Platelet production method and device

Abstract

The present invention provides a platelet production method comprising the steps of: (a) culturing megakaryocyte cells for at least 6 days in a platelet production medium in which turbulence is generated; and (b) injecting the medium containing the megakaryocyte cells after the step (a) into a predetermined platelet production device to expose the megakaryocyte cells to a laminar flow.

Description

[Technical Field]

[0001] The present invention relates to a platelet production device and a platelet production method. [Background technology]

[0002] Platelet preparations are administered to patients who experience massive bleeding during surgery or injury, or who experience bleeding tendencies associated with thrombocytopenia after anticancer drug therapy, for the purpose of treating and preventing these symptoms. Currently, the production of platelet preparations relies on blood donations, but there is a need for a safer, more stable supply of platelets with regard to infection risk. To meet this need, methods for producing platelets from in vitro cultured megakaryocytes have been developed. The present inventors have established a method for establishing immortalized megakaryocyte progenitor cell lines (imMKCL) using pluripotent stem cells as a source.

[0003] It is known that by culturing the iPS cell-derived megakaryocytic cell line imMKCL Clone 7 established by the present inventors in a turbulence-dependent vertical stirred culture device (VerMES 8L culture vessel), human platelets with functionality equivalent to donated platelets can be produced at a level of over 100 billion, which is the amount actually used for clinical transfusions (see, for example, Non-Patent Document 1 and Patent Document 1).

[0004] Furthermore, a shear stress-dependent microfluidic chip-type platelet production bioreactor is known (see, for example, Patent Document 2), which was developed based on the concept that shear stress in the bloodstream is important for in vivo platelet production (see, for example, Non-Patent Documents 2, 3, and 4). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2018 / 164040 [Patent Document 2] WO2017 / 061528

Non-Patent Literature

[0006]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

[0007] However, even with the culture method disclosed in Non-Patent Document 1, it was not observed that 100% of megakaryocytes produced platelets within the 6-day culture period, and there was a problem in that many megakaryocytes did not reach the platelet-producing form (mode).

[0008] Furthermore, platelet production was attempted using a shear stress-dependent microfluidic chip-type platelet production bioreactor disclosed in Patent Document 2, but only inferior data were obtained in terms of functionality and efficiency compared to the production methods using the turbulence-dependent culture vessels disclosed in Non-Patent Document 1 and Patent Document 1.

[0009] There is a need for a method for efficiently producing platelets with sufficient functionality by sufficiently maturing megakaryocytes to a platelet-producing form, and for an apparatus capable of realizing this method. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have found that platelets with sufficient functionality can be produced with high efficiency by performing turbulence-dependent culture of megakaryocytes for a predetermined period of time and then subjecting the medium containing the megakaryocytes to a shear stress-dependent microfluidic chip-type platelet production bioreactor, and have thus completed the present invention.

[0011] That is, the present invention includes the following aspects. [1] (a) culturing megakaryocyte cells in a turbulent platelet-producing medium for at least 6 days; (b) injecting the medium containing the megakaryocytes that have been subjected to step (a) into a platelet production device and exposing the megakaryocytes to laminar flow; A method for producing platelets, comprising: the platelet producing device comprising: an inlet for injecting megakaryocyte cells; Platelet collection area and a flow path extending from the inlet to the collection section; Equipped with The flow path is the height of the flow channel at the end on the injection port side is greater than the maximum diameter of the megakaryocyte cells to be injected; a height of the flow channel at the end on the recovery section side is smaller than the minimum diameter of the megakaryocytes to be injected and larger than the maximum diameter of the platelets; A flow path height is configured to decrease from the inlet toward the collection section, This allows the platelet production device to expose the megakaryocytes to laminar flow while trapping them within the flow path, and is configured to release platelets produced by the megakaryocytes from the flow path to a collection section, thereby achieving a platelet production method. [2] The method according to [1], wherein the width of the flow channel changes from the inlet to the collection section, and the change is correlated with the diameter distribution of the megakaryocytes to be injected. [3] The distance of the channel from the end of the inlet side is x, the height of the channel at the distance x is h(x), the width of the channel at the distance x is w(x), and the diameter of the megakaryocyte cell is x d When w(x) is determined according to the frequency of megakaryocytes with a diameter of h(x), and the diameter x d The method according to [2], wherein the greater the frequency of megakaryocyte cells having h(x), the larger w(x) is configured. [4] The method according to any one of [1] to [3], wherein the platelet production device comprises a plurality of pillars rising from the bottom surface of the end of the flow channel on the side of the collection section. [5] The method according to any one of [1] to [4], comprising, before the step of culturing the megakaryocytic cells, a step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-inhibiting gene in cells less differentiated than megakaryocytic cells to obtain immortalized megakaryocytic cells. [6] The method according to any one of [1] to [5], comprising the step of recovering platelets from the recovery section of the platelet production device. [7] The method according to any one of [1] to [6], wherein the step of culturing for at least 6 days is carried out using a swing flask or a culture tank equipped with impellers that can be operated non-stationarily. [8] An injection port for megakaryocyte cells; Platelet collection area and a flow path extending from the inlet to the collection section; A platelet production device comprising: The flow path is the height of the flow channel at the end on the injection port side is greater than the maximum diameter of the megakaryocyte cells to be injected; a height of the flow channel at the end on the recovery section side is smaller than the minimum diameter of the megakaryocytes to be injected and larger than the maximum diameter of the platelets; A flow path height is configured to decrease from the inlet toward the collection section, This allows the platelet production device to expose the megakaryocytes to laminar flow while trapping them within the flow path, and is configured to release platelets produced by the megakaryocytes from the flow path to a collection section. [9] The distance of the channel from the end of the inlet side is x, the height of the channel at the distance x is h(x), the width of the channel at the distance x is w(x), and the diameter of the megakaryocyte cell is x d When w(x) is determined according to the frequency of megakaryocytes with a diameter of h(x), and the diameter x d The device according to [8], wherein the greater the frequency of megakaryocyte cells having h(x), the larger w(x) is. [Effects of the Invention]

[0012] The platelet production method of the present invention makes it possible to efficiently produce human platelets that exhibit the same functionality as donated platelets. Furthermore, the platelet production device of the present invention has a predetermined characteristic width of the flow channel, which makes it possible to maintain a constant flow state of the liquid flowing into the device even after megakaryocytes are injected and captured in the flow channel. This makes it possible to apply a constant shear stress to the megakaryocytes, reducing the variability in the number of platelets produced and enabling effective platelet production. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a diagram schematically illustrating a method for producing platelets according to one embodiment of the present invention. [Figure 2] FIG. 2 is a conceptual cross-sectional view showing an example of a megakaryocyte cell culture vessel preferably used in the platelet production method according to one embodiment of the present invention. [Figure 3] FIG. 3 is a plan view of the culture tank shown in FIG. [Figure 4] FIG. 4 is a conceptual perspective view showing an example of the platelet production device according to the second embodiment of the present invention. [Figure 5A] FIG. 5A is a diagram illustrating variables in the design of the flow channels of the platelet production device according to the second embodiment of the present invention. [Figure 5B] FIG. 5B is a graph showing an example of the diameter distribution in a megakaryocyte cell population. [Figure 5C] FIG. 5C is a graph showing an example of the distance x from the inlet end of the flow channel in the platelet production device according to the second embodiment of the present invention and the design of the flow channel h(x). [Figure 5D] FIG. 5D is a graph showing an example of the design of the distance x from the inlet end of the channel in the platelet production device according to the second embodiment of the present invention and the channel width w(x). [Figure 6] FIG. 6 is a conceptual cross-sectional view showing an example of the production of the platelet production device according to the second embodiment of the present invention. [Figure 7]Figure 7 is a graph showing the number of CD41a / CD42b-positive platelets produced when megakaryocytes on days 5, 6, 7, and 8 of Gene OFF maturation culture were introduced into a platelet production device to produce platelets. [Figure 8] Figure 8 is a graph showing the results of measuring platelet hemostatic function (PAC-1) of megakaryocytes on days 5, 6, 7, and 8 of Gene OFF maturation culture, which were introduced into a platelet production device to produce platelets, and the platelet mixed culture medium was collected. [Figure 9] Figure 9 is a graph showing the results of measuring Annexin V, a platelet aging marker, in a platelet production device that introduced megakaryocytes on days 5, 6, 7, and 8 of Gene OFF maturation culture to produce platelets, and then collecting the platelet mixed culture medium. [Figure 10] FIG. 10 is a FACS diagram showing the results of Annexin V measurement. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the embodiments described below.

