KR20110116965A - Method for assessing cultured cell - Google Patents

Abstract

The present invention relates to a method for evaluating the type and function of cultured cells, which are difficult to judge on a predetermined basis because of individual variation, and for example, provide a method for evaluating bone formation ability. The method for evaluating cultured cells according to the present invention measures a plurality of test values or indicators of cultured cells to determine target cells from the cultured cells, and precisely predicts and determines the function of the cells by a combination thereof. The evaluation method is characterized in that it is possible not only to designate a certain suitable range for each test value, but also to determine that the sample is out of the adaptation range as a target cell with reference to other test values or indicators.

Description

Evaluation method of cultured cell {METHOD FOR ASSESSING CULTURED CELL}

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

There is a demand for the realization of regenerative medicine which aims at the regeneration of defective living tissue. Regenerative medicine is a medical technology that uses cells, biomaterials, and cell growth factors to produce the same form or function as the original tissues.

In regenerative medicine, mesenchymal stem cells are generally used. It is known that these mesenchymal stem cells can be induced to differentiate into various cells such as fat cells and osteoblasts, but it was difficult to determine in advance whether the obtained cells have a desired function.

In addition, bone marrow-derived mesenchymal stem cells are mainly used in bone regeneration medicine, and other reports include fat-derived hepatocytes, periosteum-derived hepatocytes, and synovial stem-derived hepatocytes. Among them, bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are relatively easy to harvest and have very high bone formation ability, and are currently used in clinical applications of almost all bone regeneration.

However, from the present inventors' report, it became clear that the individual difference of cells is large in the osteogenic cells derived from humans. In addition, it became clear that bone formation ability is rapidly lost by culture. Therefore, it is important to examine whether the cells to be transplanted actually have bone formation ability, but the methods reported so far have proved insufficient. In particular, conventionally, alkaline phosphatase activity (ALP activity) of cells (Patent Document 1), gene expression of bone markers (Non-Patent Document 1), etc. have been used. ALP activity is a useful assessment for osteogenic cells, but it is difficult to define osteogenic cells alone. In addition, gene expression of bone markers is also useful evaluation, but the study results of the present inventors also recognized a case where the individual difference is large in expression and it is difficult when used as an evaluation. In particular, for evaluation of human-derived cells with large individual differences, it is considered that one criterion is not sufficient and it is important to increase reliability by combining a plurality of evaluation methods, but such an evaluation method has not been reported so far.

Osteoblasts are defined as cells essential for bone formation, such as osteoblasts. Herein, cells that can form bone in particular ectopically are defined as osteogenic cells. Ectopicity is the ability to form bones in the absence of bone. Ectopic bone formation capacity is verified, for example, by subcutaneous transplantation of immunocompromised animals with a carrier that maintains human cells. Actually, the site where bone regeneration is expected is in the vicinity of the bone, but since bone regeneration needs to be performed toward the non-bone area, the ability required for bone regeneration is considered to be the most appropriate evaluation method. However, there is no method for evaluating manufactured cells from this point of view.

Japanese Laid-Open Patent Publication No. 2005-58225

Nishimura et al., J Bone Miner Metab. 26, 203-212

That is, in order to perform bone formation by transplantation, it goes through the process shown in FIG. As shown in FIG. 1, osteogenic cells coexist with an implant material, for example, pseudo-granules, to decompose pseudo-granules by osteoclasts at the site of transplantation, while being active by osteoblasts and the like. Regenerate the bone (眞 性骨). At this time, the number of bone-forming cells in the graft material is of course an important factor, but it is important that the proper bone formation ability is maintained. If the number of these osteogenic cells is small or the cells having deteriorated osteogenic capacity are used, proper bone formation is not performed, but the actual confirmation of bone formation requires a very long time. If the bone formation is not performed properly, the mesenchymal stem cells should be collected, cultured, transplanted, and regenerated, and wasteful months will be wasted. The burdensome procedure is repeated. Therefore, it is important to properly evaluate the function of cultured cells.

On the other hand, even when the determination of the bone formation ability during the culture of the cells, the test during differentiation induction also takes several weeks, so it cannot be easily repeated.

However, evaluation of whether a target cell, for example, osteogenic cell, which has been conventionally performed, is carried out by a single determination method as described above. It is conceivable to improperly determine the cells, while lowering the evaluation criteria makes use of cultured cells that are incapable of bone formation for transplantation. In addition, studies by the present inventors have made it clear that almost all test values in human cells have a large variation among individuals, and it is difficult to make an accurate judgment only by a constant reference value or a single judgment criterion, and even a realistic judgment is difficult.

