KR101632756B1 - 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 based on a certain standard because of deviation due to an individual. For example, the present invention provides a method for evaluating bone shape performance. The method for evaluating a cultured cell according to the present invention is a method for evaluating a cell in which a plurality of test values or indicators of a cultured cell are measured in order to determine a target cell from the cultured cells, The present invention is characterized in that it is possible to determine that a target cell is a target cell by referring not only to a certain suitable range for each test value but also to another test value or indicator even if the sample is out of the adaptation range.

Description

METHOD FOR ASSESSING CULTURED CELL [0002]

The present invention relates to a new evaluation method of cultured osteogenic cells (osteogenic cells) in a method for evaluating cultured cells.

And realization of regenerative medicine for regenerating defective tissue is demanded. Regenerative medicine is a medical technology that uses the cells, biomaterials, and cell growth factors to produce the same shape and function as the original tissue.

Mesenchymal stem cells (mesenchymal stem cells) are generally used for regenerative medicine. It is known that these hepatic hepatic cells can induce differentiation into various cells such as adipocytes and osteoblasts. However, it is difficult to judge in advance whether or not the obtained cells have a desired function.

In bone regeneration medicine, bone marrow-derived hepatic stem cells are mainly used, and other fat-derived hepatocytes, periosteum-derived hepatocytes, and synovial-derived hepatocytes are reported. Among them, mesenchymal stem cells or bone marrow stromal cells derived from bone marrow are used for clinical application of almost all bone regeneration since they are relatively easy to harvest and have very high bony performance.

However, in the reports of the present inventors so far, it has become clear that the osteogenic cells derived from humans have large individual differences in cells. It has also become clear that bone morphology is rapidly lost by culture. Thus, although it is important to examine whether the cells that actually transplant have a bony performance, it has been found that the methods reported so far are not sufficient. In particular, conventionally, alkaline phosphatase activity (ALP activity) of cells (Patent Document 1) and gene expression of bone marker (Non-Patent Document 1) have been used. Although ALP activity is an evaluation useful for osteogenic cells, it is difficult to define osteogenic cells alone. In addition, gene expression of the osteogenic marker is also a useful evaluation, but the present inventors have also recognized that the results of the present inventors indicate that the individual difference in expression is so large that it is difficult to use it as an evaluation. In particular, in order to evaluate human-derived cells having a large individual difference, it is considered that it is not sufficient as a single criterion and it is important to improve reliability by combining a plurality of evaluation methods. However, such an evaluation method has not been reported so far.

An osteogenic cell is defined as a cell essential for bone formation such as an osteoblast. Herein, cells capable of forming osteoblasts, in particular, are defined as osteogenic cells. Ectopicity is the ability to form a bone in a region that is originally free of bone. Ectopic osseointegration performance is verified by implanting, for example, subcutaneously into an immunodeficient animal with a carrier that maintains human cells. In fact, it is necessary to perform bone regeneration toward a bone-free portion in the vicinity of the bone, in which bone regeneration is expected in the vicinity of the bone. However, there is no method for evaluating cells produced from this viewpoint.

Japanese Laid-Open Patent Publication No. 2005-58225

Nishimura et al., ≪ / RTI > J Bone Miner Metab. 26, 203-212

That is, in order to perform bone formation by implantation, the process shown in Fig. 1 is carried out. As shown in FIG. 1, osteogenic cells are coexisted with a graft material, for example, pseudo-bone granules (pseudo bone granules) to decompose the osteoclast granules by osteoclasts at the graft site, Regenerates the genuine bone. At this time, the number of osteogenic cells in the graft material is of course an important factor, but it is important that the proper osseous performance is maintained. If the number of these osteogenic cells is small or the cells with degraded osteoid performance are used, adequate bone formation is not performed, but a considerably long period of time is required for confirmation of actual osteogenesis. If bone formation is not properly carried out, it is necessary to collect and cultivate hepatic mesenchymal stem cells again, to perform transplantation surgery and regeneration confirmation, and to waste the period of several months in a useless manner, And it is repeated. Therefore, it is important to properly evaluate the function of cultured cells.

