WO2017077964A1 - 往復動撹拌装置を用いた血小板の製造方法 - Google Patents
往復動撹拌装置を用いた血小板の製造方法 Download PDFInfo
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Definitions
- the present invention relates to a method for producing platelets using a stirring device in which a stirring blade reciprocates.
- the platelet preparation is administered for the purpose of treatment and prevention of symptoms to a patient who exhibits massive bleeding at the time of surgery or injury, or a bleeding tendency associated with thrombocytopenia after anticancer drug treatment.
- the production of platelet products relies on blood donation by healthy volunteers.
- the number of blood donors is decreasing due to the population composition, and it is estimated that there will be insufficient blood donation for about 1 million people in 2027. Therefore, one of the objects in the technical field of the present invention is to stably supply platelets.
- the present inventors have succeeded in producing platelets having high physiological activity by using a shaking culture system using a shaker flask, that is, a culture method in which the culture vessel itself is shaken.
- a shaking culture system using a shaker flask
- the production of platelets CD41a + CD42b +
- the physiological activity of platelets PAC1 binding, P-selectin positive
- an object of the present invention is to provide a method for producing high-quality platelets that can be transplanted into a living body in a large-scale amount.
- the present inventors do not shake the culture vessel, but reciprocate the stirring blade disposed in the culture vessel to stir the culture solution while stirring the culture solution. It has been found that culturing cells can increase the production efficiency and physiological activity of platelets, suppress the deterioration of platelets, and reduce abnormal platelets. Furthermore, the present invention was completed by examining the cell density and other conditions during stirring culture using a reciprocating stirring device.
- the present invention is a method for producing platelets, comprising a step of culturing megakaryocyte cells in a culture solution in a culture vessel, wherein the stirring blade is not reciprocated or reversed in one direction.
- the culture solution is stirred using a steady stirring device.
- the culture vessel is a sealed bioreactor.
- the megakaryocyte cell is an undifferentiated cell than the megakaryocyte cell, and at least one gene selected from the group consisting of an oncogene, a polycomb gene, and an apoptosis inhibitor gene is used. It is also preferred that the cell is a cell from which the forced expression has been canceled after the forced expression.
- the present invention also relates to a method for producing a platelet preparation, comprising the step of producing platelets in megakaryocytes by the method described above, collecting the platelets from the culture, and a blood cell component other than platelets from the platelets. And a removing step.
- the present invention is a method for producing a blood product, which comprises a step of producing a platelet product by the method described above, and a step of obtaining the blood product by mixing the platelet product with other components. is there.
- the present invention also relates to platelets produced by any of the above methods.
- the present invention is a platelet preparation produced by any of the above methods, or a platelet preparation containing the above platelets.
- the present invention also relates to a blood product produced by the above method or a blood product containing the above platelets.
- the production efficiency of platelets can be improved as compared with the conventional shake culture. Furthermore, the produced platelets have a higher physiological activity than platelets produced by conventional shaking culture.
- the method of the present invention it is possible to suppress the deterioration reaction of platelets (decrease in the CD42b positive rate) and to suppress the production of abnormal platelets (annexin V positive platelets).
- FIG. 1 shows a bioreactor that is an embodiment of the present invention.
- FIG. 2 shows an installation example of a seal, a drive shaft, and a stirring blade in a bioreactor that is an embodiment of the present invention.
- FIG. 3 shows the structure of a stirring blade in a bioreactor that is an embodiment of the present invention.
- FIG. 4 shows the structure of the VMF culture apparatus used in the examples of the present invention.
- FIG. 5 shows the results of flow cytometry using platelets obtained by stirring or shaking culture of megakaryocytes using anti-CD42b antibody and anti-CD41a antibody.
- FIG. 6 shows flow cytometry using anti-CD42b antibody and anti-PAC-1 antibody before and after PMA or ADP stimulation on platelets obtained by stirring or shaking culture of megakaryocytes.
- FIG. 7 shows the results of flow cytometry using anti-CD42b antibody and anti-CD62p antibody on platelets obtained by stirring or shaking culture of megakaryocytes before and after stimulation with PMA or ADP.
- FIG. 8 shows the results of measuring the ratio of annexin V binding by flow cytometry on platelets obtained by stirring or shaking culture of megakaryocytes. It is a figure which shows roughly the structural example of the bioreactor provided with the multistage stirring blade.
- the method for producing platelets according to the present invention includes a step of culturing megakaryocyte cells in a culture solution in a culture vessel.
- an unsteady stirring device in which a stirring blade reciprocates or reverses in one direction instead of one direction.
- the culture medium is used and stirred.
- the culture solution may be stirred by reciprocating the stirring blade so that the main surface receives resistance from the fluid on the main surface.
- the stirring blade is reciprocated in the support shaft direction. More preferably, the culture vessel is allowed to stand still, the support shaft that supports the stirring blade is reciprocated in the axial extension direction, and the stirring blade is moved in the vertical direction.
- the “culture vessel” is not particularly limited as long as it is a vessel capable of culturing megakaryocyte cells while stirring the culture solution with a reciprocating stirring device.
- the culture container include an open culture dish, a closed flask with a screw cap, and a bioreactor (including a closed bioreactor).
- the “stirring blade” may be any one that can be placed in the culture medium and can directly stir the culture liquid.
- the stirring blade for example, a flat plate or a bent structure is used.
