WO2017170090A1 - Étalon destiné à un essai de chaîne lourde de myosine - Google Patents

Étalon destiné à un essai de chaîne lourde de myosine Download PDF

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Publication number
WO2017170090A1
WO2017170090A1 PCT/JP2017/011609 JP2017011609W WO2017170090A1 WO 2017170090 A1 WO2017170090 A1 WO 2017170090A1 JP 2017011609 W JP2017011609 W JP 2017011609W WO 2017170090 A1 WO2017170090 A1 WO 2017170090A1
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mhc
cell
cells
standard
culture
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PCT/JP2017/011609
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English (en)
Japanese (ja)
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進弥 佐野
枝莉 野口
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テルモ株式会社
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Priority to JP2018509156A priority Critical patent/JP6779981B2/ja
Publication of WO2017170090A1 publication Critical patent/WO2017170090A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • 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

Definitions

  • the present invention relates to a standard for measuring and / or assaying myosin heavy chain (MyHC) expression rate in a cell population, a cell culture for producing the standard, a method for producing the standard or cell culture, and the like. .
  • MyHC myosin heavy chain
  • Non-Patent Documents 1 and 2 fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells for the repair of myocardial tissue damaged by ischemic heart disease such as angina pectoris and myocardial infarction or dilated cardiomyopathy Etc. have been tried (Non-Patent Documents 1 and 2).
  • Patent Document 1 Non-Patent Document 2
  • sheet cell culture For the application of sheet cell culture to the treatment, use of cultured epidermis sheet for skin damage caused by burns, use of corneal epithelial sheet cell culture for corneal injury, oral mucosa sheet for endoscopic resection of esophageal cancer Studies on the use of cell cultures are underway, and some of them are in the clinical application stage.
  • An object of the present invention is to provide an assay standard for evaluating the quality of a sheet-shaped cell culture, a cell culture for producing the standard, a method for producing the standard or the cell culture, and the like. To do.
  • the present inventors faced a new problem that there is no assay standard that uses MHC as an index that can be obtained easily and in large quantities, and that highly expresses MHC and that can be easily prepared at the time of use. .
  • As a result of diligent research to solve such problems by culturing undifferentiated cells that are easy to grow and preserve for a predetermined period under predetermined conditions, the number of MHC-expressing cells can be increased with high efficiency, and a desired cell population can be obtained.
  • the present invention was completed by finding that it can be prepared.
  • one aspect of the present invention relates to: ⁇ 1> A cell culture in which the content of cells positive for myosin heavy chain (MHC) expression is 1% or more.
  • MHC myosin heavy chain
  • ⁇ 4> The test standard according to ⁇ 3>, which is used for validation of an anti-MHC antibody.
  • ⁇ 5> The test standard according to ⁇ 3> or ⁇ 4>, which is used for flow cytometry.
  • ⁇ 6> A method for producing a cell culture containing 1% or more of MHC expression positive cells, comprising culturing skeletal myoblasts in a low serum medium with low serum for 3 to 6 days.
  • the low serum medium is a serum-free medium.
  • the low serum medium is a serum-free medium containing DMEM, 0.1 to 10 mg / ml bovine serum albumin and 0.005 to 0.5 ⁇ g / ml insulin-like growth factor-1.
  • ⁇ 9> The method according to ⁇ 6> to ⁇ 8> above, comprising pre-culturing in a growth medium before culturing.
  • a cell population having a high MHC expression positive cell rate can be obtained easily and in large quantities. Since the cell population obtained by the method of the present invention has a high MHC expression positive cell rate, it is possible to prepare a standard having a wide testable range by preparing an assay standard using the cell population. .
  • the present invention relates to a cell culture characterized in that the content of cells expressing myosin heavy chain is 1% or more.
  • the “cell culture” means a cell population obtained by culturing cells in vitro.
  • the culture may be a proliferation culture or a non-proliferation culture, and may be a primary culture or a subculture, but it may be collected from a living body and suspended in a culture medium or a culture substrate. Only seeded is not intended. That is, the “cell culture” in the present invention means a cell in which a cell suspended in a medium or seeded on a culture substrate is incubated for a predetermined time under a predetermined condition.
  • optimum conditions can be selected depending on the purpose of incubation, and examples include 37 ° C. and 5% CO 2 atmosphere.
  • an optimal time can be selected depending on the purpose of incubation, and examples thereof include 12 hours or more, 24 hours or more, 48 hours or more, 3 days or more, 5 days or more, and the like.
  • cultured cells in particular, when only cells are meant, it is referred to as “cultured cells”.
