JP2006194908A - Method for manufacturing blood filtration film, and filtration method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly acquiring serum or plasma, globule, and further leukocyte from a trace amount of whole blood. <P>SOLUTION: Problems are solved by a blood filtering method that uses a blood filtration film made of a film, in which a pore size is 0.5-40 μm and the variation coefficient of the pore size is 20% or smaller, and at least two films having the same average pore size or a different average pore size. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a filtration membrane for removing blood cells from whole blood, and a method for capturing and collecting red blood cells or white blood cells from whole blood using the filtration membrane.

  Measurement of the types and concentrations of constituents in blood such as metabolites, proteins, lipids, electrolytes, enzymes, antigens, and antibodies is usually performed using serum or plasma obtained by centrifuging whole blood as a specimen. However, it takes time and labor to centrifuge, and in particular, when it is desired to process a small number of samples in a hurry or on-site inspection, a centrifuge method that requires a centrifuge using electricity as power is not suitable. Therefore, methods for separating and collecting serum or plasma from whole blood by filtration have been studied.

  A method for obtaining serum or plasma from whole blood by filtration is commercially available as “Plasma Filter PF” manufactured by Fuji Photo Film Co., Ltd., and as known in Japanese Patent Laid-Open No. 2000-81432, 3-6 sheets There is a method of loading whole glass fiber filter paper into a plastic holder and filtering whole blood by suction. However, although this filtration method can filter plasma by about 150 μL, it requires about 3 mL of whole blood, and a trace amount of whole blood cannot be filtered.

  As a method for capturing blood cells from whole blood by filtration to obtain serum or plasma, there is a method using a three-dimensional porous material described in JP-A-10-185910, such as a polysulfone membrane or a cellulose acetate membrane. Are known. However, it is difficult to create a uniform pore size for this membrane.

  On the other hand, in recent years, it has become a popular practice to capture and collect leukocytes from whole blood, collect genes based on these, and use them for gene analysis or genetic diagnosis. Technology to collect is needed.

  A method for removing only white blood cells from whole blood is a polyurethane porous filter that has been put into practical use in "Immunoguard (registered trademark) III-RC" manufactured by Terumo Corporation, sold by Nippon Pole Co., Ltd. A polyester filter that has been put to practical use in "Pole Transfusion Filter PL1J" is known. However, this filter is for removing white blood cells from whole blood for transfusion, and it is difficult to capture white blood cells from a small amount of whole blood and collect white blood cells.

  There is a need for a method of rapidly obtaining serum or plasma from a small amount of whole blood, such as when a very small amount of whole blood can be collected, such as a newborn baby. In addition, in a method for capturing blood cells from whole blood by filtration, a method for fractionating and collecting only white blood cells is required for use in genetic diagnosis and the like.

  By using a thin membrane with a uniform pore size, it is possible to collect only blood cells from a small amount of whole blood and collect serum or plasma, and to capture and collect only white blood cells with a uniform pore size. I found out that I can do it.

  According to the present invention, serum or plasma and blood cells can be separated from a small amount of whole blood, and only leukocytes can be captured and separated.

  As a material of the membrane, a polymer compound that can be dissolved in a water-insoluble solvent such as poly-ε-caprolactone, poly-3-hydroxybutyrate, agarose, poly-2-hydroxyethyl acrylate, polysulfone, etc. can be used. It is preferable to use poly-ε-caprolactone.

  A film having a honeycomb-like structure can be formed using only these materials, but it is preferable to add an amphiphilic material. Examples of the amphiphilic material include amphiphilic polyacrylamide.

  The mixing ratio of the membrane material to the amphiphilic material is preferably used in a weight ratio range of 0: 1 to 1: 0. More preferably, the weight ratio is in the range of 5: 1 to 20: 1.

  The solvent to be cast may be any water-insoluble solvent that can dissolve a polymer compound serving as a film material, such as chloroform, dichloromethane, carbon tetrachloride, and cyclohexane. The polymer concentration at the time of casting may be a concentration capable of forming a polymer film. In order to mass-produce industrially, it is desirable to form a film at a concentration as high as 0.1 wt% or more.

  Since water-insoluble membrane material, such as poly-ε-caprolactone, is dissolved in a water-insoluble solvent, such as chloroform, moisture in the air is condensed by heat of vaporization when the solvent is evaporated by high-humidity air. Then, it gradually grows with the evaporation of the solvent and becomes water droplets having a diameter of about 0.5 to 40 μm. Since the water-insoluble poly-ε-caprolactone cannot be dissolved in the water droplets, a film having pores in the portions can be obtained. For example, since it is cast two-dimensionally on a petri dish, the grown water droplets are regularly arranged like a two-dimensional close-packed structure of spheres, and as a result, a honeycomb structure film is obtained.