[0015] [1. Spherical culture] According to one embodiment, the present invention relates to a method for producing platelets, the method comprising at least the following steps: (a) culturing megakaryocyte cells in a turbulent platelet-producing medium for at least 6 days; (b) injecting the medium containing the megakaryocytes that have been subjected to step (a) into a platelet production device and exposing the megakaryocytes to laminar flow;

[0016] In the platelet production method of the present invention, the megakaryocytes to be cultured in step (a) refer to megakaryocytes as defined below. The term "megakaryocytes" refers to the largest cells present in the bone marrow in vivo and characterized by the ability to release platelets. Megakaryocytes are also characterized by the positivity of cell surface markers CD41a, CD42a, and CD42b, and may further express at least one marker selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD122, CD131, and CD203c. When "megakaryocytes" are multinucleated (polyploidized), they have a genome 16 to 32 times larger than that of normal cells. However, in this specification, the term "megakaryocytes" refers to both multinucleated and unmultinucleated megakaryocytes, as long as they have the above characteristics. The term "pre-multinucleated megakaryocytic cells" is also synonymous with the terms "immature megakaryocytic cells" and "proliferative megakaryocytic cells." The term "pre-multinucleated megakaryocytic cells" refers to, for example, mononucleated or binucleated cells that are less differentiated than multinucleated megakaryocytic cells, are CD41a-positive, CD42a-positive, and CD42b-positive, and have not undergone nuclear polyploidization. Pre-multinucleated megakaryocytic cells can be obtained by various known methods, for example, by isolation from bone marrow, umbilical cord blood, or peripheral blood, or by induction of differentiation from pluripotent stem cells such as ES cells and iPS cells. Megakaryocytic cells can be obtained by various known methods and are not particularly limited, and may be megakaryocytic cells obtained from any source by any method. For example, megakaryocytic cells may be obtained by further inducing differentiation of the pre-multinucleated megakaryocytic cells described above. Furthermore, when the term "megakaryocyte" is used simply in this specification, it may refer not only to a single megakaryocyte but also to a megakaryocyte cell population composed of multiple megakaryocyte cells. A megakaryocyte cell population is generally a population composed of heterogeneous cells with a specific diameter distribution.

[0017] In one embodiment, the method for producing platelets according to the present invention includes, prior to the step (a), a step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-inhibiting gene in cells less differentiated than megakaryocytic cells to obtain immortalized megakaryocytic cells.

[0018] A non-limiting example of a method for producing such immortalized megakaryocytic cells is the method described in International Publication No. 2011 / 034073. In this method, immortalized megakaryocytic cell lines capable of indefinite proliferation can be obtained by forcibly expressing an oncogene and a polycomb gene in "cells less differentiated than megakaryocytic cells." Furthermore, immortalized megakaryocytic cell lines can also be obtained by forcibly expressing an apoptosis-inhibiting gene in "cells less differentiated than megakaryocytic cells" according to the method described in International Publication No. 2012 / 157586. These immortalized megakaryocytic cell lines undergo multinucleation and begin to release platelets by deactivating the forced gene expression. Therefore, the culturing step in the present invention can also be referred to as a culturing step in which the forced gene expression is deactivated.

[0019] In the step of obtaining immortalized megakaryocytic cells, which can be performed before step (a), the methods described in the above-mentioned literature may be combined to obtain megakaryocytic cells. In this case, the forced expression of the oncogene, polycomb gene, and apoptosis inhibitor gene may be performed simultaneously or sequentially. For example, the oncogene and polycomb gene may be forcedly expressed, their forced expression may be inhibited, and then the apoptosis inhibitor gene may be forcedly expressed and inhibited to obtain multinucleated megakaryocytic cells. Alternatively, the oncogene, polycomb gene, and apoptosis inhibitor gene may be simultaneously expressed and their forced expression may be simultaneously inhibited to obtain multinucleated megakaryocytic cells. The oncogene and polycomb gene may be first forcedly expressed, followed by the apoptosis inhibitor gene, and their forced expression may be simultaneously inhibited to obtain multinucleated megakaryocytic cells. In this specification, the forced expression of the genes may be referred to as the expansion culture step, the proliferation phase, or the proliferative state, and the suppression of the forced expression may be referred to as the maturation culture step or the maturation phase.

[0020] As used herein, the term "cells less differentiated than megakaryocytes" refers to cells capable of differentiating into megakaryocytes, and refers to cells at various stages of differentiation ranging from hematopoietic stem cells to megakaryocytes. Non-limiting examples of cells less differentiated than megakaryocytes include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, and megakaryocytic progenitor cells (MEPs). These cells can be obtained by isolation from, for example, bone marrow, umbilical cord blood, or peripheral blood, or by inducing differentiation from pluripotent stem cells, such as ES cells and iPS cells, which are even less differentiated cells.

[0021] As used herein, the term "oncogene" refers to a gene that induces cell transformation in vivo, and examples include MYC family genes (e.g., c-MYC, N-MYC, L-MYC), SRC family genes, RAS family genes, RAF family genes, and protein kinase family genes such as c-Kit, PDGFR, and Abl.

[0022] The term "Polycomb genes" refers to genes that negatively regulate the CDKN2a (INK4a / ARF) gene and prevent cellular senescence (Ogura et al., Regenerative Medicine, Vol. 6, No. 4, pp. 26-32; Jesus et al., Nature Reviews Molecular Cell Biology, Vol. 7, pp. 667-677, 2006; Proc. Natl. Acad. Sci. USA, Vol. 100, pp. 211-216, 2003). Non-limiting examples of Polycomb genes include BMI1, Mel18, Ring1a / b, Phc1 / 2 / 3, Cbx2 / 4 / 6 / 7 / 8, Ezh2, Eed, Suz12, HDAC, and Dnmt1 / 3a / 3b.

[0023] The term "apoptosis-suppressing gene" refers to a gene that has the function of suppressing cellular apoptosis, and examples thereof include the BCL2 gene, BCL-xL gene, Survivin gene, and MCL1 gene.

[0024] Forced gene expression and deactivation of forced expression can be performed by the methods described in WO 2011 / 034073, WO 2012 / 157586, WO 2014 / 123242, or Nakamura S et al., Cell Stem Cell. 14, 535-548, 2014, or other known methods or methods equivalent thereto. For example, when a drug-responsive gene expression induction system such as the Tet-on (registered trademark) or Tet-off (registered trademark) system is used for forced gene expression and deactivation, the forced expression may be suppressed (deactivated) by adding a corresponding drug, such as tetracycline or doxycycline, to the medium in the forced expression step and removing the drug from the medium.

[0025] The megakaryocyte culture conditions for forced gene expression and suppression (removal) of forced gene expression can be standard conditions, such as a temperature of about 35°C to about 42°C, about 36°C to about 40°C, or about 37°C to about 39°C, and 5 to 15% CO2 and / or 20% O2.

[0026] Specifically, the step of forcibly expressing the above genes in cells less differentiated than megakaryocytic cells can be carried out according to the usual methods of those skilled in the art, for example, by introducing into cells less differentiated than megakaryocytic cells a vector that expresses these genes, or a protein or RNA encoding these genes.Furthermore, it can be carried out by contacting cells less differentiated than megakaryocytic cells with a low molecular weight compound that induces the expression of these genes.

[0027] Examples of vectors that can express these genes include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses, as well as animal cell expression plasmids (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo). Retroviral and lentiviral vectors are preferred because they can be expressed by a single transfection. Examples of promoters that can be used in expression vectors include the EF-α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter. In addition to the promoter, the expression vector may optionally contain an enhancer, poly(A) addition signal, a selectable marker gene, an SV40 replication origin, and the like. Useful selection marker genes include, for example, the dihydrofolate reductase gene, the neomycin resistance gene, and the puromycin resistance gene.

[0028] A drug-responsive vector may be used as the expression vector. For example, a drug-responsive vector having a tetracycline-responsive element in the promoter region may be used to control the expression of the gene with tetracycline or doxycycline. Alternatively, an expression vector may be used in which loxP sequences are placed so as to flank the gene, the promoter region, or both, in order to excise the gene from the vector using the Cre-loxP system.