  • the cultured cells in the invention are preferably mammalian cells, more preferably human cells.
  • Myosin heavy chain is a fibrous protein that constitutes myosin, which is an ATP-driven motor protein, and is also referred to as MHC or MyHC.
  • MHC myosin heavy chain
  • MyHC MyHC
  • the MHC in the present invention is preferably a mammalian MHC, more preferably a human MHC.
  • the “content ratio of myosin heavy chain-expressing cells” means the proportion of cells expressing myosin heavy chain among all cells in a certain cell population, and “myosin heavy chain (expression) positive cells” It is synonymous with "rate”.
  • the present inventors studied the sheet-like skeletal myoblast culture, and when producing a clinical sheet-like skeletal myoblast culture, the sheet was used as part of the quality evaluation of the cells in the production process. It is necessary to confirm the proportion of MHC-expressing cells in the cells constituting the cell culture.
  • the performance of reagents such as anti-MHC antibodies used for such confirmation and the accuracy of the confirmation method are based on certain standards. Faced with the challenge of having no validation standards for this validation test, which must be validated, we examined how to prepare such standards. However, in creating such a standard, we faced the challenge that it was very difficult to prepare a cell population containing high levels of MHC expression positive cells.
  • typical cells that express MHC in a living body include muscle cells.
  • the cells do not have proliferative ability, a large amount of cell populations containing high-level MHC expression positive cells can be obtained. To obtain it, it would be necessary to collect a large amount of myocytes.
  • myocytes since it is preferable to use human myocytes as a standard for quality inspection of sheet-like cell cultures using human skeletal myoblasts, there are various restrictions on the collection of human-derived cells.
  • a cell population containing a high percentage of MHC expression positive cells is not commercially available.
  • the cell culture of the present invention contains MHC expression positive cells at a high level.
  • the cell culture of the present invention contains 1% or more of MHC expression positive cells, preferably 45% or more, more preferably 50% or more, still more preferably 60% or more, and particularly preferably 70% or more.
  • Examples of the cell population containing a high level of MHC expression positive cells include a cell population containing a high level of myocytes collected from skeletal muscle and the like, but the present invention is such a naturally occurring cell population. The purpose is to artificially create a cell population containing a high level of MHC expression positive cells by culture or the like.
  • the cell population of the present invention is preferably not purified for MHC by cell separation methods such as flow cytometry and immunopanning, particularly cell separation methods using antibodies against MHC.
  • the cell population of the present invention preferably also does not include cells artificially introduced with the MHC gene.
  • the cell population of the present invention is preferably obtained only by culturing for a predetermined period under substantially predetermined conditions.
  • the present invention relates to a method for producing a cell culture containing MHC expression positive cells at a high level, for example, 45% or more, comprising culturing skeletal myoblasts in a low serum medium for 3 to 6 days. It is.
  • a basal medium usually used in the art can be used, and the basal medium is not limited to this, but for example, DMEM, RPMI -1640, Ham's F-10, Ham's F-12, Eagle basal medium and the like. As what is generally used, DMEM is preferably used.
  • the serum content of the low serum medium in the method of the present invention is preferably about 0 to 5%, more preferably a serum-free medium, that is, a medium not containing serum.
  • serum protein may be added instead of serum.
  • serum proteins include, but are not limited to, serum albumin and transferrin.
  • the low serum medium used in the method of the present invention is preferably serum-free and is supplemented with serum albumin.
  • Serum albumin is preferably added to a concentration of about 0.05 to 20 mg / ml, more preferably about 0.1 to 10 mg / ml, and still more preferably about 0.6 to 2 mg / ml.
  • the low serum medium used in the method of the present invention preferably contains an insulin family peptide.
  • Insulin family peptides include insulin, insulin-like growth factor (IGF) -1 and IGF-2, and more preferably IGF-1 from the viewpoint of the ability to induce MHC expression.
  • IGF-1 is preferably about 0.001 to 5 ⁇ g / ml, more preferably about 0.002 to 2 ⁇ g / ml, and still more preferably 0.005 to 0.005. It is added to a concentration of about 5 ⁇ g / ml.
  • the low serum medium used in the methods of the invention comprises DMEM, 0.1-10 mg / ml bovine serum albumin (BSA) and 0.005-0.5 ⁇ g / ml insulin-like growth. It is a medium consisting of factor (IGF) -1.
  • the method includes a step of pre-culturing skeletal myoblasts in a growth medium before culturing in a low serum medium.
  • skeletal myoblasts are unlikely to adhere to the culture substrate, but the cells can be sufficiently adhered to the culture substrate by performing pre-culture.
