JP4484790B2 - Cell adsorption material - Google Patents

Description

  The present invention relates to a cell adsorbent and a cell recovery method using the same. The cell adsorbent of the present invention can be used as a basic scientific experimental instrument or a medical instrument in clinical medicine. Furthermore, this invention relates to the screening method of the metal seed | species for using with the said cell adsorption material.

  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 have safety problems 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 surface of a stem cell membrane 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 only in terms of the separation of T lymphocytes. This technique can only be applied to T lymphocytes and is not versatile for other cell separations.

  By the way, in the field of protein separation, separation techniques by liquid chromatography using various packing materials have been developed. Gel filtration chromatography, which is size separation, and normal phase / reverse phase chromatography using a hydrophilic / hydrophobic interaction. There are two types: chromatography, ion exchange chromatography utilizing the difference in electrical properties, and affinity chromatography utilizing the difference in biological specificity. Here, as in the case of cell separation, some affinity chromatography uses an antigen-antibody reaction by immobilizing an antibody on a carrier, but in recent years, attention has been focused on the interaction between metal and protein. This is immobilized metal chromatography utilizing the action (for example, Non-Patent Document 2). More recently, attempts have been made to apply this principle to cell separation. Non-Patent Document 3 discloses that a material in which copper is fixed to polyacrylamide via iminodiacetic acid can be separated from a mixture of Gram-negative bacteria (E. Coli) and Gram-positive bacteria (B. halodurans). Yes. However, this document is limited to the separation of bacteria, so-called microbial cells, and there is no description about the separation and recovery of useful cells in organs and tissues of animals (including humans). Furthermore, the only metal used is copper, and it is not taught to screen for various metals. In addition, the patent on which Non-Patent Document 3 is based is considered to be Patent Document 3, but this patent document is the main technical idea of an optimal carrier manufacturing method. There is no description of separation and recovery of useful cells in organs and tissues (including), and screening of various metals is not taught.

JP 08-033708 Japanese Patent Laid-Open No. 02-227070 Special Table 2005-504972 Supervised by Yoshito Tsuji, the latest in regenerative medicine engineering, CMC Publishing, 2002 Porath, J. et al., "Metal chelate affinity chromatography, a new approach to protein fractionation", Nature, December 1975, vol. 258, p598-599 Dainiak, MB. Et al., “Cell Chromatography: Separation of Different Microbial Cells Using IMAC Supermacroporous Monolithic Columns”, Biotechnol. Prog., February 2005, vol. 21, 644-649

  An object of the present invention is to provide a cell adsorbent for separating and recovering useful cells in animal (including human) organs and tissues and removing unnecessary cells in a safe, simple and low cost, and a cell recovery method using the same Is to provide. Another object of the present invention is to provide a screening method for metal species used in the cell separation material of the present invention described above.

  The present inventors proceeded diligently to solve the above-mentioned problems. First, it was decided in advance that biological substances such as antibodies having safety problems should not be used, and many studies have been conducted in the field of cell separation. There is a risk that organic substances such as amino acids, peptides, nucleic acids, and carbohydrates cannot solve the problem of low cost, so further exploration from the viewpoint of low cost, although there is a track record as immobilized metal affinity chromatography in protein separation We focused on metals that have hardly been studied for cell separation. Furthermore, in immobilized metal affinity chromatography for protein separation, nickel and cobalt (both of which are commercially available) are often used as metals, and zinc and copper are rarely used. Decided to remove these limitations and study a wide range of metal species. Further, the present invention has been completed by devising a method for selecting a suitable metal species and examining various metals using the method.

  That is, according to the present invention, there is provided a cell adsorbent comprising a water-insoluble carrier and a metal, wherein the metal is present on the surface of the water-insoluble carrier.

Preferably the metal is a heavy metal.
Preferably, the cells to be adsorbed are pancreatic cells.
Preferably, the metal is at least one metal selected from aluminum, titanium, chromium, iron, cobalt, copper, zinc, silver, indium, tin, and lanthanide.