This invention is performed in view of the said subject, Comprising: It aims at providing the reliable evaluation method which can determine the target cell. Although the evaluation method according to the present invention can be suitably used for osteogenic cells, the problem of deviation of human cells is considered to be a common problem for most cells that are widely targeted for regenerative medicine or cell therapy in addition to osteogenic cells. do. Therefore, the effect of the present invention is to provide an effective evaluation method for whether or not osteogenic cells as well as cells widely obtained in the treatment of cells using human cells are the cells of interest and whether they have the necessary functions for treatment. .

As a result of earnestly research by the present inventors in order to solve the said subject, it is possible to prepare a reliable evaluation method by measuring a plurality of test values or indicators of cultured cells and combining them in order to determine the cells of interest in the cultured cells. I found something. In addition, for osteogenic cells (osteoblastic (+) cells), the bone formation ability test method shown in FIG. The present invention was completed by discovering that useful methods for evaluating osteogenic cells can be prepared.

That is, the method for evaluating cultured cells according to the present invention measures a plurality of test values or indicators of cultured cells in order to determine the cells of interest in the cultured cells, and accurately predicts and determines the function of the cells by a combination thereof. As an evaluation method to

In addition to designating a certain suitable range for each test value, it is possible to determine that the sample is a target cell by referring to other test values or indicators even if the sample is out of the adaptation range.

In addition, the method for evaluating cultured osteogenic cells of the present invention is a method for evaluating cultured cells having a first criterion, a second criterion, and a third criterion.

By further performing a second judgment on the cells that were subjected to the first judgment, bone forming (+) cells were determined,

It is characterized in that bone forming (+) cells are determined by further performing third order determination of cells that are difficult to determine whether they are osteogenic (+) cells.

In the primary judgment, the criterion for determination was among ALP activity (alkali phosphatase activity), ALP index (ALP activity of differentiation inducing group / ALP activity of non-differentiation inducing group), and cell proliferation capacity (cell number after differentiation induction / initial seeding cell number). It is preferable whether or not which one satisfies more than the reference value of the primary determination.

In the second judgment, the criterion is whether all of the ALP activity, ALP index, and cell proliferation capacity satisfy or exceed the reference value of the second judgment.

The cells satisfying the criteria of both the primary and secondary judgments are determined to be osteogenic (+) cells,

For cells that did not meet the criteria in the first judgment but met the criteria in the second judgment, the procedure proceeds to the third judgment.

It is preferable to determine that cells which do not satisfy the criteria in the second judgment are osteogenic (-) cells.

In the third determination, the criterion is based on whether the surface antigen (HLA-DR) analysis by TR flow cytometry, TRAP staining, or alizarin red staining satisfy the criteria.

It is preferable to determine that cells satisfying this criterion are osteogenic (+) cells and unsatisfactory cells are osteogenic (-) cells.

In the first judgment, the reference value is preferably each (mean value + 2SD) of osteogenic (-) cells.

In the second judgment, the reference value is preferably the lower limit of each measured value of the osteogenic (+) cells (mean value-2SD) or each measured value of the osteogenic (+) cells.

The criteria for surface antigen (HLA-DR) interpretation in the third determination is preferably about 6% or more of the measured HLA-DR positive cells.

In the first determination, the reference value of ALP activity is preferably about 166 units (μmol prepared p-nitrophenol / min / μg protein).

And wherein the reference value of the ALP index in the first judgment is about 3.95.

In the first determination, the reference value of cell proliferation ability is preferably about 9.7.

In the second determination, the reference value of ALP activity is preferably about 67 units (μmol prepared p-nitrophenol / min / μg protein).

In the second determination, the reference value of the ALP index is preferably about 1.01.

In the second determination, the reference value of the cell proliferation ability is preferably about 5.6.

In the method for evaluating osteogenic cells according to the present invention, ALP activity used as a criterion is recognized for high activity on osteogenic cells, for example, osteoblasts. If the ALP activity after induction of differentiation is low, it may be considered that the cells do not contain osteogenic cells or do not have osteogenic ability. When phosphorus ALP index rises, it turns out that it is osteogenic cell and may have osteogenic ability. On the other hand, if ALP activity is sufficiently high, it is very likely that the induction of differentiation of osteogenic cells is good. However, even if ALP activity is high, if cell proliferation is lost, sufficient bone formation cannot be expected after bone transplantation.

