JP2006333850A - Base material for separating biological particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a base material for separating biological particles, capable of conducting separation and/or removal of the biological particles from a suspension containing the biological particles by a simple and safe operation conducted at a low cost in a short time, and to provide a method for producing the same. <P>SOLUTION: This method for producing the base material for separating the biological particles comprises sequentially conducting processes (1) to (3) as follows: (1) a process for preparing a water-insoluble surface of the base material having negative electric charge or positive electric charge; (2) a process for soaking the base material prepared in the process (1) in an aqueous solution of a polymer having electric charge opposite to the surface of the base material prepared in the process (1); and (3) a process for washing the base material with an aqueous solution not containing a polymer having electric charge. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a bioparticle separation substrate or a bioparticle separation filter using the same. The bioparticle separation filter using the bioparticle separation substrate produced according to the present invention can be used for a basic scientific experimental instrument or a medical instrument in clinical medicine.

  In recent years, so-called regenerative medicine that actively uses cells to treat lesions and / or defects in living organ tissues has attracted a great deal of attention, and research and development has been actively conducted in various countries around the world (for example, Non-patent document 1). Usually, unnecessary contaminating cells are often mixed in a site where these cells exist, and it is necessary to remove the contaminating cells and concentrate and separate useful cells. Usually, these cells are separated by a specific gravity centrifugation method using Ficoll-Hypaque or the like, or a method using a monoclonal antibody immobilized on magnetic particles or the like using an antigen-antibody reaction. The latter also has problems such as higher costs and the need to create a new antibody if the antibody is not commercially available. Furthermore, although these cell separation methods are usually carried out in a clean bench, they are difficult to sterilize due to the operation of a completely open system, and thus have a safety problem for clinical application to humans. Further, the method using a monoclonal antibody usually has a safety problem due to the fact that the monoclonal antibody is a non-human animal protein.

  On the other hand, in recent years, technological development for overcoming these problems has become common. For example, Patent Document 1 discloses a stem cell separation material obtained by immobilizing an antibody against an antigen on the membrane surface of a stem cell on a carrier in which a carboxyl group and a hydrophilic chain are introduced into a nonwoven fabric composed of fibers. Certainly, if the stem cell separation material of this technology is packed in a container and a filter is made and used for cell separation, the problem of complicated operation is solved, but the problem of high cost and safety in terms of using antibodies Is unresolved. Patent Document 2 discloses a cell separation material for separating and recovering T cells from lymphocytes, characterized by comprising a polyvinyl acetal resin having continuous pores having an average pore diameter of 10 to 1000 μm. This technology is epoch-making in that it uses a synthetic polymer without using an antibody that has a safety problem, and it can be said that it is a very excellent technology in terms of T lymphocyte separation. This technique can only be applied to T lymphocytes and is not versatile for other cell separations.

  By the way, in the field of transfusion medicine, cell separation technology using a filter has been developed for relatively simple and safe operation. For example, Patent Document 3 discloses a unit derived from a hydrophobic polymerizable monomer and a basic property. A leukocyte removal filter material coating polymer comprising a unit derived from a polymerizable monomer containing a nitrogen-containing portion and a unit derived from a polymerizable monomer containing a protic neutral hydrophilic portion is disclosed. The invention is superior in that it uses a synthetic polymer without using an antibody that is still problematic in terms of safety, but it refers only to the removal of leukocytes, and refers to a production method that is widely accepted by cells in general. Therefore, the technical idea is completely different from the present invention. In addition, the biggest problem of the technology according to the invention of a novel functional polymer (in this case, for leukocyte removal) is that for the purpose of investigation, it is necessary to synthesize a variety of new polymers with different copolymer compositions for coating. Yes, this is very problematic in terms of cost and effort.

  Furthermore, Patent Document 4 describes a method for separating components such as blood and body fluid by an electrostatic charge pattern, in which the following is described: “The cell is a mucopolysaccharide protein complex on a cell membrane, Alternatively, it is known that it has a negative charge as a whole based on sialic acid at the end of the sugar chain, and has −30 to −40 mV in the epithelial cell system, and the charged state of the substrate is said to affect its adhesion and proliferation. (2 pages 15-20 lines). However, the present invention does not refer to a method for producing a cell separation material, but claims `` (1) a photoreceptor having a photoconductive layer formed on a support with a conductive layer interposed therebetween, '' A charge holding medium in which an insulating layer is formed on a support with a conductive layer interposed therebetween is arranged oppositely, and image exposure is performed from the photoreceptor side while applying a voltage between the photosensitive layer and the conductive layer of the charge holding medium. A method for separating components such as blood and body fluids by means of an electrostatic charge pattern, wherein charge separation is performed by accumulating charges in a holding medium in an image-like manner and growing cells according to the charge pattern of the charge holding medium. As can be seen from the description, the present invention relates to a novel cell separation method, and there is no description of a method for producing a cell separation substrate as in the present invention, and the technical idea is completely different.

