TWI856228B - Separation base, cell separation filter, and method of preparing platelet - Google Patents

Abstract

The subject of the present invention is to provide a separation substrate with high megakaryocyte removal rate and platelet recovery rate and long filtration life, as well as a cell separation filter and platelet manufacturing method using the separation substrate. The separation substrate of the present invention is composed of a porous body used to separate platelets from a cell suspension containing megakaryocytes and platelets. In the aforementioned separation substrate, the separation substrate has a region in which a coarse filter layer and a fine filter layer are sequentially arranged from the inflow side of the cell suspension, the coarse filter layer includes one or more coarse filter membranes, and the fine filter layer includes one or more fine filter membranes. At least one of the coarse filter membranes is a coarse filter membrane X having an average pore size of 5.0 μm or more and 30.0 μm or less and a peak value of the pore diameter distribution of less than 30%, and at least one of the fine filter membranes is a fine filter membrane Y having an average pore size of 2.0 μm or more and 20.0 μm or less and a peak value of the pore diameter distribution of more than 30%.

Description

Separation substrate, cell separation filter, and platelet manufacturing method

The present invention relates to a separation substrate, a cell separation filter and a method for manufacturing platelets.

Platelets are cells that play a leading role in the formation of blood clots and exhibit hemostatic functions in the living body. Therefore, if platelets are reduced during bleeding or when using anticancer drugs, it may lead to death in severe cases. In addition, the only definite treatment for platelet reduction is transfusion of platelet products. Current platelet products rely on blood donations from volunteers. Although the shelf life is a very short 4 days, it is expected that it will be difficult to maintain a balance between demand and supply in the medical field as the population of the age group that can donate blood decreases due to the declining birth rate and the population of the elderly with high demand for blood products increases. Therefore, the development of platelet sources that replace blood donations has attracted much attention.

In recent years, a technique for producing a large number of platelets in vitro by culturing megakaryocytes using pluripotent stem cells, hematopoietic progenitor cells, mesenchymal cells, etc. as sources has been reported. In this technique, platelets are produced by cleavage of the cytoplasm of megakaryocytes, so a large number of megakaryocytes are contained in the culture medium after platelet production. Therefore, from the perspective of suppressing immunogenicity, it is necessary to develop a technique for isolating megakaryocytes and producing platelets from megakaryocytes.

As such a separation technology, for example, Patent Document 1 describes: "A separation substrate, which is composed of a non-woven fabric for separating platelets from a cell suspension containing megakaryocytes and platelets, wherein the average pore size of the separation substrate is greater than 2.0 μm and less than 15.0 μm, and the thickness of the separation substrate is greater than 10 μm and less than 500 μm." ([Claim 1]).

[Patent Document 1] International Publication No. 2018/207564

The inventors of the present invention have studied the separation substrate described in Patent Document 1 and found that the removal rate (blocking rate) of megakaryocytes is high and the recovery rate (permeability) of platelets is high, but it has been found that there is room for improvement in the filtration life.

Therefore, the subject of the present invention is to provide a separation substrate with high megakaryocyte removal rate and platelet recovery rate and long filtration life, as well as a cell separation filter and platelet manufacturing method using the separation substrate.

As a result of in-depth research to achieve the above-mentioned topic, the inventors of the present invention have completed the present invention by discovering the following: in the separation substrate, a region in which a coarse filter layer and a fine filter layer are sequentially arranged from the inflow side of the cell suspension, thereby increasing the removal rate of megakaryocytes and the recovery rate of platelets and prolonging the filtration life, and the coarse filter layer includes a coarse filter membrane that meets predetermined parameters, and the fine filter layer includes a fine filter membrane that meets predetermined parameters. That is, the inventors of the present invention have discovered that the above-mentioned topic can be achieved by the following structure.

[1] A separation substrate, which is composed of a porous body for separating platelets from a cell suspension containing megakaryocytes and platelets, wherein the separation substrate has a region in which a coarse filter layer and a fine filter layer are arranged in order from the inflow side of the cell suspension, the coarse filter layer includes one or more coarse filter membranes, the fine filter layer includes one or more fine filter membranes, and the coarse filter membrane includes one or more fine filter membranes. At least one of the coarse filter membranes is a coarse filter membrane X having an average pore size of 5.0 μm or more and 30.0 μm or less as measured by a semi-dry method and a peak value of the pore diameter distribution of less than 30%, and at least one of the fine filter membranes is a fine filter membrane Y having an average pore size of 2.0 μm or more and 20.0 μm or less as measured by a semi-dry method and a peak value of the pore diameter distribution of more than 30%.

[2] A separation substrate as described in [1], wherein the coarse filter membrane X is a membrane satisfying the following formula (1). 150 ≤ the value of the average pore size (μm) × the value of the thickness (μm) ≤ 1500 (1) [3] A separation substrate as described in [1] or [2], wherein the porosity of the fine filter membrane Y is greater than 40% and less than 90%. [4] A separation substrate as described in any one of [1] to [3], wherein the porosity of the coarse filter membrane X is greater than 40% and less than 90%. [5] A separation substrate as described in any one of [1] to [4], wherein the coarse filter layer comprises more than 3 coarse filter membranes. [6] A separation substrate as described in any one of [1] to [5], wherein all the coarse filter membranes contained in the coarse filter layer and all the fine filter membranes contained in the fine filter layer are membranes having a critical wetting surface tension of 72 mN/m or more.

[7] A separation substrate as described in any one of [1] to [6], wherein the porous body is a nonwoven fabric. [8] A separation substrate as described in [7], wherein the nonwoven fabric comprises at least one resin selected from the group consisting of a cellulose resin and a polyolefin resin. [9] A separation substrate as described in [8], wherein the cellulose resin is a cellulose acylate or cellulose. [10] A separation substrate as described in [8], wherein the polyolefin resin is polypropylene. [11] A separation substrate as described in [10], wherein the polypropylene is hydrophilized polypropylene.