  • the pre-culture time may be a time sufficient for the cells to adhere to the culture substrate, and those skilled in the art can appropriately select the culture time according to the seeded cell density, but preferably about 12 ⁇ 48 hours, more preferably about 14 to 24 hours.
  • growth medium used for the pre-culture any medium known in the technical field that can be used for growing and culturing skeletal myoblasts can be used.
  • growth media include, but are not limited to, for example, growth factors, antibiotics, etc., if necessary, in a medium in which 10-20% serum (FBS, FCS, etc.) is added to the basal medium.
  • FBS 10-20% serum
  • the cell culture obtained by the above method is a cell containing 1% or more of MHC expression positive cells, preferably 45% or more, more preferably 50% or more, still more preferably 60% or more, particularly preferably 70% or more. Become a group. Since such a cell culture is produced by culturing skeletal myoblasts as described above, it substantially consists of cells derived from skeletal myoblasts.
  • “consisting essentially of A” means that the composition may contain a component other than A as a component, but the content of components other than A is about a detection error or affects the effect of the present invention.
  • the cell culture of the present invention is preferably a cell population obtained by culturing a cell population substantially consisting of skeletal myoblasts in a low serum medium, the obtained cell population includes skeletal myoblasts. It can be considered that cells other than those derived from are not included.
  • the present invention also relates to an MHC expression inducing medium containing a basal medium and 0 to 5% serum.
  • the MHC expression induction medium of the present invention preferably further comprises a component selected from the group consisting of serum proteins and insulin family peptides. The types and contents of serum proteins and insulin family peptides are as described above for the production method of the present invention.
  • the present invention also relates to an assay standard comprising the cell culture of the present invention.
  • “certification” means that a criterion is set, and whether or not an inspection target satisfies the criterion is determined to determine pass / fail or grade. Therefore, the “standard for test” in the present invention refers to a standard indicating the standard for the test.
  • the “certification” in the present invention includes not only validation for ensuring the quality of products such as pharmaceuticals and regenerative medicine, but also accuracy of quality confirmation tests in these manufacturing stages and performance of reagents used in the quality confirmation tests. Validation for security is also included.
  • the assay standard of the present invention has a predetermined MHC expression positive cell content rate.
  • a predetermined MHC expression is obtained by adding MHC expression negative cells to a cell population having a known MHC expression positive cell content. It is common to adjust to a positive cell content.
  • MHC expression negative cell any cell may be used as long as it does not express MHC and does not adversely affect MHC expression positive cells.
  • fibroblasts preferably human fibroblasts, are preferably used as MHC expression-negative cells from the standpoint of easy acquisition, strain growth and maintenance.
  • the assay standard of the present invention quantitatively measures the MHC expression positive cell content in the cell culture of the present invention, and an appropriate amount of fibroblasts, preferably human fibers, in the cell culture.
  • a predetermined MHC expression positive cell content rate is prepared.
  • the assay standard adjusted to have a predetermined MHC expression positive cell content can be stored frozen as it is, and thawed before use and used for the assay. Since the MHC expression positive content in the assay standard of the present invention hardly changes even after cryopreservation, the assay standard after thawing can be used for the assay without confirming the MHC expression positive content by measurement. . Accordingly, the assay standard of the present invention may be provided in a frozen state. Cryopreservation and thawing may be performed by any known technique, and a cryoprotectant or the like may be used.
  • Another aspect of the present invention relates to a method for assaying the performance of an anti-MHC antibody using the assay standard of the present invention (hereinafter sometimes referred to as “anti-MHC antibody assay method”).
  • the assay method includes a step of causing an anti-MHC antibody to be assayed to act on a cell population having a known positive rate of MHC cells, a step of quantifying an anti-MHC antibody bound to a cell contained in the cell population, Comparing the MHC cell positive rate with the quantified amount of anti-MHC antibody.
  • the test method determines that the anti-MHC antibody to be tested has passed the test.
  • the process of carrying out may be included.