On the other hand, polyelectrolyte complexes formed by reacting a positively charged polyelectrolyte with a negatively charged polyelectrolyte have been used for a long time in the fields of medicine and biology. An immunoisolation membrane prepared by reacting poly-L-lysine, which is a polyelectrolyte having a positive charge, with alginic acid, which is a polyelectrolyte having a negative charge, is disclosed for artificial islets for the treatment of diabetes. This technique is for microencapsulating cells in order to shield transplanted cells (in this case, islets) from immune reactions and has nothing to do with the cell separation substrate. Patent Document 5 discloses a cell culture substrate prepared by reacting chitosan as a polyelectrolyte having a positive charge with a cellulose derivative as a polyelectrolyte having a negative charge. And in the specification, there is the following description (here PEC is an abbreviation for polyelectrolyte complex) "In the present invention, by changing the combination and blending ratio of chitosan and cellulose derivative, The charge balance of the produced PEC can be easily changed and adjusted, that is, by using the PEC having various charge balances, the surface charge of the cell culture substrate can be adjusted, and the surface characteristics can be adjusted according to the use conditions. It becomes possible to select appropriately. This is a concept that is partially incorporated in the present invention, but in the present invention, only a reference to a cell culture substrate is found, and no reference to an application to a cell separation substrate is found.

JP 08-033708 Japanese Patent Laid-Open No. 02-227070 WO2003 / 011924 (Reprinted 03/011924) Publication Japanese Patent Laid-Open No. 02-245182 Japanese Unexamined Patent Publication No. 06-277038 SMC Publishing Co., Ltd., “Cutting-edge of Regenerative Medical Engineering”, 2002 Chemical Dojin, "Chemical Frontier 3 Regenerative Medical Engineering", 2001

  An object of the present invention is to provide a simple and low-cost production method of a biological particle separation substrate for separating biological particles such as simple and safe cells, and a biological particle separation filter using the same.

  The present inventors have intensively studied to solve the above problems. First, it was determined in advance that an artificial object would be used without using an antibody having a safety problem, and among these artificial objects, it was next determined to use a material that recognizes the charge difference on the surface of a biological particle. Here, the present inventors have studied a combination of charged functional groups, designed and synthesized a novel polymer, and used a conventional method of creating a biological particle separation base material in a simple manufacturing method. We thought that it was not possible to reach it and further studied. As a result, the use of polyelectrolyte complex, which is an unprecedented technology in the base material for biological particle separation, has been achieved. In fact, the same method has various surface charges, and various biological particle separation behaviors according to the charge. The present invention has been completed by successfully producing a substrate for separating biological particles having

That is, the present invention
(1) A method for producing a biological particle separating base material, comprising sequentially performing the following steps. (A) a step of preparing a water-insoluble substrate surface having a negative charge or a positive charge, (b) the aqueous solution of the polymer having a charge opposite to that of the substrate surface prepared in (a) above (a ) Step of immersing the substrate prepared in (c), (c) washing the substrate with an aqueous solution not containing a charged polymer.
(2) Further, after the completion of the step (c), (d) the polymer having the opposite charge to the polymer used in the step (b) is immersed in an aqueous solution of the polymer (e) having the charge. The method for producing a bioparticle separation substrate according to (1), wherein the substrate is washed with an aqueous solution not containing the aqueous solution
(3) The method for producing a bioparticle separation substrate according to (2), wherein the steps (d) and (e) are repeated a plurality of times.
(4) The method for producing a bioparticle separation substrate according to (1), wherein the substrate surface prepared in (a) is negatively charged.
(5) The method for producing a bioparticle separation substrate according to (2), wherein the substrate surface prepared in (a) has a positive charge.
(6) A bioparticle separation substrate produced by the method according to any one of (1) to (4), wherein a polymer having a positive charge forms a polyion complex on the surface of the substrate. bound and, and, and wherein the surface charge when measuring surface charge by the colloid titration method is -200μ equivalent / m ² ~ + 100μ equivalents / m ^2, the biological particle separation base material.
(7), wherein the surface charge when measuring the surface charge is -30μ equivalent / m ² ~ + 10 [mu] eq / m ² by the colloid titration method, the biological particle separation substrate according to (6) .
(8) A biological particle separation filter in which the biological particle separation substrate according to (6) or (7) is housed in a container having a liquid inlet and a liquid outlet.
(9) A biological particle separation method, wherein the biological particle-containing liquid is filtered with the biological particle separation filter according to (8).

  ADVANTAGE OF THE INVENTION According to this invention, the simple and low-cost manufacturing method of the base material for biological particle separation for performing simple and safe biological particle separation can be provided.