[0029] The production of megakaryocytic cells may include at least one of the following steps: (i) treating cells cultured under forced expression of an apoptosis-inhibiting gene with an actomyosin complex function inhibitor; or (ii) treating cells with a ROCK inhibitor. These treatments can promote more stable proliferation and multinucleation.

[0030] Those skilled in the art can determine the optimal concentration of an inhibitor of actomyosin complex function, a ROCK inhibitor, or the like, through preliminary experiments. Furthermore, the treatment period and method can be appropriately selected by those skilled in the art. For example, in the case of treatment with blebbistatin, a myosin heavy chain II ATPase inhibitor, 2 to 15 μg / ml or 5 to 10 μg / ml is added to the culture medium, and the culture period is preferably about 5 to 10 days, particularly about 6 to 7 days. Furthermore, the ROCK inhibitor Y27632 can be used at a concentration of 5 to 15 μM, 8 to 12 μM, or preferably about 10 μM. The treatment period with Y27632 is 10 to 21 days, preferably about 14 days.

[0031] Examples of ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitors include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·HO (Y27632). In some cases, antibodies that inhibit Rho kinase activity or nucleic acids (e.g., shRNA) can also be used as ROCK inhibitors.

[0032] After the forced expression step, the megakaryocytes or megakaryocyte progenitor cells obtained in this step are cultured in a platelet-producing medium. In the culturing step, suppression or termination of forced expression can be achieved, for example, by preventing contact of the cells with the corresponding drug if forced expression was achieved using a drug-responsive vector in the previous step. Specifically, when forced gene expression is achieved using doxycycline or tetracycline, forced expression can be suppressed by culturing the cells in a medium from which these drugs have been removed. Alternatively, when a vector containing the above-mentioned LoxP is used, suppression can be achieved by introducing Cre recombinase into the cells. Furthermore, when a transient expression vector, or RNA or protein introduction is used, suppression can be achieved by terminating contact with the vector. The medium used in this step can be the same as that described above.

[0033] The platelet-producing medium used in step (a) is not particularly limited, and any known medium suitable for platelet production from megakaryocytic cells or a medium equivalent thereto can be used as appropriate. For example, a medium used for culturing animal cells can be prepared as the basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixtures thereof.

[0034] The medium may contain serum or plasma, or may be serum-free. If necessary, the medium may also contain one or more substances, such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and cytokines. Cytokines are proteins that promote hematopoietic differentiation, and examples include vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, stem cell factor (SCF), ITS (insulin-transferrin-selenite) supplements, and ADAM inhibitors. A preferred medium in the present invention is IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. The solution may further contain SCF and / or heparin. The concentrations of each are not particularly limited, but for example, TPO can be about 10 ng / mL to about 200 ng / mL, or about 50 ng / mL to about 100 ng / mL, SCF can be about 10 ng / mL to about 200 ng / mL, or about 50 ng / mL, and heparin can be about 10 U / mL to about 100 U / mL, or about 25 U / mL. A phorbol ester (e.g., phorbol-12-myristate-13-acetate; PMA) may also be added.

[0035] In the production method of the present invention, the megakaryocyte cell culture step may be performed under serum-free and / or feeder cell-free conditions. Preferably, the method involves culturing megakaryocytes produced according to the method of the present invention in a medium containing TPO. If the platelet production step can be performed in a serum-free and feeder cell-free environment, immunogenicity problems are less likely to occur when the obtained platelets are used clinically. Furthermore, if platelets can be produced without the use of feeder cells, there is no need to attach feeder cells, and suspension culture can be performed in flasks, etc., thereby reducing production costs and making the medium suitable for mass production. When feeder cells are not used, a conditioned medium may be used. The conditioned medium is not particularly limited and can be prepared by methods known to those skilled in the art. For example, the conditioned medium can be obtained by appropriately culturing feeder cells and removing the feeder cells from the culture using a filter.

[0036] A ROCK inhibitor and / or an actomyosin complex function inhibitor may be added to the platelet production medium. The ROCK inhibitor and actomyosin complex function inhibitor may be the same as those used in the above-mentioned method for producing multinucleated megakaryocytes. An example of a ROCK inhibitor is Y27632. An example of an actomyosin complex function inhibitor is blebbistatin, a myosin heavy chain II ATPase inhibitor. A ROCK inhibitor may be added alone, or a ROCK inhibitor and an actomyosin complex function inhibitor may be added alone, or they may be added in combination.

[0037] The ROCK inhibitor and / or actomyosin complex function inhibitor is preferably added at 0.1 μM to 30 μM, and may be, for example, 0.5 μM to 25 μM, 5 μM to 20 μM, etc. The culture period after addition of the ROCK inhibitor and / or actomyosin complex function inhibitor can be 1 to 15 days, and may also be 3 days, 5 days, 7 days, etc. The addition of the ROCK inhibitor and / or actomyosin complex function inhibitor can further increase the proportion of CD42b-positive platelets.

[0038] Regarding the conditions of the culture composition and the like of the platelet production method in step (a), reference can be made to US2012 / 0238023A1 (International Publication No. 2011 / 034073), US2014 / 0127815A1 (International Publication No. 2012 / 157586), and US2016 / 0002599A1 (International Publication No. 2014 / 123242), which disclose non-limiting examples of methods for producing megakaryocytes and methods for producing platelets; these patent applications are incorporated herein by reference.

[0039] The culture period specified in step (a) is at least 6 days. At least 6 days means about 144 hours or more. Therefore, the culture period may be, for example, 6 days, 6.5 days (about 156 hours), 7 days (about 168 hours), 7.5 days (about 180 hours), 8 days (about 192 hours), 8.5 days (about 204 hours), or 9 days (about 216 hours). In one embodiment, the culture period is at least 6 days but shorter than 8 days (about 192 hours). In another embodiment, the culture period is at least 6 days but shorter than 7 days (about 168 hours). It is desirable to perform subculture as appropriate during the culture period.

[0040] During the culture period, turbulence is generated in the medium containing megakaryocytes. Culturing megakaryocytes in the presence of turbulence "educates" the megakaryocytes and improves both the quality and quantity of platelet production from the megakaryocytes. Turbulence may be generated continuously or intermittently from the start to the end of the culture period. In one embodiment, culture is performed for at least 6 days (approximately 144 hours) under conditions where turbulence is continuously present. The culture period may be, for example, 6 days, 6.5 days (approximately 156 hours), 7 days (approximately 168 hours), 7.5 days (approximately 180 hours), 8 days (approximately 192 hours), 8.5 days (approximately 204 hours), or 9 days (approximately 216 hours). It is particularly preferable to culture for 6 days under conditions where turbulence is continuously present. Furthermore, turbulence is generated when the turbulence energy is about 0.00016 m 2 / s 2 ~approx. 0.02m 2 / s2 It is preferable to perform the culture so that the shear stress is a value of about 0.1 Pa to about 6.0 Pa. It is more preferable to perform the culture so that the shear stress is a value of about 0.1 Pa to about 6.0 Pa. The turbulence energy can be constant from the start to the end of the culture period, or it can be varied. Thus, in one embodiment, the present invention relates to a method for improving the function (i.e., the ability to produce platelets) of megakaryocytes, which comprises culturing megakaryocytes in the presence of turbulence for at least 6 days. In another embodiment, the present invention relates to a method for producing megakaryocytes with improved function (i.e., the ability to produce platelets), which comprises culturing megakaryocytes in the presence of turbulence for at least 6 days.

[0041] The method for generating turbulence in a medium containing megakaryocytes is not particularly limited. For example, as shown in FIG. 1, it can be performed using a flask 1. In this case, it can be performed by filling the flask 1 with a medium containing megakaryocytes and shaking the flask to culture. For example, the rotation radius and rotation speed of a shaker that can generate the above-mentioned preferred turbulence energy can be determined by a preliminary experiment, and the flask can be shaken according to the obtained conditions.