Therefore, the evaluation method according to the present invention also evaluates cell proliferation ability.

Even if these three evaluations (ALP activity, ALP index, and cell proliferation capacity) are combined, more careful evaluation is required for the group having difficulty in determining the presence or absence of osteogenic cells. The present inventors further positively identified one of these groups using the results of analysis of surface antigen (HLA-DR) by flow cytometry, TRAP staining, and alizarin red staining (positive HLA-DR fraction by flow cytometry). , TRAP positive, Alizarin red positive) and secondary bone formation cells are judged to be adjuvant, giving a reasonable basis to almost all individual samples.

Although the direct relationship between these HLA-DR positive cells and bone formation ability is unclear, the inventors confirmed a clear correlation between HLA-DR activity and bone formation ability. TRAP staining (tartrate-resistant acidic phosphatase staining) is used as an osteoclast marker, but hepatocytes differentiate into osteogenic cells because the maturation from osteoclast progenitor cells to osteoclasts requires interaction with osteoblasts (osteoblasts). It is thought to be used as an indirect indicator of the process. In addition, alizarin red staining shows the coalification ability in vitro and stained the deposition of calcium. As confirmed by the present inventors, it was found that the cells showing osteogenic ability became positive for alizarin red staining, and that the cells in which passage was lost in most cells were stained negative.

As described above, according to the present invention, a relatively simple and clear method of selecting ALP activity, ALP index, and cell proliferative capacity clearly selects those having bone formation ability and those without bone formation ability. DR analysis, TRAP staining, and alizarin red staining are carried out to prevent destruction by "no bone formation ability" even though the bone formation ability can be sufficiently expected.

According to the present invention, there can be provided a method capable of evaluating the type and function of a target cell, which has been difficult to judge based on a certain standard because of variation by an individual. In addition, when evaluating osteogenic cells, it becomes possible to almost certainly evaluate the osteogenic capacity of cultured cells, and can provide a method for the evaluation essential for treatment with cells.

1 is a bone formation test schedule,
2 is a flowchart showing a method for evaluating osteogenic cells according to the present invention,
3 is an explanatory diagram of a culture step, differentiation induction process of osteogenic cells to which the present invention is preferably applied,
4 is a diagram showing the distribution of ALP activity of human cultured osteoblast-like cells;
5 shows the distribution of the ALP index of human cultured osteoblast-like cells;
6 is a diagram showing the distribution of proliferative capacity of human cultured osteoblast-like cells;
7 shows the results of ALP activity measurement,
8 is a result of measuring cell proliferation ability,
9 is a surface antigen analysis result by flow cytometry,
10 shows the results of TRAP staining, and
11 shows alizarin red staining results.

EMBODIMENT OF THE INVENTION Hereinafter, preferred embodiment of this invention is described. Hereinafter, the case where the target cell is a osteogenic cell is demonstrated as an example. First, the manufacturing method of the granular cultured bone which can be effectively applied to a treatment by the evaluation method of the osteogenic cell of this invention is demonstrated using FIG. Granular 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.

In addition, although the following process shows the form using a bone marrow-derived cell, the evaluation method of this invention is produced by isolate | separating culture from periosteum, adipose, peripheral blood, etc. in addition to bone marrow, and inducing differentiation into cultured osteoblast-like cells by the same method. It can also be preferably used in granular cultured bone.

Collection of bone marrow

Bone marrow fluid is collected approximately one month before surgery. First, local anesthesia is applied to the bone marrow fluid collection unit, which is aseptically aspirated and recovered from the posterior iliac bone function.

Cultivation of bone marrow-derived mesenchymal stem cells

Bone marrow solution 10 diluted four-fold in the cell culture medium is sprinkled on the cell culture flask 12, and culture is started as shown in FIG. 3 (A), and the whole medium is exchanged on day 4 after the start of culture. As the cell culture medium, both αMEM or serum-free medium containing serum may be used for both primary culture and differentiation culture.

Confirmation of infection with normal bacteria and fungi in the culturing process is carried out by observing the medium every time the medium is changed (the medium becomes turbid if infected), and also at the start of the culture, before induction of differentiation and at the time of recovery of cultured osteoblast-like cells. Is confirmed by a sterility test (whether the criteria of the Japanese pharmacy room are satisfied).