Preferably, the metal present on the surface of the water-insoluble carrier is a metal having an adsorption rate to cells of 50% or more.
Preferably, the metal present on the surface of the water-insoluble carrier is a metal in which the ratio of the adsorption rate to the cells to be separated and recovered and the adsorption rate to the cells to be separated and removed is 2 or more.
Preferably, the metal present on the surface of the water-insoluble carrier is a metal having a ratio of the adsorption rate to the cells to be separated and recovered and the adsorption rate to the cells to be separated and removed of 0.5 or less.

  According to another aspect of the present invention, the method includes a step of bringing the cell adsorbent of the present invention into contact with a cell, and a step of recovering cells adsorbed on the cell adsorbent or cells not adsorbed on the cell adsorbent. A method for recovering cells is provided.

  According to still another aspect of the present invention, screening of metal species for use in a cell adsorbent comprising a water-insoluble carrier and a metal, wherein the metal is present at least on the surface of the water-insoluble carrier. A method comprising: (1) a step of determining one or a plurality of cells to be adsorbed on a cell adsorbent; and (2) contacting the cells determined in the step (1) with a metal ion-containing liquid, There is provided the above screening method comprising a step of selecting an affinity metal.

  Preferably, in the step (2), a metal having an adsorption rate of 50% or more is selected as a metal having affinity for cells.

  According to still another aspect of the present invention, (1) a step of determining a cell to be separated and recovered by a cell separation material and a cell to be separated and removed, and (2) a metal ion-containing liquid comprising the cell determined in the step (1) There is provided a method for screening a metal species used for a cell separation material, which comprises a step of selecting a metal having a difference in adsorption rate to cells to be separated and recovered and adsorption rate to cells to be separated and removed.

  Preferably, in the step (2), adsorption of a metal having a ratio of the adsorption rate to the cell to be separated and recovered to the cell to be separated and removed to a cell to be separated and recovered is 0.5 or less. Select a metal that has a difference in rate and the rate of adsorption to cells to be separated and removed.

  According to the cell adsorbent of the present invention and the method for recovering cells using the same, it is possible to separate and recover useful cells from animal (including human) organs and tissues and remove unnecessary cells safely, simply and at low cost. Can do. Moreover, according to the screening method of the metal seed | species used for the cell separation material of this invention, the metal seed | species suitable for using for the cell adsorption material of this invention can be screened simply.

The present invention will be specifically described below.
The cell adsorbent of the present invention comprises a water-insoluble carrier and a metal, and at least the metal is present on the surface of the water-insoluble carrier. Here, the water-insoluble carrier is substantially insoluble in water, and examples thereof include a polymer material, an inorganic material, and a metal. Examples of polymer materials include polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane and other synthetic polymers (including not only single polymers but also polymer alloys), agarose, cellulose, cellulose acetate, And natural polymers such as chitin, chitosan, alginic acid, pectin (including their respective derivatives). Examples of inorganic materials include hydroxyapatite, fluoroapatite, glasses of various compositions, alumina, titania, zirconia, silica, carbon, etc. (including not only a single material but also a mixture). Examples of the metal include stainless steel, titanium, tantalum, gold, platinum, and various alloys having various compositions.

  In the cell adsorbent of the present invention, metal is present on the surface of the water-insoluble carrier. The metal to be present on the surface of the water-insoluble carrier is preferably selected by a screening method to be described later. However, from the viewpoint of ease of preparation (eg, immobilization on a water-insoluble carrier, eg, alkali metal is difficult to immobilize). It is preferably a heavy metal, and more preferable in terms of cost, availability, and safety are aluminum, titanium, chromium, iron, cobalt, copper, zinc, silver, indium, tin, lanthanide (lanthanum). (The present inventors have found that these metals are particularly preferred in cells derived from the pancreas). Moreover, the metal to be used is not limited to one type, and a plurality of metals may be used. However, in this case, the preparation of the cell adsorbing material is appropriately selected in consideration of the effect because it is somewhat troublesome as compared with the case of using one kind of metal.

