JP2011000040A - Method for evaluating shear force influence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating a shear force influence, which evaluates the influence of shear force on cells more simply than before.SOLUTION: The method for evaluating the shear force influence includes a shear force generation step of making a liquid of prescribed flow rate flow into a micro channel in which cells are arranged so as to generate shear force to cells and a cell crushing evaluation step of evaluating whether the cells after flowing into the micro channel in the shear force generation step are crushed or not in response to the shear force by liquid inflow.

Description

  The present invention relates to a shear force influence evaluation method for evaluating the influence of shear force on cells.

  The mixing operation in the mixing tank is one of important operations in the chemical industry and the like. That is, in the field of foods, cosmetics, medicines, pharmaceuticals, paints, etc., mixing operations are utilized in various processes such as reaction, extraction, absorption, and mixing. For example, as one of the mixing operations, in an animal cell culture tank such as a bioreactor, the inside of the tank is agitated in order to uniformly mix the bubbles and nutrients of the oxygen supply source supplied to the tank. ing.

  In such an animal cell culture tank, especially when the culture tank becomes large and the height in the vertical direction becomes high, in order to mix the inside of the tank more uniformly, a large axial flow is generated in the culture tank by stirring. It is necessary to obtain a high degree of mixing. In general, when it is desired to uniformly mix materials having a density difference, these materials are easily separated in the vertical direction due to the density difference. Therefore, it is necessary to generate a large axial flow in the mixing tank by stirring. In particular, in the case of solid-liquid mixing, since the density difference becomes large and the solid content tends to settle, it is further necessary to generate an axial flow by stirring and to stir the inside of the tank vertically by this axial flow.

  And in a mixing tank, in order to obtain a high degree of mixing, it is necessary to use a high performance thing as a stirring apparatus. However, in the animal cell culture tank, animal cells are destroyed / died by shearing force generated by the stirring blades of the stirring device, and therefore it is necessary to measure the balance between stirring efficiency and shearing force. In the stirring device, in order to determine the upper limit of the rotational speed of the stirring blade, the proliferation rate of the cells when the stirring blade is rotated at an arbitrary rotational speed is measured, or the whole cells are crushed by microscopic observation. By measuring the ratio of the cells having a small diameter and the diameter of the cells, the influence of the shearing force on the cells at each rotation speed of the stirring blade has been determined.

  As described above, in Patent Document 1 below, as a measure of the shear force generated by a fluid, a flat plate having a heat transfer surface formed on a surface is suspended vertically in a liquid, and the heat transfer surface is heated uniformly by heat flux. Measure the shear force acting on the flat plate by natural convection around the flat plate of the fluid generated by the electronic balance, and measure the viscosity of the liquid by calculation from the measured value, flat plate dimensions, calorific value, and thermophysical property value of the fluid A viscosity measuring device is disclosed.

JP 2003-149114 A

By the way, in the above prior art, in order to determine the upper limit of the rotational speed of the stirring blade, the proliferation rate of the cells when the stirring blade is rotated at an arbitrary rotational speed is measured, or the whole cells are disrupted by microscopic observation. The shearing force at each rotation speed of the stirring blade is determined by measuring the ratio of the formed cells and the small-sized cells. However, in this method, while changing the rotation speed of the stirring device, it is necessary to measure the cell growth rate at each rotation speed, or to measure the ratio of crushed cells and small diameter cells. It takes a very large amount of labor to determine the influence of the shearing force on the cells for each rotation speed of the stirring blade, and it takes a long time to obtain the determination result of the shearing force.
Moreover, in the said patent document 1, although the shear force is measured by the physical method, it is difficult to measure the influence which a shear force has on a cell.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a method for evaluating the influence of shearing force, which can more easily evaluate the influence of shearing force on cells than in the past.

  In order to achieve the above object, in the present invention, as a first solving means related to the shear force effect evaluation method, a liquid having a predetermined flow rate is caused to flow into the micro flow channel in which the cells are arranged, whereby A shearing force generating step for generating a shearing force, and evaluating whether or not the cells after the liquid has flowed into the microchannel in the shearing force generating step are crushed according to the shearing force due to the inflow of the liquid The cell disruption evaluation step is employed.