[0042] One example of a method for generating turbulence in a medium containing megakaryocytic cells is a method using a culture tank capable of unsteady stirring. More specifically, a culture tank equipped with impellers capable of unsteady operation can be used. The impellers capable of unsteady operation are preferably impellers capable of up-and-down reciprocation, left-and-right reciprocation, and / or rotary reciprocation. A specific example of a culture tank that can be used is the VerMES reactor manufactured by Satake Machinery Co., Ltd. The VerMES reactor is described in detail in Patent Document 1, Non-Patent Document 1, WO2017 / 077964, and WO2019 / 009364, and the conditions and means described therein can be used.

[0043] An example of a culture vessel capable of unsteady stirring will be briefly described with reference to FIGS. 2 and 3. The illustrated culture vessel and its operation are merely examples. As long as the culture vessel can generate the predetermined turbulent energy, it is not limited to a specific structure or operation. As shown in FIGS. 2 and 3, the culture vessel capable of unsteady stirring includes a container 11 that contains a medium C containing megakaryocytes and a stirring mechanism 12 having a stirring blade 121 that stirs the medium C in the container 11. The stirring mechanism 12 is configured to reciprocate the stirring blade 121. In FIG. 2, the direction of reciprocation of the stirring blade 121 is indicated by arrow R. Furthermore, the stirring mechanism 12 controls the reciprocation of the stirring blade 121 so as to generate a desired turbulent energy in the medium C. In the stirring mechanism 12, it is preferable to control the stroke, reciprocation speed (e.g., average reciprocation speed), and reciprocation frequency of the stirring blade 121. In particular, it is preferable to control the reciprocation of the stirring blade 121 in an unsteady pattern. The desired turbulence energy can be calculated using known simulation techniques.

[0044] Furthermore, the culture tank is preferably configured as follows. The container 11 of the culture tank is hollow, and in Figures 2 and 3, the container 11 is formed in a substantially cylindrical shape, as an example. In the present invention, the container may be formed in a shape other than a substantially cylindrical shape, as long as it is hollow. The container 11 has a top wall portion (or top portion) 11a and a bottom wall portion (or bottom portion) 11b that face each other in a substantially vertical direction, and a peripheral wall portion (or peripheral portion) 11c that extends between the outer peripheral edges of the top wall portion 11a and the bottom wall portion 11b. Furthermore, the container 11 is preferably formed in an elongated shape that extends in a substantially vertical direction.

[0045] In FIG. 2, top wall 11a is configured as a lid for container 11, separate from peripheral wall 11c. Culture medium C can be introduced into container 11 with top wall 11a removed. In the present invention, an inlet for introducing the culture medium may be drilled in the container. In this case, the top wall may be integrally formed with the peripheral wall. Furthermore, in the present invention, the container may be formed to open upward depending on the platelet production conditions. In this case, it is preferable that an opening is formed in the top wall, or that the container does not have a top wall. The volume of container 11 can be any value as long as platelets can be produced. However, from the viewpoint of increasing the amount of platelets produced, the volume of container 11 is preferably about 300 mL or more, about 1 L or more, about 50 L or more, about 200 L or more, about 500 L or more, about 1000 L or more, or about 2000 L or more.

[0046] As shown in FIG. 2, the agitator blade 121 of the stirring mechanism 12 of the culture tank is arranged along an intersecting plane that intersects with the reciprocating direction at a predetermined intersecting angle θ1. The intersecting angle θ1 is approximately 90°. In other words, the agitator blade 121 is arranged along an intersecting plane that is approximately perpendicular to the reciprocating direction. The agitator blade 21 is formed in a substantially flat plate shape. The outer peripheral edge 121a of the agitator blade 121 is formed in a substantially circular shape when viewed from a direction perpendicular to the intersecting plane. As shown in FIGS. 2 and 3, the agitator blade 121 is arranged at intervals from the top wall portion 11a, bottom wall portion 11b, and peripheral wall portion 11c of the vessel 11. Such an agitator blade 121 is sometimes called an "agitator blade." The other shape of the agitator blade 121 and the distance between the peripheral wall portion 11c of the vessel 11 and the outer peripheral edge 121a of the agitator blade 121 may be determined depending on the desired turbulence energy.

[0047] However, in the agitator blade of the present invention, the crossing angle of the agitator blade may be other than approximately 90° depending on the desired turbulence energy. The crossing angle is preferably within the range of approximately 0° to approximately 180°. Furthermore, the agitator blade may be formed into a shape other than an approximately flat plate depending on the desired turbulence energy. For example, the agitator blade may be formed into an approximately hemispherical shell shape, an approximately bowl shape, an approximately curved plate shape, an approximately corrugated plate shape, or the like. Furthermore, the outer periphery of the agitator blade may be formed into a shape other than an approximately circular shape when viewed perpendicular to the crossing plane depending on the desired turbulence energy. For example, the outer periphery of the agitator blade may be formed into an approximately semicircular shape, an approximately elliptical shape, an approximately semi-elliptical shape, an approximately sector shape, an approximately polygonal shape such as an approximately square shape, or an approximately star-shaped polygonal shape when viewed perpendicular to the crossing plane. The agitator blade may also have at least one hole penetrating in the reciprocating direction, and the shape, number, and position of the hole may be determined depending on the desired turbulence energy.

[0048] 2, the stirring mechanism 12 has a driving source 122 for reciprocating the stirring blade 121 and a connecting member 123 that connects the stirring blade 121 and the driving source 122. The driving source 122 is configured to reciprocate the connecting member 122, thereby causing the stirring blade 121 to reciprocate. In addition to the reciprocating motion, the driving source 122 may also be configured to rotate the stirring blade 121 and the connecting member 123 around the axis 123a of the connecting member 123. In this case, in addition to controlling the reciprocating motion of the stirring blade 121, the stirring mechanism 12 may also control the rotation speed and rotation direction of the stirring blade 121. In particular, it is preferable that the reciprocating motion and rotation of the stirring blade 121 be controlled in a non-steady state.

[0049] Furthermore, connecting member 123 is formed in a generally shaft shape extending along its axis 123a. A longitudinal tip 23b of connecting member 123 is attached to mixing impeller 121, and a longitudinal base end 122c of connecting member 123 is held by drive source 22 so as to be able to reciprocate. As shown in Fig. 2, tip 123b of connecting member 123 is attached at a position that generally coincides with the center of gravity of mixing impeller 121. Note that the tip of the connecting member may be attached at a position offset from the center of gravity of the mixing impeller depending on the desired turbulence energy.

[0050] Such stirring mechanism 12 is attached to the top wall 11a of vessel 11. As for the specific attachment structure of stirring mechanism 12, an insertion hole 11d penetrating in the reciprocating direction is formed in top wall 11a of vessel 11, and stirring mechanism 12 is attached to top wall 11a of vessel 11 with connecting member 123 inserted into insertion hole 11d and stirring blade 121 housed inside vessel 11. Note that in the present invention, the stirring mechanism may be attached to the bottom wall or peripheral wall of the vessel using the specific attachment structure of the stirrer described above, instead of to the top wall of the vessel.

[0051] To improve the airtightness of the vessel 11, the culture tank may have a sealing member 13 configured to seal the gap between the periphery of the insertion hole 11d of the vessel 1 and the connecting member 123 of the stirring mechanism 12 while allowing the connecting member 123 to reciprocate. For example, the sealing member 13 may have a flexible structure that can follow the reciprocating movement of the connecting member 123. Furthermore, the flexible structure may be a membrane structure made of a flexible material such as rubber, or a bellows structure made of metal, Teflon (registered trademark), or the like. In the present invention, the sealing member may be configured to slidably hold the connecting member in the reciprocating direction.

[0052] In such a culture tank, the impeller 121 of the stirring mechanism 12 reciprocates within a predetermined range of movement within the vessel 11. This range of movement is set within the vessel 11 or in the medium C so as to obtain the desired turbulent energy. In particular, the length of the reciprocating direction of the range of movement, i.e., the maximum stroke of the reciprocating movement of the impeller 21 and the center position of the reciprocating direction of the range of movement, are preferably determined in accordance with the length of the vessel 11 in the reciprocating direction, the distance from the bottom wall 11b of the vessel 11 to the liquid surface c1 of the medium C, the capacity of the vessel 11, and the desired turbulent energy.

[0053] The above-mentioned culture tank is one example of an apparatus for carrying out the method of the present invention, and step (a) of the present invention is not particularly limited as long as it can impart a predetermined amount of turbulence energy to a platelet-producing medium containing megakaryocytes.