[12] A cell separation filter comprising a container having a first liquid port and a second liquid port and a filter material filled between the first liquid port and the second liquid port, wherein the filter material is a separation substrate as described in any one of [1] to [11]. [13] A method for producing platelets, comprising: a step of bringing a culture medium containing at least megakaryocytes into contact with a separation substrate as described in any one of [1] to [11]; a culturing step of culturing megakaryocytes to produce platelets at least one of before and after the contacting step; and a recovery step of recovering the culture medium containing the produced platelets after the contacting step and the culturing step. [Effect of the invention]

According to the present invention, a separation substrate having high megakaryocyte removal rate and platelet recovery rate and long filtration life, as well as a cell separation filter and platelet manufacturing method using the separation substrate can be provided.

The present invention is described in detail below. The description of the constituent elements described below is completed based on the representative implementation of the present invention, but the present invention is not limited to the implementation. In addition, in this specification, the numerical range represented by "～" refers to the range that includes the numerical values recorded before and after "～" as the lower limit and upper limit. In addition, in this specification, each component can use a single substance equivalent to each component, or two or more substances can be used in combination. Here, for each component using two or more substances in combination, unless otherwise specified, the content of the component refers to the total content of the substances used in combination.

Generally, a separation substrate refers to a structure having a large number of small pores inside, such as a fiber structure, a porous membrane, a bead-filled column, and a laminate composed of the above. Among them, a fiber structure refers to a structure formed by entangled fibers, such as a woven fabric (mesh), a knitted fabric, a braided rope, a non-woven fabric, and a column filled with fibers, etc. Among them, non-woven fabrics are particularly preferred in terms of wide pore size distribution, complex flow paths, and ease of production. In addition, as a method for making nonwoven fabrics, for example, dry method, wet method, spunbond method, melt spraying method, electrostatic spinning method, needle punching method, etc. can be cited. Among them, from the perspective of productivity and versatility, wet method, melt spraying method, and electrostatic spinning method are preferred. Porous film refers to a plastic body having countless through holes. As a method for making it, phase separation method, foaming method, etching method by irradiation with radiation or laser light, pore forming method, freeze drying method, plastic sintering method, etc. can be cited. However, from the perspective of complex flow paths and ease of production, porous films using phase separation method are particularly preferred. A ball-packed column is a column in which pores are formed between the balls by packing the column with balls. It is desirable that the particle size of the balls be uniform, and the pore size between the balls can be easily controlled by the particle size of the balls.

Examples of the porous body constituting the separation substrate of the present invention include nonwoven fabrics, fiber structures, sponges, and porous membranes. Among them, nonwoven fabrics are preferred from the viewpoint of productivity and controllability of filtering performance.

[Separation substrate] The separation substrate of the present invention is a separation substrate composed of a porous body for separating platelets from a cell suspension containing megakaryocytes and platelets. In addition, the separation substrate of the present invention has a region where a coarse filter layer and a fine filter layer are arranged in order from the inflow side of the cell suspension, the coarse filter layer includes one or more coarse filter membranes, and the fine filter layer is one or more fine filter membranes. Furthermore, in the separation substrate of the present invention, at least one of the coarse filter membranes is a coarse filter membrane X having an average pore size of 5.0 μm or more and 30.0 μm or less as measured by a semi-dry method and a peak value of the pore diameter distribution of less than 30%, and at least one of the precision filter membranes is a precision filter membrane Y having an average pore size of 2.0 μm or more and 20.0 μm or less as measured by a semi-dry method and a peak value of the pore diameter distribution of more than 30%.

Here, "average pore size measured by semi-dry method" refers to the value evaluated by blowing air into a sample completely wetted in GALWICK (manufactured by Porous Materials, Inc.) and increasing the pressure by 1 kPa/min in a pore size distribution measurement test using a perm-porometer (CFE-1200AEX manufactured by Seika Corporation). Specifically, air is blown onto one side of a membrane sample completely wetted in GALWICK to increase the pressure to 1 kPa/min, and the pressure is measured while measuring the flow rate of air passing through the opposite side of the membrane. In this method, first, the pressure and air flow rate data (hereinafter also referred to as the "wet curve") are obtained for the film sample moistened in GALWICK. Then, the same data as the dry film sample that has not been moistened (hereinafter also referred to as the "dry curve") are measured, and the pressure at the intersection of the curve (half-dry curve) equivalent to half the flow rate of the dry curve and the wet curve is calculated. After that, the surface tension (γ) of GALWICK, the contact angle (θ) with the substrate, and the air pressure (P) can be introduced into the following formula (I) to calculate the average pore size. Average pore size = 4γcosθ/P……(I)

In addition, the "peak value of the pore diameter distribution" refers to the value evaluated by the method shown below using the pore diameter distribution calculated using the wet curve and dry curve obtained in the pore diameter distribution measurement test when calculating the above-mentioned average pore diameter. The pore diameter distribution is a histogram with the x-axis being the pore diameter and the y-axis being the pore diameter distribution, the x-axis being the logarithmic axis, the interval of the data interval being 0.05, that is, the interval up to the pore diameter of the x-axis multiplied by 10 being the logarithmic value, and being divided into 20 equal intervals, and then the histogram is drawn so that the sum of the degrees of all intervals becomes 100%, and the evaluation value of the maximum degree of the histogram is set as the peak value.