- the bioreactor 10 of this embodiment includes a culture tank 11, a seal 12, a drive shaft (stirring shaft) 13, and a stirring blade 14.
- the installation condition of the drive shaft 13 and the like with respect to the culture tank 11 may be a bottom attachment attached to the bottom of the culture tank 11 or a side attachment attached to the side of the culture tank 11.
- the culture tank 11 has a three-dimensional body part obtained by extending a planar shape such as a circle or a corner in the vertical direction.
- the seal 12 is made of a flexible material such as rubber that follows the movement of the drive shaft 13 or a bellows structure of a material such as metal or Teflon (registered trademark), and covers the upper end opening of the culture tank 11. And airtight.
- the drive shaft 13 is connected to a vertical movement device such as a reciprocating drive motor 15 at an upper portion thereof, and penetrates the seal 12 at an intermediate portion of the drive shaft 13 and is airtightly fixed to the seal 12.
- the stirring blade 14 has a flat plate shape or a bent structure, and the planar shape has a reduced area perpendicular to a circular shape (FIG. 3A) as shown in FIG. 3, for example, an ellipse (FIG. 3B). ), A rectangle (FIG.
- FIG. 3C a hole structure
- FIG. 3D a hole structure in which a hole 16 is provided in a disk, and one or more stirring blades 14 are fixed to the drive shaft 13. Is done.
- the punching hole 16 may be configured with a large hole provided in the stirring blade 14 (FIG. 3D), or may be configured with a plurality of small holes provided in a predetermined region (FIG. 9). ).
- the stirring blade 14 may have a plurality of stages.
- the bioreactor 10 shown in FIG. 9 has a configuration of a two-stage blade including an upper stirring blade 14 attached to the drive shaft 13 and a lower stirring blade 14 provided with a hole 16. The shearing effect in the stirring blade 14 is further improved.
- the stirring blades attached perpendicularly to the drive shaft (stirring shaft) 13 may be rotated, and the stirring blades may be rotated forward and backward by control by a servo motor.
- the servo motor is controlled to rotate forward and backward by a driver to constitute a forward / reverse drive motor 15 to enable forward / reverse control (control of acceleration, waveform, speed, etc.) by the servo motor.
- the bioreactor 10 is suitable for optimal cell growth, cell metabolism, megakaryocyte differentiation and maturation, megakaryocyte multinucleation, formation of propretlets, platelet production, maintenance of platelet bioactivity, and the like.
- a ventilation device an exhaust device, a temperature control device, a pH control device, a dissolved oxygen pressure (DOT) control device, a baffle, a sparger, a port, and the like may be provided.
- DOT dissolved oxygen pressure
- the shape of the culture tank 11 of the bioreactor 10 is not particularly limited, but may be, for example, a vertically long tubular shape, and may have a flat surface at the top and bottom of the tank.
- the volume of the culture tank 11 is at least 300 mL, preferably at least 1 L, 50 L, more preferably at least 200 L, more preferably at least 500 L, even more preferably at least 1000 L, even more preferably at least 2000 L.
- the “megakaryocyte” is the largest cell present in the bone marrow in vivo and is characterized by releasing platelets.
- the cell surface markers CD41a, CD42a, and CD42b are positively characterized, and in addition, a marker selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c May be further expressed.
- a “megakaryocyte” has a genome 16 to 32 times that of a normal cell when multinucleated (multiploidy). In the present specification, the term “megakaryocyte” has the above characteristics.
- pre-multinucleated megakaryocyte includes both multinucleated megakaryocytes and pre-multinucleated megakaryocytes.
- Pre-multinucleated megakaryocyte is also synonymous with “immature megakaryocyte” or “proliferative megakaryocyte”.
- Megakaryocyte cells can be obtained by various known methods.
- Non-limiting examples of the method for producing megakaryocyte cells include the method described in International Publication No. 2011/034073.
- an immortalized megakaryocyte cell line that proliferates indefinitely can be obtained by forcibly expressing an oncogene and a polycomb gene in “an undifferentiated cell than a megakaryocyte”.
- immortalized megakaryocyte cells can also be obtained by forcibly expressing an apoptosis-inhibiting gene in “cells that are undifferentiated from megakaryocyte cells” according to the method described in International Publication No. 2012/157586.
- These immortalized megakaryocyte cells are polynucleated by releasing the forced expression of the gene and release platelets.
- the methods described in the above documents may be combined.
- the forced expression of the oncogene, polycomb gene, and apoptosis-suppressing gene may be performed simultaneously or sequentially.
- a multinucleated megakaryocyte cell may be obtained by forcibly expressing an oncogene and a polycomb gene, suppressing the forced expression, then forcibly expressing an apoptosis suppressing gene, and suppressing the forced expression.
- multinucleated megakaryocyte cells can be obtained by forcibly expressing an oncogene, a polycomb gene, and an apoptosis-suppressing gene at the same time and simultaneously suppressing the forced expression.
- an oncogene and a polycomb gene are forcibly expressed, followed by forcibly expressing an apoptosis-inhibiting gene, and the forcible expression is simultaneously suppressed to obtain multinucleated megakaryocyte cells.
- cells that are undifferentiated from megakaryocyte cells means cells that have the ability to differentiate into megakaryocytes and that have various differentiation stages ranging from hematopoietic stem cell lines to megakaryocyte cells.