[0054] The megakaryocytes obtained after the completion of step (a) are a cell population with heterogeneous cell diameters and a cell population with a predetermined cell diameter distribution. Generally, the cell diameter distribution curve of the cell population has a single peak and roughly shows a log-normal distribution curve. In such a megakaryocyte cell population, the maximum diameter of the megakaryocyte cells is the maximum value obtained from the actual measurements of the diameters of the megakaryocyte cell population contained in the medium after step (a). Similarly, the minimum diameter of the megakaryocyte cells is the minimum value obtained from the actual measurements of the diameters of the megakaryocyte cell population contained in the medium after step (a). The shape of the cell diameter distribution curve in a megakaryocyte cell population, as well as the maximum and minimum diameters of the cells, are generally the same in megakaryocyte cell populations cultured under the same conditions. The diameter of the megakaryocyte cells is, for example, about 5 to about 160 μm.

[0055] After step (a) is completed and before step (b) is performed, a step of removing contaminants from the medium used in step (a), for example, using a filter, or a step of replacing the medium may be performed. Alternatively, the platelet-producing medium that has been subjected to step (a) may be subjected to step (b). After step (a) is completed, step (b) may be performed, for example, within approximately 2 hours, preferably within 1 hour.

[0056] Next, step (b) will be described. Step (b) is a step of injecting megakaryocytes that have undergone the culturing step into a predetermined platelet production device 2 and exposing the megakaryocytes to laminar flow. This applies mainly shear stress to the megakaryocytes, thereby promoting the production of platelets from the megakaryocytes.

[0057] Here, a platelet production device capable of carrying out step (b) will be described with reference to Fig. 1. Fig. 1 is a cross-sectional view schematically showing a platelet production device 2 according to a first embodiment of the present invention. As shown in Fig. 1, the platelet production device 2 comprises an inlet 21 for a megakaryocyte cell population, a flow path 22, and a platelet collection section 23. One end 22a of the flow path 22 is connected to the inlet 21, and the other end 22b is connected to the collection section 23. In Fig. 1, X indicates the flow direction in the flow path 22 of the platelet production device 2, and Z indicates the height direction of the flow path 22. Flow indicates the direction of the flow added to the megakaryocyte cells.

[0058] Inlet 21 of platelet production device 2 is disposed corresponding to upstream end 22a of flow path 22. Inlet 21 is an open part that opens toward the outside of the device, and is configured so that a medium containing megakaryocytes can be applied to platelet production device 2 through inlet 21. The shape and size of inlet 21 are not particularly limited, but it can have a shape suitable for sending a medium containing megakaryocytes into device 2 using a liquid sending means, for example, a microtube or a pump.

[0059] The collection section 23 of the platelet production device 2 is disposed adjacent to the downstream end 22b of the flow channel 22. The collection section 23 can also be configured as an open section that opens toward the outside of the device. The collection section 23 has a space capable of storing the medium and platelets PL that flow in from the flow channel 22. The collection section 23 can also be shaped to be suitable for collecting the stored platelets from the device 2 using a collection means, such as a pipette, a microtube, or a pump.

[0060] The flow channel 22 is a space extending from the inlet 21 toward the collection section 23 and configured to allow a fluid to pass through. The flow channel 22 has a height defined by the distance between the bottom surface 22d and the top surface 22e, and may have a substantially rectangular cross-sectional shape perpendicular to the flow direction. The flow channel height at the end 22a on the inlet side is configured to be larger than the maximum diameter of megakaryocyte cells. The flow channel height at the end 22b on the collection section side is at least smaller than the minimum diameter of megakaryocyte cells and larger than the maximum diameter of platelets. The flow channel height decreases from the inlet 21 to the collection section 23. The flow channel height at the end 22a on the inlet side is not limited to a specific value. In one embodiment, the flow channel height can be determined based on approximately the top 0.05% probability of a log-normal distribution curve for the particle diameter of megakaryocyte cells. The flow channel height is configured to decrease from the inlet 21 toward the collection section 23. Therefore, in this specification, the flow channel height at the end 22a on the inlet side is referred to as the maximum flow channel height (h _max ), and the flow path height at the end 22b on the recovery section side is the minimum flow path height (h _min ) The channel height preferably decreases monotonically from the inlet 21 toward the recovery section 23, and may decrease linearly or exponentially. However, the channel height is preferably constant across the width of the channel. That is, the length l of the channel from the end 22a on the inlet side to the end 22b on the recovery section side is c In this case, the channel height h(x) at a predetermined distance x from the end 22a on the injection port side along the length direction of the channel is set to a constant value in the width direction. The width of the flow path may be constant or may vary from the inlet end 22a to the collection end 22b. The variable width will be described in detail later in the platelet production device according to the second embodiment.

[0061] Optionally, the flow channel 22 preferably includes a plurality of capture pillars 22c rising from the bottom surface 22d near the end 22b on the collection section side. When megakaryocytes produce platelets, the megakaryocytes form elongated platelet precursors (PPLTs). These platelet precursors may be cut by shear force. By providing the capture pillars 22c at the above locations, the elongated string-like platelet precursors are caught and captured, preventing them from flowing out of the flow channel 22 to the collection section 23, allowing for continued platelet production. The vicinity of the end 22b on the collection section side particularly refers to a portion where the channel height is smaller than the size of megakaryocytes. However, the capture pillars 22c may be provided in other locations.

[0062] The size and spacing of the capture pillars 22c are not particularly limited as long as they can capture platelet precursors, and can be determined appropriately taking into consideration the flow rate of the liquid flowing through the flow channel 22, etc. If the spacing between the capture pillars 22c is too large, platelet precursors may slip through without being caught, but if the spacing is narrowed and the capture pillars 22c are formed densely, the resistance of the fluid flowing through the flow channel 22 may increase. The spacing between the capture pillars 22c can be determined appropriately taking into consideration the pressure of the liquid flowing through the flow channel 22, etc. When forming the capture pillars 22c, they may be designed so that the flow rate of the liquid flowing through the portion where the capture pillars 22c are arranged is the same, so that the same shear force is applied to the captured platelet precursors.

[0063] Use of the platelet production device 2 equipped with the capturing pillars 22c enables more efficient production of platelets.

[0064] As shown in FIG. 1 , the platelet production device 2 may have a flow channel that flows in one direction from the inlet 21. Alternatively, the platelet production device 2 may have a plurality of flow channels that extend radially from the inlet 21 toward the periphery of the inlet 21. Furthermore, the platelet production device 2 may have a flow channel that extends 360 degrees from the circular inlet 21 toward the periphery of the inlet 21. A specific example of a flow channel that extends 360 degrees toward the periphery of the inlet 21 is the platelet production device disclosed by the present inventors in Patent Document 2. The platelet production device disclosed in Patent Document 2 also has a predetermined flow channel shape and is capable of applying shear stress to megakaryocytes for a predetermined period of time while the megakaryocytes are trapped therein. The platelet production device disclosed in Patent Document 2 can be used in step (b) of the present invention. The material of the platelet production device is not particularly limited, but examples include synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, epoxy resin, silicone resin, polycarbonate, and polyvinyl chloride; inorganic materials such as glass (borosilicate glass, etc.), silicon, alumina, and titania; metals such as stainless steel, titanium, and aluminum; and photoresist (photosensitive resin).

[0065] A method for carrying out step (b) using the above-described platelet production device will now be described. Step (b) can be mainly composed of the following substeps. (i) a loading step of injecting a medium containing megakaryocytes cultured in step (a) into the platelet production device 2; (ii) a producing step of injecting a medium containing no megakaryocytes or a fluid capable of forming a laminar flow into the platelet production device 2; and (iii) a flushing step. The flushing step is an optional step and may not be performed.

[0066] In substep (i), a medium containing megakaryocytes is injected into the platelet production device 2. As a result, the megakaryocytes are captured in the channel near a location where the channel height is appropriate for their diameter. The medium injection can be performed under pressure so that the medium flows at a predetermined rate within the channel of the platelet production device. A preferred flow rate in substep (i) is approximately 0.1 to 5 mm / s. Since the pressure required varies significantly depending on the shape and specifications of the platelet production device, the pressure should be appropriately adjusted to achieve a desired flow rate for the device being used. For example, when using a device described below with reference to Figures 4 and 5A to 5D, the pressure can be approximately 1 to 200 KPa, but is not limited to a specific value. Substep (i) is preferably performed while the device is maintained at approximately 37°C. The time required for the loading step can be appropriately determined by those skilled in the art, depending on factors such as the total amount of medium containing megakaryocytes to be injected into the platelet production device 2. For example, the time can be approximately 10 to 20 minutes, but is not limited to a specific time.