Thereafter, the whole medium is replaced twice a week. The passage is judged based on the state of the cells, but is performed approximately 21 to 28 days after the start of the culture. At passage, live cell counts are counted.

Sowing of mesenchymal stem cells

Passage is performed as follows. After the medium of the flask is absorbed, it is washed with darbecholine acid buffer (D-PBS), and then the D-PBS is absorbed to add a cell dissociating agent and incubated at 37 ° C for 10 minutes. After confirming that the cells have been dissociated, the cells are recovered by adding D-PBS or medium, and then centrifuged. Suspension again in the medium to measure the cell number. After the measurement, the bone marrow fluid 10 cultured in the deep bottom container 16 into which the 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.

Induce differentiation into cultured osteoblast-like cells

The cells are seeded into the porous pseudo bone granules 14, and then left overnight to restore the function of the cells. After 1 day, as shown in Fig. 3D, cells such as cells involved in bone formation and the porous pseudo bone granules 14 are deposited at the bottom of the deep bottom container 16. After confirming this, the medium is replaced with a differentiation inducing medium. Differentiation induction period is 1 to 3 weeks, medium change is about twice a week. As the differentiation induction proceeds, the morphology of the cultured osteoblast-like cells attached around the pseudo bone granules 14 is changed as shown in FIG. 3E, and the bone matrix proteins are secreted to form a mass.

On the other hand, cells are seeded in 12-well culture dishes for evaluation in parallel with this procedure. Cells are 2 × 10 ⁴ per well.

Hereinafter, the evaluation method of the osteogenic cell which concerns on this invention is demonstrated using the flowchart of FIG. As shown in FIG. 2, osteogenic cells are determined based on the primary criteria, the secondary criteria, and the tertiary criteria. In addition, the ratio in the parentheses of FIG. 2 shows the ratio with respect to the original cell. However, these ratios are not particularly limited as an example, and vary depending on the selection of cells to be used.

Primary criterion

Primary determinations are made based on ALP activity, ALP index, and cell proliferation ability.

In the primary judgment, if the reference value of any one of the above three evaluation methods is exceeded, the cell is determined as the primary judgment criterion (○), and if not exceeding all the reference values, the cell is referred to as the primary judgment criterion (×). Is determined.

Second judgment standard

Like the primary determination, the secondary determination is performed based on ALP activity, ALP index, and cell proliferation ability. However, as shown below, these evaluation reference values differ from a primary determination.

The primary judgment criteria (○) cells are determined to be osteogenic (+) cells if they exceed the reference values in all three evaluation methods in the second judgment, and if the cells do not exceed any of the reference values, It is determined to be osteogenic (-) cells.

If the primary criterion (×) cell satisfies all of the above three evaluation methods in the secondary judgment, the cell proceeds to the tertiary judgment criterion, and if the cell does not exceed any one of the reference values, the cell is formed into bone formation (- Cell).

3rd judgment standard

Tertiary determination is performed based on surface antigen analysis, TRAP staining, and alizarin red staining by flow cytometry.

Cells are determined to be osteogenic (+) cells if either one is positive in the third judgment, and cells are osteogenic (-) cells if all negative.

In the method for evaluating osteogenic cells according to the present invention, a plurality of judgment criteria are combined as described above to determine whether the osteogenic cells are osteogenic (+) cells. The importance of combining these methods is as follows.

As described above, ALP activity has been used to confirm the bone formation ability so far. However, as shown in Fig. 4, the distribution of ALP activity is close to cells with osteogenic ability and cells not having, and it is difficult to set clear boundaries. When set to low, cells that do not have osteogenic ability are incorporated a lot, and when set to high, cells with osteogenic capacity are excluded.

In fact, when considering the criterion of determination using ALP activity, it is conceivable to set the boundary at, for example, the mean value -2SD of the ALP activity of cells showing bone formation. If it is assumed that the ALP activity value is normally distributed, 95.45% of cells having bone formation ability can be recovered. However, referring to FIG. 4, most cells having no bone formation ability can be recovered.

It is also conceivable to set the lower limit of the osteogenic (+) cells. However, in FIG. 4, even though the lower limit of the osteogenic (+) cells is present, many cells do not have osteogenic capacity.

As described above, the inventors have reported the usefulness of the ALP index in determining bone formation ability. However, as shown in FIG. 5, the distribution of the ALP index is also close to cells with osteogenic ability and cells without, and it is difficult to set clear boundaries.