  In the present invention, as a second solving means related to the shear force effect evaluation method, in the first solving means, in the shearing force generation step, a cell culture carrier to which cells are attached is arranged in the microchannel. Is adopted.

  In the present invention, as a third solving means relating to the shear force effect evaluation method, in the second solving means, in the shear force generation step, a plurality of the above-described liquid flows so as to be parallel to the flow of the liquid that has flowed in. A means is employed in which a cell culture carrier is disposed in the microchannel.

  In the present invention, as a fourth solving means related to the method for evaluating the influence of shearing force, in the first solving means, in the shearing force generation step, a plurality of single cells are arranged in parallel with the flow of the liquid that has flowed in. Is employed in the microchannel.

  In the present invention, as a fifth solving means related to the shear force effect evaluation method, in the first to fourth solving means, in the shear force generation step, a membrane potential change detection dye is dissolved in the liquid, In the cell disruption evaluation step, means for evaluating cell disruption by detecting a change in the potential of the cell membrane using the membrane potential change detection dye is employed.

  In the present invention, as a sixth solving means relating to the shear force effect evaluation method, in the first to fifth solving means, in the shearing force generation step, the liquid is caused to flow into the microchannel by a syringe pump. Adopt the means to let.

  According to the present invention, the method for evaluating the influence of shear force includes a shear force generation step for generating a shear force to a cell by causing a liquid having a predetermined flow rate to flow into a microchannel in which the cell is arranged, and a shear force generation. A cell crushing evaluation step for evaluating whether or not the cells after the liquid has flowed into the microchannel in the step are crushed according to the shearing force caused by the inflow of the liquid.

  As described above, in the shear force effect evaluation method, the cell growth rate is measured at each rotation speed while changing the rotation speed of the stirring device of the animal cell culture tank. Since it is not necessary to measure the ratio, it is possible to evaluate the influence of shear force on cells more easily than in the past.

It is a perspective view which shows arrangement | positioning of the microcarrier 1 and the microchannel 2 of the shear force generation process of the shear force influence evaluation method which concerns on one Embodiment of this invention. It is a top view which shows the position of the microcarrier 1, the microchannel 2, and the syringe pump 3 of the shear force generation process of the shear force influence evaluation method which concerns on one Embodiment of this invention.

  Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The present embodiment relates to a shear force effect evaluation method for evaluating the effect of shear force on cells.

The process in the shear force influence evaluation method according to the present embodiment will be described.
The shear force effect evaluation method according to the present embodiment is a method for evaluating the influence of shear force generated by rotation of a stirring blade of an animal cell culture tank on cells, and includes a shear force generation step and a cell disruption evaluation step. Prepare.

Each step in the shear force influence evaluation method will be described along the procedure.
First, in the shear force effect evaluation method, in the shear force generation step, a microcarrier (cell culture carrier) 1 to which a plurality (about 10) of cells C are attached is placed in an open microchannel 2 on the upper surface side. As shown in FIG. 1, the upper surface side of the microchannel 2 is closed by covering the upper surface with a transparent flat plate 2a. FIG. 1 is a perspective view showing the arrangement of microcarriers 1 and microchannels 2 in the shearing force generation step of the shearing force effect evaluation method according to the present embodiment.

  The microcarrier 1 is used in cell culture as a carrier for culturing cells, and has a fine bead shape of a micrometer level of about 50 μm to 200 μm. In the shearing force generation step, the microcarriers 1 of various sizes are properly used according to the number of cells to be attached. In addition, the size per cell is about 10 μm to 20 μm.

  As the microcarrier 1, glass beads, plastic beads, or those obtained by binding anion exchange groups to these are used as substrate beads. Further, there is a microcarrier 1 in which the surface of the substrate bead is coated with cellulose, collagen, or hydroxyapatite.

  The microchannel 2 is constituted by a substrate made of an inert member in the culture medium L used for animal cell culture. This inert material is a material that is not reactive with the solvent, solute, and the compound produced by the chemical reaction of the culture solution L, such as glass, quartz, silica, magnesia, zirconia, alumina, apatite, silicon nitride, Ceramic members such as oxides, carbides, nitrides, borides and silicides of metals such as silicon, titanium, aluminum, yttrium and tungsten. In addition, metals, plastics, and the like can be used as the substrate of the microchannel 2 as long as they are inert materials.