The present invention will be specifically described below.
The biological particle separation substrate referred to in the present invention is a material used for separation of biological particles, but the biological particles referred to here are human and animal cells (including bacteria and fungi) and viruses. To tell. Moreover, not only living cells and viruses, but also dead cells, inactivated viruses, and fragments thereof (for example, DNA as a cell fragment and lipopolysaccharide as a bacterial fragment) are included. Examples of cells referred to in the present invention include the following, but are not limited thereto. Nucleated cells, mononuclear cells, multinucleated cells, leukocytes, erythrocytes, granulocytes, neutrophils, basophils, eosinophils, myelospheres, erythroblasts, lymphocytes, T lymphocytes, helper T lymphocytes, cells Injury T lymphocytes, suppressor T lymphocytes, B lymphocytes, NK cells, NKT cells, monocytes, macrophages, dendritic cells, osteoclasts, osteoblasts, bone cells, hematopoietic stem / progenitor cells (hereinafter hematopoietic cells) Abbreviated stem cells), fibroblasts, chondroblasts, mesenchymal stem / progenitor cells (stroma stem cells or mesenchymal stem cells), megakaryocytes, platelets, adipocytes, liver parenchymal cells, liver non-parenchymal cells, endothelial cells, Epithelial cells, smooth muscle cells, myoblasts, endocrine cells, exocrine cells, pancreatic β cells, pancreatic α cells, pancreatic δ cells, pancreatic PP cells, renal mesangial cells, renal tubular cells, renal glomerular cells, nerve cells, Adult stem cells (SP cells, MAPC, etc.), ES cells, various tumor cells Etc. Also included are not only single cells, but also islets that are aggregates of cells, such as aggregates of endocrine cells.

  The separation of biological particles as used in the present invention refers to separation / recovery or removal of desired biological particles from a biological particle-containing liquid containing at least one kind of biological particles. The desired biological particle may be one type or plural types. Examples of biological particle-containing liquids are classified according to whether they are derived from living organisms. Examples of biological sources include bone marrow, umbilical cord blood (including not only cord blood but also collected from placental blood vessels), peripheral blood (including blood collected by administration of hematopoietic factors such as granulocyte colony-stimulating factor) Those subjected to any treatment such as centrifugation, culture (differentiation induction, amplification), freeze-thawing, gene transfer, etc., or various organs, muscles, blood vessels such as pancreas, liver, kidney, heart, stomach, intestine, spleen In addition, there are those obtained by resuspending cells extracted from various tissues such as bone and skin by an enzyme digestion method or the like in some liquid, and those obtained by subjecting them to any of the aforementioned treatments.

  Examples of those that are not derived from living organisms include solutions containing useful substances produced by cells (especially bacteria, fungi, and viruses are separated) when, for example, pharmaceuticals and foods are produced using useful substances produced by cells. The subject is often this, in which case it is not clear whether it contains bioparticles such as bacteria, fungi or viruses, but here it is treated as a bioparticle-containing fluid). Therefore, the material used for the separation of the biological particles, that is, the biological particle separation substrate referred to in the present invention refers to a material that comes into contact with the biological particles contained in the biological particle-containing liquid and realizes the separation operation. In addition, as a form of the biological particle separation operation, the desired biological particles are adsorbed, the other biological particles are allowed to pass through without being adsorbed, and then the adsorbed desired biological particles are recovered by some method. In addition, both of those which adsorb unnecessary biological particles and collect other biological particles (including desired biological particles) are collected.

  In the production method according to the present invention, first, a water-insoluble substrate surface having a negative charge or a positive charge is prepared, and as this adjustment method, a method of creating a substrate with a material itself having a negative charge or a positive charge or The material constituting the raw material substrate itself does not have negative charge or positive charge, but there is a method of introducing negative charge or positive charge by some surface treatment. As an example of the former, in the case of a negative charge, a polymer polymerized from a monomer containing a carboxyl group, a sulfonic acid group, a sulfate ester group or a phosphate group, a natural polymer containing these groups, a positive charge If present, polymers polymerized from monomers containing primary amino groups, secondary amino groups, tertiary amino groups, imino groups, quaternary ammonium groups, quaternary phosphonium groups, and natural polymers containing these groups Is given. Examples of the latter include graft polymerization, chemical treatment with a silane coupling agent, hydrogen sulfide gas or hydrogen chloride gas, or plasma treatment on a polymer having no negative charge or positive charge, such as polyethylene. And surface modification to negative charge or positive charge by physical treatment by corona treatment. Next, the substrate is immersed in an aqueous solution of a polymer having a charge opposite to the surface of the substrate prepared here.

  For example, if the first prepared substrate surface has a negative charge, the polymer used here has a positive charge, and the first prepared substrate surface has a positive charge. If so, the polymer used here is negatively charged. Examples of polymers having negative charges include polymers polymerized from monomers containing carboxyl groups, sulfonic acid groups, sulfate ester groups, and phosphoric acid groups, and natural polymers containing these groups.