[0067] In substep (ii), a medium or other fluid not containing megakaryocytes is injected into the platelet production device 2. The other fluid is not particularly limited as long as it can form a laminar flow within the flow channel and does not adversely affect the function of megakaryocytes. Examples of the other fluid include, but are not limited to, saline and phosphate-buffered saline. Examples of the medium not containing megakaryocytes include, but are not limited to, the medium obtained by removing megakaryocytes and platelets from the supernatant of the medium used in step (a) using a filter or the like. The medium or other fluid can be injected under pressure so that the medium or other fluid flows at a predetermined velocity within the flow channel of the platelet production device. In substep (ii), the preferred flow velocity is approximately 0.1 to 5 mm / s, as in substep (i). Therefore, the pressure can be determined in the same manner as in substep (i). For example, when using a device described below with reference to Figures 4 and 5A-D, the pressure range may be the same as in substep (i). Substep (ii) can also be performed for, for example, 1 to 10 hours, or 4 to 6 hours, while the device is maintained at approximately 37°C, but is not limited to a specific time range. During the manufacturing process of substep (ii), the megakaryocytes are captured in the flow channel and exposed to a medium or other fluid. This applies shear stress to the megakaryocytes, which stretches them to form platelet precursors, which then produce platelets. The produced platelets may flow through the flow channel and reach the collection section. Alternatively, some may flow through the flow channel in the form of platelet precursors. Optionally, trapping pillars 22c may be provided downstream of the flow channel to capture the platelet precursors and promote platelet production. Note that some megakaryocytes may not exhibit the behavior described in this paragraph.

[0068] In the optional substep (iii), a fluid similar to that used in substep (ii) is injected into the platelet production device 2 at a pressure equal to or greater than that used in substep (ii). The preferred flow rate of the fluid in the flow path is approximately 5 to 50 mm / s. For example, when using a device described below with reference to Figure 4 and Figures 5A to 5D, the pressure required to achieve this flow rate can be approximately 50 to 200 KPa, but is not limited to a specific pressure value. Substep (iii) can also be performed for approximately 10 to 20 minutes, for example, while the device is maintained at approximately 37°C, but this time period is not limited to a specific range. Substep (ii) typically allows platelet production and recovery, but this operation can also be performed additionally. In Figure 4, "Flow" indicates the direction of fluid flow near the inlet.

[0069] Next, a second embodiment of the platelet production device can be a device having the above configuration and further having a configuration in which the width of the channel is variable. Another embodiment of the platelet production device will be described with reference to Fig. 4. The device 3 shown in Fig. 4 is similar to the platelet production device 2 shown in Fig. 1 in the configuration including an inlet 31 for megakaryocyte cells, a collection section 33 for platelets, and a channel 32 extending from the inlet to the collection section, as well as in the channel height. The device 3 also has the same trapping pillars 33c, which may be optionally provided, as the platelet production device 2 shown in Fig. 1. In Fig. 4, X indicates the flow direction of the channel 32 of the platelet production device 3, Y indicates the width direction of the channel 32, and Z indicates the height direction of the channel 32.

[0070] In this embodiment, the width of the channel 32 changes from the inlet 31 toward the collection section 33, and this change correlates with the frequency distribution of the diameter of the megakaryocyte cell population to be injected. More specifically, the diameter of the megakaryocyte cell is expressed as diameter x d , where the distance from the inlet end is x, the height of the flow channel at distance x is h(x), and the width of the flow channel at distance x is w(x), w(x) is determined according to the diameter distribution of megakaryocyte cells with a diameter of h(x), and the diameter x dThe greater the frequency of megakaryocytes with h(x), the larger w(x) is configured. By configuring device 3 as described above, it is possible to provide the same functions as those described with reference to Fig. 1, and further to maintain constant fluid conditions, such as a constant flow rate, even after the megakaryocyte cell population is captured in flow channel 32.

[0071] The design of the width of the channel will be described in more detail with reference to Figures 5A to 5D. d is the probability density function P(x d ) is considered to follow a log-normal distribution and is expressed by the following equation (1):

number

[0072] In this embodiment, the width of the channel is designed to reflect the cell size distribution. c , with a maximum length of l c Using a flow channel with a diameter of x d Consider the case where you are trying to capture a group of megakaryocytes x d is x d_min or more, and x d_max The values ​​are in the following range. Here, the cross-sectional area of ​​the flow channel at a distance x from the inlet side end 33a of the flow channel 33 is defined as A(x), the height of the flow channel is defined as h(x), and the width of the flow channel is defined as 2w(x). FIG. 5A is a diagram illustrating the defined variables. In FIG. 5A, the flow channel is symmetrical with respect to the axis x. Although not shown, the starting points of the arrows x, w(x), and h(x) correspond to the inlet side end 33a of the flow channel 33.

[0073] diameter x dConsidering that megakaryocytes having the above structure are captured at a location having a specific flow channel cross-sectional area at a distance x from the inlet end 33a, the height h(x) of the flow channel is expressed by the following equation (2):

number

[0074] Consider the case where the height of the flow channel, h(x), decreases linearly with the length of the flow channel, x. d is obtained by the following equation (3).

number

[0075] In formula (3), S lope is the gradient of the flow channel in the height direction, and is expressed by the following equation (4).

number

[0076] In this configuration, the megakaryocyte cell population injected into the device has a diameter x d This results in large megakaryocytes x d_max From small megakaryocyte cells x d_min The cross-sectional area A(x) at the position of distance x is the diameter of the captured particle x d The cross-sectional area is reduced due to the megakaryocytes. dec Then, the effective cross-sectional area A through which the medium of megakaryocyte cells can pass is ef (x) is expressed by the following equation (5).

number

[0077] The effective cross-sectional area is the reduction in channel width caused by the capture of megakaryocytes, w dec Since it changes depending on (x), the effective flow path width w ef (x) is expressed by the following equation (6).

number

[0078] Here, the probability density function P(x d ), the reduction in flow path width w dec (x) is expressed by the following equation (7).

number

[0079] To keep the flow rate in the flow path constant, the effective cross-sectional area A ef If (x) is designed to be constant, the following equation (8) is obtained.

number

number

[0080] Therefore, the channel width can be derived as a function of the channel length as shown in the following equation (10).

number

number

number

[0081] Figure 5B shows an example of the diameter distribution of megakaryocyte cells differentiated from human pluripotent stem cells after culturing in step (a), and is the measurement result for 10,314 cells cultured in five dishes. The results were fitted to the log-normal distribution of equation (1) by the least squares method. Then, μ d is 2.99 μm, σ d was 0.38 μm. Next, based on the fact that the diameter of normal platelets is less than 3 μm, x d_min was set to 5 μm. Also, based on the probability of the top 0.1% of the normal distribution, x d_max Next, for device manufacturing reasons, c 10mm, l c was set to 20 mm. The three-dimensional shape of the channel can be obtained from these values ​​and equations (11) and (12). Figure 5C shows a graph of h(x), and Figure 5D shows a graph of w(x), which were designed based on the diameter distribution of the megakaryocyte cell group shown in Figure 5B.

[0082] The platelet production device designed as described above can be manufactured using a 3D printer or photoresist formation technology.

[0083] In this way, the platelet production device according to the second aspect of the present invention can be designed to be compatible with a specific population of megakaryocyte cells cultured under specific conditions. Therefore, the production method of the present invention may optionally include, prior to step (a), a step of designing and manufacturing a platelet production device to be compatible with a desired population of megakaryocyte cells.

[0084] The method for carrying out step (b) using the platelet production device according to the second aspect of the present invention may be the same as that described in the first aspect.

[0085] The platelet production device according to the second aspect of the present invention, having the above-described characteristics regarding the channel width, can maintain a constant fluid flow condition (flow rate) in the channel even after megakaryocytes are trapped in the channel. Therefore, by using the platelet production device, it is possible to maintain a constant flow condition for the medium, etc., particularly in the producing step, which is a substep of step (b), thereby reducing the variability in the number of platelets produced.

