JP2009296934A - Method for recovering cell or virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for recovering a cell or a virus, which simply and efficiently recovers a cell or a virus while maintaining its biological activity. <P>SOLUTION: The method for recovering a cell or a virus comprises isolating a cell or virus 2 attached to the surface of a carrier 1 having at least the vicinity of the surface constituted mainly of a calcium phosphate-based compound from the carrier 1 and recovering it, and bringing a recovery solution 400 containing a protein having higher affinity to the carrier 1 than that of the cell or virus 2 into contact with the carrier 1 to which the cell or virus 2 is attached and isolating the cell or virus 2 in the recovery solution 400 to recover the cell or the virus 2. Casein is preferable as the protein contained in the recovery solution 400. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for recovering cells or viruses.

  For example, cells and viruses can be collected and isolated from samples such as culture media and body fluids such as blood, and their physiological mechanisms can be examined, or biological substances such as specific antibodies against these can be detected using cells and viruses. Or has been done. In these cases, the cells and viruses to be collected must be collected efficiently (at a high concentration) without being destroyed or damaged by various treatments for collection. It becomes.

  Examples of methods for recovering (isolating) cells and viruses from a sample include: First, by bringing a sample into contact with a solid support, cells and viruses are attached to the surface of the solid support. Next, after separating the solid support and the sample, there is a method in which the solid support is brought into contact with the eluent, whereby the cells and viruses attached to the solid support are eluted and recovered in the eluent ( For example, see Patent Documents 1 and 2.)

  Here, as the solid support, a substrate made of glass, silica, latex, a polymer substance, or a substrate obtained by applying a positive charge coating or a negative charge coating to a base material made of these materials is used.

  However, in the conventional cell recovery method using such a solid support, there are problems that cells and viruses are destroyed or their biological activity is inhibited by the reagent or solid support used.

  In addition, depending on the combination of the solid support and cells or viruses, the affinity of the solid support to cells or viruses may be insufficient, or conversely, once cells or viruses adhere to the solid support. Due to the strong binding, there has been a problem that it is difficult to recover cells and viruses in a high concentration state while maintaining their biological activity.

Japanese translation of PCT publication No. 2002-507116 JP 2005-531304 gazette

  An object of the present invention is to provide a method for recovering cells or viruses that can be recovered easily and with high yield while maintaining the biological activity of the cells or viruses.

Such an object is achieved by the present inventions (1) to (15) below.
(1) A cell or virus recovery method for recovering cells or viruses attached to the surface of a carrier composed mainly of a calcium phosphate compound at least near the surface, by separating the cells or viruses from the carrier,
Contacting the carrier to which the cells or viruses are attached with a recovery solution containing a protein having a higher affinity for the carrier than the cells or viruses, thereby removing the cells or viruses from the carrier and collecting them. A method for recovering a characteristic cell or virus.

  As a result, cells or viruses can be recovered with high yield by a simple operation of bringing the recovered liquid into contact with the carrier while maintaining its biological activity.

(2) A cell or virus recovery method for recovering cells or viruses attached to the surface of a carrier composed mainly of a calcium phosphate compound at least near the surface, by separating the cells or viruses from the carrier,
The cell or virus is further removed from the carrier by bringing the recovered solution containing the protein into contact with the carrier to which the cell or virus and a protein having a higher affinity for the carrier than the cell or virus are attached. A method for recovering cells or viruses, comprising separating and recovering cells.

  As a result, cells or viruses can be recovered with high yield by a simple operation of bringing the recovered liquid into contact with the carrier while maintaining its biological activity.

  Further, in the method for recovering cells or viruses having such a structure, since a protein having an affinity for the carrier larger than that of the cell or virus is previously attached to a part of the surface of the carrier, a relatively small amount of the cell or virus It is suitably applied when collecting viruses.

  (3) The method for recovering cells or viruses according to (1) or (2) above, wherein the protein is a metalloprotein.

  Since the metal protein has a high affinity for the calcium phosphate compound, cells or viruses can be efficiently removed from the carrier by using a recovery solution containing the metal protein.

  (4) The method for recovering cells or viruses according to (3) above, wherein the metalloprotein is casein.

  Casein is considered to bind strongly to calcium ions on the surface of the carrier (calcium phosphate compound) at its phosphate group. For this reason, casein exhibits an excellent adsorption ability (high affinity) for the carrier. Furthermore, casein is a protein contained in mammalian milk, and is excellent in that damage to cells and viruses is extremely small.

  (5) The method for recovering cells or viruses according to any one of (1) to (4) above, wherein the concentration of the protein in the recovery solution is 0.1 to 100 mg / mL.

  Thereby, cells or viruses can be efficiently removed from the carrier.

(6) The method for recovering a cell or virus according to any one of (1) to (5) above, wherein the time for contacting the recovery liquid with the carrier is 1 to 15 minutes.
As a result, many cells or viruses can be collected in a single treatment.

(7) The method for recovering cells or viruses according to any one of (1) to (6) above, wherein the temperature of the recovery liquid when the recovery liquid is brought into contact with the carrier is 2 to 40 ° C.
Thereby, cells or viruses can be efficiently removed from the carrier.

  (8) The method for recovering cells or viruses according to any one of (1) to (7) above, wherein vibration or impact is applied to the carrier when the recovery liquid is brought into contact with the carrier.

  Thereby, cells or viruses that adhere more firmly to the surface of the carrier can also be removed.

  (9) The cell or virus recovery method according to any one of (1) to (8), wherein the treatment of bringing the recovery solution into contact with the carrier is performed a plurality of times.

  A sufficient number of cells or viruses can be recovered by performing the treatment with the recovery solution a plurality of times.

  (10) The method for recovering a cell or virus according to any one of (1) to (9), wherein the recovery solution does not substantially contain a degrading enzyme and a chelating agent.

  By using a recovery solution that does not substantially contain a degrading enzyme, the number of viable cells can be more reliably prevented. By using a recovery solution that does not substantially contain a chelating agent, a calcium phosphate system can be used. It can be reliably prevented that the compound is decomposed and the surface of the carrier is crushed.