Moreover, although it has not been used for the determination of bone formation ability so far, it is clear by the present inventors that the proliferative ability of cell proliferation ability, especially at the time of differentiation induction, has a relationship with bone formation ability. However, as shown in Fig. 6, the distribution of cell proliferation ability is also close to cells having no bone formation ability and cells which do not have a clear boundary.

Since it is as mentioned above, it is understood that proper determination of bone forming (+) cells is difficult with the conventionally used indices and simple combinations thereof.

Therefore, the present inventors have a strict criterion (primary criterion) capable of recovering only osteogenic (+) cells because of the overlap of data of osteogenic (+) cells and osteogenic (-) cells. It was the first time.

However, since the primary determination criteria include a large number of osteogenic (+) cells in the unselected portions, these reliefs are performed using the secondary determination criteria and the tertiary determination criteria.

In addition, there is a possibility that slightly osteogenic (-) cells are also included in cells that satisfy the primary criterion. For this reason, the secondary determination standard is implemented also about these cells.

As described above, in the method for evaluating osteogenic cells according to the present invention, first to third judgments are performed, but the osteogenic (+) cells determined as a result are almost 100% osteogenic (+) by the present inventors. It is clear that it is a cell.

Subsequently, the criteria used in the first and second judgments will be described. First, the reference value and the evaluation method (ALP activity, ALP index, cell proliferation ability) will be described in detail.

In the present invention, the reference value of the primary judgment was set to each (mean value + 2SD) of osteogenic (-) cells. At this time, assuming that the sample data have a normal distribution, the probability of accidentally including cells without bone formation ability is (100-95.45) /2=2.275%.

In addition, the reference value of the secondary determination was set to the lower limit of each measured value of the bone formation (+) cells (average value -2SD) or each measurement value of the bone formation (+) cells.

It is also conceivable that the reference value changes depending on the population (gender, age, race, etc.) of the patient taking the cells. In other words, it is important to select an appropriate value for the reference value for the patient. Therefore, the reference values shown below are determined by the cells of the patient collected by the present inventors, and are not limited thereto. It is also possible to set this value more precisely with most data.

In addition, although the specific reference value of the secondary determination shown below takes the lower limit of each measured value of bone formation (+) cells as an example, the valid result is obtained even if each (average value-2SD) of bone formation (+) cells is used. .

ALP activity

For the measurement of ALP activity, for example, p-nitrophenylphosphate purification set (Sigma-Aldrich) and WST-8 (produced by Dojindo Research Institute), which are cell aggregation kits, can be used.

Hereinafter, the measurement method is shown to the example where ALP activity measurement is performed with the said product. First, 100 μl of WST-8 solution is added to each well. After the color reaction is carried out for 1 to 4 hours in a carbon dioxide incubator, the absorbance is measured using a microplate reader. After WST-8 analysis, cell membrane proteins were dissolved and extracted, p-nitrophenylphosphate solution was added to the lysate, and after standing at room temperature for 10 minutes, the reaction was stopped with NaOH and p-nitrophenol (p-nitrophenyl Absorbance of the phosphate degradation product by ALP) is measured. ALP activity is expressed in moles of protein per hour of p-nitrophenol / mass of protein (μ-mol prepared p-nitrophenol / min / μg protein). Or p-nitrophenol absorbance (OD; 405 nm) / WST-8 absorbance (OD; 450 nm).

7 shows an example of the result of ALP activity measurement.

The mean ALP activity + 2SD of the osteogenic (-) cells was 2.31 {p-nitrophenol absorbance (OD; 405 nm) / WST-8 absorbance (OD; 450 nm)}. This value corresponds to 166 units (μmol prepared p-nitrophenol / min / μg protein). For this reason, the present inventors set the reference value of the primary determination of ALP activity to about 166 units (micromol-made p-nitrophenol / min / microg protein). That is, the reference value of the primary determination of ALP activity is preferably 166 ± 5 units (μmol prepared p-nitrophenol / min / μg protein).

The conversion formula for this unit of ALP activity per cell (μmol prepared p-nitrophenol / min / μg protein) is required by the present inventors by the cells used in this study.

The lower limit of the ALP activity measurement of osteogenic (+) cells was 0.93 {p-nitrophenol absorbance (OD; 405 nm) / WST-8 absorbance (OD; 450 nm)}. This value corresponds to 67 units (μmol prepared p-nitrophenol / min / μg protein). For this reason, the present inventors set the reference value of the secondary determination of ALP activity to about 67 units (p-nitrophenol / min / microg protein manufactured by micromol). That is, it is preferable that the reference value of the secondary determination of ALP activity is 67 ± 5 units (μmol prepared p-nitrophenol / min / μg protein).