- Non-limiting examples of cells that are undifferentiated from megakaryocytes include hematopoietic stem cells, hematopoietic progenitor cells, CD34 positive cells, megakaryocytes / erythroid progenitor cells (MEP). These cells can be obtained by isolation from, for example, bone marrow, umbilical cord blood, and peripheral blood, and further obtained by inducing differentiation from pluripotent stem cells such as ES cells and iPS cells, which are more undifferentiated cells. You can also.
- oncogene refers to a gene that induces canceration of a cell in a living body, such as a MYC family gene (eg, c-MYC, N-MYC, L-MYC), SRC family.
- MYC family gene eg, c-MYC, N-MYC, L-MYC
- protein kinase family genes such as genes, RAS family genes, RAF family genes, c-Kit, PDGFR, and Abl.
- the “polycomb gene” is known as a gene that negatively regulates the CDKN2a (INK4a / ARF) gene and functions to avoid cell aging (Ogura et al., Regenerative Medicine vol.6, No .4, pp26-32; Jseus et al., Jseus et al., Nature Reviews Molecular Cell Biology vol.7, pp667-677, 2006; Proc. Natl. Acad. Sci. USA vol.100, pp211-216, 2003 ).
- Non-limiting examples of polycomb genes include BMI1, Mel18, Ring1a / b, Phc1 / 2/3, Cbx2 / 4/6/7/8, Ezh2, Eed, Suz12, HADC, Dnmt1 / 3a / 3b .
- apoptosis-suppressing gene refers to a gene having a function of suppressing cell apoptosis, and examples thereof include a BCL2 gene, a BCL-xL gene, a Survivin gene, and an MCL1 gene.
- platelet is one of cell components in blood and is characterized by CD41a positive and CD42b positive. Platelets play an important role in thrombus formation and hemostasis, and are also involved in tissue regeneration after injury and pathophysiology of inflammation. When platelets are activated by bleeding or the like, receptors for cell adhesion factors such as IntegrinIntegr ⁇ IIB ⁇ 3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) are expressed on the membrane. As a result, platelets aggregate and fibrin is coagulated by various blood coagulation factors released from the platelets, thereby forming a thrombus and promoting hemostasis.
- IntegrinIntegr ⁇ IIB ⁇ 3 glycoprotein IIb / IIIa; a complex of CD41a and CD61
- Platelet function can be measured and evaluated by known methods. For example, the amount of activated platelets can be measured using an antibody against PAC-1 that specifically binds to Integrin ⁇ IIB ⁇ 3 on the activated platelet membrane.
- CD62P P-selectin
- flow cytometry can be used by gating with an antibody against the activation-independent platelet marker CD61 or CD41, and then detecting the binding of anti-PAC-1 antibody or anti-CD62P antibody. These steps may be performed in the presence of adenosine diphosphate (ADP).
- ADP adenosine diphosphate
- the evaluation of platelet function can be performed by checking whether or not it binds to fibrinogen in the presence of ADP. Binding of platelets to fibrinogen results in integrin activation required early in thrombus formation.
- evaluation of platelet function can also be performed by visualizing and observing thrombus formation ability in vivo as shown in FIG. 6 of International Publication No. 2011/034073.
- CD42b in platelets when the expression rate of CD42b in platelets is low or the annexin V positive rate is low, it is evaluated that the platelets are deteriorated or abnormal. These platelets do not have thrombus formation or hemostasis function and are not clinically useful.
- degradation of platelets refers to a decrease in CD42b (GPIb ⁇ ) on the platelet surface. Therefore, degraded platelets include platelets with reduced expression of CD42b and platelets whose extracellular region of CD42b has been cleaved by a shedding reaction. When CD42b on the platelet surface is depleted, it cannot associate with von Willebrand factor (VWF), resulting in loss of platelet blood clotting function. Platelet degradation can be evaluated using the CD42b negative rate (or CD42b negative particle number) relative to the CD42b positive rate (or CD42b positive particle number) in the platelet fraction as an index.
- VWF von Willebrand factor
- CD42b positive rate means the proportion of platelets that can be bound by anti-CD42b antibody among platelets contained in the platelet fraction
- CD42b negative rate means that anti-CD42b antibody binds among platelets contained in the platelet fraction. Means the proportion of not platelets.
- abnormal platelets refers to platelets in which phosphatidylserine, which is a negatively charged phospholipid, is exposed from the inside to the outside of the lipid bilayer.
- phosphatidylserine which is a negatively charged phospholipid
- Abnormal platelets always have a lot of phosphatidylserine exposed on the surface, and when such platelets are administered to a patient, they cause an excessive blood clotting reaction and severe pathological conditions such as disseminated intravascular coagulation syndrome.
- annexin V binds to phosphatidylserine
- phosphatidylserine on the platelet surface can be detected using a flow cytometer with the binding amount of fluorescently labeled annexin V as an index. Therefore, the amount of abnormal platelets can be evaluated by the annexin V positive rate in the platelet fraction, that is, the ratio or number of platelets to which annexin binds. The higher the annexin V positive rate or the higher the number of annexin V particles, the more abnormal platelets.
- the culture conditions for megakaryocyte cells in the present invention can be normal conditions.
- the temperature can be about 35 ° C. to about 42 ° C., about 36 ° C. to about 40 ° C., or about 37 ° C. to about 39 ° C., and can be 5-15% CO 2 and / or 20% O 2. .