(11) The cell or virus according to any one of (1) to (10), wherein the carrier to which the cells or viruses are attached is washed at least once prior to the treatment of bringing the recovered solution into contact with the carrier. Collection method.
Thereby, the processing effect by protein can be improved more.

(12) The cell or virus recovery method according to (11), wherein the cleaning solution used for the cleaning is an isotonic solution.
Thereby, destruction of the cell or virus by the difference in osmotic pressure can be prevented.

  (13) The method for recovering cells or viruses according to any one of (1) to (12) above, wherein the carrier is granular.

  Thereby, the surface area of a support | carrier can be increased and a cell or a virus can be efficiently attached to the surface of a support | carrier.

  (14) The cell or virus recovery method according to (13), wherein the granular carrier has an average particle diameter of 1 to 1000 μm.

  Thereby, a sufficient surface area of the carrier can be secured, and cells or viruses can be more efficiently attached to the surface of the carrier.

  (15) The method for recovering cells or viruses according to any one of (1) to (14), wherein the calcium phosphate compound is composed mainly of hydroxyapatite.

  Hydroxyapatite is used as a biomaterial, can attach cells or viruses very efficiently, and has a particularly low possibility of causing damage to cells or viruses. For this reason, since at least the vicinity of the surface of the carrier is mainly composed of hydroxyapatite, the cell or virus can be reliably attached to the surface of the carrier while reliably maintaining its biological activity.

  According to the present invention, cells or viruses can be easily and efficiently recovered while maintaining their biological activity.

  In addition, by using a metal protein, particularly casein or basic protein, as the protein to be contained in the recovered liquid, cells or viruses can be more efficiently detached from the carrier.

  Hereinafter, the cell or virus recovery method of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.

  The cell or virus recovery method of the present invention is a method in which cells or viruses attached to the surface of a carrier mainly composed of a calcium phosphate compound at least near the surface are separated from the carrier and recovered. The cell or virus is detached from the carrier and recovered by contacting the carrier with the attached protein with a recovery solution containing a protein having a higher affinity for the carrier than the cell or virus.

  First, prior to describing the method for recovering cells or viruses of the present invention, the carrier used in the present invention will be described.

FIG. 1 is a cross-sectional view showing an example of a carrier used in the present invention.
The carrier 1 shown in FIG. 1 is granular (preferably substantially spherical). The carrier 1 can be used in various shapes such as a block shape (lump shape), a pellet shape, and a sheet shape, but by using a granular material, its surface area can be increased and more efficiently. Cells or viruses can be attached.

  As shown in FIG. 1, the carrier 1 includes a base material 11 mainly composed of a resin material, and a calcium phosphate compound layer 12 that is provided so as to cover the surface of the base material 11 and mainly composed of a calcium phosphate compound. Have. This facilitates adjustment of the shape, size (average particle diameter, etc.), physical properties (density, etc.), etc. of the carrier 1. The carrier 1 may be entirely composed of a calcium phosphate compound. The carrier 1 composed entirely of a calcium phosphate compound can capture cells or viruses more efficiently.

The specific surface area of the carrier 1 is preferably from 5 to 100 m ² / g, and more preferably 15~80m ² / g. When the value is below the lower limit, there is a possibility that cells or viruses cannot be attached efficiently. Moreover, when the upper limit value is exceeded, the carrier 1 may be crushed due to a decrease in the mechanical strength of the carrier 1.

  When the substrate 11 is made of a resin material, various thermosetting resins and various thermoplastic resins can be used as the resin material. Specifically, examples of the thermoplastic resin include polyamide, polyethylene, Examples of thermosetting resins such as polypropylene, polystyrene, polyimide, acrylic resin, and thermoplastic polyurethane include epoxy resin, phenol resin, melamine resin, urea resin, unsaturated polyester, alkyd resin, thermosetting polyurethane, and ebonide. 1 type or 2 types or more of these can be used in combination.

As the calcium phosphate-based compound constituting the calcium phosphate-based compound layer 12 is not particularly limited, Ca / P ratio can be used various compounds of 1.0 to 2.0, for _{example, Ca 10 (PO 4) 6} ( _{OH) 2, Ca 10 (PO} 4) 6 F 2, Ca 10 (PO 4) 6 Cl 2, Ca 3 (PO 4) 2, Ca 2 P 2 O 7, Ca (PO 3) 2, CaHPO 4 , etc. One or two or more of them can be used in combination.

Among these, as the calcium phosphate-based compound, those having hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) as the main component are optimal. Hydroxyapatite is used as a biomaterial, can attach cells or viruses very efficiently, and has a particularly low possibility of causing damage to cells or viruses.

  These calcium phosphate compounds can be synthesized by a known wet synthesis method, dry synthesis method, or the like. In this case, the calcium phosphate compound may contain a substance (raw material, etc.) remaining during the synthesis or a secondary reaction product generated during the synthesis.

  In addition, as shown in FIG. 1, in the calcium phosphate compound layer 12, a part of particles 13 (hereinafter simply referred to as “particles 13”) composed of a calcium phosphate compound penetrates near the surface of the substrate 11. It is preferable that it is formed by doing so. Thereby, the adhesiveness of the calcium-phosphate type compound layer 12 and the base material 11 can be made excellent. For this reason, peeling from the surface of the base material 11 of the calcium-phosphate-type compound layer 12 can be prevented suitably, ie, the intensity | strength of the support | carrier 1 can be made excellent.

  In this case, the calcium phosphate compound layer 12 is formed by, for example, colliding porous particles mainly composed of a calcium phosphate compound (hereinafter simply referred to as “porous particles”) on the surface of the substrate 11. be able to. According to this method, the calcium phosphate compound layer 12 can be formed easily and reliably.

  The collision between the base material 11 and the porous particles can be performed by a dry method using, for example, a commercially available hybridization apparatus. The conditions at this time are, for example, a mixing ratio of the base material 11 and the porous particles of about 100: 5 to 100: 30 in a weight ratio, and the temperature in the apparatus used as the main material of the base material 11 Or less (usually 80 ° C. or less).

