JP2018153097A - Cell culture vessel, device, and separation recovery method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture vessel, device, and separation recovery method which improve the quality of cultured cells and reduces complications in subculture operations.SOLUTION: A method of efficiently separating and recovering cells involves culturing cells using a cell culture vessel that uses, secured to a support body 1, a separation recovery film 10 of a thin film 2 comprising a pH/temperature responsive polymer the hydrophilicity 2c and hydrophobicity 2a of which changes in response to the temperature and pH due temperature and pH responsive sites therein and comprises: a pH/temperature changing step in which the pH and temperature of the separation recovery film 10 are changed; and a cell recovery step of supplying film treatment liquid to the separation recovery membrane 10 the pH and temperature of which were changed in the pH/temperature changing step, recovering the film treatment liquid that was supplied by a cell recovery device, and recovering cells 20 adsorbed to the cell binding site 2b.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a cell culture container, and more particularly, to a culture technique for easily collecting adherent cells and the like.

  Cell culture is the separation of cells from living tissue and the growth and maintenance of the cells outside the body, and is used for applications such as basic biological research, biopharmaceutical production, and regenerative medicine. The period from separation from the living body to the first replanting is called primary culture, and the transfer of existing cultured cells to a new culture vessel to proliferate and maintain is called subculture. Cultured cells can be broadly classified into two forms. Cells that adhere to and proliferate in culture vessels are called adherent cells, or adherent cells, and cultured cells that proliferate in a floating state in a culture medium. Call it.

  In order to grow such cells, the cells are seeded at a low density and grown by cell division, before nutrient components in the culture are depleted or before cell growth stops due to accumulation of cell secretions. Then, it is necessary to dilute and re-seed the cells in a new culture vessel by passage. In this subculture work, there is a risk of contamination of biological factors other than the intended biological factor, that is, contamination risk, and a clean bench and sterilized reagents and instruments that can be aseptically operated to prevent it, as well as skilled work Operation by personnel is required.

  In the subculture process in the adherent cells, for example, the old medium is removed, the cell surface attached to the culture vessel is washed with a solution such as PBS (−), and the solution is discarded. Repeat this procedure several times, then wash the cell surface with 0.25% trypsin, immediately remove it, drop a drop, let stand for a few minutes, tap the dish horizontally to peel off the cells. Then, after confirming that it has been peeled off with a microscope, a serum-containing medium is added, trypsin activity is stopped and pipetting is performed, and the cells are peeled off from the dish and simultaneously become isolated cells. Place the medium in a new dish, dilute the cells with the medium to the desired density, and inoculate in a new culture vessel.

  Examples of techniques relating to such exfoliation of cultured cells include Patent Document 1 and Patent Document 2. In Patent Document 1, in the production of a cell culture support, the amount of the monomer in the coating composition comprising a monomer capable of forming a stimulus-responsive polymer, a prepolymer obtained by polymerizing the monomer, and an organic solvent. It is disclosed that cell adhesion, cell sheet and single cell detachment properties are optimized by selecting according to the type of substrate. Further, Patent Document 2 discloses a polymer site fixed to a support and having a structure that changes depending on temperature, and is bonded to the polymer site and exposed to the outer surface to be contacted with a liquid to be treated containing target cells. A cell separation / recovery membrane comprising a cell binding site capable of specifically adsorbing target cells when exposed and a hydrophilic site exposed to the outer surface and in contact with the liquid to be treated is disclosed.

JP 2013-162696 A WO2015 / 033457

  As described above, when transplanting cultured cells to a new culture container (passaging), the process of peeling the cells from the bottom of the culture container by enzyme treatment and removing the enzyme by centrifugation treatment not only burdens the operator but also removes germs. Various investigations have been made to further increase the risk of contamination, but rapid cell detachment technology has not yet been established.

  An object of the present invention is to provide a cell culture container, an apparatus, and a separation and recovery method that achieve the above-mentioned problems, improve the quality of cells that can be produced, and reduce complicated passage operations. It is in.