- the medium for culturing megakaryocyte cells is not particularly limited, and a known medium suitable for producing platelets from megakaryocyte cells or a medium equivalent thereto can be appropriately used.
- a medium used for animal cell culture can be prepared as a basal medium.
- basal media include IMDM medium, MediumMedi199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer Life's medium, Neurosal's medium ) And mixed media thereof.
- the medium may contain serum or plasma, or may be serum-free. If necessary, the medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (eg L-glutamine), ascorbic acid It may also contain one or more substances such as heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like. Cytokines are proteins that promote blood cell differentiation.
- VEGF vascular endothelial growth factor
- TPO thrombopoietin
- SCF Stem Cell Cell Factor
- ITS insulin-transferrin-selenite Supplements
- ADAM inhibitors etc.
- a preferable medium in the present invention is an IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO.
- SCF may be included, and heparin may also be included.
- TPO can be about 10 ng / mL to about 200 ng / mL, or about 50 ng / mL to about 100 ng / mL
- SCF is about 10 ng / mL to about 200 ng / mL
- Heparin can be about 10 U / mL to about 100 U / mL, or about 25 U / mL.
- Phorbol esters eg, phorbol-12-myristate-13-acetate; PMA may be added.
- human serum When using serum, human serum is desirable. Further, human plasma or the like may be used instead of serum. According to the method of the present invention, even if these components are used, platelets equivalent to those obtained using serum can be obtained.
- Tet-On® or Tet-Off® for forced gene expression and release
- a drug-responsive gene expression induction system such as a system
- the forced expression step the corresponding drug, for example, tetracycline or doxycycline is contained in the medium, and the forced expression is suppressed by removing them from the medium. May be.
- the step of culturing megakaryocytes in the present invention is carried out by suspension culture, it can be carried out without feeder cells.
- the present invention also includes platelets produced by the method according to the present invention.
- the method for producing a platelet preparation according to the present invention comprises a step of culturing megakaryocyte cells by the method according to the present invention to produce platelets, recovering a fraction rich in platelets from the culture, and from the platelet fraction. Removing blood cell components other than platelets.
- the step of removing blood cell components is performed by removing blood cell components other than platelets including megakaryocytes using a leukocyte removal filter (eg, Terumo, Asahi Kasei Medical). Can do.
- a leukocyte removal filter eg, Terumo, Asahi Kasei Medical
- the method for producing a blood product according to the present invention includes a step of producing a platelet product by the method according to the present invention, and a step of mixing the platelet product with other components.
- other components include red blood cells.
- TKDN SeV2 human fetal skin fibroblast-derived iPS cells established using Sendai virus
- the gene transfer system utilized a lentiviral vector system.
- the lentiviral vector is a Tetracycline-controlled Tet-On (registered trademark) gene expression induction system vector.
- the mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G was prepared by recombination with c-MYC, BMI1, and BCL-xL.
- LV-TRE-c-Myc-Ubc-tTA-I2G LV-TRE-BMI1-Ubc-tTA-I2G
- LV-TRE-BCL-xL-Ubc-tTA-I2G LV-TRE-BCL-xL-Ubc-tTA-I2G
- the virus particles were prepared by gene transfer of the above lentiviral vector into 293T cells by any method.
- BMI1, MYC, and BCL-xL genes are introduced into the genomic sequence of the target cell by infecting the target cell with such virus particles. These genes stably introduced into the genome sequence can be forcibly expressed by adding doxycycline (clontech # 631311) to the medium.
- Medium is basal medium (15% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin-Glutamine (GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO), 0.45mM 1-Thioglycerol (Sigma -Aldrich), 50 ⁇ g / mL L-Ascorbic Acid (Sigma-Aldrich) containing IMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich)) 50ng / mL Human thrombopoietin (TPO) (R & D SmL A medium (hereinafter referred to as differentiation medium) supplemented with Stem® Cell® Factor® (SCF) ® (R & D® SYSTEMS) and 2 ⁇ g / mL Doxycycline® (Dox) and further added Protamine at a final concentration of 10 ⁇ g / mL was used.
- Infection day 12 to infection day 13 The same operation as on infection day 2 was performed. After counting the number of cells, the cells were seeded on C3H10T1 / 2 feeder cells in a 3 ⁇ 105 cells / 10 mL / 100 mm dish (100 mm dish).
- BCL-xL virus infection of megakaryocyte self-propagating strain BCL-xL was introduced into the megakaryocyte self-propagating strain on day 14 of infection by the lentiviral method. Viral particles were added to the medium so as to have MOI of 10, and the cells were infected by spin infection (centrifugation at 32 ° C., 900 rpm, 60 minutes).
- the forced expression of the BCL-xL gene was performed by adding 1 ⁇ g / mL of doxycycline (clontech # 631311) to the medium.
- the obtained SeV2-MKCL was statically cultured in a 10 cm dish (10 mL / dish).
- the medium used was IMDM as a basic medium, and the following components were added (the concentration was the final concentration).
- FBS Sigma # 172012 lot.12E261) 15% L-Glutamin (Gibco # 25030-081) 2mM ITS (Gibco # 41400-045) 100 times diluted MTG (monothioglycerol, sigma # M6145-25ML) 450 ⁇ M Ascorbic acid (sigma # A4544) 50 ⁇ g / mL Puromycin (sigma # P8833-100MG) 2 ⁇ g / mL SCF (Wako Pure Chemical # 193-15513) 50ng / mL TPO-like active substance 200ng / mL
- the culture conditions were 37 ° C. and 5% CO 2.