Such a carrier 1 preferably has a density (specific gravity) close to that of water. Specifically, the density of the carrier 1 is preferably from 0.8 to 1.4 g / cm ³ or so, and more preferably 0.9~1.2g / cm ³ order. Thereby, the support | carrier 1 can be suspended more uniformly in the to-be-processed liquid and collection | recovery liquid mentioned later. As a result, these liquids and the carrier 1 can be brought into contact with each other more reliably and uniformly, and the efficiency of attaching cells or viruses to the carrier 1 and the yield of cells or viruses in the collected liquid can be further improved. Can do.

  The average particle diameter of the carrier 1 is preferably about 1 to 1000 μm, more preferably about 2 to 100 μm, and further preferably about 4 to 10 μm. Thereby, since the surface area of the support | carrier 1 can be ensured enough, the adhesion rate of the cell or virus to the support | carrier 1 can be improved more. If the particle size is too small, the liquid may not easily flow between the carriers 1.

  Next, an example of a collection method for collecting cells or viruses using the carrier shown in FIG. 1 (the method for collecting cells or viruses of the present invention) will be described.

  In the present embodiment, [1] cells or viruses are attached to a carrier, [2] the carrier to which the cells or viruses are attached is washed, and then [3] cells or viruses are detached from the carrier and collected. The case where the treatment is performed and then, further, [4] the carrier from which the cells or viruses are collected will be described as a representative.

  2 and 3 are process diagrams schematically showing the cell or virus recovery method of the present embodiment.

  As shown in FIGS. 2 and 3, these series of steps (operations) are performed in a container such as a beaker or a test tube. Hereinafter, each process will be described sequentially.

[1] Step of attaching a cell or virus to a carrier First, a cell or virus to be attached to the carrier 1 (hereinafter, cell or virus is represented as “cell / virus 2”) is prepared, and a treatment liquid 200 containing the cell or virus is prepared. Get.

  Examples of the cell / virus 2 include various cells such as bacterial cells, plant cells, and animal cells, viruses that cause infectious diseases such as influenza, Japanese encephalitis, dengue fever, rubella, measles, and mumps.

The cell / virus 2 may be freeze-dried, cultured in a body fluid such as blood or saliva, or a culture solution, or various waters (living bath water, river water, well water, It may be suspended in a liquid such as water in a pool.
In addition, when the cell / virus 2 is freeze-dried, the liquid 200 to be treated is obtained by suspending the cell / virus 2 in a dispersion. Further, when the cell / virus 2 is suspended in a liquid, this suspension may be used as it is as the liquid 200 to be processed, and a solution diluted with a diluent is used as the liquid 200 to be processed. It may be used.

  Here, an isotonic liquid (a liquid having an osmotic pressure substantially equal to the osmotic pressure of the intracellular liquid) is preferable as the dispersion and the diluting liquid. Thereby, the destruction of the cell / virus 2 due to the difference in osmotic pressure when the dispersion and the diluted solution contact the cell / virus 2 can be prevented.

  As this isotonic solution, for example, Dulbecco solution (PBS: phosphate buffered saline), Locke solution, Ringer solution, Tyrode solution, Earle solution, Krebs solution, physiological saline and the like can be used.

  Next, the treatment liquid 200 is brought into contact with the carrier 1 to obtain the cell / virus adhesion carrier 3 in which the cells / viruses 2 are adhered to the carrier 1.

  Specifically, the carrier 1 is accommodated in the container 100 and the liquid 200 to be treated is supplied (see (a) in FIG. 2). Then, the carrier 1 is dispersed (suspended) in the liquid to be treated 200 by shaking (shaking), stirring, applying ultrasonic waves, or the like (see (b) in FIG. 2). As a result, the cells / viruses 2 come into contact with and adhere to the surface of the carrier 1.

  Next, the to-be-treated liquid 200 containing the carrier 1 is allowed to stand so that the carrier 1 is settled in the container 100, and the to-be-treated liquid 200 in the supernatant is removed and discarded (see FIG. 2C). .

  Here, at least the vicinity of the surface of the carrier 1 is mainly composed of a calcium phosphate compound. Since the calcium phosphate compound has a high affinity for the cell / virus 2, the cell / virus 2 adheres efficiently.

  As will be described later, a protein having a higher affinity for the carrier 1 than that of the cell / virus 2 is obtained by electrostatically binding calcium (site), which is one of the constituent elements of the calcium phosphate compound, or phosphate (site). The cell / virus 2 to be recovered by adapting the present invention is a protein having a higher affinity for calcium phosphate than the virus or cell intended for recovery. If is selected, the cells / viruses 2 can be recovered more efficiently.

  As described above, the cells / viruses 2 attached to each site include various cells as described above, and further, flaviviridae to which dengue virus and Japanese encephalitis virus belong, orthomyxoviridae to which influenza virus belongs, and rubella virus Envelope viruses such as Paramyxoviridae viruses to which Togaviridae, Measles virus and Mumps virus belong are conceivable.

  That is, it is considered that the recovery method of the present invention can be suitably applied to recovery of viruses having various cells and envelopes.

  The time for which the liquid 200 to be treated is brought into contact with the carrier 1 is preferably about 1 to 240 minutes, and more preferably about 10 to 180 minutes. If this time is too short, the number of cells or viruses adhering to the carrier 1 may decrease, and there is a possibility that the cells / viruses 2 cannot be sufficiently recovered. On the other hand, even if this time is increased beyond the upper limit, Further increase in the effect cannot be expected, and the cell / virus 2 may collapse, making it difficult to use the cell / virus 2 for the intended purpose.

  Further, the temperature of the liquid 200 to be treated at this time (during treatment) is preferably 70 ° C. or less, more preferably 50 ° C. or less, and further preferably about 5 to 40 ° C. If the temperature of the liquid 200 to be treated is too low, depending on the type of the cells / viruses 2 or the like, the cells / viruses 2 may not be efficiently attached to the carrier 1. Even if the temperature of the liquid 200 exceeds the upper limit, not only an increase in the effect cannot be expected, but the cell / virus 2 may be destroyed (destroyed). It becomes difficult to use it for the purpose intended.