  Examples of positively charged polymers include polymers polymerized from monomers containing primary amino groups, secondary amino groups, tertiary amino groups, imino groups, quaternary ammonium groups, and quaternary phosphonium groups. Examples include natural polymers containing these groups. These polymers are made into an aqueous solution and the above-mentioned base material is immersed therein. The aqueous solution here means a solution containing at least 50% by weight of water as a solvent. Further, an organic solvent may be included as long as a uniform solution can be obtained. Furthermore, if necessary, a water-soluble inorganic substance or organic substance may be added. For example, since chitosan is insoluble in pure water, an inorganic acid such as hydrochloric acid, sulfuric acid or nitric acid or an organic acid such as citric acid, acetic acid or malic acid is added to water and dissolved to obtain an aqueous solution. In the production method according to the present invention, the substrate is then washed with an aqueous solution not containing a charged polymer. Here, pure water is generally used as an aqueous solution that does not include a charged polymer, but a solution containing a water-soluble substance such as a nonionic surfactant may be used as necessary for cleaning. .

  In the production method according to the present invention, if necessary, further dipping steps may be repeated thereafter. For example, a substrate having a negatively charged surface is first prepared, and the substrate is immersed in an aqueous solution of a polymer having a positive charge opposite to the surface of the substrate, so that the aqueous solution does not contain a charged polymer. After washing the substrate with the solution, the substrate is immersed in an aqueous solution of a negatively charged polymer opposite to the immersed polymer, and the substrate is washed with an aqueous solution that does not contain a charged polymer. is there. These may be repeated a plurality of times as necessary. As an advantage of repeating a plurality of times, it is conceivable that the film thickness becomes thicker and stronger due to the lamination, but on the other hand, it becomes complicated by repeating the operation, so it is appropriately selected.

  Next, the bioparticle separation substrate according to the present invention will be described. In the production method of the present invention, both a biological particle separation substrate having a negative charge on the outermost surface and a biological particle separation substrate having a positive charge on the outermost surface can be easily produced. It is considered that the combination is more preferably a polymer having a positive charge. In this case, the polymer having a positive charge forms a so-called polyion complex, and the surface of the substrate (the first substrate having a negative charge if not laminated, the polymer having the most negative charge in the case of multiple lamination) The polyion complex referred to here is a water-insoluble substance formed by binding a negatively charged polymer and a positively charged polymer by ionic bonds. . In the present invention, polyion complex (PIC) and polyelectrolyte complex (PEC) are used as the same meaning.

Preferably the surface charge is -200μ equivalent / m ² ~ + 100 microns equivalent / m ² when the surface charge of the biological particle separation base material measured by the colloid titration method according to the invention, more preferably -30μ equivalent / m ² ˜ + 10 μeq / m ² . The colloidal titration method used here is a titration method of a polymer electrolyte created by Terayama in 1946. The principle is based on the fact that a polymer having a positive charge and a polymer having a negative charge are ionically associated to form a complex (the aforementioned polyion complex) instantaneously. To detect the end point of titration, polydidimethylammonium chloride is often used as a standard substance for a positively charged polymer, and potassium polyvinyl sulfate is often used as a standard substance for a negatively charged polymer. For example, when a 1/400 N polydidimethylammonium chloride solution is titrated with a 1/400 N potassium polyvinyl sulfate standard solution, it gradually becomes cloudy, and at the end of the titration, toluidine blue added as an indicator changes from blue to reddish purple. Discolor. TB is a positively charged dye and maintains its original blue color as long as it is a positively charged colloid. However, when even one drop of potassium potassium sulfate is excessive at the end of titration, it is adsorbed by the negatively charged colloid and sensitive to reddish purple. The color changes. Based on this principle, the charge amount is measured.
In addition to the colloid titration method, there are methods using a zeta potential measuring device and an electrophoresis device to define the charge amount of the biological particle separation substrate according to the present invention. The colloid titration method is most preferable because it can be measured regardless of the properties of the surface of the material such as nonwoven fabric or particles.

  Having a positive charge means that an animal cell can survive in an aqueous solution having an organic solvent content of less than 50% and in a normal atmosphere with a humidity of 10% RH to 100% RH, that is, a pH of 5 to 10, more preferably A state in which the charge of the polymer is positive when the pH is in the range of 6.5 to 8.5. In the aqueous solution, inorganic salts such as sodium chloride may be present. Having negative charge means a state in which the polymer is negatively charged under the above-mentioned conditions.

In this invention, the calculation method of the theoretical effective surface area of the nonwoven fabric surface is a surface area when the fiber which comprises a nonwoven fabric is assumed to be a cylinder, and is shown by the following formula | equation [Formula 1].

Effective surface area (m ² / g) = 4 / Specific gravity of nonwoven material (d) × Average fiber diameter of nonwoven fabric (μm) [Formula 1]

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

(Preparation of positively charged polymer)
N, N, N ′, N-tetramethylethylenediamine (2.32 g, 20 mmol) and α-α′-dichloro-p-xylene (3.50 g, 20 mmol) were dissolved in 100 ml of DMSO and stirred at room temperature for 12 hours. The resulting white precipitate was filtered off with a glass filter, washed with acetone, and then dried under reduced pressure to obtain poly [(dimethylimino) ethylene (dimethylimino) methylene-1,4-phenylenemethylenedichloride] (quaternary ammonium group-containing polymer). (Hereinafter referred to as polymer 1) 4.37 g was obtained. The molecular weight of this polymer 1 was Mw = 12,500 as determined from the amount of proton at the polymerization terminal by NMR.

