bench, add 5 ml of 50 mM tartaric acid-containing buffer to the chromogenic substrate of the TRAP staining kit, and mix by vortex to prepare the chromogenic substrate. After preparing the chromogenic substrate, distilled water is removed, 100 μl / well of chromogenic substrate is added, and the mixture is incubated for 1 hour at 5% CO ₂ and 37 ° C. (humidity 95% rH or more). After the incubation, the chromogenic substrate is removed, and distilled water is added at 250 μl / well and washed to remove the distilled water. This washing with distilled water is repeated twice. After washing, add distilled water at 50 μl / well and examine the cells for staining under a microscope, and take a cell photograph.

As shown in FIG. 10A, when the measured TRAP staining is positive, it is determined that the fraction contains osteogenic (+) cells.
However, as shown in FIG. 10B, when the measured TRAP staining is negative, it is determined that the fraction is highly likely not to contain osteogenic (+) cells.

-Alizarin red staining In order to perform alizarin red staining, cells are seeded in a 12-well dish at a density of 2.0 × 10 ⁴ / well. The next day, the medium is changed to a differentiation-inducing medium, and thereafter the medium is changed twice a week to induce differentiation for three weeks.
The medium is then aspirated and the cells are washed twice with D-PBS. Thereafter, the cells are fixed with a fixing solution (70% ethanol) at minus 20 degrees for 1 hour. Aspirate the fixative and wash twice with distilled water.
Next, Alizarin Red S solution (40 mM, pH 4.2) is added, and staining is performed at room temperature for 10 minutes. The pH is adjusted with a 25% aqueous ammonium solution. Aspirate the staining solution and wash the cells with distilled water until non-specific staining is completely eliminated. Then macro and micrographs are taken.

FIG. 11 shows the results of staining with alizarin red. The left side is a sample that has not been induced to differentiate (control), and the right side is a sample after differentiation induction.
As shown in FIG. 11A, when the measured alizarin red staining is positive, it is determined as a fraction containing osteogenic (+) cells.
However, as shown in FIG. 11B, when the measured alizarin red staining is negative, it is determined that the fraction is highly likely not to contain osteogenic (+) cells.

In the method for evaluating cultured cells according to the present invention, a plurality of test values or an adaptation order for indices and determination criteria are provided, and then a flow chart of them is used to accurately form bone formation of cells regardless of experience. It is preferable to determine the performance. In particular, it is preferable to predict and determine the function of cells with high accuracy, including at least three test values and indices, and combining them with a statistical basis. In the present invention, a plurality of test values and indices are evaluated in a flow chart for human cells having variations, thereby making it possible to improve the influence of the variations to such an extent that there is no clinical problem.
In the method for evaluating cultured cells according to the present invention, for each test value, even if the sample is included in the applicable range, it is determined that the cell is not the target cell by referring to another index. There is also.

Claims (14)

1. In order to determine a target cell from a cultured cell, a plurality of test values or indices of the cultured cell are measured, and a combination thereof, an evaluation method for accurately predicting and determining the function of the cell,
   For each test value, not only can you specify a certain compatible range, but even if the sample is outside the applicable range, you can determine the target cell by referring to another test value or index. A method for evaluating cultured cells, wherein
2. In a method for evaluating cultured cells having a primary determination criterion, a secondary determination criterion, and a tertiary determination criterion,
   The cells that have undergone the primary determination are further determined as bone forming (+) cells by performing a secondary determination.
   A method for evaluating cultured osteogenic cells, characterized in that a bone-forming (+) cell is determined by performing a third determination on a cell that is difficult to determine whether it is a bone-forming (+) cell.
3. In the primary determination according to claim 2, the determination criteria are ALP activity (alkaline phosphatase activity), ALP index (ALP activity of differentiation induction group / ALP activity of non-differentiation induction group), cell proliferation ability (cells after differentiation induction) (The number of cells / the number of initially seeded cells) is a value that satisfies a primary determination reference value or more.
4. In the secondary determination according to claim 2, the determination criterion is whether or not all of ALP activity, ALP index, and cell proliferation ability satisfy the reference value of the secondary determination,
   A cell that satisfies both the primary determination and the secondary determination is determined as a bone-forming (+) cell,
   For cells that did not meet the criteria in the primary judgment but met the criteria in the secondary judgment, the cells moved to the tertiary judgment criteria,
   A method for evaluating cultured osteogenic cells, wherein cells that do not satisfy the criteria in the secondary determination are determined as osteogenic (−) cells.
5. In the tertiary determination according to claim 2, the determination criterion is whether or not any of surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining, or alizarin red staining satisfies the criterion,
   A method for evaluating cultured osteogenic cells, characterized in that cells satisfying this criterion are determined as osteogenic (+) cells, and cells not satisfying the criteria are determined as osteogenic (−) cells.
6. 4. The method for evaluating cultured osteogenic cells according to claim 3, wherein the reference value is each (average value + 2SD) of osteogenic (−) cells.
7. 5. The secondary determination according to claim 4, wherein the reference value is a lower limit value of each measurement value of bone forming (+) cells (average value −2SD) or each value of bone forming (+) cells. Method for evaluating osteogenic cells.
8. 6. The tertiary determination according to claim 5, wherein the surface antigen (HLA-DR) analysis criterion is about 6% or more of measured HLA-DR positive cells. Evaluation method.
9. 7. The method for evaluating cultured osteogenic cells according to claim 6, wherein the reference value of ALP activity is about 166 units (μmol p-nitrophenol produced / min / μg protein).
10. 7. The method for evaluating cultured osteogenic cells according to claim 6, wherein the reference value of the ALP index is about 3.95.
11. 7. The method for evaluating cultured osteogenic cells according to claim 6, wherein the reference value of the cell growth ability is about 9.7.
12. 8. The method for evaluating cultured osteogenic cells according to claim 7, wherein the reference value of ALP activity is about 67 units (μmol p-nitrophenol produced / min / μg protein).
13. 8. The method for evaluating cultured osteogenic cells according to claim 7, wherein the reference value of the ALP index is about 1.01.
14. 8. The method for evaluating cultured osteogenic cells according to claim 7, wherein the reference value of the cell proliferation ability is about 5.6.

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