[2] First Washing Step for Washing the Carrier with Cells or Virus Attached Next, the cell / virus adhesion carrier 3 with the cells / viruses 2 attached to the carrier 1 is washed with the washing liquid 300.

Specifically, the cleaning liquid 300 is supplied into the container 100 (see (d) in FIG. 2). Then, the cell / virus adhesion carrier 3 is dispersed (suspended) in the cleaning liquid 300 by gently stirring the cleaning liquid 300 (see (e) in FIG. 2). Next, by allowing to stand, the cell / virus adhesion carrier 3 is allowed to settle in the container 100, and the supernatant washing solution 300 is removed and discarded (see (f) in FIG. 2).
Thereby, the to-be-processed liquid 200 remaining on the surface of the cell / virus adhesion carrier 3 is removed.

  The treatment liquid 200 may contain, for example, an inhibitor that inhibits the adhesion of a high-affinity protein, which will be described later, to the carrier 1. In this step [2], the cell / virus attachment carrier 3 By removing the liquid 200 to be treated remaining on the surface as much as possible, the treatment effect by the high affinity protein in the next step [3] can be further improved.

As the cleaning solution 300 used in this step [2], various types can be used. When the purpose is to recover cells / viruses 2 as in this embodiment, an isotonic solution (intracellular solution) is used. A liquid having an osmotic pressure approximately equal to the osmotic pressure is preferred. Thereby, destruction of the cell and the virus 2 by the difference in osmotic pressure can be prevented.
Examples of the isotonic solution include those described in the above step [1].

  In addition, this step [2] may be repeated a plurality of times as necessary. Thereby, the to-be-processed liquid 200 which remain | survives on the surface of the cell and virus adhesion support | carrier 3 can be removed more reliably.

  In this case, the cleaning liquid 300 to be used may be the same or different types (conditions) may be used each time.

  In addition to the above-described method, for example, the cell / virus adhesion carrier 3 is placed on a filter, and the washing liquid 300 is supplied to the cell / virus adhesion carrier 3 (step 2). For example, a method of spraying with a pipette or the like) can also be used.

  In addition, this process can also be abbreviate | omitted when the inhibitory substance mentioned above is not contained in the to-be-processed liquid 200, or is contained even if it is trace amount.

[3] Treatment Step for Collecting Cells or Virus Adhered to Carrier Next, the washed cell / virus adhering carrier 3 is a protein having a higher affinity for carrier 1 than cell / virus 2 (hereinafter referred to as “high affinity”). A process of contacting the recovered liquid 400 containing “protein” is performed.

  Specifically, a recovery liquid 400 containing a high affinity protein is supplied into the container 100 (see (a) in FIG. 3). Then, the recovered solution 400 is stirred to disperse (suspend) the cell / virus attachment carrier 3 in the recovered solution 400 as shown in FIG. Contact with the virus attachment carrier 3. As a result, the cells / viruses 2 are detached (released) from the cell / virus adhesion carrier 3 into the collected liquid 400. Next, the cell / virus adhesion carrier 3 containing the cell / virus adhesion carrier 3 is allowed to stand to settle the cell / virus adhesion carrier 3 in the container 100, and the supernatant recovery solution 400 is collected ((f in FIG. 3) )reference).

  Thereby, the cell / virus 2 is detached from the cell / virus attachment carrier 3 while maintaining its biological activity by a simple operation of bringing the collection solution 400 into contact with the cell / virus attachment carrier 3, and the collection solution is recovered. It can be recovered with good yield while floating in 400.

  Here, the cell / virus 2 adhering to the carrier 1 is detached (released) by contacting the cell / virus adhesion carrier 3 with the recovery liquid 400 containing the high affinity protein as follows. It is assumed that this is due to a mechanism.

  That is, in the cell / virus adhesion carrier 3, it is considered that the cell / virus 2 is adhered using the calcium phosphate compound layer 12 as a scaffold. On the other hand, such high-affinity proteins attached to the cell / virus attachment carrier 3 attach calcium (sites) and phosphates (sites), which are constituent elements of calcium phosphate compounds, via electrostatic binding or the like. It is thought to do.

  When the high affinity protein comes into contact with the cell / virus adhesion carrier 3, the high affinity protein adheres to the cell 1 based on the fact that the affinity for the carrier 1 is greater than that of the cell / virus 2. The virus 2 adheres to the carrier 1 so that it can be pushed away. On the other hand, the cells / viruses 2 lose their scaffolds so that they can be pushed away by the high affinity protein, and further detach (separate) from the carrier 1 by being affected by the steric hindrance of the high affinity protein. Presumed to be liberated inside.

  The recovery solution used for releasing the cell / virus 2 from the cell / virus attachment carrier 3 includes a decomposing enzyme such as trypsin or ethylenediaminetetraacetic acid in addition to the recovery solution 400 containing a high affinity protein. Other chelating agents are also conceivable.

  However, a degrading enzyme such as trypsin liberates the cell / virus 2 from the cell / virus attachment carrier 3 by degrading the protein contained in the cell / virus 2, but in the method using such a degrading enzyme, However, there is a problem that the cell / virus 2 itself is destroyed (dissolved) to adversely affect the characteristics of the cell / virus. On the other hand, the chelating agent has an action of capturing and extracting calcium, which is one of its constituent elements, as an ion from the calcium phosphate compound layer 12. -Virus 2 can be released. However, in the method using such a chelating agent, there is a problem that the calcium phosphate compound is decomposed and the calcium phosphate compound layer 12 is crushed when calcium ions are extracted from the calcium phosphate compound layer 12.

  On the other hand, the high affinity proteins include those exemplified below, but these all have little damage to the cells / viruses 2 and adhere to the cells / viruses while maintaining their physiological activity. The cell / virus 2 can be detached from the carrier 3. Further, since the high affinity protein adheres to the surface of the calcium phosphate compound layer 12 in a state where calcium is taken in, the calcium taken into the protein is retained in the calcium phosphate compound layer 12 and the calcium phosphate compound layer 12 is decomposed. There is an advantage that the problem does not occur.