As described above, the separation substrate of the present invention is provided with a region including a coarse filter layer including a coarse filter membrane X satisfying predetermined parameters and a fine filter layer including a fine filter membrane Y satisfying predetermined parameters in order from the inflow side of the cell suspension, so that the removal rate of megakaryocytes and the recovery rate of platelets are both high and the filtration life is prolonged. Although the details of the reason for exerting such an effect are unclear, the inventors of the present invention speculate as follows. First, in the coarse filter membrane X, the average pore size measured by the semi-dry method is 5.0 μm or more and 30.0 μm or less and the peak value of the pore diameter distribution is less than 30%, so it can be said that it has a large number of pores with various pore sizes compared with the fine filter membrane Y described later. Furthermore, by providing the coarse filter layer including the coarse filter membrane X on the inflow side of the cell suspension than the fine filter layer including the fine filter membrane Y, it is considered that the number of megakaryocytes reaching the fine filter layer can be reduced, thereby maintaining the megakaryocyte removal rate and platelet recovery rate at a high level and extending the filtration life. In particular, by providing the coarse filter layer including the coarse filter membrane X, a liquid flow in a direction parallel to the membrane surface is sufficiently generated at the interface between the membranes in the laminated membrane, and the filtration object is effectively dispersed on the membrane surface of each membrane, and as a result, it is considered that the effective gaps of the membrane can be utilized to the maximum extent, thereby extending the filtration life.

[Coarse filter layer] The coarse filter layer of the separation substrate of the present invention is a filter layer comprising one or more coarse filter membranes. Herein, the coarse filter membrane refers to a filter membrane having a peak value of pore diameter distribution of less than 30%. In the present invention, at least one of the coarse filter membranes is a coarse filter membrane X having an average pore diameter of 5.0 μm or more and 30.0 μm or less measured by a semi-dry method and a peak value of pore diameter distribution of less than 30%. It is preferred that all coarse filter membranes contained in the coarse filter layer are coarse filter membranes X. When the coarse filter layer includes two or more coarse filter membranes, the same coarse filter membranes may be laminated or different coarse filter membranes may be laminated. When different coarse filter membranes are laminated, it is preferred that the adjacent coarse filter membranes are laminated in such a manner that the average pore size of the coarse filter membrane disposed on the inflow side of the cell suspension is equal to or larger than the average pore size of the coarse filter membrane disposed on the outflow side.

The thickness of one sheet of the coarse filter membrane is not particularly limited, but is preferably 10 to 1000 μm, more preferably 15 to 400 μm, and even more preferably 20 to 200 μm. Here, the "thickness of the coarse filter membrane" refers to the value obtained by measuring the thickness of the coarse filter membrane at 10 locations using a micrometer (manufactured by Mitutoyo Corporation) and averaging the measured values.

In the present invention, the coarse filter layer preferably includes 3 or more coarse filter membranes, and more preferably includes 3 to 20 coarse filter membranes, because the filtration life of the separation substrate becomes longer.

<Coarse filter membrane X> The coarse filter membrane X is a coarse filter membrane having an average pore size of 5.0 μm or more and 30.0 μm or less as measured by the semi-dry method as described above, and a peak value of the pore size distribution of less than 30%. Moreover, the average pore size of the coarse filter membrane X is preferably 7.5 μm or more and 26.0 μm or less, and more preferably 9.0 μm or more and 23.0 μm or less. Moreover, the peak value of the pore size distribution of the coarse filter membrane X is preferably 5% or more and less than 30%, and more preferably 10% or more and less than 30%.

From the perspective of extending the filtration life of the separation substrate, the coarse filter membrane X is preferably a membrane that satisfies the following formula (1), and more preferably a membrane that satisfies the following formula (2). 150≤Number of average pore size (μm) ×Number of thickness (μm)≤1500 (1) 150≤Number of average pore size (μm) ×Number of thickness (μm)≤1200 (2)

In order to increase the recovery rate of platelets and prolong the filtration life of the separation substrate, the porosity of the coarse filter membrane X is preferably 40% or more and 90% or less, and more preferably 50% or more and 90% or less. Here, "porosity" refers to the value calculated by the following formula. Porosity (%) = [1-{m/ρ/(S×d)}]×100 m: sheet weight (g) ρ: resin density (g/cm ³ ) S: sheet area (cm ² ) d: sheet membrane thickness (cm)

[Precision filter layer] The precision filter layer of the separation substrate of the present invention is a filter layer comprising one or more precision filter membranes. Here, the precision filter membrane refers to a filter membrane having a peak value of pore diameter distribution of 30% or more. In the present invention, at least one of the precision filter membranes is a precision filter membrane Y having an average pore diameter of 2.0 μm or more and 20.0 μm or less measured by a semi-dry method and a peak value of pore diameter distribution of 30% or more. It is preferred that all precision filter membranes contained in the precision filter layer are precision filter membranes Y. Furthermore, when the precision filter layer includes two or more precision filter membranes, the same precision filter membranes may be laminated or different precision filter membranes may be laminated. Furthermore, when different precision filter membranes are laminated, it is preferred that the adjacent precision filter membranes are laminated in such a manner that the average pore size of the precision filter membrane disposed on the inflow side of the cell suspension is equal to or larger than the average pore size of the precision filter membrane disposed on the outflow side.

The thickness of one precision filter membrane is not particularly limited, but is preferably 10 to 500 μm, more preferably 10 to 250 μm, and even more preferably 10 to 200 μm. Here, the "thickness of the precision filter membrane" refers to the value obtained by measuring the thickness of the precision filter membrane at 10 locations using a micrometer (manufactured by Mitutoyo Corporation) and averaging the measured values.

In the present invention, from the perspective of extending the filtration life of the separation substrate, the precision filter layer preferably includes two or more precision filter membranes, and more preferably includes 2 to 10 precision filter membranes.