- the immortalized megakaryocyte cell line (SeV2-MKCL) obtained by the above method was washed twice with PBS ( ⁇ ) and suspended in the following medium at a seeding density of 1.0 ⁇ 10 5 cells / mL.
- the medium is IMDM containing the following components (concentration is final concentration).
- the immortalized megakaryocyte cell line (SeV2-MKCL) obtained by the above method was suspended in the above medium to prepare a cell suspension.
- 2.4 L of the cell suspension was added to the bioreactor 10 and 25 mL of the cell suspension was added to a 125 mL volume shaker flask.
- the bioreactor 10 has at least one stirrer blade 14 and used a 3.0 L volume VMF culture apparatus (hereinafter referred to as VMF) capable of reciprocating the stirrer blade 14 in the vertical direction.
- VMF 3.0 L volume VMF culture apparatus
- VMF a 2.4 L immortalized megakaryocyte cell line suspension was cultured.
- the culture environment was 37 ° C. and 5% CO 2.
- the stirring speed was 1.6 Hz, and the stirring stroke length was 3 cm.
- 3-1 Measurement of platelets
- 900 mL of diluent was added to a 1.5 mL microtube, and 100 mL of culture supernatant was added thereto and mixed.
- 200 mL of the diluted culture supernatant was dispensed into a FACS tube, and the following labeled antibody or protein was added for staining.
- dispense 100 mL of the culture supernatant into a FACS tube add the following labeled antibody or protein, stain, and use annexin V binding buffer (BD) immediately before flow cytometer analysis. Diluted 5 times and analyzed.
- BD annexin V binding buffer
- the antibodies used are as follows. Measurement of normal and deteriorated platelets 0.5 ⁇ L Anti-CD41 antibody APC label (Bio Legend 303710) 0.5 ⁇ L anti-CD42a antibody PB labeling (eBioscience 48-0428-42) 0.5 ⁇ L anti-CD42b antibody PE label (eBioscience 12-0428-42) Measurement of physiological activity of platelets 0.5 ⁇ L Anti-CD42a antibody PB labeling (eBioscience 48-0428-42) 0.5 ⁇ L anti-CD42b antibody PE label (eBioscience 12-0428-42) 0.5 ⁇ L anti-CD62p antibody APC labeling (Bio Legend 304910) 10 ⁇ L anti-PAC-1 antibody FITC labeling (BD 303704) Measurement of abnormal platelet count 0.5 ⁇ L Anti-CD41 antibody APC label (Bio Legend 303710) 0.5 ⁇ L anti-CD42a antibody PB labeling (eBioscience 48-0428-42) 5 (L Annexin V FITC sign (BD, 556419)
- FIG. 5B The measurement results of normal platelet production are shown in FIG. Normal platelet production was higher in VMF culture than in shaker flask culture (FIG. 5B). In addition, when the number of normal platelets per megakaryocyte number was calculated as the normal platelet production efficiency, the normal platelet production efficiency was about 6.0 to 7.7 times higher in the culture with VMF than in the culture with the shaker flask (FIG. 5). (C)). In other words, the production of normal platelets per megakaryocyte number could be increased by culturing with VMF, compared with culturing with a conventional shaker flask.
- 3-3 Measurement of deteriorated platelets
- 3-1 3-1.
- each treated sample was analyzed using a flow cytometer, and the number of particles of CD41a positive CD42b positive and CD41a positive CD42b negative was measured.
- the ratio of the number of deteriorated platelets to the number of normal platelets was calculated using the number of CD41a positive CD42b positive particles as the normal platelet number and the CD41a positive CD42b negative particle number as the deteriorated platelet number. The results are shown in FIG.
- the ratio of the number of deteriorated platelets to the number of normal platelets in the culture with VMF decreased to about 0.31 to 0.39 times the ratio of the number of deteriorated platelets to the number of normal platelets in the culture with the shaker flask (FIG. 5 (A)).
- the production of deteriorated platelets could be reduced by culturing with VMF as compared with the culturing with a conventional shaker flask.
- Platelet stimulation was performed at room temperature with PMA 0.2 mM (Phorbol 12-myristate 13-acetate, sigma # P1585-1MG) or ADP 100 ⁇ M (sigma # A2754) and Thrombin 0.5 U / mL (sigma). I went there. Measurement was carried out 30 minutes after stimulation with FACSverce from BD. The PAC-1 positive rate and CD62p positive rate before and after stimulation in the CD42a positive platelet fraction were measured and evaluated. The results are shown in FIG. 6 and FIG.
- the production of platelets with higher bioactivity could be increased by culturing with VMF than by culturing with conventional shaker flasks.
- the present invention can obtain high-quality platelets that cannot be achieved by shaking culture in a shaker flask. Therefore, the present invention can contribute to the realization of industrial-scale mass production of platelets.