  From the above, the recovery liquid 400 is substantially made of, for example, a degrading enzyme (digestive enzyme) such as trypsin, pepsin or papain, and a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or ethylenediaminediacetic acid (EDDA). It is preferable that they are not included in the above. As described above, by using the recovery liquid 400 that substantially does not contain a decomposing enzyme and a chelating agent, the above problem can be reliably solved.

  The high affinity protein is not particularly limited as long as the affinity for the carrier 1 is larger than that of the cell / virus 2 attached to the carrier 1 in the step [1]. For example, casein, viterin, phosvitin , Cytochrome c, metalloprotein such as transferrin, albumin and myoglobin.

  Among such proteins, metal proteins are preferably used. Since the metal protein has a metal ion in its molecule, it has a high affinity for the metal ion. Since the calcium phosphate compound has calcium ions and phosphate ions, which are metal ions, the metal protein has an excellent adsorptive capacity (high affinity) for the carrier 1 provided with the calcium phosphate compound layer 12 near the surface. ) Is obtained.

  The metal protein is preferably a phosphorylated protein in which a part of the protein is phosphorylated, such as casein, vitellin, and phosvitin. Here, phosphorylated proteins such as casein, vitellin, and phosvitin contain calcium as a metal ion that binds to a phosphate group. And when this metal (calcium) ion leaves | separates from metal protein, it is thought that phosphorylated protein couple | bonds strongly with the calcium ion on the surface of a support | carrier (calcium phosphate type compound) in the phosphate group. For this reason, the phosphorylated protein exhibits an excellent adsorption ability (high affinity) for the carrier 1 provided with the calcium phosphate compound layer 12 in the vicinity of the surface. Furthermore, among the phosphorylated proteins having metal (calcium) ions, casein is a protein contained in mammalian milk, and is excellent in that damage to cells and viruses is extremely small.

  When a protein having a metal ion such as a metal protein is used as the high-affinity protein, the high-affinity protein is subjected to a treatment for removing (decreasing) the held metal ion. Is preferred. Accordingly, the affinity of the high affinity protein for the carrier 1 can be made higher, and the cell / virus 2 can be efficiently released from the cell / virus attachment carrier 3.

  Examples of the method for removing the metal ions include a method of treating the solution (recovered solution 400) containing such a high affinity protein with a chelating agent such as ethylenediaminetetraacetic acid (EDTA). In this case, as a method of treating a solution containing a high affinity protein with a chelating agent, for example, after adding the chelating agent to a solution containing a high affinity protein, for example, gel filtration, ultrafiltration, dialysis, etc. And a method of removing a chelating agent from a solution containing a high affinity protein.

  The concentration of the high affinity protein in the collection liquid 400 is not particularly limited, but is preferably about 0.1 to 100 mg / mL, and more preferably about 0.2 to 10 mg / mL. If the concentration of the high affinity protein is too low, the cell / virus 2 may not be efficiently removed from the carrier 1 depending on the type of the high affinity protein. Even if it exceeds the value, not only an increase in the effect cannot be expected, but depending on the type of the high affinity protein, there is a possibility that the cell / virus 2 may be damaged (adverse effect).

  Moreover, as a solvent which melt | dissolves high affinity protein, the isotonic liquid similar to the dispersion liquid or dilution liquid demonstrated by the said process [1] is suitable, for example. Thereby, destruction of the cell and virus 2 due to the difference in osmotic pressure can be surely prevented.

  The treatment time with the recovered liquid 400 is preferably about 1 to 15 minutes, and more preferably about 3 to 5 minutes. If the treatment time is too short, the number of cells or viruses released in the collection liquid 400 decreases, and the number of treatments must be increased to obtain the desired number of cells or viruses. The amount of the recovered liquid 400 is undesirably increased unnecessarily. On the other hand, even if the treatment time is increased beyond the upper limit, not only an increase in the effect cannot be expected, but the cell / virus 2 may be damaged.

  The temperature of the recovered liquid during treatment with the recovered liquid 400 is preferably about 2 to 40 ° C, more preferably about 4 to 37 ° C. If the temperature of the recovered liquid 400 at the time of processing is too low, depending on the type of high-affinity protein, etc., there is a possibility that the cells / viruses 2 cannot be efficiently removed from the cell / virus-adhering carrier 3. Even if the temperature of the recovered liquid 400 is increased beyond the upper limit, not only an increase in the effect can be expected, but there is a possibility that the cell / virus 2 may be damaged (adverse effect).

  In this step [3], it is preferable to apply vibration or impact to the cell / virus adhesion carrier 3 when the recovered liquid 400 is brought into contact with the cell / virus adhesion carrier 3. Thereby, the cells / viruses 2 that adhere more firmly to the surface of the carrier 1 can also be removed.

  As this operation, for example, a pipetting operation (see (c) to (e) in FIG. 3) in which the cell / virus adhesion carrier 3 is sucked with the pipette 600 together with the recovery liquid 400 and discharged is used, or a pipette is used. Then, the supernatant recovered liquid 400 is sucked and sprayed onto the carrier 1, a method of applying vibration to the carrier 1 with a shaker, a method of applying ultrasonic waves to the recovered liquid 400, and the like. In particular, according to the pipetting operation as shown in the figure, there is an advantage that the operation can be performed in a short time while preventing the cell / virus 2 from being damaged.

  The treatment of the cell / virus adhesion carrier 3 with the collected liquid 400 in this step [3] may be performed once, but is preferably performed a plurality of times, particularly about 2 to 3 times. Is more preferable. In this way, a sufficient number of cells / viruses 2 can be recovered by performing the treatment with the recovery solution 400 a plurality of times. It should be noted that even if the number of treatments with the recovery liquid 400 is increased beyond the upper limit, further increase in the recovery rate of cells / viruses 2 cannot be expected, and the amount of liquid to be recovered may increase, which is not preferable.