  By adjusting the concentration and amount of the liquid to be cast and supplying it to a support layer such as a petri dish, the temperature and humidity of the atmosphere or the air to be blown are controlled to control the evaporation speed and condensation speed of the solvent. Can be controlled. Furthermore, by adding a small amount of a surfactant to suppress the fusion of water droplets and stabilize the film, a more regular honeycomb structure film can be formed.

  The high-humidity air to be blown onto the membrane was mainly those having a relative humidity of 30% and 80%, but any humidity that can condense moisture in the air on the surface of the membrane may be used, depending on the temperature. Relative humidity may be sufficient, and not only air but relatively inert gas such as nitrogen or argon may be used.

  The flow rate of the high-humidity air sprayed on the film may be any flow rate that can condense moisture in the air on the surface of the film and evaporate the solvent used for casting.

The temperature of the atmosphere when high-humidity air is blown may be a temperature at which the solvent used for casting can evaporate, and is 15 to 32 ° C. at the laboratory level and 5 to 80 ° C. at the production level. Is desirable.

  By changing the concentration of the solution to be cast, the amount of the solution, the type of solvent, the relative humidity / temperature / flow rate of the air to be blown, the condensation, water droplet growth, and the evaporation rate of the solvent are controlled. A membrane with a structure can be obtained, and blood components such as platelets with a diameter of about 3 μm, leukocytes with a diameter of about 15 μm, and red blood cells with a large deformability but a diameter of about 7 μm are separated by changing the pore size. Can be used as a filter.

  Moreover, the ability to separate as a filter can be enhanced by laminating membranes having substantially the same pore diameter. Furthermore, several biological materials can be separated or fractionated simultaneously by laminating a plurality of membranes having different pore diameters. For example, by laminating a membrane having a pore size of 5.5 to 8.5 μm and a membrane having a pore size of 3.5 μm or less and supplying whole blood from the membrane side having a large pore size, leukocytes are captured by the membrane having a large pore size, Capturing red blood cells with a small membrane can be achieved simultaneously.

  Furthermore, by setting the pore diameter to the submicron order, it can be expected as a technique for selectively separating and recovering a target biological substance as represented by blood purification such as dialysis.

  In the present invention, the honeycomb-like structure refers to a structure in which a plurality of holes having a substantially constant hole diameter are regularly arranged and such holes penetrate the film. There is no particular limitation on the cross section of the hole, and it may be circular, elliptical, hexagonal, rectangular, square or the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

Example 1 Preparation of a honeycomb-like structure film (1)
Chloroform solution (0.1-2 wt as the polymer concentration) in which poly-ε-caprolactone (compound 1) having an average molecular weight of 70,000 to 100,000 and amphiphilic polyacrylamide (compound 2) are mixed at a weight ratio of 10: 1. %) Is cast on a petri dish with a diameter of 10 cm, high-humidity air with a relative humidity of 30 to 80% is blown at a flow rate of 1 to 20 L / min, and the chloroform solvent is evaporated, thereby having flexibility and elasticity. As a result, a film having a honeycomb-like structure with high mechanical strength was obtained.

  When the structure of the film prepared under the various conditions described above was observed with an optical microscope and a scanning electron microscope, it was a film having a honeycomb-like structure with a pore diameter of 0.5 to 40 μm. It was a structure penetrating through. The hole diameter was a clean circle over the entire cast surface, and the size was almost the same. When the hole diameter was measured from the photograph taken and the distribution of the hole diameter was determined, it was found that the coefficient of variation was CV 10% or less. In addition, it was found that diffracted light of the 10th order or higher was observed over the entire surface of the cast film by light scattering evaluation using a laser, and thus it was found that a highly regular porous film could be produced.

Example 2 Production of a honeycomb-like film (2)
For the substrate to be cast, an amphiphilic polyacrylamide composed of compound 2 is used as a material to be a film, the solvent to be cast is changed to chloroform, benzene, toluene, xylene, the solution concentration is 1.0 g / L, the cast amount is 30 μL. Using glass, the solvent dependence of a film that can be produced when the flow rate of high-humidity air was 0.09 L / min, the relative humidity was 85%, and the temperature was 20 ° C. was evaluated. The form observation result at this time is shown in FIG.
In FIG. 1, the pore diameter is 0.5 μm to 10 μm from the top.