  • One or more cell populations can be used in the assay method. By using a plurality of cell populations having different MHC cell positive rates, a more accurate assay can be performed. Further, by using a cell population having a high MHC cell positive rate, the range of MHC expression that can be appropriately detected by the anti-MHC antibody (for example, the range of the MHC cell positive rate) can be broadened.
  • the anti-MHC antibody that has passed the test can be used for quality control of clinical sheet-like skeletal myoblast cultures.
  • the quantification of the anti-MHC antibody bound to the cells contained in the cell population can be performed by any known quantification technique such as flow cytometry.
  • Another aspect of the present invention is a method for assaying the accuracy of a method for confirming the expression of MHC in cells constituting a sheet-like cell culture using the assay standard of the present invention (hereinafter referred to as “accuracy assay method”). ”).
  • the assay method comprises a step of subjecting a cell population having a known MHC cell positive rate to the MHC expression confirmation method to be assayed, the MHC cell positive rate of the cell population, and the expression of MHC confirmed by the MHC expression confirmation method Comparing the amount.
  • the MHC expression confirmation method to be assayed is The process of determining with having passed may be included.
  • the MHC expression confirmation method that has passed the test can be used for quality control of clinical sheet-like skeletal myoblast cultures.
  • the MHC expression confirmation method may be based on any known MHC expression confirmation method, for example, an immunostaining method using an anti-MHC antibody, particularly flow cytometry.
  • a method for assaying skeletal myoblasts used in the production of a sheet-shaped cell culture using the assay standard of the present invention By this assay method, the quality of skeletal myoblasts used for the production of sheet-like skeletal myoblast cultures can be examined.
  • Such an assay method includes the following steps: (A) a step of collecting a part of proliferated and cultured skeletal myoblasts and measuring the expression level of MHC; (B) a step of measuring the MHC expression level of the test standard of the present invention having a predetermined MHC positive cell rate in the same manner as in (a); and (c) the result obtained in (a) and the result obtained in (b). Comparing the obtained results.
  • “proliferated and cultured skeletal myoblast” means a skeletal myoblast collected from a transplant subject and subjected to proliferating culture for production of a sheet-like skeletal myoblast culture.
  • the MHC expression level of the skeletal myoblast culture is measured before being used for the production of the sheet-shaped cell culture.
  • the “standard for assay having a predetermined MHC positive cell rate” means a standard for assay having a threshold value in the present assay method. For example, if the MHC expression level in the skeletal myoblast to be tested is lower than the MHC expression level in the test standard, it can be determined that the test has passed.
  • any measurement technique known in the technical field can be used, and the measurement method is not limited thereto.
  • a quantitative method using immunostaining specifically, Examples include flow cytometry. From the viewpoint of ease of work, etc., measurement is preferably performed by flow cytometry.
  • the measurement methods in steps (a) and (b) may not be the same measurement method as long as the respective measured values (actually measured values) can be directly or indirectly compared, but the step (c) From the standpoint of ease of comparison, etc., the same technique is preferably used.
  • it is not always necessary to use a quantitative technique and for example, a semi-quantitative technique such as measuring fluorescence intensity after immunostaining with an anti-MHC antibody may be used.
  • step (c) the result obtained in step (a) is compared with the result obtained in step (b).
  • the fluorescence intensity obtained in step (a) is compared with the fluorescence intensity obtained in step (b).
  • the fluorescence intensity in step (b) is stronger, the MHC positive cell rate of the skeletal myoblast culture in step (a) is less than the predetermined MHC positive cell rate.
  • a skeletal myoblast culture having a MHC positive cell rate equal to or lower than a predetermined MHC positive cell rate is used for producing a sheet-like cell culture. Select as myoblasts.
  • a method for assaying a sheet-shaped cell culture using the assay standard of the present invention is also provided.
  • the quality of the produced sheet-shaped cell culture can be inspected.
  • Such an assay method includes the following steps: (A) collecting a part of the sheet-shaped cell culture and dissociating the cell-cell adhesion to form a cell suspension; (B) measuring the MHC expression level of the cell population contained in the cell suspension; (C) a step of measuring the MHC expression level of the test standard of the present invention having a predetermined MHC positive cell rate; and (d) comparing the result obtained in (b) with the result obtained in (c). Process.
  • the “part of the sheet cell culture” may be a part of the sheet cut out, or one of a plurality of sheets produced by one production lot. There may be.
  • the quality of the original sheet can be inspected by the verification method of the present invention.