[0086] After step (b), a platelet collection step can be performed. In this step, the platelet-containing medium stored in the collection section is collected using a pipette, pump, or other means, and platelets are collected from the medium using a conventional method such as FACS. Platelets are a cellular component of blood and are characterized by CD41a and CD42b positivity. Platelets play an important role in thrombus formation and hemostasis, as well as in the pathophysiology of tissue regeneration and inflammation after injury. When platelets are activated by bleeding or other factors, receptors for cell adhesion molecules such as integrin αIIBβ3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) are expressed on their membranes. As a result, platelets aggregate, and various blood coagulation factors released from the platelets coagulate fibrin, forming a clot and promoting hemostasis.

[0087] 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, which specifically binds to integrin αIIBβ3 on the membrane of activated platelets. Similarly, the amount of activated platelets can be measured by detecting CD62P (P-selectin), a platelet activation marker, with an antibody. For example, flow cytometry can be used to gate with antibodies against the activation-independent platelet markers CD61 or CD41, followed by detecting the binding of anti-PAC-1 or anti-CD62P antibodies. These steps may be performed in the presence of adenosine diphosphate (ADP).

[0088] Platelet function can also be evaluated by observing whether or not platelets bind to fibrinogen in the presence of ADP. Platelet binding to fibrinogen activates integrins, which are required for the early stage of thrombus formation. Furthermore, platelet function can also be evaluated by a method for visualizing and observing thrombus formation in vivo, as described in International Publication No. 2011 / 034073.

[0089] Platelets obtained by the production method of the present invention can be administered to patients as a pharmaceutical preparation. For administration, platelets obtained by the method of the present invention may be stored and formulated in, for example, human plasma, infusion solutions, citrate-containing physiological saline, glucose-added acetate Ringer's solution as the main ingredient, or PAS (platelet additive solution) (Gulliksson, H. et al., Transfusion, 32:435-440, (1992)). The storage period is approximately 3 to 7 days, for example, about 4 days. Storage conditions include preferably shaking and stirring at room temperature (approximately 20 to 24°C).

[0090] The present invention was completed based on the discovery that turbulence-dependent megakaryocyte maturation requires at least six days of culture, not five days, and that if pre-culture for megakaryocyte maturation is performed, functional platelets can be efficiently produced in the subsequent shear stress-dependent platelet production process.The production method of the present invention makes it possible to efficiently produce platelets that are sufficiently endowed with properties that allow them to be administered as blood products. [Example]

[0091] The present invention will be described in more detail below with reference to examples, which are not intended to limit the scope of the present invention.

[0092] 1. Fabrication of the Platelet-producing Device The platelet production device is outlined in Figure 4 and designed according to Figures 5A-D. It was fabricated as follows. Figure 6 is a schematic diagram illustrating the device fabrication. The platelet production device was fabricated to consist of four layers: a cover layer, a 3D flow channel layer, a holder layer, and a polydimethylsiloxane (PDMS) layer. The fabrication process is as follows. In Figure 6, (i) to (iv) are cross-sectional views illustrating the fabrication of the 3D flow channel layer, (v) and (vi) are the cover layer, and (vii) and (viii) are the holder layer. (ix) shows a platelet production device in which the four layers are integrated and packaged. PMER, SU-8, NCM-250, Si, Glass, and PMDS represent the materials that make up each layer in the figure, and details are provided below.

[0093] (i) For the 3D flow channel layer, a positive photoresist PMER (Tokyo Ohka Kogyo Co., Ltd.) was patterned on the surface of a Si substrate using grayscale lithography. In this process, a pattern designed in 8-bit grayscale by laser scanning was directly exposed by changing the laser intensity. (ii) The Si substrate was etched using deep reactive ion etching with controlled selective ratio (D-RIE-CSR), and the 3D surface of the photoresist was transferred to the Si substrate according to the selectivity. (iii) Next, a negative photoresist, SU-8 3010 (Microchem Co. Ltd., Japan), was patterned on the Si substrate. The SU-8 layer was used as an etching mask for the D-RIE process to fabricate the inlet and collection area of ​​the platelet production device. (iv) The injection port and collection port were fabricated using D-RIE, after which the remaining photoresist was removed by a cleaning process. (v) For the cover layer, a negative photoresist, NCM-250 (Nikko-Materials Co. Ltd, Japan), was patterned on borosilicate glass as an etching mask for sandblasting. (vi) The borosilicate glass was etched by sandblasting. This process produced the inlet and the collector. (vii) For the holder layer, SU-8 3010 was patterned on borosilicate glass. (viii) Borosilicate glass is etched using RIE, d_min A height of 5 μm, corresponding to the micropillar region, was obtained. (ix) Finally, the three fabricated layers—the cover, the 3D channel, and the holder layer—were bonded together using anodic bonding. The PDMS parts of the inlet and collection chamber were bonded as a PDMS layer on the cover layer.

[0094] 2. Platelet production Platelets were produced by the production method of the present invention, and their properties were evaluated.

[0095] [imMKCL (megakaryocytic cell line) Gene ON proliferation culture] imMKCLs were cultured in imMKCL differentiation medium (IMDM medium containing 15% FBS, L-glutamine, insulin-transferrin-selenium, ascorbic acid, and l-thioglycerol) supplemented with 50 ng / mL SCF, 200 ng / mL TA-316, and 1 μg / mL doxycycline at 37°C in a 5% CO2 environment.

[0096] [imMKCL (megakaryocytic cell line) Gene OFF maturation culture (step a)] ImMKCLs were cultured in imMKCL differentiation medium supplemented with 50 ng / mL SCF, 200 ng / mL TA-316, 15 mM KP-457, 0.75 mM SR-1, and 10 mM Y27632 at a concentration of 1 × 10e cells using a Lab-Therm shaker. 5 The cells were cultured at a cell density of / ml in 125 mL Corning_Erlenmeyer cell culture flasks at 100 rpm, 37℃, and 5% CO2 for 5, 6, 7, or 8 days.

[0097] [Platelet production using the platelet production device (step b)] The platelet production device used was manufactured using the method described above and shown in Figure 4. This platelet production device 3 consists of an inlet 31 for introducing megakaryocytes and culture medium, a channel 32 for capturing megakaryocytes, capture micropillars 32c for capturing platelet precursor cells that occasionally break off from megakaryocytes, and a collection section 33 for collecting produced platelets. Culture medium containing megakaryocytes on days 5, 6, 7, and 8 of Gene OFF maturation culture was loaded into the inlet at 10 kPa for 15 minutes (substep (i) loading step). This resulted in the introduction of 2,500 megakaryocytes into the platelet production device. In the experiment to evaluate the production number, after introducing the culture medium containing megakaryocytes, the pressure was maintained at 10 kPa, and only the culture medium for the production process, which did not contain megakaryocytes, was allowed to flow for 6 hours, thereby applying shear stress to the megakaryocytes (substep (ii) production step). The medium used for the manufacturing process was the culture supernatant used in the Gene OFF maturation culture step, from which megakaryocytes and platelets had been removed using a 0.22 μm filter. Therefore, when megakaryocytes that had undergone Gene OFF maturation culture for 5 days were applied to the platelet production device, the medium used for manufacturing in substep (ii) of the manufacturing process was the culture supernatant after 5 days of Gene OFF maturation culture, from which megakaryocytes and platelets had been removed. Similarly, when megakaryocytes that had undergone Gene OFF maturation culture for 6, 7, and 8 days were applied to the platelet production device, the medium used was the culture supernatant after 6, 7, and 8 days of Gene OFF maturation culture, from which megakaryocytes and platelets had been removed, respectively. The platelet-mixed culture medium that had accumulated in the collection compartment at the rear of the channel was then collected, and the platelet count was measured using a FACS Verse. In the platelet function measurement experiment, after loading the medium containing megakaryocytes, the pressure was maintained at 10 kPa, and the same manufacturing process medium used in the platelet count evaluation experiment was passed through the medium for 1 hour, applying shear stress to the megakaryocytes. Thereafter, the platelet mixed culture solution accumulated in the collection section at the rear of the flow channel was collected, and the platelet hemostatic function was measured. In this example, the substep (iii) flushing step was not performed.