  Dimethylaminoethyl methacrylate: DMAEMA (6.29 g, 40 mmol) was dissolved in 20 g of ethanol, azobisisobutyronitrile: AIBN (33 mg, 0.20 mmol) was added, and the inside of the container was purged with argon while dissolving under ice cooling. The container was sealed and reacted at 70 ° C. for 8 hours. The reaction solution was added to a large amount of n-hexane, and the resulting precipitate was dried under reduced pressure to obtain 4.70 g of polydimethylaminoethyl methacrylate (hereinafter referred to as polymer 2) which is a tertiary amino group-containing homopolymer. The molecular weight of this polymer 2 was Mw = 88,000 in terms of PMMA.

  In the same manner as above, dimethylaminoethyl methacrylate: DMAEMA (6.29 g, 40 mmol) is dissolved in 20 g of ethanol, azobisisobutyronitrile: AIBN (2 mg, 0.01 mmol) is added, and the mixture is dissolved under ice cooling. Argon substitution was performed. The container was sealed and reacted at 70 ° C. for 8 hours. The reaction solution was added to a large amount of n-hexane and the resulting precipitate was dried under reduced pressure to obtain 4.70 g of polydimethylaminoethyl methacrylate (hereinafter referred to as polymer 3) which is a tertiary amino group-containing homopolymer. The molecular weight of this polymer 3 was Mw = 500,000 in terms of PMMA.

(Preparation of biological particle separation substrate)
Polypropylene nonwoven fabric P03040 (manufactured by Asahi Kasei Fibers Co., Ltd., average fiber type 20 μm) was introduced with sulfonic acid groups on the surface by SO ₃ gas using a sulfonation apparatus described in JP-A-3-269160. The sulfonic acid content at this time was 0.54% as the total sulfur (S) content by a fluorescent X-ray analyzer. This sulfonated polypropylene non-woven fabric was immersed in 1N NaOH solution for 30 minutes to make the surface sulfonic acid group Na type. After washing 6 times with deionized water, after confirming that the pH of the washing solution is neutral, polymer 1 is dissolved in deionized water so that the final concentration is 0.2 wt%. It was immersed in the aqueous solution adjusted to 1 and reacted at room temperature for 1 hour. After washing with deionized water four times, the nonwoven fabric was freeze-dried to produce a bioparticle separation base material on which the positively charged polymer 1 was immobilized on the surface by a polyion complex.

(Quantification of substrate surface charge by colloid titration method)
The biological particle separation base material produced as in Example 2 was punched out to 12 mmφ, placed in a transparent test tube, and after adding 1 ml of standard polycation liquid polydiallyldimethylammonium chloride: PDAC (Wako Pure Chemical Industries) adjusted to 1/4000 N, 30 μl of toluidine blue indicator solution (Wako Pure Chemical Industries) was added. Titration was performed with a standard polyanion solution adjusted to 1/4000 N: potassium polyvinylsulfate: PVSK (Wako Pure Chemical Industries, Ltd.). The end point of the titration was that the toluidine blue liquid color changed from blue to reddish purple. By calculating the surface charge of the bioparticle separation substrate 12mmφ of the present invention from the difference between the titration amount of the PVSK solution and the pre-added PDAC solution, and further normalizing it per theoretical effective surface area of the nonwoven fabric surface, The surface charge was quantified. The surface charge of the sulfonated polypropylene nonwoven fabric of Example 2 is −51.18 μequivalent / m ² , and the biologically charged polymer 1 having the positive charge prepared in Example 2 immobilized on the surface by a polyion complex is used. The surface charge of the substrate was -13.72 μeq / m ² .

(Production of base material-filled filter for bioparticle separation)
A biological particle separation base material prepared as in Example 2 is punched out to a diameter of 8.5 mm and filled with 10 pieces in a 2.5 ml syringe cylinder (manufactured by Terumo), thereby having a liquid inlet and a liquid outlet. The base material for biological particle separation was accommodated in the container. By the above operation, a substrate-filled filter for separating biological particles was produced.

(Fibroblast removal experiment)
NIH-3T3 (ATCC: CRL-1658; mouse cell line fibroblast), HEPES-NaOH (20 mM) (Sigma), penicillin-streptomycin (GIBCO-BRL, USA), and 10% FBS (GIBCO Subcultured in DMEM medium (pH 7.4) (GIBCO BRL 11995-065) containing -BRL. At the time of the experiment, both cells on the tissue culture dish were washed with physiological phosphate buffered saline pH = 7.4 (hereinafter referred to as PBS (−)), and then kept at 37 ° C. 0.05% trypsin-0.53 mM EDTA-4Na A solution (hereinafter referred to as trypsin solution; manufactured by GIBCO-BRL) was added and treated with trypsin digestion conditions (2 min; 37 ° C.) as a proteolytic enzyme. Thereafter, serum (FBS) -containing medium was added to stop the action of trypsin, and the cells on the bottom surface of the tissue culture dish were detached and collected with a water flow using a pipette (hereinafter referred to as pipetting).