  • the quality of all sheets manufactured by the same production lot can be verified by verifying one sheet. Can be inspected.
  • any treatment may be performed as long as it does not cause damage to cells.
  • the dissociation method include, but are not limited to, a method of treating with an enzyme that dissociates cell-cell adhesion, such as trypsin, and a method of physically dissociating with a pipette or the like. It is done.
  • the same method as the measurement in the steps (a) and (b) of the method for assaying skeletal myoblasts used in the production of the sheet-like cell culture is used. it can.
  • step (d) the result obtained in step (b) is compared with the result obtained in step (c). Thereby, it can be judged whether the MHC expression positive cell rate in the manufactured sheet-like cell culture is higher or lower than a predetermined MHC expression positive cell rate.
  • a sheet-like skeletal myoblast culture having an MHC positive cell rate equal to or lower than a predetermined MHC positive cell rate is accepted.
  • quality ranking can be performed by using a plurality of test standards.
  • the quality of a sheet-like cell culture can be classified into four ranks by using three kinds of assay standards having different MHC expression positive cell rates. That is, for example, when the three standards for testing are the A standard, B standard, and C standard in descending order of MHC expression positive cell rate, if the MHC expression positive cell rate is higher than the A standard, it is ranked A, and the B standard If it is less than A standard, it can be rank B, if it is C standard or more and less than B standard, it can be rank C, and if it is less than C standard, it can be rank D.
  • Example 1 Preparation of cell culture with high MHC expression positive cell rate (1) Preparation of MHC expression cells After suspending human skeletal myoblasts in 12 mL growth medium (MCDB131 + 20% FBS), 8.0 ⁇ 10 4 cells / cm 2 was seeded in a culture dish at a density of 37 ⁇ 1 ° C. and pre-cultured under conditions of 37 ⁇ 1 ° C. and 5 ⁇ 1% CO 2 . 12-24 hours after the start of preculture, the medium was changed to a low serum medium (DMEM + bovine serum albumin (BSA) 1 mg / mL + insulin-like growth factor 0.05 ⁇ g / mL) and cultured for 6 days.
  • DMEM + bovine serum albumin (BSA) 1 mg / mL + insulin-like growth factor 0.05 ⁇ g / mL
  • the medium After culturing, the medium is discarded, and the surface of the formed sheet-shaped cell culture is washed with HBSS ( ⁇ ), 6 mL of TrypLE Select (manufactured by Thermo Fisher Scientific) is added, and the cells are incubated at 37 ° C. for 10 minutes. Dissociated. The dissociated cells were collected in a centrifuge tube, washed with HBSS ( ⁇ ), centrifuged, and the supernatant was discarded to obtain a skeletal myoblast culture containing MHC-expressing cells.
  • Human fibroblasts were suspended in 75 mL of medium (MCDB131 + 20% FBS), and then seeded at a density of 1000 cells / cm 2 , 37 ⁇ 1 ° C., 5 ⁇ 1% CO 2. For 7 days. After the culture, the medium was discarded and the cell culture surface was washed with HBSS ( ⁇ ). 50 mL of a cell dissociation agent was added and incubated at 37 ° C. for 15 minutes to dissociate the cells. The dissociated cells were collected in a centrifuge tube, washed with HBSS ( ⁇ ), centrifuged, and the supernatant was discarded to obtain cultured human fibroblasts.
  • medium MCDB131 + 20% FBS
  • Example 2 Preparation of assay standard (1) Preparation of MHC-expressing cells Suspended skeletal myoblasts in 12 mL of growth medium (MCDB131 + 20% FBS) and seeded on a culture dish at a density of 8.0 ⁇ 10 4 cells / cm 2. And 37 ⁇ 1 ° C. and 5 ⁇ 1% CO 2 . Twenty-six hours after the start of preculture, the medium was changed to a low serum medium (DMEM + bovine serum albumin (BSA) 1 mg / mL + insulin-like growth factor 0.05 ⁇ g / mL) and cultured for 5 days.
  • DMEM + bovine serum albumin (BSA) 1 mg / mL + insulin-like growth factor 0.05 ⁇ g / mL
  • the medium After culturing, the medium is discarded, and the surface of the formed sheet-shaped cell culture is washed with HBSS ( ⁇ ), 6 mL of TrypLE Select (manufactured by Thermo Fisher Scientific) is added, and the cells are incubated at 37 ° C. for 10 minutes. Dissociated. The dissociated cells were collected in a centrifuge tube, washed with HBSS ( ⁇ ), centrifuged, and the supernatant was discarded to obtain a skeletal myoblast culture containing MHC-expressing cells. In this skeletal myoblast culture, the number of MHC-expressing cells and the total number of cells were measured using a flow cytometer in the same manner as in Example 1 above.
  • the assay standard was prepared by mixing the skeletal myoblast culture obtained in (1) with human fibroblasts prepared in the same manner as in Example 1 (2). The mixed cells are centrifuged, and the supernatant is discarded. Then, a preservation solution (MCDB131 medium containing L-glutamine, DMSO, and human serum albumin at final concentrations of 8.7 mmol / L, 10% v / v, and 7 mg / mL, respectively) Were added and suspended and cryopreserved.
  • MCDB131 medium containing L-glutamine, DMSO, and human serum albumin at final concentrations of 8.7 mmol / L, 10% v / v, and 7 mg / mL, respectively
  • the assay standard produced using this can be assayed in a wide range of concentrations.