On the other hand, even when the bone-shape performance is determined during culturing of the cells, it takes several weeks for the examination during induction of differentiation, so that it can not be easily repeated.

However, evaluation of whether or not the cell is a target cell that has been conventionally performed, for example, an osteogenic cell is carried out by a single judgment method as described above. If the evaluation standard is raised, the regeneration rate after transplantation is somewhat increased, It is considered that the cultured cells can be judged to be inadequate. On the other hand, when the evaluation standard is lowered, cultured cells unable to undergo osteogenesis are used for transplantation. Further, studies by the present inventors have revealed that, in human cells, the deviation between individuals is large in almost all of the test values, and it is difficult to accurately judge only with a constant reference value or a single determination criterion, and it is clear that even realistic judgment is difficult.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an object thereof 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 deviation of human cells is considered to be a problem common to many cells that are widely used for regenerative medicine or cell therapy in addition to osteogenic cells do. Therefore, the effect of the present invention provides an effective evaluation method for whether or not the obtained cells are the target cells and whether or not they have the functions required for treatment, in cell therapy using widely bone cells as well as osteogenic cells will be.

As a result of intensive studies conducted by the present inventors in order to solve the above-described problems, in order to determine a target cell in a cultured cell, a plurality of test values or indicators of the cultured cells are measured and combined to prepare a reliable evaluation method I found that I could. For the osteogenic cells (osteogenic (+) cells), the bone morphometric performance test method shown in Fig. 2 was developed and the prediction and evaluation as to whether or not the osteogenesis was appropriately performed after the implantation was evaluated before the implantation operation It is possible to prepare a method for evaluating a useful osteogenic cell by carrying out the method of the present invention precisely. Thus, the present invention has been completed.

That is, the method for evaluating cultured cells according to the present invention is a method for evaluating a cultured cell by measuring a plurality of test values or indicators of cultured cells to determine a target cell in the cultured cells, As an evaluation method for determining,

It is possible to determine that the target cell is a target cell by referring not only to a certain suitable range for each test value but also to a different test value or indicator even if the sample is out of the adaptation range.

Further, the method for evaluating cultured osteogenic cells of the present invention is a method for evaluating cultured cells having a primary determination standard, a secondary determination standard, and a tertiary determination standard,

Cells subjected to the primary determination are further subjected to secondary determination to determine bone formation (+) cells,

(+) Cells are judged by further performing a tertiary judgment on cells which are difficult to judge whether they are osteogenic (+) cells.

In the primary determination, the criteria are ALP activity (ALP activity), ALP index (ALP activity value of differentiation inducing group / ALP activity value of non-differentiation inducing group) and cell proliferation ability (number of cells after induction of differentiation / Number) satisfies at least the reference value of the primary determination.

In the secondary determination, the determination criterion is whether or not all the ALP activity, the ALP index, and the cell proliferation ability satisfy the reference value of the secondary determination or more,

Cells satisfying both criteria of the first judgment and the second judgment were judged to be osteogenic (+) cells,

Cells that did not meet the criteria in the primary determination but met the criteria in the secondary determination proceeded to the tertiary determination criteria,

It is preferable to determine that the cells that do not satisfy the criteria in the secondary determination are osteogenic (-) cells.

In the tertiary determination, the criteria are whether or not at least one selected from the group consisting of surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining and alizarin red staining satisfies the criteria,

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

In the primary determination, it is preferable that the reference value is the angle (average value + 2SD) of osteogenic (-) cells.

In the secondary determination, the reference value is preferably a lower limit value of each measured value of the osteogenic (+) cells (average value -2 SD) or osteogenic (+) cells.

In the tertiary determination, it is preferable that the HLA-DR-positive cells measured by the surface antigen (HLA-DR) analysis are about 6% or more.

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

Wherein in the primary determination, the reference value of the ALP index is about 3.95.

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

In this secondary determination, the reference value of ALP activity is preferably about 67 units (μmol p-nitrophenol / min / ug protein produced).