To the filter prepared above, NIH-3T3 cells, which are fibroblasts, were suspended in DMEM medium containing 10% FBS, 2.5 ml each of the cell suspension (5 × 10E5 cells / ml) passed by natural dropping, The filtered solution was collected. The time required for this filter was less than 1 minute, and it was possible to perform processing in a short time and a simple method of dropping by gravity.
The number of cells before and after passing through the filter was calculated with CyQUANT (Molecular Probes) according to the instructions. The cell removal rate calculated on the basis of this was 73.2% for a bioparticle-separating base material-filled filter in which polymer 1 having a positive charge was immobilized on the surface by a polyion complex. For comparison, 24.7% was obtained when a similar experiment was performed using a filter filled with a sulfonated polypropylene nonwoven fabric in which the polymer 1 was not immobilized on the surface. Thus, the NIH-3T3 cells, which are fibroblasts, can be effectively removed by the substrate-filled filter for separating bioparticles in which the positively charged polymer 1 of the present invention is immobilized on the surface by a polyion complex. It became clear. Also, when considered in conjunction with the quantitative value of the surface charge of Example 3, and wherein the surface charge when measuring surface charge by the colloid titration method is -30μ equivalent / m ² ~ + 10 [mu] eq / m ² It has been found that a biological particle separating substrate is particularly preferable.

(Non-fibroblast removal experiment)
RINr cells (rat pancreatic islet tumor-derived insulinoma cells; reference: Proceedings National Academy of Science, USA 77 (6) p3519-3523), HEPES-NaOH (20 mM) (Sigma), penicillin-streptomycin (GIBCO- BRL) and 5% FBS (GIBCO-BRL) RPMI1640 medium (pH 7.4) (SIGMA R-8758). During the experiment, the cells on the tissue culture dish were washed with PBS (−), treated with a trypsin solution kept at 37 ° C. for 2 minutes, and then the serum (FBS) -containing medium was added to stop the action of trypsin. Pipetting was performed to separate and collect the cells on the bottom of the tissue culture dish.

  Cell suspension (5 × 10E5 cells / ml), in which RINr cells are suspended in DMEM medium containing 10% FBS, is passed through the substrate-filled filter for separating biological particles prepared in Example 4 by natural dropping. Then, the same operation as in Example 4 was performed except that the filtered solution was recovered.

  The cell removal rate calculated based on this was 29.6% for the bioparticle-separating substrate-filled filter in which the positively charged polymer 1 was immobilized on the surface by a polyion complex. In addition, in the case where the same experiment was performed using a filter filled with a sulfonated polypropylene non-woven fabric in which the polymer 1 was not immobilized on the surface, it was 17.9%. In this way, the substrate-filled filter for separating biological particles in which the positively charged polymer 1 of the present invention is immobilized on the surface by a polyion complex substantially eliminates RINr cells that are not fibroblasts but are insulinoma cells. do not do. This indicates that the separation selectivity between fibroblasts and insulinoma cells is recognized together with the results of Example 4, and the biological particle separation substrate of the present invention is a cell suspension containing two types of cells. It proved useful for the separation of biological particles from the liquid.

(Preparation of biological particle separation substrate)
As in Example 2, polypropylene nonwoven fabric P03040 (manufactured by Asahi Kasei Fibers Co., Ltd., average fiber system: 20 μm) was introduced with sulfonic acid groups on the surface by SO ₃ gas using a sulfonation apparatus described in JP-A-3-269160. This sulfonated polypropylene non-woven fabric was immersed in 1N NaOH solution for 30 minutes to make the surface sulfonic acid group Na type. After confirming that the pH of the cleaning solution is neutral after washing 6 times with deionized water, the tertiary amino group-containing homopolymer prepared in Example 1 was used so that the final concentration was 0.2 wt%. A certain polymer 2 or 3 was dissolved in deionized water and immersed in an aqueous solution adjusted between pH = 6 and 8, and reacted at room temperature for 1 hour. After washing with deionized water four times, the nonwoven fabric was freeze-dried to produce a bioparticle separation base material on which the positively charged polymer 2 or polymer 3 was immobilized on the surface by a polyion complex.

(Quantification of substrate surface charge by colloid titration method)
The biological particle separation base material produced in Example 6 was punched out to 12 mmφ, placed in a transparent test tube, and the substrate surface charge was quantified by the colloid titration method in the same manner as in Example 3. The surface charge of the sulfonated polypropylene non-woven fabric prepared in Example 6 is −34.98 μeq / m ² , and the biological particle separation in which the positively charged polymer 2 prepared in Example 6 is immobilized on the surface by a polyion complex. The surface charge of the base material for bioparticles is −4.58 μequivalent / m ² , and the surface charge of the base material for biological particle separation in which the polymer 3 having positive charge is immobilized on the surface by the polyion complex is −8.02 μequivalent / m ² .

