WO2010084943A1 - Procédé pour évaluer des cellules cultivées - Google Patents

Procédé pour évaluer des cellules cultivées Download PDF

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WO2010084943A1
WO2010084943A1 PCT/JP2010/050765 JP2010050765W WO2010084943A1 WO 2010084943 A1 WO2010084943 A1 WO 2010084943A1 JP 2010050765 W JP2010050765 W JP 2010050765W WO 2010084943 A1 WO2010084943 A1 WO 2010084943A1
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cells
cell
determination
osteogenic
bone
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PCT/JP2010/050765
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English (en)
Japanese (ja)
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秀明 各務
秀樹 縣
祐輔 堀
聡志 大島
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国立大学法人東京大学
株式会社Ｔｅｓホールディングス
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Priority to JP2010547527A priority Critical patent/JP5701614B2/ja
Publication of WO2010084943A1 publication Critical patent/WO2010084943A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the present invention relates to a method for evaluating cultured cells, and more particularly to a new method for evaluating cultured osteogenic cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ALP activity cell alkaline phosphatase activity
  • Non-Patent Document 1 gene expression of bone markers
  • Non-Patent Document 1 gene expression of bone markers
  • ALP activity is a useful assessment for osteogenic cells, it alone is difficult to define osteogenic cells.
  • 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.
  • Osteogenic cells are defined as cells essential for bone formation, such as osteoblasts.
  • 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.
  • 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.
  • a transplant material such as pseudo bone granules
  • the pseudo bone granules are decomposed by osteoclasts at the transplant site, while the true bone is regenerated by osteoblasts and the like.
  • 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.
  • the conventional evaluation of target 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.
  • 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.
  • a target cell from the cultured cell 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.
  • bone-forming cells bone-forming (+) cells
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the reference value is preferably each (average value + 2SD) of bone-forming ( ⁇ ) cells.
  • 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.
  • the standard for surface antigen (HLA-DR) analysis is preferably about 6% or more of measured HLA-DR positive cells.
  • the reference value for ALP activity is about 166 units ( ⁇ mol). p-nitrophenol produced / min / ⁇ g protein) is preferred.
  • the reference value of the ALP index is about 3.95, The method for evaluating cultured osteogenic cells.
  • the reference value of the cell proliferation ability is about 9.7.
  • the reference value for ALP activity is about 67 units ( ⁇ mol). p-nitrophenol produced / min / ⁇ g protein) is preferred.
  • the reference value of the ALP index is about 1.01.
  • the reference value of the cell growth ability is about 5.6.
  • 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.
  • 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.
  • ALP activity evaluation 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.
  • 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
  • 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.
  • 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 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.
  • 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.
  • the cell culture medium either serum-containing ⁇ MEM or serum-free medium can be used for both primary culture and differentiation culture.
  • ⁇ 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.
  • D-PBS darbecolate buffer
  • osteogenic cells are determined based on a primary determination criterion, a secondary determination criterion, and a tertiary determination criterion.
  • the ratio in the parenthesis in FIG. 2 indicates the ratio to the initial cell.
  • these ratios are merely examples and are not particularly limited, and vary depending on the selection of cells to be used.
  • 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 ( ⁇ ).
  • 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.
  • 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.
  • 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.
  • 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. .
  • the bone-forming cell evaluation method 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.
  • ALP activity has also been used for confirmation of bone formation ability so far.
  • 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.
  • ALP activity 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.
  • the usefulness of the ALP index has been reported by the present inventors for the determination of bone forming ability.
  • 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.
  • the present inventors have clarified that, among cell proliferating ability, the proliferating ability particularly during differentiation induction is related to the osteogenic ability. .
  • 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.
  • 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.
  • the relief is performed using the secondary determination criterion and the tertiary determination criterion.
  • cells that satisfy the primary determination criteria may contain a small amount of osteogenic ( ⁇ ) cells.
  • the secondary determination standard is performed also about these cells.
  • 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.
  • the reference value for primary determination was set as each (average value + 2SD) of bone-forming ( ⁇ ) cells.
  • 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.
  • 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.
  • WST-8 cell counting kit
  • the measurement method will be described by taking ALP activity measurement with the above product as an example. First, 100 ⁇ l 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.
  • ALP activity is expressed in moles of p-nitrophenol per hour / mass of protein ( ⁇ mol p-nitrophenol produced / min / ⁇ g 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 ( ⁇ mol p-nitrophenol produced / min / ⁇ g protein).
  • the present inventors set the reference value for primary determination of ALP activity as about 166 units ( ⁇ mol p-nitrophenol produced / min / ⁇ g protein). That is, the reference value for primary determination of ALP activity is preferably 166 ⁇ 5 units ( ⁇ mol p-nitrophenol produced / min / ⁇ g protein).
  • the conversion formula for the unit of ALP activity per cell 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 ( ⁇ mol p-nitrophenol produced / min / ⁇ g protein).
  • the present inventors set the reference value for secondary determination of ALP activity as about 67 units ( ⁇ mol p-nitrophenol produced / min / ⁇ g protein). That is, the reference value for secondary determination of ALP activity is preferably 67 ⁇ 5 units ( ⁇ mol p-nitrophenol produced / min / ⁇ g protein).
  • 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.
  • 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.
  • the lower limit value of the measured ALP index value of the bone-forming (+) cells was 1.01.
  • 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 can be calculated
  • the cell proliferation rate during the differentiation induction period is defined as the cell proliferation ability.
  • the determination can be made with the ratio of the OD values of the cell counting kit (WST-8).
  • 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.
  • 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.
  • 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.
  • the lower limit of the proliferative ability measurement value of the bone forming (+) cells was 5.6.
  • 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.
  • the criteria and evaluation methods surface antigen (HLA-DR) analysis by flow cytometry, TRAP staining, alizarin red staining
  • HLA-DR surface antigen
  • TRAP staining alizarin red staining
  • 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.
  • 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.
  • 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.
  • the measured HLA-DR positive cells when the measured HLA-DR positive cells are 6% or more, it is determined as a cell fraction containing osteogenic (+) cells.
  • 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.
  • 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 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.
  • 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, ⁇ glycerophosphate, and ascorbic acid in ⁇ -MEM.
  • 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.
  • the medium in the 96-well plate during culture is removed, D-PBS is added at 250 ⁇ l / well and washed, and D-PBS is removed.
  • 50 ⁇ l / 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 ⁇ l / well for washing to remove the distilled water. This washing with distilled water is repeated three times.
  • 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.
  • a fixing solution 70% ethanol
  • 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.
  • FIG. 11A when the measured alizarin red staining is positive, it is determined as a fraction containing osteogenic (+) cells.
  • FIG. 11B when the measured alizarin red staining is negative, it is determined that the fraction is highly likely not to contain osteogenic (+) cells.
  • 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.
  • 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.
  • 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.