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

In the secondary 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, the ALP activity used as a judgment standard is recognized to be high in an osteogenic cell, for example, an osteoblast. When the ALP activity after induction of differentiation is low, it may be considered that the cells do not contain osteogenic cells or have no osteoid performance. However, the inventors of the present invention have found that the ALP activity of the differentiation inducing group and the non- In the case of an increase in the ALP index as a ratio indicates that the osteogenic cell has a bony function. On the other hand, if ALP activity is sufficiently high, induction of differentiation of osteogenic cells is highly likely to be good. However, even if the activity of ALP is high, loss of cell proliferative ability does not result in sufficient bone formation after bone grafting.

Therefore, in the evaluation method according to the present invention, the cell proliferation ability is also evaluated.

A more careful evaluation is required for a group in which it is difficult to determine the presence or absence of osteogenic cells by combining the above three evaluations (ALP activity, ALP index, cell proliferation ability). The inventors of the present invention found that any of these groups were positive (flow cytometry HLA-DR by flow cytometry, analysis of surface antigen (HLA-DR), TRAP staining and alizarin red staining) DR fraction positive, TRAP positive, alizarin red positive) and adjuvant osteogenic cells, thus providing a reasonable basis for samples with almost all individual differences.

The direct relationship between this HLA-DR positive cell and the bone morphology is unclear, but the present inventors have found a clear correlation between HLA-DR activity and bone morphology. In addition, although TRAP staining (tartaric acid-resistant acid phosphatase staining) is used as an osteoclast marker, since maturation from osteoclast precursor cells to osteoclasts requires interaction with osteoblasts (osteogenic cells) And can be used as an indirect indicator of the process of differentiation into forming cells. In addition, alizarin red staining indicates the ability to calcify in vitro and stains the deposition of calcium. As a result of the inventors' observation, it was found that the cells exhibiting the osteosynthetic performance were negative for alizarin red staining, and the oocyte-laden cells showed negative staining on the cells that had undergone the loss.

As described above, in the present invention, those having clearly osteoform performance and those having no osteoform performance are selected by the ALP activity evaluation, the ALP index, and the cell proliferation ability determination, which are comparatively simple and clear methods, HLA-DR analysis, TRAP staining, and alizarin red staining are performed to prevent the bone structure from being destroyed as " no bony structure "

According to the present invention, it is possible to provide a method for evaluating the type and function of a target cell in which it is difficult to judge based on a certain criterion because there is a deviation due to an individual. In addition, when the evaluation of osteogenic cells is carried out, it becomes possible to almost completely evaluate the bone morphology of the cultured cells, and it is possible to provide a method for evaluation necessary for treatment using cells.

FIG. 1 is a diagram illustrating a bone structure test schedule,
2 is a flowchart showing a method for evaluating osteogenic cells according to the present invention,
Fig. 3 is an explanatory diagram of a process for culturing osteogenic cells to which the present invention is preferably applied, a process for inducing differentiation,
FIG. 4 shows the distribution of ALP activity in human cultured osteoblast-like cells,
5 shows the distribution of ALP indices of human cultured osteoblast-like cells,
6 shows the distribution of the ability of human cultured osteoblast-like cells to proliferate,
7 shows the results of ALP activity measurement,
8 shows the results of measurement of cell proliferation ability,
FIG. 9 shows the result of surface antigen analysis by flow cytometry,
Figure 10 shows the results of TRAP staining, and
Fig. 11 shows the result of alizarin red staining.

Hereinafter, preferred embodiments of the present invention will be described. Hereinafter, description will be given taking as an example the case where the target cells are osteogenic cells. First, a method for producing a granular culture bone which can be effectively applied for treatment by a method for evaluating an osteogenic cell of the present invention will be described with reference to Fig. Granule type cultured bone can be prepared 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 steps show a form using bone marrow derived cells, the evaluation method of the present invention is produced by separately culturing from bone marrow, fat, peripheral blood, etc. in addition to bone marrow, and inducing differentiation into cultured osteoblast-like cells by the same method It can be preferably used also in a granular culture.