<Precision filter membrane Y> Precision filter membrane Y is a precision filter membrane having an average pore size of 2.0 μm or more and 20.0 μm or less as measured by the semi-dry method as described above, and a peak value of pore diameter distribution of 30% or more. Preferably, the average pore size of precision filter membrane Y is 2.0 μm or more and 17.5 μm or less, and more preferably, 2.0 μm or more and 15.0 μm or less. Preferably, the peak value of pore diameter distribution of precision filter membrane Y is 30% or more and 99% or less, and more preferably, 45% or more and 99% or less.

From the perspective of increasing the recovery rate of platelets and extending the filtration life of the separation substrate, the porosity of the precision filtration membrane Y is preferably 40% to 90%, and more preferably 50% to 90%.

In the present invention, from the perspective of extending the filtration life of the separation substrate, the critical wetting surface tension (Critical Wetting Surface Tension: CWST) of all the coarse filter membranes included in the coarse filter layer and all the fine filter membranes included in the fine filter layer is preferably 72 mN/m or more, and 72 to 110 mN/m is more preferably. Here, "critical wetting surface tension" refers to the value calculated by the following procedure. First, using solutions with different surface tensions, 1 drop of the solution is gently dropped on a horizontal filter membrane and left for 10 minutes. When the filter membrane is wetted, a solution with a higher surface tension than the solution being wetted is added dropwise in the same manner, and this process is repeated until no more wetting occurs. Then, the average of the surface tensions of the solution with the largest surface tension in the wetted solution and the solution with the smallest surface tension in the unwetted solution is calculated as the critical wetting surface tension.

As a method for adjusting the critical wetting surface tension within the above range, various surface treatment methods can be adopted. As surface treatment methods, for example, surface coating of hydrophilic raw materials, conversion or addition of hydrophilic functional groups based on alkali saponification, etc. can be cited. In addition, as for the coating material, known materials can be used, and it is particularly preferred that the coating material does not have platelet adsorption.

In the present invention, regarding the above-mentioned coarse filter layer and fine filter layer, it is preferred that the average pore size on the outflow side of the coarse filter layer is equal to or larger than the average pore size on the inflow side of the fine filter layer.

The separation substrate of the present invention has a region in which the above-mentioned coarse filter layer and fine filter layer are arranged in order from the inflow side of the cell suspension, but the total volume of the region is preferably 50% or more, and more preferably 65-100% relative to the total volume of the separation substrate.

The thickness of the separation substrate of the present invention is preferably 100 to 3000 μm, and more preferably 150 to 1000 μm. Here, "the thickness of the separation substrate" refers to the value obtained by measuring the film thickness of the separation substrate at 10 locations using a micrometer (manufactured by Mitutoyo Corporation) and averaging the measured values.

In the present invention, the average fiber diameter of the fibers constituting the porous body (especially, nonwoven fabric) is preferably 800 nm or more and 3500 nm or less, from the perspective of being able to take into account both sufficient fiber strength and good separation performance. In addition, the average fiber length of the fibers constituting the nonwoven fabric is preferably 1 mm or more and 1 m or less, from the perspective of being able to prevent fiber peeling when using a separation substrate (for example, during filtration). In addition, the average fiber diameter and the average fiber length can be adjusted by adjusting the manufacturing conditions such as the concentration of the raw material (for example, cellulose acetate, etc.) solution when making the nonwoven fabric.

Here, the average fiber diameter refers to the value measured as follows. The surface of a nonwoven fabric composed of fibers is observed using a transmission electron microscope (TEM) image or a scanning electron microscope (SEM) image. The electron microscope image is observed at a magnification selected from 1000 to 5000 times depending on the size of the fibers. The sample, observation conditions, or magnification are adjusted to meet the following conditions. (1) A straight line X is drawn at any position in the observation image, and 20 or more fibers intersect the straight line X. (2) A straight line Y is drawn in the same image, which intersects the straight line X at right angles, and 20 or more fibers intersect the straight line Y. For the electron microscope observation image as described above, read the width (short diameter of the fiber) of at least 20 fibers (i.e., at least 40 fibers in total) for each fiber intersecting with the straight line X and the straight line Y. Observe at least 3 groups of electron microscope images as described above in this way, and read the fiber diameters of at least 40 fibers × 3 groups (i.e., at least 120 fibers). As described above, the fiber diameters read are averaged to obtain the average fiber diameter.

In addition, the average fiber length refers to the value measured as follows. That is, the fiber length of the fiber can be obtained by analyzing the electron microscope observation image used when measuring the above-mentioned average fiber diameter. Specifically, for the electron microscope observation image as described above, the fiber lengths of at least 20 fibers (that is, at least 40 fibers in total) are read for each fiber intersecting with the straight line X and the fiber intersecting with the straight line Y. In this way, at least 3 groups of electron microscope images as described above are observed, and the fiber lengths of at least 40 fibers × 3 groups (that is, at least 120 fibers) are read. As described above, the average fiber length is obtained by averaging the read fiber lengths.

The separation substrate of the present invention preferably includes a resin material. As the resin material, specifically, for example, cellulose resins such as cellulose acylate and cellulose; polyacrylonitrile resin; polyester resin; fluororesin; polyether polyester resin; polyamide resin; polypropylene and other polyolefin resins, etc., can be cited. A single type of these can be used, or two or more types can be used in combination. Among these, it is preferred to include at least one resin selected from the group including cellulose resins and polyolefin resins.

As the above-mentioned cellulose resin, cellulose acylation or cellulose is preferred. In addition, cellulose acylation may be a partially saponified cellulose acylation. In addition, as the acyl group possessed by the cellulose acylation, specifically, for example, acetyl, propionyl, and butyryl can be mentioned. In addition, the substituted acyl group may be only one type (for example, only acetyl) or may be two or more types.

As the polyolefin resin, polypropylene is preferred, and hydrophilized polypropylene is more preferred.