(Production of base material-filled filter for bioparticle separation)
In the same manner as in Example 4, the biological particle separation base material produced in Example 6 was punched out to a diameter of 8.5 mm, and 10 sheets were filled in a 2.5 ml syringe cylinder (manufactured by Terumo) to fill the liquid inlet. And a biological particle separation base material in a container having a liquid outlet. By the above operation, a substrate-filled filter for separating biological particles was produced.

(Fibroblast removal experiment)
As in Example 4, 2.5 ml each of cell suspensions (5 × 10E5 cells / ml) in which NIH-3T3 cells, which are fibroblasts, were suspended in DMEM medium containing 10% FBS were added to the filter prepared above. The solution was passed by natural dropping, and the filtered solution was recovered. The number of cells before and after passing through the filter was calculated with CyQUANT (Molecular Probes) according to the instructions. The cell removal rate calculated based on this was 98.9% for the biological particle separation base material-filled filter in which the positively charged polymer 2 was immobilized on the surface by a polyion complex, and the positively charged polymer 3 was 98.6% of a substrate-filled filter for separating biological particles immobilized on the surface by a polyion complex. For comparison, 19.7% was obtained when a similar experiment was performed using a filter filled with a sulfonated polypropylene nonwoven fabric in which neither the polymer 2 nor the polymer 3 was immobilized on the surface. Thus, NIH-3T3 cells, which are fibroblasts, can be effectively treated with the substrate-filled filter for separating biological particles of the present invention, in which the polymer 2 or polymer 3 having a positive charge is immobilized on the surface by a polyion complex. It became clear that it can be removed. Moreover, when considered together with the value of the molecular weight of each polymer of Example 1, it was revealed that the molecular weight of the polymer for polyion complex does not affect the separation performance. Also, when considered in conjunction with the quantitative value of the surface charge of Example 7, wherein a surface charge when measuring surface charge by the colloid titration method is -30μ equivalent / m ² ~ + 10 [mu] eq / m ² It was revealed that the biological particle separating substrate is particularly preferable.

(Preparation of biological particle separation substrate)
In the same manner as in Example 2, polypropylene nonwoven fabric P03040 (manufactured by Asahi Kasei Fibers Co., Ltd., average fiber system: 20 μm) was introduced with sulfonic acid groups on the surface by SO ₃ gas using a sulfonation apparatus described in JP-A-3-269160. This sulfonated polypropylene non-woven fabric was immersed in 1N NaOH solution for 30 minutes to make the surface sulfonic acid group Na type. After washing with deionized water 6 times, after confirming that the pH of the washing solution is neutral, the final concentration is 0.2wt%, 0.04wt%, 0.008wt%, 0.0016wt%, respectively. Polymer 2 which is a tertiary amino group-containing homopolymer prepared in Example 1 was dissolved in deionized water and immersed in an aqueous solution adjusted between pH = 6 and 8, and reacted at room temperature for 1 hour. After washing with deionized water four times, the nonwoven fabric is freeze-dried, so that the polymer 2 having a positive charge is immobilized on the surface by the polyion complex, and a series of biological particle separation substrates having a different surface charge are formed. Manufactured. These base materials are represented in the following order as polymer 2-0.2 wt%, polymer 2-0.04 wt%, polymer 2-0.008 wt%, and polymer 2-0.0016 wt%, respectively.

(Quantification of substrate surface charge by colloid titration method)
The biological particle separation base material produced in Example 9 was punched out to 12 mmφ, placed in a transparent test tube, and the substrate surface charge was quantified by colloid titration as in Example 3. The surface charge of the sulfonated polypropylene non-woven fabric prepared in Example 9 is −30.24 μeq / m ² , and the biological particle separation in which the positively charged polymer 2 prepared in Example 9 is immobilized on the surface by a polyion complex. The surface charge of the base material for the polymer is −0.25 μeq / m ² for the polymer 2-0.2 wt%, −0.73 μeq / m ² for the polymer 2-0.04 wt%, and the polymer 2-0.008 wt%. % Was −3.89 μeq / m ² , and the polymer 2-0.0016 wt% was −15.08 μeq / m ² .

(Production of base material-filled filter for bioparticle separation)
In the same manner as in Example 4, the biological particle separation base material prepared in Example 9 was punched out to a diameter of 8.5 mm, and 10 sheets were filled in a 2.5 ml syringe cylinder (manufactured by Terumo) to fill the liquid inlet. And a biological particle separation base material in a container having a liquid outlet. By the above operation, a substrate-filled filter for separating biological particles was produced.