· Collection of bone marrow fluid

Bone marrow fluid is collected about one month before surgery. First, local anesthesia is performed on the bone marrow harvesting section, and aseptic suction is performed from the posterior supernumerary (posterior superior iliac crest) to recover.

· Culture of hepatocytes from mesenchymal stem cells from bone marrow

When the bone marrow fluid 10 diluted four times with the cell culture medium is sprayed on the cell culture flask 12, the culture is started as shown in (A) of Fig. 3, and the whole medium is changed on the fourth day after the start of the culture. In the cell culture medium, either αMEM containing serum or serum-free medium can be used for both the primary culture and the differentiation culture.

Confirmation of the state not infected with general bacteria and fungi in the culture process is carried out by observing the medium every time the medium is exchanged (if the medium becomes infected, the medium becomes turbid), and at the start of culture, before the induction of differentiation, (As to whether it meets the Japanese Pharmacopoeia criteria).

After that, the entire medium is exchanged twice a week. The timing of the passage is determined by observing the state of the cells, but is performed about 21 to 28 days after the initiation of culturing. Measure the number of living cells in the passage.

· Sowing of Hepatocyte

The passages are carried out as follows. After removing the culture medium from the flask, the plate is washed with Dulbecco's phosphate buffer (D-PBS), then D-PBS is removed, a cell dissociating agent is added, and the plate is incubated at 37 ° C for 10 minutes. After confirming that the cells are disassociated, D-PBS or medium is added to recover the cells, followed by centrifugation. The cells are suspended again in the medium to measure the number of cells. After the measurement, the cultured bone marrow fluid 10 is introduced into the basal vessel 16 into which the porous doctor bone granules 14 as shown in Fig. 3 (B) are previously charged. 3 (C), the bone marrow fluid 10 and the pseudo-bone graft 14 float when the bone marrow fluid 10 is injected into the basal vessel 16 containing the pseudo-bone grains 14 therein.

· Induction of differentiation into cultured osteoblast-like cells

After sowing the cells in the porous doctor bone graft 14, the cells are allowed to stand overnight for restoration of cell function. After one day, as shown in Fig. 3 (D), cells such as cells involved in bone formation and the porous pseudo-bone grains 14 are settled in the lower portion of the basal vessel 16. After confirming this, the medium is replaced with a differentiation induction medium. The differentiation induction period is 1 to 3 weeks, and the medium exchange is performed twice a week. As the induction of differentiation proceeds, the morphology of cultured osteoblast-like cells attached around the pseudo-bone granules 14 is changed as shown in (E) of FIG. 3, and the osteoblastic protein is secreted into a lump.

On the other hand, the cells are sown in a 12-well culture dish for evaluation in parallel with this process. Cells should be 2 x 10 ⁴ per well.

Hereinafter, a method of evaluating osteogenic cells according to the present invention will be described with reference to the flowchart of Fig. As shown in Fig. 2, the osteogenic cells are judged on the basis of the primary judgment standard, the secondary judgment standard and the tertiary judgment standard. In addition, the ratio in parentheses in Fig. 2 represents the ratio to the original cells. However, these ratios are not particularly limited as an example and differ depending on the selection of the cell used.

Primary judgment criteria

Primary determinations are based on ALP activity, ALP index, and cell proliferative capacity.

In the primary determination, when the cell exceeds the reference value of any one of the above three evaluation methods, the cell is determined as the primary determination reference (O), and when the cells do not exceed all of the reference values, .

Secondary Criteria

The secondary determination is also carried out on the basis of ALP activity, ALP index, and cell proliferation ability as in the primary determination. However, as shown below, these evaluation reference values are different from the primary determination.

In the secondary determination, the cells are judged to be osteogenic (+) cells when they exceed the reference value in all of the above three evaluation methods, and when they do not exceed any of the reference values, (-) cells.

Cells of the first judgment criterion (x) are subjected to the third criterion when the whole of the above three evaluation methods are satisfied in the second judgment, and when the cells do not exceed any one of the reference values, -) cells.

Tertiary decision criteria

The tertiary determination is carried out on the basis of surface antigen analysis by flow cytometry, TRAP staining, and alizarin red staining.