In the present invention, from the perspective of inhibiting platelet adsorption to the separation substrate and further improving the permeability of platelets, the surface of the separation substrate can be hydrophilized to chemically or physically modify the platelet low adsorption material. As a platelet low adsorption material, a polymer having a hydrophilic group in the side chain is preferred, for example, 2-methacryloyloxyethyl phosphocholine, ethylene glycol, methyl methacrylate, hydroxyethyl methacrylate, vinyl alcohol, N-vinyl-2-pyrrolidone, and a polymer of a sulfobetaine monomer can be cited. As the hydrophilic group, specifically, for example, there can be cited a hydroxyl group, an ether group, a nitro group, an imino group, a carbonyl group, a phosphate group, a methoxydiethylene glycol group, a methoxytriethylene glycol group, an ethoxydiethylene glycol group, an ethoxytriethylene glycol group, an amino group, a dimethylamino group, a diethylamino group, a carboxyl group, a phosphoyl group, a phosphocholine group, a sulfate group or salts thereof. In addition, as a platelet low adsorption material and a modification method thereof, the materials and methods described in WO87/05812, Japanese Patent Laid-Open No. 4-152952, Japanese Patent Laid-Open No. 5-194243, WO2010/113632, etc. can be used.

[Cell suspension] The cell suspension used for separation of platelets using the separation substrate of the present invention is a cell suspension containing megakaryocytes and platelets. Among them, megakaryocytes and platelets are not particularly limited, and examples thereof include megakaryocytes and platelets collected from adult tissues; megakaryocytes and platelets differentiated from cells having differentiation totipotency such as pluripotent stem cells, hematopoietic progenitor cells, and mesenchymal cells; megakaryocytes and platelets produced by direct reprogramming technology from cells that do not have differentiation totipotency to megakaryocytes in conventional methods; combinations of these megakaryocytes and platelets; etc.

Examples of pluripotent stem cells include embryonic stem cells [ES (embryonic stem) cells], nuclear transfer embryonic stem cells [nt (nuclear transfer) ES cells], and artificial pluripotent stem cells [iPS (induced pluripotent stem) cells], among which artificial pluripotent stem cells (iPS cells) are preferred. Examples of hematopoietic progenitor cells include cells from bone marrow, cord blood, mobilized [G-CSF (Granulocyte-colony stimulating factor)] peripheral blood, mid-lobar cells derived from ES cells, and cells from peripheral blood, but are not limited to these. Examples of such hematopoietic progenitor cells include cluster of differentiation (CD) 34 positive cells (e.g., CD34+ cells, CD133+ cells, SP cells, CD34+CD38- cells, c-kit+ cells, or CD3-, CD4-, CD8-, and CD34+ cells) (International Publication No. WO2004/110139). Examples of such mesenchymal cells include mesenchymal stem cells, adipose progenitor cells, bone marrow cells, fat cells, and synovial cells, among which adipose progenitor cells are preferred. In conventional methods, as cells that do not have the totipotency to differentiate into megakaryocytes, for example, fibroblasts can be cited, but the present invention is not limited to these.

[Cell separation filter] The cell separation filter of the present invention comprises: a container having a first liquid port and a second liquid port; and a filter material filled between the first liquid port and the second liquid port, wherein the separation substrate of the present invention is used in the filter material.

The shape, size, and material of the container used in the cell separation filter are not particularly limited. The container may be in any shape, such as a sphere, a container, a box, a bag, a tube, a column, etc. The container may be in any shape, such as a horizontal type or a columnar type.

[Method for producing platelets] The method for producing platelets of the present invention comprises: a contacting step of bringing a culture medium containing at least megakaryocytes into contact with the above-mentioned separation substrate of the present invention; a culturing step of culturing megakaryocytes to produce platelets before and/or after the contacting step; and a recovery step of recovering the culture medium containing the produced platelets after the contacting step and the culturing step. The contacting mechanism in the contacting step can be appropriately selected according to the amount of the culture medium and the concentration of megakaryocytes, but for example, a method of supplying a cell suspension to a tower or column filled with the separation substrate of the present invention can be cited. In addition, the method of producing platelets in the culture step includes, for example, a method of applying shear stress by a fluid, and specifically, a method of stirring a culture medium containing megakaryocytes, etc. In addition, the megakaryocytes cultured in the culture step may be megakaryocytes supplemented by the separation substrate of the present invention when the culture step is performed after the contacting step. Furthermore, when there is a culture step after the contact step, as in the embodiment described later, when the cell suspension containing megakaryocytes and platelets is brought into contact with the separation substrate, the megakaryocytes replenished in the early stage are considered to also produce platelets due to the shear stress of the cell suspension (i.e., fluid) that is subsequently contacted. In addition, as a recovery method in the recovery step, for example, a method of passing the culture solution containing the produced platelets through a tower or column filled with the separation substrate of the present invention can be cited. [Example]

The present invention is further described in detail below based on the embodiments. The materials, usage amounts, ratios, processing contents, processing steps, etc. shown in the following embodiments can be appropriately changed as long as they do not deviate from the purpose of the present invention. However, the scope of the present invention is not limited to the embodiments shown below.

[Preparation of coarse filter membrane] Using polypropylene (PP) as a raw material, a nonwoven fabric was prepared by melt-blowing, and after calendering treatment as needed, a hydrophilic coating treatment was performed to prepare coarse filter membranes R1 to R13 composed of nonwoven fabrics. Here, regarding the calendering treatment, the temperature, pressure, and conveying speed were adjusted to achieve the porosity described in Table 1 below. In addition, regarding the hydrophilic coating, the macromolecular monomer of methoxy polyethylene glycol methacrylate was graft-polymerized under the conditions described in Japanese Patent No. 3250833 to achieve the CWST shown in Table 1 below. In addition, for coarse filter membranes with different average pore diameters, thicknesses, peak values of pore distribution, and porosity, the coarse filter membranes listed in Table 1 below were obtained by adjusting the manufacturing conditions of the nonwoven fabric such as weight, polymer extrusion speed, or temperature. In addition, for the coarse filter membranes R1 to R13 prepared, the average pore diameter, thickness, CWST, peak value of pore diameter distribution, and porosity were measured by the above method, and the product of the average pore diameter and thickness (the value of the above formula (1)) was calculated. The results are shown in Table 1 below.