  In the cell-adsorbing material of the present invention, metal is present on at least the surface of the water-insoluble carrier, but the metal may or may not be present in a portion other than the surface of the water-insoluble carrier. Good. Examples of the method for allowing the metal to be present on the surface of the water-insoluble carrier include physical methods such as vapor deposition and sputtering, and methods using chemical reactions. Hereinafter, methods using chemical reactions will be described in more detail. As a method by chemical reaction, (1) a method for bonding a metal after binding a metal to the surface of a water-insoluble carrier by some method, and (2) adding a metal when preparing a water-insoluble carrier. And (3) a method of bringing a water-insoluble carrier containing another metal into contact with a desired metal ion-containing liquid. As an example of (1), a chelating group having a binding property to a metal such as iminodiacetic acid is immobilized on the surface of a water-insoluble carrier by graft polymerization or the like. The method of fixing a metal by making it contact is mentioned. Examples of (2) include a method in which a sodium alginate aqueous solution is reacted with various metal salt aqueous solutions to form a water-insoluble carrier containing the metal, or a known hydroxyapatite wet synthesis method using a calcium hydroxide suspension and a phosphoric acid aqueous solution. In which a desired metal compound is allowed to coexist in a calcium hydroxide suspension. As an example of (3), a desired metal ion aqueous solution is brought into contact with hydroxyapatite, and calcium in hydroxyapatite is substituted with a desired metal.

  Further, at least the metal itself present on the surface may be a water-insoluble carrier. In other words, the cell adsorbent may be made of the metal itself present on the surface (in this case, there is a single metal that is not water-insoluble, and even if it is water-insoluble, there are other factors in terms of formability and cost). It is slightly disadvantageous compared to the method). The present inventors consider that the method of reacting a sodium alginate aqueous solution or a pectin aqueous solution with various metal salt aqueous solutions is most preferable because it is simple and low cost.

  The cell adsorbent referred to in the present invention is a material that adsorbs cells for some purpose such as cell removal, cell concentration, and cell recovery, but the cells referred to here are cells present in human or animal organs or tissues, or This refers to those with some kind of manipulation (differentiation induction, gene transfer, etc.) and includes not only living cells but also dead cells. Examples of cells include, but are not limited to: 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, neurons, 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. Organs / tissues are various organs such as pancreas, liver, kidney, heart, stomach, intestine, spleen, etc., and solid organs / tissues such as muscle, blood vessels, bones and skin (often these are cells that are released by enzymatic digestion. ) As well as bone marrow that is liquid, umbilical cord blood (including not only cord blood but also collected from placental blood vessels), peripheral blood (those collected by administering hematopoietic factors such as granulocyte colony-stimulating factor) (Including)).

  Examples of the shape of the cell adsorbent of the present invention include flat membranes, particles, meshes, woven fabrics, non-woven fabrics, fiber clusters (short fibers, long fibers), sponge-like structures, particle aggregates, etc. A laminated body, a bag shape, a three-dimensional molded body (block shape) or the like is housed in a container having a liquid inlet and a liquid outlet and used as a cell separation filter.