In the third criterion, the cells are judged to be osteogenic (+) cells when one is positive and the cells are judged to be osteogenic (-) cells when all are negative.

In the method for evaluating osteogenic cells according to the present invention, it is judged whether osteogenic (+) cells are formed by combining a plurality of criteria. The importance of combining these methods is as follows.

As described above, ALP activity has also been used to confirm the bone shape performance so far. However, as shown in Fig. 4, the distribution of ALP activity is close to that of cells having and without osteosynthesis, making it difficult to set clear boundaries. If set low, cells that do not have osteogenesis performance will be incorporated in large numbers, and if set high, osteogenesis cells will be excluded.

In fact, when considering the criteria based on the ALP activity, it is conceivable to set the boundary to the mean value -2SD of the ALP activity of the cells showing bone formation, for example. Assuming that the ALP activity value is a normal distribution, 95.45% of the cells having the bony performance can be recovered. However, as shown in Fig. 4, most of the cells having no bony performance are likely to be recovered.

It is also conceivable to set the lower limit value of the osteogenic (+) cells. However, as shown in FIG. 4, there are many cells that do not have a bony function even at the lower limit of the bone formation (+) cell.

As described above, the usefulness of the ALP index has been reported by the present inventors in the determination of the bony performance. However, as shown in FIG. 5, the distribution of the ALP index is close to that of the cells having the bony performance, and it is difficult to set clear boundaries.

Further, although it has not yet been used for the determination of the bone shape performance, it has become clear by the inventors of the present invention that the cell growth ability, particularly the ability to proliferate upon induction of differentiation, is related to the bone shape performance. However, as shown in Fig. 6, the distribution of cell proliferation ability is also close to that of cells having and without osteosynthesis, and it is difficult to set clear boundaries.

As described above, it can be seen that it is difficult to appropriately determine the osteogenic (+) cells using the conventionally used indicators or simple combinations thereof.

Therefore, the inventors of the present invention have found that since there are many portions overlapping in the data of osteogenic (+) cells and osteogenesis (-) cells in the above determination method, strict criteria for collecting only osteogenic (+) cells ) For the first time.

However, since the primary selection criteria includes a large number of osteogenic (+) cells in the non-selected areas, these are rescued using the secondary and tertiary criteria.

In addition, cells satisfying the primary criteria may contain some osteogenic (-) cells. For this reason, secondary determination criteria are also applied to these cells.

As described above, in the method for evaluating osteogenic cells according to the present invention, the first to third determination are carried out. As a result, the osteogenic (+) cells determined as a result are almost 100% It is becoming clear that it is a cell.

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

In the present invention, the reference value of the primary determination is set to the angle (average value + 2SD) of osteogenic (-) cells. At this time, if the sample data is assumed to have a normal distribution, the probability of erroneously including non-osseous cells is (100-95.45) / 2 = 2.275%.

The reference value of the secondary determination was set as the lower limit value of each measured value of the osteogenic (+) cells (mean value -2 SD) or osteogenic (+) cells.

It is also conceivable that the reference value is changed according to the population (sex, age, race, etc.) of the patient from which the cells are to be collected. That is, 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 numerical value more strictly by a large amount of data.

Although a specific reference value of the secondary determination shown below is a lower limit value of each measured value of osteogenic (+) cells as an example, it is also possible to obtain a valid result by using the angle (mean value -2SD) Loses.

· ALP activity

For measurement of ALP activity, for example, a p-nitrophenyl phosphate purification kit (Sigma-Aldrich) and a cell aggregation kit WST-8 (manufactured by Tojin Kagaku Corporation) can be used.

Hereinafter, a measurement method will be described as an example of the case where the ALP activity measurement is performed with the above product. First, 100 μl of WST-8 solution is added to each well. After performing a color reaction for 1 to 4 hours in a carbonic acid gas incubator, the absorbance is measured using a microplate reader. After the WST-8 analysis, the cell membrane protein was dissolved and extracted, and a p-nitrophenyl phosphate solution was added to the solution. After standing at room temperature for 10 minutes, the reaction was stopped with NaOH and p-nitrophenol The decomposition product of phosphate by ALP) is measured. ALP activity is expressed as moles of p-nitrophenol per hour / mass of protein (produced μmol p-nitrophenol / min / μg protein). Or as p-nitrophenol absorbance (OD; 405 nm) / WST-8 absorbance (OD; 450 nm).