ALP index

The ALP index measures ALP activity on cells to which differentiation induction medium is added (differentiation induction group) and cells into which medium is normally added (non-differentiation induction group) as a control (ALP activity / differentiation induction group of differentiation induction group). ALP activity).

The mean ALP index + 2SD of osteogenic (-) cells was 3.95. For this reason, the present inventors set the reference value of the primary determination of an ALP index to about 3.95. That is, the reference value of the primary determination of the ALP index is preferably 3.95 ± 0.15.

In addition, the lower limit of the ALP index measurement value of osteogenic (+) cells was 1.01. For this reason, the present inventors set the reference value of the secondary determination of an ALP index to 1.01. That is, it is preferable that the reference value of the secondary determination of the ALP index is 1.01 ± 0.15.

Cell proliferation ability

The cell proliferation capacity can be determined by the ratio of the number of cells before and after the culture, that is, the number of cells after the induction of differentiation / initial seeding cell number. Although actually obtained by measuring the number of cells after the end of the differentiation induction period, it is possible to make the seeded cells constant and count as (the number of recovered cells / the number of seeded cells). Herein, the cell proliferation rate in the differentiation induction period is defined as cell proliferation capacity. For example, it can determine with the ratio of the OD value of the said cell aggregation kit (WST-8). That is, after performing the color reaction, the absorbance (OD value) of 450 nm is measured, and the cell proliferation rate during induction of differentiation can be determined by dividing by the OD value of WST-8 with respect to the number of cells before induction of differentiation. In addition, the determination of cell proliferation capacity according to the present invention can be applied to the first to fourth passages.

8 shows an example of cell proliferation measurement results.

The mean proliferative capacity of osteogenic (-) cells + 2SD was 9.7. For this reason, the present inventors set the reference value of the primary determination of cell proliferation ability to about 9.7. That is, the reference value for the primary determination of cell proliferation ability is preferably 9.7 ± 0.3.

In addition, the lower limit of the proliferative capacity measurement value of the osteogenic (+) cells was 5.6. For this reason, the present inventors set the reference value of the secondary determination of cell proliferation ability to about 5.6. That is, the reference value for the primary determination of cell proliferation ability is preferably 5.6 ± 0.3.

Subsequently, the criteria and evaluation methods (surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining and alizarin red staining) used in the third determination will be described in detail.

In the present invention, the criterion for the third judgment was that surface antigen (HLA-DR) positive cells were 6% or more, TRAP positive, and alizarin red positive.

Surface antigen analysis by flow cytometry

Six-color flow cytometry analysis can use a conventional flow cytometer, but here we use a FACS Aria flow cytometer (BDIS).

The following substances were used for the antibody. Fluorescent isothiocyanate complex (FITC-), phycoerythrin complex (PE-), peridinine chlorophyll protein complex (PerCP-Cy5.5-), aropicocyanine complex (APC-), Alexa Fluor 405 complex used It became.

In addition to the complex, biotinylated antibodies against HLA-ABC, HLA-DR, CD3, CD14, CD19, CD34, CD73, CD90, CD106, CD146, mouse-IgG1k, mouse-IgM (all from BD Pharmingen), CD10 And a biotinylated antibody against CD 29 (Dako) and a biotinylated antibody against CD45 (Invitrogen) were also used. A CD105 antibody (Immunotech) covalently bound to FITC was also used. The biotinylated antibody was detected by Streptoavidin Pacific Blue (Invitrogen) or Streptoavidin PerCP-Cy5.5 (BD Pharmingen) complex.

STRO-1 antibody (manufactured by R & D Systems) was detected with PE-binding anti mouse IgM. In addition, propidium iodide (Dojindo Research Institute) was used to detect dead cells.

The surface antigen analysis by flow cytometry was performed using the said several dozen types of antibodies. As a result of the investigation by the present inventors, it has become clear that the cells having bone formation ability may or may not be obtained even when cultured by bone marrow-derived mesenchymal stem cells, but the above-mentioned antibodies other than the antibody against HLA-DR In the expression of, no difference was found in cells with osteogenic capacity and cells without. However, it became clear that only the expression of antibodies to HLA-DR differed significantly. For this reason, in the surface antigen analysis which concerns on this invention, the biotinylated antibody to HLA-DR is used as an antibody.