(Fibroblast removal experiment)
As in Example 4, 2.5 ml each of cell suspensions (5 × 10E5 cells / ml) in which NIH-3T3 cells, which are fibroblasts, were suspended in DMEM medium containing 10% FBS were added to the filter prepared above. The solution was passed by natural dropping, and the filtered solution was recovered. The number of cells before and after passing through the filter was calculated with CyQUANT (Molecular Probes) according to the instructions. The cell removal rate calculated based on this was a series of base material-filled filters for separating biological particles, in which a polymer 2 having a positive charge was immobilized on the surface by a polyion complex. % Was 90.1%, polymer 2-0.04 wt% was 88.9%, polymer 2-0.008 wt% was 84.7%, and polymer 2-0.0016 wt% was 71.5%. For comparison, the same experiment was conducted with a filter filled with a sulfonated polypropylene nonwoven fabric in which the polymer 2 was not immobilized on the surface, and the ratio was 10.5%.

(Non-fibroblast removal experiment)
In the same manner as in Example 5, a cell separation experiment was performed using RINr cells, which are insulinoma cells. The cell suspension (5 × 10E5 cells / ml) in which RINr cells are suspended in DMEM medium containing 10% FBS is passed through the substrate-filled filter for separation of biological particles prepared in Example 11 by natural dropping. Then, the same operation as in Example 11 was performed except that the filtered solution was recovered.

  The removal rate of cells is 30.6% at 2-0.2 wt% of polymer, which is a series of substrate-filling filters for separating biological particles, in which polymer 2 having a positive charge is immobilized on the surface by a polyion complex, The polymer 2-0.04 wt% was 37.9%, the polymer 2-0.008 wt% was 57.1%, and the polymer 2-0.0016 wt% was 12.2%. For comparison, 7.1% was obtained when a similar experiment was performed using a filter filled with a sulfonated polypropylene nonwoven fabric in which the polymer 2 was not immobilized on the surface.

  NIH-3T3 cells, which are fibroblasts, are effectively removed by a series of surface-charged bioparticle-separating substrate-filled filters in which the positively charged polymer 2 is immobilized on the surface by a polyion complex. This can be seen in Example 11. In addition to this, in Example 12, RINr cells, which are insulinoma cells that are not fibroblasts, also change the surface charge by using the production method of the present invention, thereby separating biological particles that exhibit optimal cell separation behavior for RINr cells. It became clear that a base material for use can be easily created. As described above, the present invention can provide an optimum surface-charged base material for each target biological particle simply, safely, inexpensively, and quickly.

Also, when considered in conjunction with the quantitative value of the surface charge of Example 10, wherein a surface charge when measuring surface charge by the colloid titration method is -30μ equivalent / m ² ~ + 10 [mu] eq / m ² It was revealed that the biological particle separating substrate is particularly preferable.

  The substrate for separating bioparticles according to the present invention is used in laboratory instruments in basic science, clinical medicine, and industrial process filters. The cell suspension from which specific biological particles have been separated is subjected to further separation and purification (including washing), culture, activation, amplification, gene transfer, cryopreservation, etc., as is or as necessary. Later, it is used for basic science experiments such as transplantation to living bodies, cell biology and immunology. Alternatively, by removing biological particles from a useful protein-containing solution including viruses and bacteria, it can be used for the production of highly safe pharmaceuticals.

Claims (9)

  The manufacturing method of the base material for biological particle separation characterized by performing the following process one by one. (1) a step of preparing a water-insoluble substrate surface having a negative charge or a positive charge, (2) the aqueous solution of the polymer having a charge opposite to that of the substrate surface prepared in (1) above (1 ) Step of immersing the substrate prepared in (3), (3) step of washing the substrate with an aqueous solution not containing a charged polymer.   Further, after completion of the step (3), (4) immersion in an aqueous solution of a polymer having a charge opposite to the polymer used in the step (2), and (5) an aqueous solution not containing a charged polymer. The manufacturing method of the base material for biological particle separation of Claim 1 which performs the process of wash | cleaning a base material with a solution. 3. The method for producing a bioparticle separation substrate according to claim 2, wherein the steps (4) and (5) are repeated a plurality of times.   2. The method for producing a bioparticle separation substrate according to claim 1, wherein the surface of the substrate prepared in (1) is negatively charged.   3. The method for producing a bioparticle separation substrate according to claim 2, wherein the surface of the substrate prepared in (1) is positively charged. A bioparticle separation substrate produced by the method according to any one of claims 1 to 4, wherein a polymer having a positive charge forms a polyion complex and is bonded onto the substrate surface, and, wherein the surface charge when measuring surface charge by the colloid titration method is -200μ equivalent / m ² ~ + 100μ equivalents / m ^2, the biological particle separation base material. Wherein the surface charge when measuring surface charge by the colloid titration method is -30μ equivalent / m ² ~ + 10μ equivalents / m ^2, the biological particle separation substrate of claim 6.   A biological particle separation filter in which the biological particle separation substrate according to claim 6 or 7 is housed in a container having a liquid inlet and a liquid outlet. A biological particle separation method, wherein the biological particle-containing liquid is filtered with the biological particle separation filter according to claim 8.