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Abstract

La présente invention décrit : un étalon d'essai destiné à l'évaluation de la performance d'un réactif à utiliser dans la production d'une culture de cellules en forme de feuille ou la précision d'un test d'évaluation de qualité ; une culture de cellules destinée à la production de l'étalon ; un procédé destiné à la production de l'étalon ou à la culture de cellules, etc. Ce but est atteint grâce à un procédé de production d'une culture de cellules, ledit procédé comprenant la culture de myoblastes squelettiques dans un milieu de culture à faible teneur en sérum contenant du sérum sous une concentration faible durant 3 à 6 jours.
PCT/JP2017/011609 2016-03-31 2017-03-23 Étalon destiné à un essai de chaîne lourde de myosine WO2017170090A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011520A1 (fr) * 1989-03-28 1990-10-04 Yamasa Shoyu Kabushiki Kaisha Anticorps contre une chaine lourde de myosine des muscles lisses
JP2013198438A (ja) * 2012-03-26 2013-10-03 Terumo Corp 細胞培養装置
JP2014042536A (ja) * 2013-12-11 2014-03-13 Terumo Corp シート状細胞培養物の製造方法
WO2015129764A1 (fr) * 2014-02-26 2015-09-03 テルモ株式会社 Procédé de production d'une population cellulaire ayant une grande pureté des cellules cibles

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Publication number Priority date Publication date Assignee Title
WO2014148321A1 (fr) * 2013-03-19 2014-09-25 学校法人東京女子医科大学 Stratifié de tapis de cellules contenant des myoblastes et son procédé de production

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011520A1 (fr) * 1989-03-28 1990-10-04 Yamasa Shoyu Kabushiki Kaisha Anticorps contre une chaine lourde de myosine des muscles lisses
JP2013198438A (ja) * 2012-03-26 2013-10-03 Terumo Corp 細胞培養装置
JP2014042536A (ja) * 2013-12-11 2014-03-13 Terumo Corp シート状細胞培養物の製造方法
WO2015129764A1 (fr) * 2014-02-26 2015-09-03 テルモ株式会社 Procédé de production d'une population cellulaire ayant une grande pureté des cellules cibles

Non-Patent Citations (2)

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Title
HAUGK, L. KATHLEEN ET AL.: "Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF", ENDOCRINOLOGY, vol. 141, no. 1, pages 100 - 110, XP002939650 *
MEN'EKI SOSHIKI SENSHOKU TROUBLE SHOOTING, 24 April 2012 (2012-04-24), Retrieved from the Internet <URL:http://www.abcam.co.jp/blog/index.cfm/2012/4/24/IHC_troubleshooting> *

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