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Abstract
Description
本実施形態のバイオリアクター10は、図1に示す通り、培養槽11と、シール12と、駆動軸(撹拌軸)13と、撹拌羽根14と、を有する。培養槽11に対する駆動軸13等の設置条件は、図2に示す通り、培養槽11の底部に取り付けるボトム取付、若しくは培養槽11の側部に取り付けるサイド取付でもよい。培養槽11は、円若しくは角等の平面形を縦方向に伸ばした3次元の胴部を有している。シール12は、駆動軸13の動きに追従するゴム等のフレキシブルな材料の膜状体、又は、金属若しくはテフロン(登録商標)等の素材のベローズ構造からなり、培養槽11の上端開口部を覆って気密に設けられている。駆動軸13は、その上部において上下動装置例えば往復駆動式モータ15に連結されていて、尚かつ、駆動軸13の中間部でシール12を貫通すると共に、シール12に気密に固定されている。撹拌羽根14は、平板状、若しくは折り曲げ構造で、平面形状は、図3に示す通り、円形(図3(A))に対して直交する面積を減じた、例えば、楕円(図3(B))、矩形(図3(C))、若しくは円盤に抜き穴16が設けられた抜き穴構造(図3(D))を有しており、駆動軸13に1段以上の撹拌羽根14が固定される。撹拌羽根14を穴あきとすることで、該抜き穴16を通過する流体や細胞への剪断作用が強まる(図9参照)。抜き穴16は撹拌羽根14に大きく設けられた穴で構成されていてもよいし(図3(D))、所定の領域に設けられた複数の小さな穴で構成されていてもよい(図9)。
システムのような薬剤応答性の遺伝子発現誘導システムを用いる場合、強制発現する工程においては、対応する薬剤、例えば、テトラサイクリンまたはドキシサイクリンを培地に含有させ、これらを培地から除くことによって強制発現を抑制してもよい。
1-1.iPS細胞からの造血前駆細胞の調製
ヒトiPS細胞(TKDN SeV2:センダイウイルスを用いて樹立されたヒト胎児皮膚繊維芽細胞由来iPS細胞)から、Takayama N., et al. J Exp Med. 2817-2830 (2010)に記載の方法に従って、血球細胞への分化培養を実施した。即ち、ヒトES/iPS細胞コロニーを20ng/mL VEGF (R&D SYSTEMS)存在下でC3H10T1/2フィーダー細胞と14日間共培養して造血前駆細胞(Hematopoietic Progenitor Cells;HPC)を作製した。培養条件は20% O2、5% CO2で実施した(特に記載がない限り、以下同条件)。
遺伝子導入システムは、レンチウイルスベクターシステムを利用した。レンチウイルスベクターは、Tetracycline制御性のTet-On(登録商標)遺伝子発現誘導システムベクターである。LV-TRE-mOKS-Ubc-tTA-I2G(Kobayashi, T., et al. Cell 142, 787-799 (2010))のmOKSカセットをc-MYC、BMI1、BCL-xLに組み替えることで作製した。それぞれ、LV-TRE-c-Myc-Ubc-tTA-I2G、LV-TRE-BMI1-Ubc-tTA-I2G、およびLV-TRE-BCL-xL-Ubc-tTA-I2Gとした。
予めC3H10T1/2フィーダー細胞を播種した6 well plate上に、上記の方法で得られたHPCを5x104cells/wellずつ播種し、レンチウイルス法にてc-MYCおよびBMI1を強制発現させた。このとき、細胞株1種類につき6 wellずつ使用した。即ち、それぞれMOI 20になるように培地中にウイルス粒子を添加し、スピンインフェクション(32℃ 900rpm、60分間遠心)で感染させた。本操作は、12時間おきに2回実施した。
上記の方法でcMYC及びBMI1ウイルス感染を実施した日を感染0日目として、以下の通り、cMYC及びBMI1遺伝子導入型巨核球細胞を培養することで、巨核球自己増殖株をそれぞれ作製した。BMI1遺伝子、c-MYC遺伝子の強制発現は、培地にドキシサイクリン (clontech #631311) 1μg/mLを加えることにより行った。
ピペッティングにて上記の方法で得られたウイルス感染済み血球細胞を回収し、1200rpm、5分間遠心操作を行って上清を除去した後、新しい分化培地で懸濁して新しいC3H10T1/2フィーダー細胞上に播種した(6well plate)。感染9日目に同様の操作をすることによって継代を実施した。細胞数を計測後1×105 cells/2mL/wellでC3H10T1/2フィーダー細胞上に播種した(6well plate)。
感染2日目と同様の操作を実施した。細胞数を計測後3×105cells/10mL/100mm dishでC3H10T1/2フィーダー細胞上に播種した(100mm dish)。
ウイルス感染済み血球細胞を回収し、細胞1.0×105個あたり、抗ヒトCD41a-APC抗体(BioLegend)、抗ヒトCD42b-PE抗体(eBioscience)、抗ヒトCD235ab-pacific blue(BioLegend)抗体をそれぞれ2μL、1μL、1μLずつを用いて抗体反応した。反応後に、FACS Verse(BD)を用いて解析した。感染14日目において、CD41a陽性率が50%以上であった細胞を、巨核球自己増殖株とした。
前記感染14日目の巨核球自己増殖株に、レンチウイルス法にてBCL-xLを遺伝子導入した。MOI 10になるように培地中にウイルス粒子を添加し、スピンインフェクション(32℃ 900rpm、60分間遠心)で感染させた。BCL-xL遺伝子の強制発現は、培地にドキシサイクリン (clontech #631311) 1μg/mLを加えることにより行った。