  In the screening method of the metal species used for the cell adsorbent according to the present invention, first, cells to be adsorbed on the cell adsorbent are determined, then the cells are brought into contact with a metal ion-containing liquid, and a metal having affinity with the cells ( Alternatively, the cells to be contacted may be living cells or cells fixed by a known fixing method such as glutaraldehyde or paraformaldehyde. The advantage of using living cells is that a complicated fixing operation is not required, but it is affected by the physiological activity of the cells. That is, there are problems such as damage to cells by the metal salt aqueous solution to be used (cytotoxicity of the element itself, pH of the aqueous solution, etc.), and it has no affinity but is taken into the cells. In addition, fixed cells have a problem in living cells, and have the advantage that they are not affected by the physiological activity of the cells, but there is a problem that the fixing operation is complicated. Therefore, it is preferable to make a comprehensive judgment by performing both live cells and fixed cells. In the screening method of the present invention, a plurality of types of metal ion-containing liquids are used as the metal ion-containing liquid. In the present invention, the cell and the metal ion-containing liquid are brought into contact with each other by adding one type of metal ion-containing liquid to one container containing cells. Therefore, in order to screen a plurality of types of metal ion-containing liquids, for example, a plurality of containers containing cells are prepared in advance, and the above-described screening is performed by adding one type of metal ion-containing liquid to each container. Or screening by pipetting a certain amount of cell fluid in one container and adding it to multiple containers containing one type of metal salt solution. Also good.

As a method for selecting a metal having affinity for cells, a predetermined number of cells are brought into contact with a metal ion-containing liquid having a predetermined concentration, and various quantitative analysis methods (atomic absorption analysis) are used to determine the metal concentration before contact and the metal concentration after contact. Quantification by a method, ICP emission or mass spectrometry, ion chromatography, etc.) and 100− (100 × concentration after contact / concentration before contact) = adsorption rate may be judged by the magnitude of the adsorption rate. The concentration of the metal ion-containing liquid used here is not particularly limited, but is generally about 0.01 ppm to 100 ppm, preferably about 0.1 ppm to 10 ppm, more preferably 0.5 ppm to 5 ppm. is there. The number of cells used here is not particularly limited, but is generally 1 × 10 ⁴ to 1 × 10 ⁸ cells / ml, preferably 1 × 10 ⁵ to 1 × 10 ⁷ cells / ml. More preferably, it is 5 × 10 ^{5 to} 5 × 10 ⁶ pieces / ml. In general, those having an adsorption rate calculated by this formula of 50% or more can be selected.

In addition, for example, when one of two different types of cells is to be collected and one of them is to be removed (in this case, it will be called a cell separation material instead of a cell adsorbent), each cell is made into various metal ion-containing liquids. Contacting each other, calculating the adsorption rate, and evaluating by the ratio of the adsorption rates. In this case, as the separation format, the cells to be collected are adsorbed, the cells to be removed are allowed to pass without being adsorbed, and then the cells to be collected to be adsorbed are collected by some method and the cells to be removed are adsorbed, Since both of the cells to be collected are collected by passing, the adsorption ratio can be selected to be 2 or more or 0.5 or less.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1
This example shows screening of metal species having high adsorptivity to pancreatic endocrine cells.

(1) Cell sample Using the rat pancreatic endocrine cell line RIN-5F as it is (“live cells”) and Becton Dickinson's cell fixative (trade name Cellfix. 1% paraformaldehyde is the main component) Therefore, fixed cells (“fixed cells”) were used as cell samples.

(2) Metal ion-containing solution A metal standard solution (concentration: 1000 ppm) manufactured by Wako Pure Chemical Industries, Ltd. was used. The composition of the stock solution is as shown in Table 1.

(3) Cell-metal ion adsorption experiment A cell suspension obtained by suspending the cells of (1) in physiological saline and a metal ion-containing solution of (2) (neutral pH with 0.01 N aqueous sodium hydroxide solution) 2 × 10 ⁶ cells in 2 ml of 1 ppm metal solution, left at 37 ° C. for 1 hour, filtered through a 0.22 μm filter, It was set as the solution after contact. In addition, the same operation was performed with physiological saline in the absence of cells to obtain a “pre-contact solution”. The same operation was performed with acrylic resin particles (manufactured by Soken Chemical Co., Ltd., particle size 10 μm) considered to have no metal ion adsorption as a comparative control.

(4) Measurement The metal concentration of the solution before contact and the solution after contact was quantified by plasma emission analysis, and the adsorption rate was calculated from the formula: 100− (100 × concentration after contact / concentration before contact) = adsorption rate.