Fig. 7 shows an example of the results of ALP activity measurement.

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

The expression for conversion of the unit of ALP activity per cell (the resulting μmol p-nitrophenol / min / ug protein) was obtained by the present inventors from the cells used in this study.

The lower limit value of the measured value of ALP activity 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 p-nitrophenol / min / μg protein produced). For this reason, the present inventors set the reference value of the secondary determination of ALP activity to about 67 units (μmol p-nitrophenol / min / μg protein produced). That is, the reference value of the secondary determination of ALP activity is preferably 67 +/- 5 units (μmol p-nitrophenol / min / μg protein produced).

· ALP index

The ALP index was determined by measuring the ALP activity on the cells to which the differentiation induction medium (differentiation induction group) and the control medium (control differentiation induction group) were differentiated (ALP activity / non-differentiation inducing group Of ALP activity).

The mean value of ALP index + 2SD of osteogenic (-) cells was 3.95. Therefore, the present inventors set the reference value of the primary determination of the 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 value of the ALP index value of osteogenic (+) cells was 1.01. Therefore, the present inventors set the reference value of the secondary determination of the ALP index to 1.01. That is, the reference value of the secondary determination of the ALP index is preferably 1.01 0.15.

· Cell growth ability

The cell proliferating ability can be determined by the ratio of the number of cells before and after the culture, that is, the number of cells after induction of differentiation / the number of seeding cells to be initially seeded. In practice, it is obtained by measuring the number of cells after the termination of the differentiation induction period, but it can be calculated as the number of seeded cells / the number of seeded cells by making the seeded cells constant. Here, the cell proliferation rate during the differentiation induction period is defined as the cell proliferation ability. Further, it can be determined by the ratio of the OD value of the cell aggregation kit (WST-8). That is, after performing the color reaction, the absorbance (OD value) at 450 nm is measured, and the cell growth rate during induction of differentiation can be determined by dividing the OD value of WST-8 with respect to the number of cells before induction of differentiation. In addition, the cell proliferation ability determination according to the present invention can be applied to the first to fourth passages.

Fig. 8 shows an example of the result of cell proliferation assay.

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

In addition, the lower limit of the measured value of the proliferative capacity of osteogenic (+) cells was 5.6. Therefore, the present inventors set the reference value of the secondary determination of the cell proliferation ability to about 5.6. That is, the reference value of the secondary determination of the cell proliferation ability is preferably 5.6 + - 0.3.

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

In the present invention, the criterion for the tertiary determination is that the surface antigen (HLA-DR) positive cells are more than 6%, TRAP positive, and alizarin red positive.

· Surface antigen analysis by flow cytometry

Six-color flow cytometry analysis can be performed using conventional flow cytometry, but here, a FACS Aria flow cytometer (BDIS) was used.

The following materials were used for the antibody. The fluorescence isothiocyanate complex (FITC-), the phycoerythrin complex (PE-), the peridinin chlorophyll protein complex (PerCP-Cy5.5-), the aripycocyanin complex (APC-) and the Alexa Fluor 405 complex .

(All BD Pharmingen) for HLA-ABC, HLA-DR, CD3, CD14, CD19, CD34, CD73, CD90, CD106, CD146, mouse- IgG1k and mouse- IgM as antibodies, Biotinylated antibodies to CD10 and CD 29 (Dako), and biotinylated antibodies against CD45 (Invitrogen) were also used. A CD105 antibody (Immunotech) with covalently conjugated FITC was also used. The biotinylated antibody was detected by streptavidin-pacific blue (Invitrogen) or streptavidin PerCP-Cy5.5 (BD Pharmingen) complex.

The STRO-1 antibody (R & D Systems) was detected with PE-conjugated anti-mouse IgM. In addition, propidium iodide (Tojin Kagaku Kenkujosa) was used to detect dead cells.