・感染14目~感染18日目
前述の方法で得られたBCL-xLを遺伝子導入した巨核球自己増殖株を回収し、1200rpm、5分間遠心操作を行った。遠心後、沈殿した細胞を新しい分化培地で懸濁した後、新しいC3H10T1/2フィーダー細胞上に2×105cells/2mL/wellで播種した(6well plate)。
細胞数を計測後、3×105 cells/10mL/100mm dishで播種した。
細胞数を計測後、1×105 cells/10mL/100mm dishで播種した。以後、4-7日毎に継代を行い、維持培養を行った。
FBS(シグマ#172012 lot.12E261)15%
L-Glutamin (Gibco #25030-081) 2mM
ITS (Gibco #41400-045) 100倍希釈
MTG (monothioglycerol, sigma #M6145-25ML) 450μM
アスコルビン酸 (sigma #A4544) 50μg/mL
Puromycin (sigma #P8833-100MG) 2μg/mL
SCF (和光純薬 #193-15513) 50ng/mL
TPO様作用物質 200ng/mL
培養条件は、37℃、5%CO2とした。
次に、ドキシサイクリンを含まない培地で培養することで強制発現を解除した。具体的には、1.の方法で得た不死化巨核球細胞株(SeV2-MKCL)を、PBS(-)で2度洗浄し、1.0x105 cells/mLの播種密度で次の培地に懸濁した。
FBS 15%
L-Glutamine (Gibco #25030-081) 2mM
ITS (Gibco #41400-045) 100倍希釈
アスコルビン酸 (sigma #A4544) 50μg/mL
SCF (和光純薬 #193-15513) 50ng/mL
TPO様作用物質 200ng/mL
ADAM阻害剤 15μM
SR1 750nM
ROCK阻害剤 10μM
VMFの仕様は、次のとおりである。本体外寸法(mm):300W×485D×890H、培養槽11の寸法(mm):内径140×深さ203、培養槽11の液量:3.0 L、撹拌羽根14:下段楕円形状折り曲げ構造+上段平板楕円形状 2段取付、直動伝達方式:リニアシャフトドライブノンシール式、温度調節範囲:室温+5~20℃、上下動振幅:10~30 mm、上下動最大翼速度:80~150 mm/s、計測制御:撹拌, 温度, pH, 溶存酸素, レベル, フィード。図4にVMFを示す。
上記2.の方法で生産された血小板を測定するために、強制発現解除後、培養6日後の培養上清サンプルを回収し、各種抗体による染色と共に、フローサイトメーターを用いた分析を行った。CD41a陽性CD42b陽性の粒子数を正常血小板数とし、CD41a陽性CD42b陰性の粒子数を劣化血小板数とした。アネキシンV陽性の粒子数を異常血小板数とした。また、サンプルをPMA又はADP/Thrombinで刺激し、刺激前後のPAC-1およびCD62pの陽性率を算出して生理活性を測定した。
3-1.血小板の測定
正常血小板、劣化血小板、血小板の生理活性の測定のために、1.5 mLマイクロチューブに希釈液 900 mLを添加し、そこに培養上清100 mLを添加し、混合した。希釈混合した培養上清200 mLをFACSチューブに分注し、以下の標識抗体或はタンパク質を添加して染色を行った。異常血小板の測定のために、培養上清 100 mLをFACSチューブに分注し、以下の標識抗体或はタンパク質を添加して染色を行い、フローサイトメーター分析直前にアネキシン V binding buffer(BD)で5倍希釈し、分析した。
使用した抗体は以下のとおりである。
正常血小板および劣化血小板の測定
0.5μL 抗CD41抗体 APC標識(Bio Legend 303710)
0.5μL 抗CD42a抗体PB標識(eBioscience 48-0428-42)
0.5μL 抗CD42b抗体PE標識(eBioscience 12-0428-42)
血小板の生理活性の測定
0.5μL 抗CD42a抗体PB標識(eBioscience 48-0428-42)
0.5μL 抗CD42b抗体PE標識(eBioscience 12-0428-42)
0.5μL 抗CD62p抗体APC標識(Bio Legend 304910)
10μL 抗PAC-1抗体FITC標識(BD 303704)
異常血小板数の測定
0.5μL 抗CD41抗体 APC標識(Bio Legend 303710)
0.5μL 抗CD42a抗体PB標識(eBioscience 48-0428-42)
5 (L Annexin V FITC標識(BD, 556419)
結果を図5に示す。正常血小板生産量は、VMFによる培養では、シェーカーフラスコによる培養に比較して高かった(図5(B))。また、巨核球細胞数当たりの正常血小板数を正常血小板生産効率として算出した場合、シェーカーフラスコによる培養に比べて、VMFによる培養では、当該正常血小板生産効率が約6.0~7.7倍高かった(図5(C))。つまり、VMFによる培養によって、従来のシェーカーフラスコによる培養よりも、巨核球細胞数当たりの正常血小板生産量を増加させることができた。
劣化血小板を測定するとき、前記3-1.の方法によって、各処理サンプルをフローサイトメーターを用いて分析し、CD41a陽性CD42b陽性、及び、CD41a陽性CD42b陰性、それぞれの粒子数を測定した。そして、CD41a陽性CD42b陽性の粒子数を正常血小板数、並びに、CD41a陽性CD42b陰性の粒子数劣化血小板数として、正常血小板数に対する劣化血小板数の割合を算出した。