  In the microchannel 2, an opaque member may be used for both side walls, but a transparent flat plate 2a is used for the lower surface in the same manner as the upper surface. This is for observing cells adhering to the microcarrier 1 with a fluorescence microscope in the cell disruption evaluation step described later. In the microchannel 2, the microcarrier 1 is fixed by being pressed by the transparent flat plates 2a on the upper surface and the lower surface.

  Then, in order to flow the culture medium L on both sides of the microcarrier 1, the microchannel 2 having a width capable of forming a space through which the culture medium L flows is used. Further, in order to fix the microcarrier 1 to the microchannel 2 by pressing the transparent flat plate 2a on the upper surface and the lower surface of the microchannel 2, the microchannel 2 having an appropriate height corresponding to the height of the microcarrier 1 is used. .

  In the shearing force generation step, after the upper surface of the micro flow path 2 is covered with the transparent flat plate 2a, the liquid feed port of the syringe pump 3 is inserted into the inflow port of the micro flow path 2 as shown in FIG. A waste liquid reservoir is disposed at the discharge port of the flow path 2, and the culture liquid L in which rhodamine 123 (membrane potential change detection dye) is dissolved is caused to flow into the micro flow path 2 by the syringe pump 3 at a desired flow rate. Then, in the shearing force generation step, the flowed culture medium L flows on both sides of the microcarrier 1, and a shearing force corresponding to the flow rate of the culture liquid L is generated.

  FIG. 2 is a plan view showing the positions of the microcarrier 1, the microchannel 2, and the syringe pump 3 in the shearing force generation step of the shearing force effect evaluation method according to the present embodiment. In addition, the arrow in FIG. 2 has shown the inflow direction F to the microchannel 2 of the culture solution L by the syringe pump 3. FIG.

  The syringe pump 3 is for sending a very small amount of liquid with high accuracy and no pulsating flow. In the syringe pump 3, a CPU (Central Processing Unit) presses the plunger of a syringe (syringe) to a motor at a constant speed based on a preset amount of liquid delivered per unit time, so that a very small amount can be obtained with high accuracy. Deliver liquid.

  Rhodamine 123 is a cationic fluorescent dye having cell permeability, and is taken into the cell membrane of cell C having a negative membrane potential, that is, taken into the cell membrane at a normal location where the membrane potential has not changed due to crushing or stress. . The rhodamine 123 is taken up into the cell membrane of the cell C relatively quickly without causing cytotoxicity.

  Then, after the shearing force generation step is completed, in the cell disruption evaluation step (not shown) of the shearing force effect evaluation method, after the culture medium L has flowed into the microchannel 2 in the shearing force generation step. By attaching the microchannel 2 to the fluorescence microscope and irradiating the fluorescence microscope to the cell C adhering to the microcarrier 1 on the microchannel 2 with blue excitation light having a wavelength of 495 nm, the cells C stained with rhodamine 123 Green fluorescence with a wavelength of 520 nm is emitted.

  When the specimen prepared by the shearing force generation step is observed with a fluorescence microscope, the membrane potential changes due to breakage or stress at a predetermined portion of the cell membrane of the cell C due to the shearing force generated by the culture medium L flowing into the microchannel 2. If this occurs, it can be confirmed that the predetermined portion of the cell membrane is not stained with rhodamine 123.

In the cell disruption evaluation step, it is evaluated whether or not the cell membrane of the cell C is disrupted according to the shearing force depending on whether or not the rhodamine 123 is stained on the cell membrane of the cell C. Note that the shearing force is the flow rate of the culture medium L flowing into the microchannel 2 in the shearing force generation process, the flow rate of the culture medium L, the shape of the microchannel 2, the size of the microchannel 2, and the microcarrier 1 It is calculated based on the shape, the size of the microcarrier 1, the shape of the cell C, the size of the cell C, and the like.
Then, by generating various shearing forces in the shearing force generation step, it is evaluated whether or not the cells C were crushed according to various shearing forces in the cell disruption evaluation step.

  As described above, in the shear force influence evaluation method according to the present embodiment, in the shear force generation step, the microcarrier 1 to which the cells C are attached is arranged in the microchannel 2 and the syringe pump 3 is connected to the microchannel 2. A shearing force is generated by flowing a culture solution L in which rhodamine 123 is dissolved into the cell, and in the cell disruption evaluation step, the cell membrane of cell C is disrupted according to the shearing force depending on whether rhodamine 123 is stained on the cell membrane of cell C By evaluating whether or not it has been done, the influence of the shearing force on the cell C is evaluated.