[Table 1] Porous body Raw materials Calendering Coating treatment Average pore size (μm) Thickness (μm) CWST (mN/m) Peak value of pore diameter distribution Porosity Average pore diameter × thickness Coarse filter membrane R1 Non-woven fabric PP without have 4.0 75 73 11% 41% 300 Coarse filter membrane R2 Non-woven fabric PP without have 5.2 28 73 twenty two% 49% 146 Coarse filter membrane R3 Non-woven fabric PP without have 5.1 30 73 16% 63% 153 Coarse filter membrane R4 Non-woven fabric PP without have 11.0 85 75 twenty three% 81% 935 Coarse filter membrane R5 Non-woven fabric PP without have 17.0 66 76 19% 80% 1122 Coarse filter membrane R6 Non-woven fabric PP without have 28.0 53 74 19% 77% 1484 Coarse filter membrane R7 Non-woven fabric PP without have 27.2 56 74 25% 68% 1523 Coarse filter membrane R8 Non-woven fabric PP without have 32.0 44 78 27% 57% 1408 Coarse filter membrane R9 Non-woven fabric PP have have 10.6 60 73 19% 36% 636 Coarse filter membrane R10 Non-woven fabric PP without have 12.3 120 73 19% 91% 1476 Coarse filter membrane R11 Non-woven fabric PP without have 17.0 66 70 19% 67% 1122 Coarse filter membrane R12 Non-woven fabric PP without have 10.9 70 73 twenty one% 42% 763 Coarse filter membrane R13 Non-woven fabric PP without have 11.2 125 73 20% 89% 1400

[Preparation of precision filter membrane] Precision filter membranes P1 to P10 composed of nonwoven fabrics were prepared using the electrospinning method under the conditions shown in Table 2 below. Specifically, nonwoven fabrics composed of cellulose acetate (hereinafter also referred to as "CA") and/or cellulose acetate propionate (hereinafter also referred to as "CAP") were prepared by the same method as Example 1 of Patent Document 1 (International Publication No. 2018/207564). In addition, precision filter membranes with different average pore diameters, thicknesses, peak values of pore distribution, and porosities were obtained by adjusting weight, heat treatment temperature, and heat treatment time to obtain precision filter membranes listed in Table 2 below. Next, an alkaline saponification treatment was performed using a water/ethanol solution of sodium hydroxide to adjust the CWST. Then, the membranes were immersed in pure water, taken out and air-dried to produce precision filter membranes P1 to P10. In addition, the average pore size, thickness, CWST, peak value of pore size distribution and porosity of the produced precision filter membranes P1 to P10 were measured by the above method. The results are shown in Table 2 below.

[Table 2] Porous body Raw materials Average pore size (μm) Thickness (μm) CWST (mN/m) Peak value of pore diameter distribution Porosity Precision filter membrane P1 Non-woven fabric CAP 7.0 45 78 80% 70% Precision filter membrane P2 Non-woven fabric CAP 1.5 30 75 32% 74% Precision filter membrane P3 Non-woven fabric CAP 2.0 150 73 32% 63% Precision filter membrane P4 Non-woven fabric CA/ CAP 18.0 80 80 66% 65% Precision filter membrane P5 Non-woven fabric CA/ CAP 21.0 70 80 66% 66% Precision filter membrane P6 Non-woven fabric CAP 5.4 20 77 80% 33% Precision filter membrane P7 Non-woven fabric CAP 7.0 100 85 39% 91% Precision filter membrane P8 Non-woven fabric CAP 7.0 45 69 80% 70% Precision filter membrane P9 Non-woven fabric CAP 6.5 25 74 78% 40% Precision filter membrane P10 Non-woven fabric CAP 6.7 90 73 63% 88%

[Preparation of cell suspension] Culture medium: RPMI1640 (manufactured by Thermo Fisher Scientific) 450 ml with 50 ml of bovine serum (manufactured by Thermo Fisher Scientific) added was used. Megakaryocytes: MEG-01 (manufactured by ATCC) was used as megakaryocytes. This was mixed with the culture medium to prepare a megakaryocyte suspension (6×10 ⁵ cells/ml). Platelet suspension: Platelets isolated from mouse peripheral blood were used as platelets. Specifically, 10 ml of whole blood collected from a mouse was collected in a 15 ml conical tube for centrifugation separation (Falcon) filled with citrate-dextrose solution (ACD) (Sigma-Aldrich). The plasma layer and the buffy coat layer were recovered after centrifugation at 300×g and room temperature for 7 minutes. The recovered solution was centrifuged and separated in the same manner, and only the plasma layer was recovered. The recovered solution was then centrifuged at 1800×g and room temperature for 5 minutes, and the supernatant was recovered to detect platelets. This was mixed with the culture medium to prepare a platelet suspension (2×10 ⁸ cells/ml). The megakaryocyte suspension and the platelet suspension were mixed in equal amounts to prepare a cell suspension.