[4] Second Washing Step for Washing the Carrier from which Cells or Viruses were Collected Next, the carrier 1 from which the cells / viruses 2 have been removed from the cell / virus adhesion carrier 3 through the step [3] is washed with a washing solution. Wash (process) with (second recovered liquid) 500.

  Specifically, the cleaning liquid 500 is supplied into the container 100 (see (g) in FIG. 3). Then, the carrier 1 is dispersed (suspended) in the cleaning liquid 500 by stirring the cleaning liquid 500 (see (h) in FIG. 3). Next, by allowing to stand, the carrier 1 is settled in the container 100, and the supernatant washing liquid 500 is collected (see (i) in FIG. 3).

  Thus, cells that have been removed from the cell / virus-adhering carrier 3 after passing through the step [3], but have not been recovered in the recovery liquid 400 by being adsorbed on the surface of the carrier 1 or the inner surface of the container 100. -Virus 2 can be recovered, and as a result, the recovery rate of cells / virus 2 can be further improved.

  As the cleaning liquid 500 used in this step [4], for example, an isotonic liquid similar to the dispersion or dilution liquid described in the above step [1] is suitable. Thereby, destruction of the cell and virus 2 due to the difference in osmotic pressure can be surely prevented.

  In addition, the cleaning liquid 500 (second recovery liquid) is not limited to such a liquid, and for example, the cleaning liquid 500 has a condition different from that of the recovery liquid 400. Those having different conditions such as concentration, type and composition can also be used.

  This step [4] can be omitted when a sufficient amount of cells / viruses 2 has been recovered in step [3], but the cell / virus 2 recovery rate can be further improved. Therefore, it may be repeated a plurality of times as necessary. Thereby, the cells / viruses 2 adsorbed and remaining on the surface of the carrier 1 or the inner surface of the container 100 can be more reliably recovered.

  In this case, the cleaning liquid 500 to be used may be the same or different types (conditions) may be used each time.

  Further, in this embodiment, a cell / virus adhesion carrier 3 in which cells / viruses 2 are adhered to a carrier 1 to which no high affinity protein is adhered is obtained, and a high affinity protein is applied to the cell / virus adhesion carrier 3. The case where the cell / virus 2 is detached from the cell / virus adhesion carrier 3 and recovered in the recovery solution 400 by bringing the recovery solution 400 into contact therewith has been described. The cell / virus 2 may be collected as follows.

  That is, the cell / virus 2 is detached from the carrier by contacting the carrier with the cell / virus 2 and the high-affinity protein 2 attached thereto with a collection solution 400 containing the high-affinity protein. You may make it collect | recover in the liquid 400. FIG.

  More specifically, prior to the step [1] in which the cells / viruses 2 are attached to the carrier 1, a solution containing a high affinity protein is supplied to the carrier 1 so that at least one of the surfaces of the carrier 1 is present. After attaching the high affinity protein to the part, the steps [1] to [4] may be performed to recover the cell / virus 2 in the recovery solution 400.

  When the cell / virus 2 is collected through this process, when the cell / virus 2 is attached to the carrier, a high affinity protein is already attached to a part of the surface of the carrier. When a relatively small amount of cells / viruses 2 are recovered, the recovery method having such a configuration is preferably applied. Furthermore, there is an advantage that the concentration of the high-affinity protein in the recovery solution 400 used in the step [3] for recovering the cells / viruses 2 attached to the carrier can be lowered, and the processing time by the recovery solution 400 can be set short. can get.

  As mentioned above, although the collection method of the cell or virus of this invention was demonstrated, this invention is not limited to these, Arbitrary processes may be added as needed.

Next, specific examples of the present invention will be described.
1. Preparation of carrier First, 50 g of nylon particles (base material) having an average particle diameter of 7 μm and a density of 1.02 g / cm ³ , and hydroxyapatite particles having a Ca / P ratio of 1.67 and an average particle diameter of 10 μm (primary particles aggregated and bonded. 10 g of porous particles) were prepared.

The hydroxyapatite particles had a specific surface area of 45 m ² / g and a pore diameter of 600 mm.

  Next, these nylon particles and hydroxyapatite particles were put into a NARA hybridization system NHS-1 (manufactured by Nara Machinery Co., Ltd., rated power 5.5 kW, rated current 23 A). Operated at ~ 50 ° C for 7 minutes. This obtained the support | carrier (refer FIG. 1) by which the surface was coat | covered with the hydroxyapatite.

The obtained carrier had an average particle size of 9 μm (average hydroxyapatite coating layer thickness of 1 μm) and a density of 1.03 g / cm ³ .

2. Examination using influenza virus (2-1) Preparation of virus suspension (treatment liquid) Influenza virus solution (B / Osaka / 72 / CCAstandard) from which supernatant was removed after centrifugation at 1500 rpm for 5 minutes was added to PBS. What was diluted 5-fold with (phosphate buffered saline) was used as a virus suspension.

(2-2) Preparation of carrier dispersion <1A> First, a 0.1 g carrier was prepared, and this carrier was stored in a tube (falcon tube). And the 10% ethanol containing PBS solution was supplied in this tube so that the whole quantity might be 10 mL.

  <2A> Next, the carrier in the tube was loosened using a vortex, and then rotated at 37 ° C. for 1 hour.

  <3A> Next, the dispersion after rotation was centrifuged at 1500 rpm for 5 minutes, and then the supernatant was discarded using an aspirator.

  <4A> Next, PBS was supplied into the tube so that the total amount became 10 mL, whereby a carrier dispersion containing 1% carrier (1% carrier dispersion) was obtained.

(2-3) Examination of virus attachment performance to carrier <1B> First, five 2 mL Eppendorf tubes (first tube to fifth tube) × 8 sets, of which the second tube to the fifth tube are prepared. 200 μL of PBS was supplied to each tube, and 200 μL of 1% carrier dispersion was supplied to the first tube.