結果を図5に示す。VMFによる培養での正常血小板数に対する劣化血小板数の割合は、シェーカーフラスコによる培養での正常血小板数に対する劣化血小板数の割合の約0.31~0.39倍に減少した(図5(A))。つまり、VMFによる培養によって、従来のシェーカーフラスコによる培養よりも、劣化血小板の生産量を低く抑えることができた。
血小板の刺激は、PMA 0.2 mM (Phorbol 12-myristate 13-acetate, sigma #P1585-1MG)、又は、ADP 100 μM(sigma #A2754)およびThrombin 0.5 U/mL(sigma)で室温にて行った。刺激30分後にBD社FACSverceにて測定を実施した。CD42a陽性の血小板画分における、刺激前後のPAC-1陽性率及びCD62p陽性率を測定し、比較評価した。
結果を図6、及び図7に示す。VMFによる培養では、シェーカーフラスコによる培養に比較して、PMAもしくはADP/Thrombin刺激時のPAC-1陽性率が約1.9~3.1倍高かった(図6)。また、VMFによる培養では、シェーカーフラスコによる培養に比較して、CD62p陽性率が約1.8~2.7倍以上高かった(図7)。つまり、VMFによる培養によって、従来のシェーカーフラスコによる培養よりも、生理活性の高い血小板の生産量を増加させることができた。
前記3-1.の方法によって、各処理サンプルをフローサイトメーターを用いて分析し、アネキシンV陽性の粒子数を測定した。CD42b陽性アネキシンV陽性の粒子数を異常血小板数とした。
結果を図8に示す。VMFによる培養では、シェーカーフラスコによる培養に比較して、CD42b陽性アネキシンV陽性の粒子数が約0.31~0.34倍未満に減少した(図8)。つまり、 VMFによる培養によって、従来のシェーカーフラスコによる培養よりも、異常血小板の生産量を低く抑えることができた。
Claims (13)
- 血小板の製造方法であって、
培養容器内の培養液中で巨核球細胞を培養する培養工程を含み、
前記培養工程において、前記培養液を往復動する撹拌羽根で撹拌する、方法。 - 前記培養工程に用いられる前記撹拌羽根を上下方向、若しくは左右方向、若しくは正逆回転方向に往復動して培養液を非定常撹拌するバイオリアクターを用いる、請求項1に記載の方法。
- 前記培養工程に用いられる前記撹拌羽根は楕円形状であって、前記培養工程において前記撹拌羽根を上下に往復して培養液を撹拌するバイオリアクターを用いる、請求項1に記載の方法。
- 前記培養工程に用いられる前記撹拌羽根は楕円形状折り曲げ構造であって、前記培養工程において前記撹拌羽根を上下に往復して培養液を撹拌するバイオリアクターを用いる、請求項1に記載の方法。
- 前記培養工程に用いられる前記撹拌羽根は折り曲げ楕円形状であって、平板楕円形状の撹拌羽根と直交して撹拌軸に1段以上取り付けられており、前記培養工程において前記撹拌羽根を上下に往復して培養液を撹拌するバイオリアクターを用いる、請求項1に記載の方法。
- 前記撹拌羽根として、抜き穴が設けられたものを用いる請求項1から5のいずれか一項に記載の方法。
- 前記培養容器が、密閉型バイオリアクターである、請求項1に記載の方法。
- 前記巨核球細胞が、
巨核球細胞より未分化な細胞において、癌遺伝子、ポリコーム遺伝子、及びアポトーシス抑制遺伝子からなる群より選択される遺伝子の少なくとも1つを強制発現した後、当該強制発現を解除した細胞である、請求項1から7のいずれか1項に記載の方法。 - 血小板製剤の製造方法であって、
請求項1から8のいずれか1項に記載の方法で巨核球細胞に血小板を産生させ、培養物から血小板を回収する工程と、
前記血小板から血小板以外の血球系細胞成分を除去する工程と、を含む方法。 - 血液製剤の製造方法であって、
請求項9に記載の方法で血小板製剤を製造する工程と、
前記血小板製剤を他の成分と混合して血液製剤を得る工程と、を含む方法。 - 請求項1から8のいずれか1項に記載の方法で製造された血小板。
- 請求項8に記載の方法で製造された血小板製剤、又は請求項11に記載の血小板を含む、血小板製剤。
- 請求項10に記載された方法で製造された血液製剤、又は請求項11に記載の血小板を含む、血液製剤。
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JPWO2017077964A1 (ja) | 2018-10-11 |
KR20180080261A (ko) | 2018-07-11 |
RU2018120143A3 (ja) | 2020-04-06 |
JP6856537B2 (ja) | 2021-04-07 |
CN108473954A (zh) | 2018-08-31 |
EP3372674A1 (en) | 2018-09-12 |
CA3003679A1 (en) | 2017-05-11 |
US20180318352A1 (en) | 2018-11-08 |
AU2016350296A1 (en) | 2018-05-31 |
AU2016350296B2 (en) | 2022-02-10 |
EP3372674B1 (en) | 2020-10-07 |
EP3372674A4 (en) | 2019-04-24 |
KR102664131B1 (ko) | 2024-05-10 |
ES2833357T3 (es) | 2021-06-15 |
RU2018120143A (ru) | 2019-12-04 |
SG11201803591UA (en) | 2018-05-30 |
DK3372674T3 (da) | 2020-10-12 |
RU2741871C2 (ru) | 2021-01-29 |
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