[Examples 1 to 26 and Comparative Examples 1 to 10] [Evaluation 1] Each coarse filter membrane and each precision filter membrane produced was cut into 25 mm φ circles, and the separation substrate produced by stacking in the manner of the stacked structure described in Table 3 below was assembled on a commercially available filter support frame. Next, a polyvinyl chloride hose with an inner diameter of 3 mm and a length of 30 cm was connected to the inlet and outlet sides of the filter support frame. A syringe with a piston removed was connected to the end of the hose on the inlet side, and the syringe part was fixed with a clamp to suspend the syringe, hose, and filter support frame in the direction of the lead hammer. In addition, with the hose connected, the filter support frame is reversed up and down so that the inflow side becomes the lower side and the outflow side becomes the upper side to prevent the hose from breaking, and the clamp is fixed at a position 20 cm below the bottom of the syringe. In addition, the part of the bottom of the syringe close to the hose is fixed with a clamp and sealed, and then the cell suspension is introduced into the syringe. Then, the clamp is opened and the cell suspension is introduced into the filter so that it contacts the separation substrate. Next, after confirming that the filter liquid flows out from the outflow side of the filter support frame, the filter support frame is removed from the clamp, and the syringe, hose, and filter support frame are returned to the arrangement arranged along the hammer direction, and the outflowing filter liquid is recovered. The filter liquid is recovered in small portions with a certain amount of liquid, and the platelet recovery rate and megakaryocyte removal rate are measured for each liquid. During filtration, the liquid flow rate until the flow rate becomes half of the value at the beginning of filtration is evaluated as the filter life. In addition, the platelet recovery rate and megakaryocyte removal rate are evaluated using the average performance until the filter life. These results are shown in Table 3 below. In Table 3 below, the membrane listed in "coarse filter membrane, etc." is placed on the inflow side of the cell suspension, and the membrane listed in "precision filter membrane, etc." is placed on the outflow side of the membrane listed in "coarse filter membrane, etc.". In addition, in the membranes listed in "coarse filter membrane, etc." and "precision filter membrane, etc.", if they are divided into two layers, the upper membrane is placed on the inflow side of the cell suspension, and the lower membrane is placed on the outflow side of the upper membrane.

[Table 3] Coarse filter membrane, etc. Precision filter membrane, etc. Platelet recovery rate Megakaryocyte removal rate Filter life Type Number of pieces Type Number of pieces (ml) Embodiment 1 R5 3 P1 3 93.1% 99.9% 56 Embodiment 2 R5 6 P1 3 95.6% 99.9% 60 Embodiment 3 R5 12 P1 3 93.8% 99.9% 67 Embodiment 4 R5 18 P1 3 92.6% 99.9% 66 Embodiment 5 R5 6 P1 3 92.7% 99.9% 47 R4 3 Embodiment 6 R5 6 P1 3 92.2% 99.9% 50 R4 5 Embodiment 7 R5 18 P1 3 91.4% 99.9% 50 R4 5 Embodiment 8 R5 6 P3 3 90.2% 99.9% 46 Embodiment 9 R5 6 P4 3 97.2% 99.3% 66 Embodiment 10 R3 4 P1 3 93.1% 99.9% 52 Embodiment 11 R6 6 P1 3 94.0% 99.9% 53 Embodiment 12 R5 6 P1 3 91.8% 99.9% 48 R3 3 Embodiment 13 R6 6 P1 3 94.7% 99.9% 57 R5 6 Embodiment 14 R2 3 P1 3 90.6% 99.9% 29 Embodiment 15 R7 6 P1 3 93.4% 99.9% 30 Embodiment 16 R5 6 P9 3 93.2% 99.9% 46 Embodiment 17 R5 6 P10 3 93.5% 99.9% 53 Embodiment 18 R12 6 P1 3 92.9% 99.9% 41 Embodiment 19 R13 6 P1 3 93.5% 99.9% 44 Embodiment 20 R5 6 P6 3 93.0% 99.9% 28 Embodiment 21 R5 6 P7 3 93.5% 99.9% 36 Embodiment 22 R9 4 P1 3 90.9% 99.9% 34 Embodiment 23 R10 4 P1 3 91.0% 99.9% 35 Embodiment 24 R5 2 P1 3 93.0% 99.9% twenty four Embodiment 25 R5 6 P8 3 91.1% 99.9% 15 Embodiment 26 R11 6 P1 3 90.4% 99.9% 16 Comparison Example 1 - - P1 1 95.4% 99.3% 4 Comparison Example 2 - - P1 3 92.3% 99.9% 2 Comparison Example 3 R5 6 - - 79.6% 55.6% 124 Comparison Example 4 R5 6 - - 94.6% 84.9% 88 R4 5 Comparison Example 5 P1 3 R5 6 92.0% 99.9% 2 Comparative Example 6 P1 3 R5 6 91.9% 99.9% 2 R4 5 Comparison Example 7 R5 6 P2 3 5.4% 99.9% 1 Comparative Example 8 R5 6 P5 3 95.3% 86.4% 20 Comparative Example 9 R1 4 P1 3 11.5% 99.9% 1 Comparative Example 10 R8 6 P1 3 94.8% 99.9% 2