  <2B> Next, 200 μL of 1% carrier dispersion is supplied to the second tube and stirred, and 200 μL of carrier dispersion is dispensed from the inside of the second tube. Feed and stir.

  By carrying out this operation in the same manner from the third tube to the fourth tube and from the fourth tube to the fifth tube, the carrier dispersion was diluted twice. As a result, carrier dispersion liquids (200 μL) with carrier concentrations of 1 wt%, 0.5 wt%, 0.25 wt%, 0.125 wt%, and 0.063 wt% were respectively placed in the first tube to the fifth tube. After obtaining, centrifuge at 1500 rpm for 5 minutes to remove the supernatant.

  <3B> Next, 200 μL of the virus suspension was dispensed into each of the first to fifth tubes, and then rotated at 37 ° C. for 1 hour to attach influenza virus to the carrier. .

  Separately, a control 2 mL Eppendorf tube (tube C1) was prepared, and 200 μL of the virus suspension was dispensed into this tube C1, and rotated at 37 ° C. for 1 hour.

  <4B> Next, the dispersion liquid in each tube was centrifuged at 1500 rpm for 5 minutes, and then a part of the supernatant in each tube was collected. And about each supernatant, the detection test of influenza virus was done using hemagglutination reaction.

  The hemagglutination reaction is a virus detection method using a reaction in which a virus binds to a sugar chain present on the surface of erythrocytes.

  More specifically, when erythrocytes are added to a well of a U-bottom microplate containing a virus-containing solution, the red blood cells spread in a sheet form via the virus and settle to the whole bottom of the well. is there. In wells without virus, red blood cells aggregate in the center of the bottom of the well.

  Therefore, it can be judged as “negative” when red blood cells aggregate at the bottom of the well, and “positive” when red blood cell aggregation is not observed or very slight even if it is observed. . In addition, the concentration of the virus can be estimated by diluting the concentration of the solution containing the virus stepwise and observing the presence or absence of the hemagglutination reaction.

  In this example, six rows of wells are used corresponding to the first to fifth tubes and the tube C1, and the supernatant of the stock solution in each tube is added to each well constituting each row. A diluted supernatant obtained by doubling the supernatant was injected, and “negative” and “positive” were determined.

  For each tube, the maximum dilution ratio of the supernatant determined as “positive” is shown in Table 1, and the relationship between the carrier content and the maximum dilution ratio of the supernatant determined as “positive” is shown in FIG. In addition, it means that there is much quantity of the influenza virus contained in a supernatant, so that this maximum dilution rate is large.

  As shown in Table 1 and FIG. 4, the maximum dilution ratio determined as “positive” was smaller in the tube having a higher carrier content. That is, the content of influenza virus contained in the supernatant decreased. This is because the carrier has a function of attaching influenza virus, and the more the carrier content, the more influenza virus is attached to the carrier. It is thought that the amount of virus is decreasing.

(2-4) Examination of virus release performance of recovered solution <1C> First, each tube (first tube to fifth tube) from which a part of the supernatant was collected in step <4B> of (2-3) above. The remaining carrier dispersion in) was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded using an aspirator.

  <2C> Next, 200 μL of casein solution (concentration 300 μg / mL) is dispensed into each tube at a time, and then rotated at 37 ° C. for 1 hour to bring the casein into contact with the carrier to which the influenza virus is attached. It was.

  <3C> Next, after centrifuging the carrier dispersion in each tube at 1500 rpm for 5 minutes, a part of the supernatant in each tube is collected and the same as described in the above step <4B>. An influenza virus detection test was conducted.

  For each tube, the maximum dilution ratio of the supernatant determined as “positive” is shown in Table 2, and the relationship between the carrier content and the maximum dilution ratio of the supernatant determined as “positive” is shown in FIG.

  As shown in Table 2 and FIG. 5, the maximum dilution factor that was determined as “positive” increased as the content of the carrier increased. That is, the content of influenza virus contained in the supernatant increased. This suggests that casein has a function of releasing influenza virus adhering to the carrier, and the amount of influenza virus released into the supernatant increases as the carrier content increases. .

3. Examination using dengue virus (3-1) Preparation of virus suspension (liquid to be treated) Mixing four inactivated serotype dengue viruses (dengue type 1, dengue type 2, dengue type 3, dengue type 4) The resulting virus solution (186 μg / mL) was diluted with PBS to prepare a 25 μg / mL dengue virus suspension.

(3-2) Preparation of carrier dispersion A carrier dispersion (5%) was prepared in the same manner as in the step (2-2) except that the concentration of the carrier contained in the carrier dispersion was adjusted to 5%. A carrier dispersion) was obtained.

(3-3) Examination of virus attachment performance of carrier <1D> First, using a method similar to that described in (2-3) above, each tube ( In the sixth tube to the fourteenth tube), the carrier concentrations are 5 wt%, 2.5 wt%, 1.25 wt%, 0.63 wt%, 0.32 wt%, 0.15 wt%, 0.08 wt%, respectively. 0.04 wt% and 0.02 wt% carrier dispersion liquid (200 μL) was obtained, followed by centrifugation at 1500 rpm for 5 minutes to remove the supernatant.

  <2D> Next, 200 μL of the virus suspension was dispensed into each of the sixth to fourteenth tubes, and then the dengue virus was attached to the carrier by rotating at 4 ° C. for 1 hour.

  Separately, 2 mL Eppendorf tubes for control (tube C2 and tube C3) were prepared, and 200 μL of the dengue virus suspension was dispensed into these tubes. And tube C2 was left still at 37 degreeC for 1 hour, and tube C3 rotated at 37 degreeC for 1 hour.

  <3D> Next, the dispersion liquid in each tube was centrifuged at 1500 rpm for 5 minutes, and then a part of the supernatant in each tube was collected. Each supernatant was subjected to a dengue virus detection test using the hemagglutination method.

  For each tube (sixth to fourteenth tubes, tubes C2 and C3), the maximum dilution ratio of the supernatant determined to be “positive” is shown in Table 3, and the carrier content and “positive” were determined. FIG. 6 shows the relationship between the maximum dilution ratio of Qing. In addition, it means that the amount of dengue virus contained in a supernatant is so large that this maximum dilution rate is large.