[0098] [Flow cytometric analysis] Flow cytometric analysis was performed using a FACS Verse. Antibodies used were anti-hCD41-APC (#303710), anti-hCD42b-PE (#303906), anti-hCD42a-PE (#558819), anti-hCD62PAPC (#304910), and FITC Annexin V (#556419). Platelet activation assays for PAC-1 / p-selectin positivity were performed using 40 mM TRAP-6 and 100 mM ADP. For Annexin V positivity assays, 20 mM ionomycin was used.

[0099] [result] Figure 7 shows the number of CD41a / CD42b-positive platelets produced when megakaryocytes on days 5, 6, 7, and 8 of Gene OFF maturation culture were introduced into a platelet production device to produce platelets. Platelet counts were measured. On day 5 of Gene OFF maturation culture, the platelet count per imMKCL was approximately 16. On day 6 of culture, the platelet count per imMKCL was approximately 59, more than three times higher, demonstrating a dramatic improvement. On day 7, the platelet count per imMKCL was approximately 54, and on day 8, the platelet count per imMKCL was approximately 55. The platelet count per imMKCL referred to here is the number produced by introducing the cells into the platelet production device and is calculated by subtracting the number of platelets already produced at the completion of Gene OFF maturation culture from the final number of platelets produced.

[0100] Figure 8 shows the results of measuring platelet hemostatic function (PAC-1 positivity) of imMKCL-derived platelets on days 5, 6, 7, and 8 of Gene OFF maturation culture. Platelets were produced by introducing megakaryocytes into a platelet production device. The platelet mixed culture medium was then collected and analyzed for platelet hemostatic function (PAC-1 positivity). The imMKCL-derived platelets on day 5 of Gene OFF maturation culture had a PAC1 positivity rate of approximately 2.3% without stimulation (NS) and approximately 5.1% with the addition of the platelet-activating factor ADP / TRAP (AT). In contrast, the imMKCL-derived platelets on day 6 of Gene OFF maturation culture had a PAC1 positivity rate of approximately 2.2% without stimulation (NS) and approximately 10% with the addition of ADP / TRAP (AT), demonstrating an improved response to platelet-activating factors. Furthermore, imMKCL-derived platelets on day 7 of Gene OFF maturation culture had a PAC1 positivity rate of approximately 2.5% without stimulation (NS) and approximately 7.6% with the addition of ADP / TRAP (AT). ImMKCL-derived platelets on day 8 of Gene OFF maturation culture had a PAC1 positivity rate of approximately 2.3% without stimulation (NS) and approximately 4% with the addition of ADP / TRAP (AT).

[0101] Figure 9 shows the results of measuring Annexin V, a platelet aging marker, in imMKCL-derived platelets on days 5, 6, 7, and 8 of Gene OFF maturation culture. Megakaryocytes on days 5, 6, 7, and 8 were introduced into a platelet production device to produce platelets. The platelet mixture was then collected and assayed for Annexin V. The Annexin V positivity rate for imMKCL-derived platelets on day 5 of Gene OFF maturation culture was approximately 41%, whereas that for imMKCL-derived platelets on day 6 of Gene OFF maturation culture was approximately 20%, indicating low Annexin V positivity. The Annexin V positivity rate for imMKCL-derived platelets on day 7 of Gene OFF maturation culture was approximately 26%, and that for imMKCL-derived platelets on day 8 of Gene OFF maturation culture was approximately 43%. Figure 10 shows FACS diagrams showing Annexin V assay results. The horizontal axis indicates Annexin V, and the Annexin V-positive gate was set based on the main population obtained when ionomycin, a positive control, was added.

[0102] As used in this specification and claims, unless otherwise required by context, singular terms include plurals, and plural terms include the singular. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. The term "about" generally refers to a range of numbers that one of ordinary skill in the art would consider equivalent to the stated value (i.e., having the same function or result). For example, the term "about" refers to ±10%, ±5%, or ±2% of the stated value. [Industrial Applicability]

[0103] The platelet production method and platelet production device according to the present invention are useful in the production of blood products. [Explanation of symbols]

[0104] 1 flask, 11 container, 12 stirring mechanism 2, 3 Platelet-producing devices, 21, 31 inlet, 22, 32 flow path, 23, 33 recovery section

Claims

1. (a) culturing megakaryocyte cells in a turbulent platelet-producing medium for 6 to 9 days; (b) injecting the medium containing the megakaryocytes that have been subjected to step (a) into a platelet production device and exposing the megakaryocytes to laminar flow; A method for producing platelets, comprising: the platelet producing device comprising: an inlet for injecting megakaryocyte cells; Platelet collection area and a flow path extending from the inlet to the collection section; Equipped with The flow path is the height of the flow channel at the end on the injection port side is greater than the maximum diameter of the megakaryocyte cells to be injected; a height of the flow channel at the end on the recovery section side is smaller than the minimum diameter of the megakaryocytes to be injected and larger than the maximum diameter of the platelets; A flow path height is configured to decrease from the inlet toward the collection section, Thus, the platelet production device is configured to expose the megakaryocytes to a laminar flow while capturing the megakaryocytes in the flow path, and to release platelets produced by the megakaryocytes from the flow path to a collection section, where x is the distance from the inlet end of the flow channel, h(x) is the height of the flow channel at the distance x, w(x) is the width of the flow channel at the distance x, and xd is the diameter of megakaryocyte cells, w(x) is determined to vary depending on the frequency of megakaryocyte cells having a diameter of h(x), and the greater the frequency of megakaryocyte cells having a diameter xd of h(x), the larger w(x) is configured to be; The method further comprises providing a plurality of pillars rising from the bottom surface of the end of the flow channel on the collection section side.

2. The method described in claim 1, wherein h(x) and w(x) are expressed by the following equations (11) and (12), and the outer shape of the flow path width is determined by w(x) and -w(x). [Equation 1] where w c is the maximum width of the channel; x d_min is the minimum value of the megakaryocyte cell diameter, x d_max is the maximum value of the megakaryocyte cell diameter, Slope is the slope of the channel in the height direction, P is the probability density function representing the probability of a megakaryocyte cell having a given diameter; N represents the total number of megakaryocyte cells injected)

3. The method according to claim 1 or 2, further comprising, prior to the step of culturing the megakaryocytic cells, a step of forcibly expressing an oncogene, a polycomb gene, and an apoptosis-inhibiting gene in cells less differentiated than megakaryocytic cells to obtain immortalized megakaryocytic cells.

4. The method according to any one of claims 1 to 3, comprising the step of recovering platelets from the recovery portion of the platelet production device.

5. The method according to any one of claims 1 to 4, wherein the step of culturing for 6 to 9 days is carried out using a swing flask or a culture tank equipped with impellers that can be operated non-stationarily.

6. an inlet for injecting megakaryocyte cells; Platelet collection area and a flow path extending from the inlet to the collection section; A platelet production device comprising: The flow path is the height of the flow channel at the end on the injection port side is greater than the maximum diameter of the megakaryocyte cells to be injected; a height of the flow channel at the end on the recovery section side is smaller than the minimum diameter of the megakaryocytes to be injected and larger than the maximum diameter of the platelets; A flow path height is configured to decrease from the inlet toward the collection section, Thus, the platelet production device is configured to expose the megakaryocytes to a laminar flow while capturing the megakaryocytes in the flow channel, and to release platelets produced by the megakaryocytes from the flow channel to a collection section, where x is the distance from the inlet end of the flow channel, h(x) is the height of the flow channel at the distance x, w(x) is the width of the flow channel at the distance x, and xd is the diameter of megakaryocyte cells, w(x) is determined to vary depending on the frequency of megakaryocyte cells having a diameter of h(x), and the greater the frequency of megakaryocyte cells having a diameter xd of h(x), the larger w(x) is configured to be; A platelet production device comprising a plurality of pillars rising from the bottom surface of the end of the flow channel on the collection section side.

7. A platelet production device as described in claim 6, wherein h(x) and w(x) are expressed by the following equations (11) and (12), and the outer shape of the flow path width is determined by w(x) and -w(x). [Equation 2] where w c is the maximum width of the channel; x d_min is the minimum value of the megakaryocyte cell diameter, x d_max is the maximum value of the megakaryocyte cell diameter, Slope is the slope of the channel in the height direction, P is the probability density function representing the probability of a megakaryocyte cell having a given diameter; N represents the total number of megakaryocyte cells injected)

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