  As described above, in the shear force effect evaluation method, the cell growth rate is measured at each rotation speed while changing the rotation speed of the stirring device of the animal cell culture tank. Since it is not necessary to measure the ratio, it is possible to evaluate the influence of shear force on cells more easily than in the past.

As mentioned above, although one Embodiment of this invention was described, this invention is not limited to the said embodiment, For example, the following modifications can be considered.
(1) In the above embodiment, the microcarrier 1 to which a plurality of cells C are attached is arranged in the microchannel 2 in the shearing force generation step, but the present invention is not limited to this.
For example, the width of the microcarrier 1 may be widened, and the plurality of microcarriers 1 may be arranged so as to be parallel to the flow of the culture solution L that has flowed into the microchannel 2. Thereby, the influence of the shearing force on more cells C can be evaluated. Further, a plurality of microcarriers 1 may be arranged so as to be in tandem with the flow of the culture solution L flowing into the microchannel 2.

  In addition, one cell C is arranged in the microchannel 2 having a size capable of arranging one cell C, and the cell C is fixed to the microchannel 2 by pressing the transparent flat plate 2a. A shearing force may be generated by flowing L. In addition, a plurality of cells C may be arranged so as to be in parallel with the flow of the culture solution L flowing into the microchannel 2. Further, a plurality of cells C may be arranged so as to be in tandem with the flow of the culture solution L flowing into the microchannel 2.

(2) In the above embodiment, in the shearing force generation step, laminar flow is generated by flowing the culture medium L through the straight microchannel 2, and the laminar flow generates shearing force. It is not limited to.
For example, by providing a turbulent flow generation plate that generates turbulent flow in the micro flow path 2, turbulent flow may be generated in the micro flow path 2, and shear force may be generated by the laminar flow. Moreover, the microchannel 2 may be configured by a curve as well as a straight line.

(3) In the above embodiment, in the shearing force generation step, the culture solution L is flowed into the microchannel 2, but the present invention is not limited to this.
For example, instead of the culture solution L, water may be allowed to flow through the microchannel 2. That is, any liquid that does not affect the character of the cell C may be used. However, from the viewpoint of evaluating the shear force generated by the rotation of the stirring blade of the stirring device of the animal cell culture tank, it is preferable to use the culture solution L stored in the animal cell culture tank.

(4) In the above embodiment, rhodamine 123 is used as the membrane potential change detection dye in the shearing force generation step, but the present invention is not limited to this.
For example, instead of rhodamine 123, trypan blue may be dissolved in the culture solution L as a dye for evaluating the integrity of the membrane.

DESCRIPTION OF SYMBOLS 1 ... Micro carrier, 2 ... Micro flow path, 2a ... Transparent flat plate, 3 ... Syringe pump

Claims (6)

A shearing force generating step for generating a shearing force to the cells by flowing a liquid at a predetermined flow rate into the microchannel in which the cells are arranged;
A cell disruption evaluation step for evaluating whether or not the cells after the liquid has flowed into the microchannel in the shear force generation step are disrupted according to the shear force due to the inflow of the liquid;
A shearing force effect evaluation method comprising:   The shear force influence evaluation method according to claim 1, wherein in the shear force generation step, a cell culture carrier to which cells are attached is disposed in the microchannel.   3. The shear according to claim 2, wherein in the shearing force generation step, the plurality of cell culture carriers are arranged in the microchannel so as to be in parallel with the flow of the liquid that has flowed in. Force impact assessment method.   The shear force influence evaluation according to claim 1, wherein in the shearing force generation step, a plurality of single cells are arranged in the microchannel so as to be parallel to the flow of the liquid that has flowed in. Method. In the shearing force generation step, a membrane potential change detection dye is dissolved in the liquid,
5. The shear force influence evaluation according to claim 1, wherein in the cell disruption evaluation step, cell disruption is evaluated by detecting a change in the potential of the cell membrane using the membrane potential change detection dye. Method. 6. The shear force influence evaluation method according to claim 1, wherein in the shearing force generation step, the liquid is caused to flow into the microchannel by a syringe pump.

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