Surface antigens were analyzed by flow cytometry using the above-mentioned dozens of kinds of antibodies. As a result of the studies conducted by the present inventors, it has become clear that the cells in the bone marrow-derived mesenchymal stem cells are cultured by the same method, but the cells having the bony performance are sometimes obtained or not. However, , There was no difference between the cells with and without osteoblastic performance. However, it was clear that only the expression of antibodies to HLA-DR was significantly different. Therefore, in the surface antigen analysis according to the present invention, a biotinylated antibody against HLA-DR is used as an antibody.

Hereinafter, a preferred method of analyzing the surface antigen when using a biotinylated antibody against HLA-DR is described. Passage number of the cells 0 and 3 is detected by the tree peusin -EDTA, of 1 × 10 ⁶ cells were ice-cooled (氷冷) a phosphate-buffered physiological saline 50㎕: was suspended in (PBS units Sui Seiya flexors). Subsequently, the cells were incubated with biotinylated antibodies against HLA-DR for 20 minutes on ice. The cells were then washed and incubated with the streptavidin complex for 20 minutes on ice. Finally, the cells were washed, resuspended in 200 [mu] l of ice cold PBS, stained with iodinated propidium and analyzed by flow cytometry. 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, CD14, respectively). The lower right figure shows the Y-axis antibody (CD14) negative and the X-axis antibody (HLA-DR) positive fraction.

As shown in Fig. 9 (A), when the measured HLA-DR positive cells are 6% or more, it is determined that the cell fraction contains osteogenic (+) cells. However, as shown in Fig. 9 (B), when the measured HLA-DR positive cells are less than 6%, it is judged as fractions which are highly likely not to contain osteogenic (+) cells.

The lower limit of 6% of the HLA-DR positive cells is an empirically determined value found by a large number of subjects of the present inventors, but the present invention is not limited to this value. In other words, it is possible to set the numerical value more strictly by the data of a larger number of samples. Specifically, in this case, the value obtained by subtracting 2SD from the average value of the cells forming the bone, and the average value of the non- Of the values obtained by adding 2SD and the like to the values of the above-mentioned values.

· TRAP staining

For TRAP staining, for example, a TRAP staining kit (Wako Junya Kogyo Co.), which is a buffer solution containing 50 mM tartaric acid (pH 5.0, color development substrate 30 mg / vial), may be used. The TRAP staining evaluation method using the above TRAP staining kit is shown below.

First, D-PBS was warmed up in a water bath and cultured in a differentiation induction medium containing 10% serum, 1% penicillin streptomycin, 1% amphotericin B, dexamethasone,? -Glycerophosphate and ascorbic acid in? -MEM do.

In order to obtain the strictness, the results were also obtained by using Osteo Clast precursor Basal medium M-CSF (-), RANKL (-), Osteo Clast precursor Basal medium M-CSF (+) and RANKL (+ It is preferable to compare them.

Subsequently, the culture medium of the 96-well plate in culture was removed, D-PBS was added at a rate of 250 μl / well, followed by washing, and D-PBS was removed. Subsequently, 50 占 퐇 / well of the fixation solution of the TRAP staining kit was added and fixed for 5 minutes. After 5 minutes, the fixing solution is removed, 250 μl / well of distilled water is added to the solution, and the distilled water is removed. The washing in the distilled water is repeated three times. Before removing the third distilled water, turn off the clean bench, add 5 ml of a buffer containing 50 mM tartaric acid to the color development substrate of the TRAP staining kit, and mix with a vortex to prepare a color development substrate. After the preparation of the chromogenic substrate, the distilled water was removed, and 100 μl / well of the chromogenic substrate was added thereto. The incubation was carried out at 5% CO ₂ and 37 ° C. (humidity 95% rH or more) for 1 hour. After incubation, the chromogenic substrate is removed, and 250 쨉 l / well of distilled water is added thereto to clean, and the distilled water is removed. The washing in the distilled water is repeated twice. After washing, distilled water was added at a rate of 50 쨉 l / well, and microscopic examination of the cells stained was carried out, and cell photographs were taken.