(5) Results Table 2 shows the results of the adsorption rate of each metal. It can be seen that the adsorption rate of copper, silver, lanthanum, cerium, and lutetium to cells is extremely high.

Example 2
This example shows screening of metal species having selectivity between pancreatic endocrine cells and pancreatic exocrine cells.
(1) Cell sample In addition to the rat pancreatic endocrine cell line RIN-5F used in Example 1, the rat pancreatic exocrine cell line AR42-J was used.

(2) Metal ion-containing solution A metal standard solution (concentration: 1000 ppm) manufactured by Wako Pure Chemical Industries, Ltd. was used. The composition of the stock solution is shown in Table 3.

(3) Cell-metal ion adsorption experiment The same operation as in Example 1 was performed. However, acrylic resin particles were not used.

(4) Measurement The same operation as in Example 1 was performed, but the selectivity was calculated from the following formula as an index of selectivity: endocrine cell line adsorption rate / exocrine cell line adsorption rate = selectivity.

(5) Results Table 4 shows the results of the selectivity of each metal. It can be seen that the selectivity of cobalt, zinc, iron, indium, tin, aluminum, and titanium is extremely high.

Example 3
In this example, a method for producing a cell adsorbent using sodium alginate is shown.
(1) Preparation of sodium alginate solution Sodium alginate (Kimika Co., Ltd., trade name I-1) was dissolved in purified water to give a 1 wt% solution.

(2) Preparation of metal salt solution The following metal salts (all manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in purified water to prepare a 0.2M aqueous solution.
Zinc chloride, copper chloride dihydrate, lanthanum chloride heptahydrate, cerium chloride heptahydrate, cobalt chloride hexahydrate, magnesium chloride hexahydrate

(3) Preparation of Cell Adsorbent 5 ml of the sodium alginate solution prepared in (1) was placed in a 20 ml disposable syringe (Terumo) with an 18 gauge blunt needle (JMS) and prepared in (2). It was dripped by pushing by hand into a 50 ml beaker containing 20 ml of 2M metal salt aqueous solution.

(4) Results Zinc chloride, lanthanum chloride heptahydrate, cerium chloride heptahydrate: White spherical particles were instantaneously formed after the sodium alginate droplets contacted the metal salt aqueous solution.
Copper chloride dihydrate: After contact of the sodium alginate droplets with the metal salt aqueous solution, light blue spherical particles were instantaneously formed.
Cobalt chloride hexahydrate: After the droplets of sodium alginate contacted the metal salt aqueous solution, light purple spherical particles were instantly formed.
Magnesium chloride hexahydrate: No change occurred when sodium alginate droplets contacted the metal salt aqueous solution. Although the sodium alginate concentration was increased to 3% and the metal salt concentration was increased to 2M, the viscosity of the solution was only slightly increased, and there was no formation of spherical particles like other metals.
In the case of producing spherical particles, the particles were taken out from the solution, put into purified water for washing, and gently stirred for one day and night with a magnetic stirrer to obtain a cell adsorbent.

  Although this method could produce a spherical cell adsorbent very easily at low cost, it could not be applied to magnesium chloride.

Example 4
In this example, a method for producing a cell adsorbent using pectin is shown.
(1) Preparation of pectin solution Low-methoxylated pectin (manufactured by Sanki Co., Ltd., product GENU pectin type LM-102AS-J, esterification degree: about 34%) was dissolved in purified water to obtain a 4 wt% aqueous solution.

(2) Preparation of metal salt solution The following metal salts (all manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in purified water to prepare a 0.2M aqueous solution.
Zinc chloride, copper chloride dihydrate, lanthanum chloride heptahydrate, cerium chloride heptahydrate, cobalt chloride hexahydrate, magnesium chloride hexahydrate

(3) Preparation of Cell Adsorbent 5 ml of the low methoxylated pectin solution prepared in (1) was placed in a 20 ml disposable syringe (manufactured by Terumo) with an 18 gauge blunt needle (manufactured by JMS) and prepared in (2). It was dripped by pushing by hand into a 50 ml beaker containing 20 ml of 0.2M metal salt aqueous solution.