Hereinafter, the surface antigen analysis method in the case of using the biotinylated antibody to HLA-DR which is preferable for this invention is shown. Passage 0 and 3 cells were detected by trypsin-EDTA, and 1 × 10 ⁶ cells were suspended in 50 μl of ice-cold phosphate buffered saline (PBS: Nissui Seiyaku Co., Ltd.). The cells were then incubated with biotinylated antibodies against HLA-DR for 20 minutes on ice. Cells were then washed and incubated with streptoavidin complex for 20 minutes on ice. Finally the cells were washed, resuspended in 200 μl of 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. X-axis and Y-axis in Fig. 9 show responses to other antibodies (HLA-DR and CD14, respectively). The lower right numerical value represents the fraction of the antibody (CD14) negative on the Y axis and the antibody (HLA-DR) positive on the X axis.

When the HLA-DR positive cells measured as shown in FIG. 9 (A) are 6% or more, it is determined that the cell fraction including the osteogenic (+) cells. However, when the HLA-DR positive cells measured as shown in FIG. 9 (B) are less than 6%, it is determined as a fraction likely to not contain osteogenic (+) cells.

The lower limit of 6% of these HLA-DR positive cells is an empirically determined value found by the present inventors in many subjects, but the present invention is not limited to this value. In other words, it is possible to set this value more precisely based on the data of most samples, in which case, the average value of the cells that formed bones was subtracted from 2SD, and the average value of cells that did not form bones. It can be approximated as a higher numerical value among the values which added 2SD etc.

TRAP staining

For TRAP staining, for example, a TRAP staining kit (Wako Pure Chemical Co., Ltd.), in which the fixative is 50 mM tartaric acid-containing buffer (pH5.0, chromogenic substrate 30 mg / vial), can be used. The TRAP staining evaluation method in the case of using the above TRAP staining kit is shown below.

First, heat D-PBS with a water bath, and in a differentiation induction medium containing 10% serum, 1% penicillin streptomycin, 1% cryptotericin B and dexamethasone, βglycerophosphate, and ascorbic acid in α-MEM. Incubate.

In addition, in order to obtain the rigor, in addition to the medium, compared the results using Osteo Clast precursor Basal Medium M-CSF (-), RANKL (-), Osteo Clast precursor Basal Medium M-CSF (+), RANKL (+) It is desirable to.

Subsequently, the medium of the 96-well plate in culture is removed, and D-PBS is added and washed by 250 μl / well, and D-PBS is removed. Subsequently, 50 microliters / well of the fixed solution of a TRAP staining kit is added, and fixation is performed for 5 minutes. After 5 minutes, the fixed solution was removed, and distilled water was added and washed by 250 µl / well, and distilled water was removed. This washing with distilled water is repeated three times. Before the third distilled water was removed, the clean bench was turned off, and 5 ml of 50 mM tartaric acid-containing buffer was added to the colored substrate of the TRAP staining kit and mixed by Voltics to prepare a colored substrate. After preparation of the chromogenic substrate, distilled water was removed, and 100 µl / well of the chromogenic substrate was added, followed by incubation for 1 hour at 5% CO ₂ and 37 ° C (95% rH or higher humidity). After incubation, the chromogenic substrate is removed, distilled water is added 250 μl / well and washed to remove distilled water. This washing with distilled water is repeated twice. After washing, distilled water is added in 50 µl / well, and the cells are photographed under the microscope for the presence of stained cells.

When TRAP staining measured as shown in Fig. 10 (A) is positive, it is determined that the fraction containing the osteogenic (+) cells.

However, when TRAP staining measured as shown in Fig. 10B is negative, it is determined that the fraction is unlikely to contain osteogenic (+) cells.

Alizarin Red Dyeing

Cells are seeded in 12-well dishes at a density of 2.0 × 10 ⁴ / well for alizarin red staining. The next day, the medium is changed to differentiation induction medium, and then the medium is changed twice a week to induce differentiation for 3 weeks.

Subsequently, the medium is aspirated and the cells are washed twice with D-PBS. Thereafter, the cells are fixed for one hour at minus 20 degrees with a fixed solution (70% ethanol). The fixed solution is aspirated and washed twice with distilled water.

Subsequently, alizarin red (S) solution (40 mM, pH4.2) was added and stained for 10 minutes at room temperature. pH is adjusted with 25% aqueous ammonium solution. The stain is aspirated and the cells are washed with distilled water until the nonspecific staining is completely lost. Thereafter, macro and micrographs are taken.