  In order to achieve the above object, in the present invention, a support is coated with a thin film containing a pH / temperature-responsive polymer having a temperature-responsive site and a pH-responsive site, and the hydrophilicity and hydrophobicity change depending on the temperature and pH. A cell culture vessel having a separation and recovery membrane is configured.

  In order to achieve the above object, the present invention comprises a support and a thin film that is fixed to the support and includes a pH / temperature-responsive polymer that changes in hydrophilicity and hydrophobicity depending on temperature and pH. A cell culture vessel equipped with a separation / recovery membrane, an impregnating device for impregnating the separation / recovery membrane of the cell container into a treatment liquid containing cells, a heating / cooling device for heating or cooling the separation / recovery membrane, and an acidic solution for the culture solution And a cell recovery device for recovering the cells adsorbed on the separation / recovery membrane, and by changing the temperature and pH of the separation / recovery membrane by the heating / cooling device and the liquid supply device, A cell culture device that allows the collection device to rapidly collect cells is configured.

  Furthermore, in order to achieve the above object, in the present invention, there is provided a method for separating and recovering cells cultured in a cell culture device, the cell culture device being fixed to a support, and a cell binding site. A cell culture container having a separation / recovery membrane, and a separation / recovery membrane in a liquid to be treated containing cells, and a thin film containing a pH / temperature-responsive polymer whose hydrophilicity and hydrophobicity change depending on temperature and pH. An impregnation apparatus for impregnation and a cell recovery apparatus for recovering cells adsorbed on the separation / recovery membrane are provided, and the separation / recovery membrane is brought into contact with a liquid to be treated containing the cells by the impregnation device to adsorb the cells to the cell binding site. Separation / recovery membrane contact step, separation / recovery membrane contact step, pH / temperature change step for changing the pH and temperature of the separation / recovery membrane, and separation in which pH and temperature are changed in the pH / temperature change step Times The membrane treatment was supplied to the surface of the membrane, the supplied film processing liquid collected by cell harvester and constitutes a target cell collection step of collecting the cells adsorbed to the cell binding domain, a separation recovery method comprising.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to exfoliate a cell rapidly, and can improve the quality of a cultured cell, the risk of contamination reduction, and a worker's load reduction.

It is a figure which shows the basic composition of the separation-and-recovery membrane which concerns on this invention and Example 1. FIG. It is a figure for demonstrating the pH and temperature-responsive polymer of this invention. It is a figure for demonstrating the effect | action of the cell culture container of this invention. It is a figure for demonstrating the effect | action of the separation-recovery membrane of this invention and Example 1. FIG. It is a figure which shows the modification of the separation collection membrane based on Example 1. FIG. 1 is a top view of a culture sheet according to Example 1. FIG. 1 is a cross-sectional view of a culture sheet according to Example 1. FIG. 6 is a block diagram showing one configuration of a cell culture device according to Example 2. FIG. It is a figure which shows the adhesion state of the cell at the time of culture | cultivating a MEF cell by changing pH and temperature. It is a figure which shows the adhesion state of the cell at the time of changing temperature and culture | cultivating an iPS cell. It is a figure which shows the attachment state of the cell at the time of cultivating iPS cell changing pH and temperature.

  Embodiments for carrying out the present invention will be described in detail with reference to the drawings. The cell culture container using the separation and recovery membrane of the present invention is particularly effective for culturing adherent cells. As the adherent culture, epithelium such as organ-derived parenchymal cells (typical cells HeLa and Vero) is mainly used. Cell-like, mainly fibroblasts such as stromal cells (typical cells WI38 / C3H / 10 1/2 clone8), and others that extend cytoplasmic processes like neurons and macrophages There are things that show amoeba-like morphology. In addition, as cells other than adhesion-type cells, there are suspension-type cells, for example, blood cell-type cells such as cells that are mainly derived from blood organs and do not adhere to the culture vessel wall (typical cell HL60), and other tumor tissue-derived cells. Some cells do not have the ability to adhere to (typical cell FM3A).