  As shown in Table 3 and FIG. 6, the maximum dilution rate determined as “positive” was smaller for tubes with a higher carrier content. That is, the content of dengue virus contained in the supernatant decreased. This means that the carrier has a function of attaching dengue virus, and the amount of dengue virus attached to the carrier increases as the carrier content increases, so the amount of dengue virus contained in the supernatant accordingly. Seems to be decreasing.

(3-4) Examination of virus release performance of recovered solution <1E> First, each tube (sixth tube to fourteenth tube) from which a part of the supernatant was collected in step <3D> of (3-3) above. The remaining carrier dispersion in) was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded using an aspirator.

  <2E> Next, 200 μL of casein solution (concentration: 300 μg / mL) was dispensed into each tube, and then rotated at 37 ° C. for 1 hour to bring the casein into contact with the carrier to which the dengue virus was attached. .

  <3E> Next, after centrifuging the carrier dispersion in each tube at 1500 rpm for 5 minutes, a part of the supernatant in each tube is collected and the same as described in the above step <4B>. Dengue virus detection test was conducted.

  For each tube, the maximum dilution ratio of the supernatant determined as “positive” is shown in Table 4, and the relationship between the carrier content and the maximum dilution ratio of the supernatant determined as “positive” is shown in FIG.

  As shown in Table 4 and FIG. 7, it was confirmed that the virus adsorbed on the carrier was released into the solution in the tube having the carrier concentration exceeding 1.25 wt%. In addition, it is considered that virus was released even in a tube having a carrier concentration of 0.63 wt% or less, but it was considered that the released virus could not be detected due to the small number of carriers.

  In addition, the amount of virus released is maximized at a carrier concentration of 1.25 wt%, and the amount of virus released decreases as the carrier concentration increases. This is probably because the amount of the carrier increases due to the large amount of the carrier, and the site where casein can bind to the binding site of the carrier surface without pushing out the virus has appeared.

  From the above results, the carrier whose surface is composed of a calcium phosphate compound has a function of attaching various viruses, and the virus attached to the carrier has a high affinity for a carrier such as casein. It was found that it can be recovered by protein release.

It is sectional drawing which shows an example of the support | carrier used by this invention. It is process drawing which shows typically the cell or virus collection | recovery method of this embodiment. It is process drawing which shows typically the cell or virus collection | recovery method of this embodiment. It is a figure which shows the relationship between the content of a support | carrier, and the maximum dilution rate of the supernatant in which the influenza virus was determined to be "positive" after making an influenza virus contact a support | carrier. It is a figure which shows the relationship between the content of a support | carrier, and the maximum dilution rate of the supernatant in which the influenza virus was determined to be "positive", after contacting casein with a support | carrier. It is a figure which shows the relationship between the content of a support | carrier, and the maximum dilution ratio of the supernatant determined to be a dengue virus "positive" after making a dengue virus contact a support | carrier. It is a figure which shows the relationship between the content of a support | carrier, and the maximum dilution rate of the supernatant determined to be dengue virus "positive" after contacting casein with a support | carrier.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Carrier 11 Base 12 Calcium phosphate compound layer 13 Particle 2 Cell / virus 3 Cell / virus attachment carrier 100 Container 200 Liquid to be treated 300 Washing liquid 400 Recovery liquid 500 Cleaning liquid 600

Claims (15)

A cell or virus recovery method for recovering cells or viruses attached to the surface of a carrier composed mainly of a calcium phosphate compound at least near the surface, by separating the cells or viruses from the carrier,
Contacting the carrier to which the cells or viruses are attached with a recovery solution containing a protein having a higher affinity for the carrier than the cells or viruses, thereby removing the cells or viruses from the carrier and collecting them. A method for recovering a characteristic cell or virus. A cell or virus recovery method for recovering cells or viruses attached to the surface of a carrier composed mainly of a calcium phosphate compound at least near the surface, by separating the cells or viruses from the carrier,
The cell or virus is further removed from the carrier by bringing the recovered solution containing the protein into contact with the carrier to which the cell or virus and a protein having a higher affinity for the carrier than the cell or virus are attached. A method for recovering cells or viruses, comprising separating and recovering cells.   The method for recovering cells or viruses according to claim 1 or 2, wherein the protein is a metalloprotein.   The method for recovering cells or viruses according to claim 3, wherein the metalloprotein is casein.   The method for recovering cells or viruses according to any one of claims 1 to 4, wherein the concentration of the protein in the recovery solution is 0.1 to 100 mg / mL.   The method for recovering cells or viruses according to any one of claims 1 to 5, wherein the time for contacting the recovery liquid with the carrier is 1 to 15 minutes.   The method for recovering cells or viruses according to any one of claims 1 to 6, wherein the temperature of the recovery liquid when the recovery liquid is brought into contact with the carrier is 2 to 40 ° C.   The method for recovering cells or viruses according to any one of claims 1 to 7, wherein when the recovery liquid is brought into contact with the carrier, vibration or impact is applied to the carrier.   The method for recovering cells or viruses according to any one of claims 1 to 8, wherein the treatment of bringing the recovery solution into contact with the carrier is performed a plurality of times.   The method for recovering cells or viruses according to any one of claims 1 to 9, wherein the recovery solution is substantially free of degrading enzymes and chelating agents.   The cell or virus recovery method according to any one of claims 1 to 10, wherein the carrier to which the cells or viruses are attached is washed at least once prior to the treatment of bringing the recovery solution into contact with the carrier.   The method for recovering cells or viruses according to claim 11, wherein the washing solution used for washing is an isotonic solution.   The method for recovering cells or viruses according to any one of claims 1 to 12, wherein the carrier is granular.   14. The method for recovering cells or viruses according to claim 13, wherein the average particle size of the granular carrier is 1-1000 μm.   The method for recovering cells or viruses according to any one of claims 1 to 14, wherein the calcium phosphate compound is mainly composed of hydroxyapatite.

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