Abstract

La présente invention concerne un procédé pour évaluer le type ou la fonction de cellules cultivées, tel qu'un procédé pour évaluer une capacité d'ostéogenèse, qui a été difficile à mettre en œuvre en se basant sur un certain critère du fait des fluctuations entre individus. Dans le procédé destiné à évaluer les cellules cultivées, dans le but d'identifier une cellule intéressante parmi les cellules cultivées, de multiples valeurs tests ou indicateurs pour les cellules cultivées sont mesurés, et la fonction de la cellule intéressante est prédite ou déterminée avec une grande exactitude en se basant sur une combinaison des multiples valeurs tests ou indicateurs. Le procédé est caractérisé en ce qu'une cellule intéressante peut être identifiée non seulement en désignant une certaine plage d'adaptation pour chacune des valeurs tests, mais en se référant également à une autre valeur test ou indicateur pour un échantillon qui sort de la gamme d'application.
PCT/JP2010/050765 2009-01-23 2010-01-22 Procédé pour évaluer des cellules cultivées WO2010084943A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005058225A (ja) * 2003-07-30 2005-03-10 Asahi Techno Glass Corp アルカリホスファターゼ活性を有する細胞の相対測定法
JP2007528706A (ja) * 2003-06-27 2007-10-18 エチコン、インコーポレイテッド 分娩後由来細胞を用いた軟骨と骨の修復と再生

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070105165A1 (en) * 2005-11-04 2007-05-10 Charles Goolsby Composite profiles of cell antigens and target signal transduction proteins for analysis and clinical management of hematologic cancers
JP5243021B2 (ja) * 2006-12-28 2013-07-24 三菱化学メディエンス株式会社 間葉系幹細胞などの細胞を検出するための遺伝子マーカー

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007528706A (ja) * 2003-06-27 2007-10-18 エチコン、インコーポレイテッド 分娩後由来細胞を用いた軟骨と骨の修復と再生
JP2005058225A (ja) * 2003-07-30 2005-03-10 Asahi Techno Glass Corp アルカリホスファターゼ活性を有する細胞の相対測定法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GUO L ET AL.: "Osteogenic differentiation of human mesenchymal stem cells on chargeable polymer-modified surfaces.", J BIOMED MATER RES A., vol. 87, no. 4, 2008, pages 903 - 912 *
ITO A ET AL.: "A new methodology of mesenchymal stem cell expansion using magnetic nanoparticles", BIOCHEMICAL ENGINEERING JOURNAL, vol. 20, no. 2-3, 2004, pages 119 - 125 *
KONDO Y ET AL.: "Role of stromal cells in osteoclast differentiation in bone marrow.", J BONE MINER METAB., vol. 19, no. 6, 2001, pages 352 - 358 *
SYED-PICARD FN ET AL.: "Three-dimensional engineered bone from bone marrow stromal cells and their autogenous extracellular matrix.", TISSUE ENG PART A., vol. 15, no. 1, January 2009 (2009-01-01), pages 187 - 195 *

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