11 shows the results of staining alizarin red. In addition, the left side is the sample (control) which did not induce differentiation, and the right side is the sample after induction of differentiation.

When alizarin red staining measured as shown in Fig. 11 (A) is positive, it is determined that the fraction containing osteogenic (+) cells.

However, when the alizarin red staining measured as shown in Fig. 11B is negative, it is determined that the fraction is unlikely to contain osteogenic (+) cells.

In the method for evaluating cultured cells according to the present invention, it is preferable to set the order of adaptation and determination criteria for a plurality of test values or indicators, and to make them flow chart to accurately determine the bone formation ability of the cells regardless of experience. . In particular, it is desirable to include at least three test values and indices, and to accurately predict and determine the function of the cells on a statistical basis by these combinations. According to the present invention, it is possible to improve the effect of the deviation to a clinically trouble-free level by evaluating a plurality of test values and indices with respect to human cells having a deviation.

In addition, in the method for evaluating cultured cells according to the present invention, it may be determined that even a sample included in an adaptation range for each test value is not a target cell with reference to other indicators.

10: bone marrow fluid
12: flask for cell culture
14: porous pseudo-granule
16: deep bottom container

Claims (14)

As an evaluation method for measuring a plurality of test values or indicators of cultured cells to determine target cells from the cultured cells, and accurately predicting and determining the function of the cells by a combination thereof.
A method for evaluating cultured cells, characterized in that it is possible not only to designate a certain suitable range for each test value, but also to determine a target cell with reference to other test values or indicators even if the sample deviates from the adaptation range. As a method for evaluating cultured cells having a primary criterion, a secondary criterion, and a tertiary criterion,
By further performing a second judgment on the cells that were subjected to the first judgment, bone forming (+) cells were determined,
A method for evaluating cultured osteogenic cells, characterized in that osteogenic (+) cells are determined by further performing a third determination of cells that are difficult to determine whether they are osteogenic (+) cells. The method of claim 2,
In the above first judgment, the criterion of determination is ALP activity (alkali phosphatase activity), ALP index (ALP activity of differentiation inducing group / ALP activity of non-differentiation inducing group), cell proliferation capacity (cell number after differentiation induction / initial seeding cell number) The evaluation method of the cultured osteogenic cells characterized in that any one of the above meets the reference value of the primary determination. The method of claim 2,
In the second judgment, the judgment criterion is whether or not all of the ALP activity, the ALP index, and the cell proliferation capacity satisfy or exceed the reference value of the second judgment,
The cells satisfying both the primary and secondary judgment criteria were determined to be osteogenic (+) cells,
For cells that did not meet the criteria in the first judgment but met the criteria in the second judgment, the procedure proceeds to the third judgment.
A method for evaluating cultured osteogenic cells, characterized in that the cells that do not satisfy the criteria in the second judgment are determined to be osteogenic (-) cells. The method of claim 2,
In the third judgment, the criterion is based on whether the surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining, or alizarin red staining satisfies the criterion,
A method for evaluating cultured osteogenic cells, wherein the cells satisfying the criteria are determined to be osteogenic (+) cells and unsatisfactory cells to be osteogenic (-) cells. The method of claim 3, wherein
In the primary judgment, the reference value is the angle of the osteogenic (-) cells (average value + 2SD). The method of claim 4, wherein
In the second judgment, the reference value is a method for evaluating cultured osteogenic cells, characterized in that the lower limit of each measurement of osteogenic (+) cells (mean value-2SD) or each measured value of osteogenic (+) cells. The method of claim 5, wherein
In a third judgment, the criterion for surface antigen (HLA-DR) interpretation is that the measured HLA-DR positive cells are at least about 6%. The method according to claim 6,
In a first judgment, the reference value of ALP activity is about 166 units (μmol prepared p-nitrophenol / min / μg protein). The method according to claim 6,
In a first judgment, the reference value of the ALP index is about 3.95. The method according to claim 6,
In a first judgment, the reference value of cell proliferation ability is about 9.7. The method of claim 7, wherein
In a second judgment, the reference value of ALP activity is about 67 units (μmol prepared p-nitrophenol / min / μg protein). The method of claim 7, wherein
In the second judgment, the reference value of the ALP index is about 1.01. The method of claim 7, wherein
In the second judgment, the reference value for cell proliferation ability is about 5.6.

Images