(4) Results Zinc chloride, lanthanum chloride heptahydrate, cerium chloride heptahydrate: White spherical particles were instantaneously formed after the low-methoxylated pectin droplets contacted the metal salt aqueous solution.
Copper chloride dihydrate: After the low methoxylated pectin droplets contacted the metal salt aqueous solution, light blue spherical particles were instantly formed.
Cobalt chloride hexahydrate: After the low methoxylated pectin droplets contacted the aqueous metal salt solution, light purple spherical particles were instantly formed.
Magnesium chloride hexahydrate: No change occurred when the low-methoxylated pectin droplets contacted the aqueous metal salt solution. Although the low methoxylated pectin concentration was increased to 5% and the metal salt concentration was increased to 2M, the viscosity of the solution was only slightly increased, and there was no formation of spherical particles like other metals.
In the case of producing spherical particles, the particles were taken out from the solution, put into purified water for washing, and gently stirred for one day and night with a magnetic stirrer to obtain a cell adsorbent.

  Although this method could produce a spherical cell adsorbent very easily at low cost, it could not be applied to magnesium chloride.

Example 5
In this example, a method for producing a cell adsorbent using agarose beads on which iminodiacetic acid is immobilized is shown.
(1) Carrier Amersham Bioscience Co., Ltd. Chelating Sepharose FF (iminodiacetic acid immobilized on agarose beads) was used.

(2) Preparation of metal salt solution The same metal salt solution as in Example 3 was prepared.

(3) Preparation of cell adsorbent The carrier of (1) was put into the metal salt solution of (2) and gently stirred for one day with a magnetic stirrer. Thereafter, as washing, the beads were settled, the supernatant was replaced with purified water, and the mixture was stirred with a magnetic stirrer. This was repeated 4 times every 2 hours to obtain a cell adsorbent.

Example 6
This example shows a method for producing a cell adsorbent using agarose beads on which iminodiacetic acid is immobilized and a method for using the same.
(1) Manufacture of cell adsorbent 0.1M copper sulfate pentahydrate on Amersham Biosciences HiTrap Chelating HP (1 ml column filled with agarose beads with an average particle size of about 34 μm fixed with imidinoacetic acid) 4 ml of the aqueous solution was injected into the column using a 5 ml disposable syringe (manufactured by Terumo). After injecting all the copper sulfate aqueous solutions, 5 ml of Kyoto Solution (registered trademark, manufactured by Kyoto Biomedical Science) was passed through the same as a washing. Thereafter, a portion of about 5 mm from the column outlet was cut using an ultrasonic cutter, a 5 ml syringe containing 5 ml of Kyoto solution was attached to the column inlet, and beads inside the column were collected by pushing the plunger of the syringe.

(2) Cell suspension The cell suspension was obtained by suspending the rat endocrine cell line RIN-5F in Kyoto Solution at a concentration of 1.4 × 10 ⁶ cells / ml.

(3) Cell adsorption experiment 10 ⁶ cell adsorbents prepared in (1) (suspended in 3 ml Kyoto solution) and 3 ml of cell suspension (that is, 4.2 × as the absolute number of cells) 10 ⁶⁾ suspended cells polystyrene culture dishes (Corning Inc., was added to a diameter 6 cm), it was adsorbed by allowing it to stand 30 minutes at room temperature, in air.

(4) Results The state of cell adsorption to the cell adsorbent was observed with an optical microscope. A photograph is shown in FIG. The large circle is the cell adsorbent, and the small circle is the cell. It is observed that a large number of cells are adsorbed on the cell adsorbent, and the cell adsorbent is adsorbed there again to form a cluster.

Comparative Example This comparative example is compared with Example 6 by using a cell adsorbent that does not fix copper.
(1) Production of cell adsorbent The same operation as in Example 6 was performed except that the aqueous copper sulfate solution was not passed.
(2) Cell suspension The same cell suspension as in Example 6 was used.