From the results shown in Table 3 above, it can be seen that the separation substrate provided with only a fine filter membrane has high megakaryocyte removal rate and platelet recovery rate, but the filtration life is short (Comparison Examples 1 and 2). In addition, it can be seen that the separation substrate provided with only a coarse filter membrane has a lower megakaryocyte removal rate (Comparison Examples 3 and 4). In addition, it can be seen that the separation substrate provided with a fine filter layer and a coarse filter layer in order from the inflow side of the cell suspension has a short filtration life (Comparison Examples 5 and 6). In addition, it can be seen that when a filter membrane with an average pore size of less than 2.0μm is used as a precision filter membrane, the platelet recovery rate becomes low and the filter life is also short (Comparative Example 7). In addition, it can be seen that when a filter membrane with an average pore size of more than 20μm is used as a precision filter membrane, the megakaryocyte removal rate becomes low and the filter life is also short (Comparative Example 8). In addition, it can be seen that when a filter membrane with an average pore size of less than 5.0μm is used as a coarse filter membrane, the platelet recovery rate becomes low and the filter life is also short (Comparative Example 9). In addition, it can be seen that when a filter membrane with an average pore size of more than 30 μm is used as a coarse filter membrane, the filtration life is short (Comparative Example 10). In contrast, it can be seen that the separation substrate in which a coarse filter layer including a coarse filter membrane X and a fine filter layer including a fine filter membrane Y are arranged in order from the inflow side of the cell suspension has a high megakaryocyte removal rate and a platelet recovery rate and a long filtration life (Examples 1 to 26). In addition, from the comparison of Examples 2, 11 and 15, it can be seen that if the coarse filter membrane X is a membrane that satisfies the above formula (1), the filtration life of the separation substrate becomes longer. Furthermore, from the comparison of Examples 10, 22, and 23, it can be seen that if the porosity of the coarse filter membrane X is 40% or more and 90% or less, the recovery rate of platelets becomes higher and the filtration life of the separation substrate becomes longer. Furthermore, from the comparison of Examples 2, 16, 17, 20, and 21, it can be seen that if the porosity of the precision filter membrane Y is 40% or more and 90% or less, the recovery rate of platelets becomes higher and the filtration life of the separation substrate becomes longer. Furthermore, from the comparison of Examples 1 and 24, it can be seen that if the coarse filter layer includes 3 or more coarse filter membranes, the filtration life of the separation substrate becomes longer.

[Examples 2, 3 and 5 to 7] [Evaluation 2] Referring to the non-patent literature [Life Support and Anesthesia, vol.8, no.11, 2001-11, p.1010-p.1013], a filter housing is prepared, and the laminate (separation substrate) prepared in Evaluation 1 of Examples 2, 3 and 5 to 7 is cut into a circular shape and assembled in the filter housing. When assembling the separation substrate in the filter housing, a filler is sealed in the outer peripheral portion of the separation substrate in the internal space of the filter housing, and the separation substrate is fixed in the filter housing. Next, the same cell suspension as in Evaluation 1 was transferred to the infusion bag and connected to the inflow side of the amplification filter. At this time, a hose clamp was used to prevent the cell suspension in the infusion bag from flowing into the amplification filter. Next, the infusion bag was hung on the bracket from the top, and the hose clamp was opened to filter the cell suspension. The evaluation of the recovery, separation and filtration life of the filtrate was carried out in the same manner as the evaluation using a commercially available filter support. The results are shown in Table 4 below.

[Table 4] Coarse filter membrane, etc. Precision filter membrane, etc. Platelet recovery rate Megakaryocyte removal rate Filter life Type Number of pieces Type Number of pieces (ml) Embodiment 2 R5 6 P1 3 96.5% 99.9% 730 Embodiment 3 R5 12 P1 3 95.2% 99.9% 780 Embodiment 5 R5 6 P1 3 94.2% 99.9% 540 R4 3 Embodiment 6 R5 6 P1 3 94.1% 99.9% 600 R4 5 Embodiment 7 R5 18 P1 3 93.0% 99.9% 610 R4 5

From the results shown in Table 4 above, it can be seen that in Examples 2, 3, and 5 to 7, the megakaryocyte removal rate and platelet recovery rate were high and the filtration life was long in the enlarged evaluation.

without.

Claims (12)

A separation substrate is composed of a porous body for separating platelets from a cell suspension containing megakaryocytes and platelets. The separation substrate has a region in which a coarse filter layer and a fine filter layer are sequentially arranged from the inflow side of the cell suspension. The coarse filter layer includes one or more coarse filter membranes, and the fine filter layer includes one or more fine filter membranes. At least one of the coarse filter membranes is formed by a semi-dry method. A coarse filter membrane X having an average pore size of 5.0 μm or more and 30.0 μm or less and a peak value of a pore size distribution of less than 30%, wherein at least one of the above-mentioned precision filter membranes is a precision filter membrane Y having an average pore size of 2.0 μm or more and 20.0 μm or less and a peak value of a pore size distribution of more than 30%, wherein the above-mentioned coarse filter membrane X is a membrane satisfying the following formula (1), 150

Average pore size (μm) × thickness (μm)

1500 (1). The separation substrate as described in claim 1, wherein the porosity of the aforementioned precision filter membrane Y is greater than 40% and less than 90%. The separation substrate as described in claim 1, wherein the porosity of the aforementioned coarse filter membrane X is greater than 40% and less than 90%. The separation substrate as described in claim 1, wherein the aforementioned coarse filter layer comprises more than 3 coarse filter membranes. The separation substrate as described in claim 1, wherein all the coarse filter membranes contained in the aforementioned coarse filter layer and all the fine filter membranes contained in the aforementioned fine filter layer are membranes with a critical wetting surface tension of 72 mN/m or more. The separation substrate as described in claim 1, wherein the aforementioned porous body is a non-woven fabric. The separation substrate as described in claim 6, wherein the nonwoven fabric comprises at least one resin selected from the group consisting of cellulose resins and polyolefin resins. The separation substrate as described in claim 7, wherein the aforementioned cellulose resin is cellulose acylate or cellulose. The separation substrate as described in claim 7, wherein the aforementioned polyolefin resin is polypropylene. The separation substrate as described in claim 9, wherein the aforementioned polypropylene is hydrophilized polypropylene. A cell separation filter comprises: a container having a first liquid port and a second liquid port; and a filter material filled between the first liquid port and the second liquid port. In the cell separation filter, the filter material is a separation substrate as described in any one of claim 1 to claim 10. A method for producing platelets, comprising: a contacting step, which is a step of bringing a culture medium containing at least megakaryocytes into contact with a separation substrate as described in any one of claim 1 to claim 10; a culturing step, in at least one of before and after the contacting step, culturing megakaryocytes to produce platelets; and a recovery step, in which after the contacting step and the culturing step, the culture medium containing the produced platelets is recovered.