Example 3 Production of a honeycomb-like structure film (3)
A poly-ε-caprolactone composed of Compound 1 and an amphiphilic polyacrylamide composed of Compound 2 are used in a weight ratio of 10: 1 as a substance to be a film, chloroform is used as a solvent, and the solution concentration is 10.0 g / L. When the casting amount is changed to 5, 10, 20 mL, glass is used for the substrate to be cast, the flow rate of high humidity air is 2.0 L / min, the relative humidity is 30%, and the temperature is 20 ° C. The cast amount dependence of the film that can be produced was evaluated. The form observation result at this time is shown in FIG.
In FIG. 2, the hole diameter is 8 μm to 35 μm from the top.

Example 4 Preparation of a honeycomb-like structure film (4)
A poly-ε-caprolactone composed of Compound 1 and an amphiphilic polyacrylamide composed of Compound 2 are used in a weight ratio of 10: 1 as a substance to be a film, chloroform is used as a solvent, and the solution concentration is 1, 5, 10 When the cast amount is 10 mL, hydrogel is used for the substrate to be cast, the flow rate of high-humidity air is 2.0 L / min, the relative humidity is 30%, and the temperature is 20 ° C. The solution concentration dependence of the membrane that can be produced was evaluated. The form observation result at this time is shown in FIG.
In FIG. 3, the pore diameter is a minimum of 15 μm and a maximum of 25 μm.

Example 5 Preparation of a honeycomb-like structure film (5)
Poly-ε-caprolactone composed of compound 1 is used as a material to be a film, chloroform is used as a solvent, a solution concentration is 1.0 g / L, a cast amount is 5 mL, and a substrate to be cast is agarose gel, glass, mica, In place of PHEMA, the dependence of the high humidity air flow rate at 2.0 L / min, the relative humidity at 30%, and the temperature produced at 20 ° C. was evaluated on the cast substrate dependence. The form observation result at this time is shown in FIG.
In FIG. 4, the pore diameter is a minimum of 7 μm and a maximum of 14 μm.

  In FIGS. 1 to 4, all the bars have a length of 20 μm.

Example 6 Evaluation of Filtration Performance (1)
Prior to evaluating the blood filtration performance of the prepared membrane having a honeycomb-like structure, a passage experiment of polystyrene particles having a known particle diameter was conducted, and the kind and passage rate of passing particles were examined by changing the pore diameter. The results are shown in Table 1. When a membrane having a pore diameter of 5.5 to 8.5 μm was used, it was revealed for the first time that particles having a diameter of 3 μm pass but particles having a diameter of 10 μm or more did not pass at all.

Example 7 Evaluation of Filtration Performance (2)
Using the prepared honeycomb-like membrane, an experiment for capturing leukocytes in human whole blood was performed. Human whole blood collected with a heparin blood collection tube was filtered using membranes with pore sizes of 5.2 μm and 9.8 μm, and the number of white blood cells was measured by casting the blood on a hemocytometer. Before the filtration, 4800 / μL It was found that the white blood cell count was 0 / μL after filtration (Table 2). Moreover, when the color of the supernatant obtained by centrifuging the filtered filtrate at 3000 rpm for 10 minutes was visually confirmed, hemolysis was not confirmed.

Example 8 Evaluation of Filtration Performance (3)
Using the prepared honeycomb-like membrane, an experiment for capturing red blood cells in human whole blood was performed. Using a membrane having a pore diameter of 3.5 to 5.5 μm, three sheets each having a diameter of 5 mm were punched out and laminated, and left on the nitrocellulose membrane. Human whole blood with a hematocrit value of 40% collected with a heparin blood collection tube was diluted with plasma obtained by centrifuging the same whole blood to obtain a whole blood prepared to a hematocrit value of 2.5%, and then laminated. When 5 μL was spotted on the membrane and allowed to stand for 10 seconds, the laminated membrane was lifted, and it was evaluated whether the red color of red blood cells remained in the plasma that was filtered and transferred to the nitrocellulose membrane. It was confirmed that red blood cells were not captured with a membrane having a pore size of 3.5 μm, and that red blood cells were captured (Table 3).

This shows the solvent dependence of the pore diameter when forming a honeycomb-like film. It shows the dependence of the hole diameter on the cast amount when forming a honeycomb-like film. This shows the concentration dependence of the pore diameter when forming a honeycomb-like film. This shows the dependence of the hole diameter on the substrate to be cast when forming a honeycomb-like film.

Claims (4)

A method for producing a membrane having a honeycomb-like structure, wherein benzene, toluene or xylene is used as a solvent, the pore size is 2 to 10 μm, and the variation in pore size is a variation coefficient of 20% or less. The manufacturing method according to claim 1, wherein the atmosphere during film formation is 80% to 100% relative humidity. 3. The production method according to claim 1, wherein an amphiphilic polyacrylamide is used as a base material of the membrane. A blood filtration method for capturing red blood cells in blood by using three or more layers of the membrane according to claim 1 in a laminated manner.

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