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

However, when the TRAP staining measured as shown in FIG. 10 (B) is negative, it is determined that there is a fragment which is likely to not contain osteogenic (+) cells.

· Alizarin red dyeing

To perform alizarin red staining, the cells are seeded in 12 wells at a density of 2.0 x 10 < ⁴ > / well. Next day, the medium is changed to the differentiation induction medium, and then the medium is changed twice a week to induce differentiation for 3 weeks.

Subsequently, the medium is removed (aspirated) and the cells are washed twice with D-PBS. Thereafter, cells are fixed with fixative (70% ethanol) at -20 ° C for 1 hour. The fixation liquid is sucked and washed twice with distilled water.

Subsequently, a solution of Alizarin Red S (40 mM, pH 4.2) was added, and the mixture was stained at room temperature for 10 minutes. The pH is adjusted with 25% aqueous ammonium solution. The staining solution is aspirated and the cells are washed with distilled water until nonspecific staining is completely eliminated. Then, the macro and microscopic photographs are taken.

Fig. 11 shows the result of staining of alizarin red. The left side is a sample (control) without induction of differentiation, and the right side is a sample after induction of differentiation.

As shown in Fig. 11 (A), when the measured aligarine red staining is positive, it is determined that the fraction contains osteogenic (+) cells.

However, as shown in Fig. 11 (B), when the measured aligarnin red stain is negative, it is judged that there is a fragile fragile fragment that does not contain osteogenic (+) cells.

In the method for evaluating cultured cells according to the present invention, after setting a plurality of test values or an adaptation order and a criterion for an indicator, the flow charts are used to accurately determine the bone form performance of cells regardless of the experience . In particular, it is desirable to include at least three test values and indicators, and to precisely predict and determine the function of the cells with a combination of these on a statistical basis. In the present invention, a plurality of test values and indicators are measured and evaluated for human cells having deviations in a flow chart, thereby making it possible to improve the influence of the deviations to a level without problems.

Further, in the method for evaluating cultured cells according to the present invention, it may be judged whether or not a sample is included in an adaptation range for each test value, by referring to another index, and not a target cell.

10: bone marrow solution
12: Flask for cell culture
14: Porous physiological bone grains
16:

Claims (14)

A method for evaluating cultured cells having a primary determination standard, a secondary determination standard, and a tertiary determination standard,
Cells subjected to the primary determination are further subjected to secondary determination to determine bone formation (+) cells,
A method for evaluating a cultured osteogenic cell in which osteogenic (+) cells are judged by subjecting a cell, in which it is difficult to judge whether or not osteogenic (+) cells are difficult,
In the primary determination, the criteria are ALP activity (ALP activity), ALP index (ALP activity value of differentiation induced group ÷ ALP activity value of non-differentiation induction group) and cell proliferation ability (number of cells after differentiation induction / And the reference values are respectively 166 ± 5 units (μmol p-nitrophenol / min / μg protein produced), 3.95 ± 0.15, and 9.7 ± 0.3,
In the secondary determination, the determination criteria is whether or not all the ALP activity, ALP index, and cell proliferation ability satisfy the reference value of the secondary determination, and each reference value is 67? 5 units (μmol p-nitrophenol / Min / 占 퐂 protein), 1.01 ± 0.15, 5.6 ± 0.3,
Cells satisfying both criteria of the first judgment and the second judgment were judged to be osteogenic (+) cells,
Cells that did not meet the criteria in the primary determination but met the criteria in the secondary determination proceed to the tertiary determination criteria,
Cells that did not meet the criterion in the secondary determination were judged to be osteogenic (-) cells,
In the tertiary determination, the criteria are whether or not at least one selected from the group consisting of surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining and alizarin red staining satisfies the criteria, Each of the measured HLA-DR positive cells was more than 6% positive, positive,
The cells satisfying this criterion are judged to be osteogenic (+) cells, and the unsatisfactory cells are judged to be osteogenic (-) cells. delete delete delete delete delete delete delete delete delete delete delete delete delete

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