(3) Cell adsorption experiment It carried out by the method similar to Example 6. FIG.
(4) Results The state of cell adsorption to the cell adsorbent was observed with an optical microscope. A photograph is shown in FIG. The cells were not adsorbed on the cell adsorbent, and the effect of Example 6 on the cell adsorption of copper was confirmed.

  The cell adsorbent according to the present invention can be used as it is or in a container and used as a cell separation filter in laboratory instruments and clinical medicine in basic science. Cells obtained using this cell-adsorbing material are subjected to further separation and purification (including washing), culture, activation, amplification, gene transfer, cryopreservation, etc., as they are or as necessary. It can be used for basic science experiments such as transplantation to living bodies, cell biology and immunology.

FIG. 1 shows the result of observation of the adsorption state of cells on the cell adsorbent of the present invention with an optical microscope. FIG. 2 shows the result of observation of the adsorption state of the cells on the cell adsorbent of the comparative example with an optical microscope.

Claims (14)

A cell adsorbent comprising a water-insoluble carrier and a metal, wherein the metal is present on the surface of the water-insoluble carrier. The cell adsorbent according to claim 1, wherein the metal is a heavy metal. The cell adsorbent according to claim 1 or 2, wherein the cells to be adsorbed are cells derived from the pancreas. The cell adsorbent according to claim 3, wherein the metal is one or more metals selected from aluminum, titanium, chromium, iron, cobalt, copper, zinc, silver, indium, tin, and lanthanide. The cell adsorbent according to any one of claims 1 to 4, wherein the metal present on the surface of the water-insoluble carrier is a metal having an adsorption rate to cells of 50% or more. The metal present on the surface of the water-insoluble carrier is a metal having a ratio of the adsorption rate to the cells to be separated and recovered to the adsorption rate to the cells to be separated and removed is 2 or more. Cell adsorption material. 5. The metal according to claim 1, wherein the metal present on the surface of the water-insoluble carrier is a metal having a ratio of the adsorption rate to the cells to be separated and recovered and the adsorption rate to the cells to be separated and removed of 0.5 or less. The cell adsorbent described in 1. Cell recovery comprising the step of bringing the cell adsorbent according to any one of claims 1 to 4 into contact with a cell, and the step of recovering cells adsorbed on the cell adsorbent or cells not adsorbed on the cell adsorbent. Method. A method for recovering cells, comprising a step of bringing the cell adsorbent according to claim 5 or 6 into contact with cells, and a step of recovering cells adsorbed on the cell adsorbent. A method for recovering cells, comprising the step of bringing the cell adsorbent according to claim 7 into contact with cells, and the step of recovering cells that have not been adsorbed to the cell adsorbent. A cell-adsorbing material comprising a water-insoluble carrier and a metal, and a screening method for metal species for use in a cell-adsorbing material in which metal is present on at least the surface of the water-insoluble carrier, comprising: (1) a cell-adsorbing material A step of determining one or a plurality of cells to be adsorbed on the cell, and (2) a step of bringing the cell determined in the step (1) into contact with a metal ion-containing liquid and selecting a metal having an affinity for the cell. A screening method as described above. The method according to claim 11, wherein in the step (2), a metal having an adsorption rate of 50% or more is selected as a metal having affinity for cells. (1) a step of determining a cell to be separated and recovered by a cell separation material and a cell to be separated and removed, and (2) contacting the cell determined in the step (1) with a metal ion-containing liquid and separating and collecting the cell. A method for screening a metal species used for a cell separation material, comprising a step of selecting a metal having a difference in rate and an adsorption rate to a cell to be separated and removed. In the step (2), the ratio of the adsorption rate to the cells to be separated and recovered and the adsorption rate to the cells to be separated and removed is 2 or 0.5 or less. The method according to claim 13, wherein the metal is selected as a metal having a difference in adsorption rate to cells to be removed.