  As a form for carrying out the present invention, in order to facilitate the isolation of cultured target cells, the cell surface is covered with a thin film containing a pH / temperature-responsive polymer having both a temperature-responsive site and a pH-responsive site. There is a culture vessel. FIG. 1 shows the basic structure of the cell culture vessel of the present invention and the separation / recovery membrane coated on the surface thereof. The separation / recovery membrane 10 according to the present invention includes a support 1 and a pH / temperature-responsive polymer composed of a pH-responsive part and a temperature-responsive part, and further a cell-binding part, which are coated on the support 1. The thin film 2 is used. When the separation and recovery membrane of the present invention is used for a cell culture container, the cell culture container itself may be the support 1. In that case, FIG. 1 shows a part of a cell culture vessel whose surface is covered with a thin film 2 containing a pH / temperature-responsive polymer or the like.

  FIG. 2A is a diagram schematically showing a method for synthesizing a pH / temperature-responsive polymer used in the present invention. As shown in the figure, the pH / temperature-responsive polymer contains a cell binding site. The temperature-responsive site material is a polymer that exhibits hydrophobicity at a temperature higher than a predetermined temperature and is hydrophilic at a temperature lower than the predetermined temperature, and the pH-responsive site material exhibits hydrophobicity at a pH higher than a predetermined pH, A polymer having hydrophilicity at a pH lower than the predetermined pH. By synthesizing these temperature-responsive sites, pH-responsive sites, and cell-binding sites that are adhesion factors such as gelatin that bind to the target cells, pH / temperature-responsive polymer 3 can be obtained.

  Hydrophobic herein refers to a property of a substance or molecule or a part thereof that has a low affinity for water, i.e., is difficult to dissolve in water or miscible with water, and conversely, hydrophilicity refers to water. It is a property with high affinity. The determination of hydrophilicity and hydrophobicity includes determination of solubility in water, determination of acid dissociation constant, which is an indicator of ionization degree, and partition coefficient, that is, an organic solvent that generally phase-separates the substance from water (generally n- There is one that obtains the ratio of the concentration (concentration in organic solvent ÷ concentration in water) when it is dissolved in octanol and mixed with water and equilibrium is reached. In addition, it is possible to quantitatively measure hydrophobicity or hydrophilicity by coating a polymer on the surface of the material, dropping water droplets on the surface, and measuring the contact angle.

  In the separation / recovery membrane according to the present invention, it is only necessary to cause a structural change depending on the temperature and pH. Therefore, the measured value may be changed more than the measurement error by any of the measurement methods described above. Since the separation / recovery membrane according to the present invention has a structure having a pH response site in addition to a temperature response site, a structural change occurs in a short time and cells can be rapidly detached.

  As the temperature response site of pH / temperature-responsive polymer, polypropylene glycol, ε-polylysine valeric acid amide, ε-polylysine butyric acid amide, N-hydroxypentyl ε-polylysine, N-hydroxybutyl ε-polylysine, polyethylene glycol, poly- Examples thereof include N-isopropylacrylamide, poly-N, N-diethylacrylamide, polyvinyl caprolactam, polyvinyl methyl ether, and polymethacrylate. In addition, pH, temperature responsive polymer pH responsive sites include amine, imine, phosphazene, guanidine, nitrogen-containing heteroaromatic compounds, carboxylic acid, phenolic hydroxyl group, boric acid, sulfonic acid, phosphonic acid, phosphinic acid. Can be mentioned. FIG. 2B schematically shows an example of a material having both a pH response site and a temperature response site.

  Subsequently, the operation of the cell culture vessel provided with the separation and recovery membrane 2 according to the present invention will be described using iPS cell culture as an example with reference to FIG. As shown on the left side of FIG. 3 (a), a thin film 2 containing a pH / temperature-responsive polymer 3 is formed on a support 1 such as a cell culture container, and feeder cells 4 and iPS cells 5 are formed at the cell binding sites. Are joined. Various types of membrane proteins are expressed in the cell membrane of cells. The cells can adhere to the culture surface of the cell culture vessel via this membrane protein. Under cell growth conditions (for example, 37 ° C.), the membrane protein and the cell binding site of the thin film 2 bind to each other, so that the cell binds on the surface of the cell culture vessel such as the nonwoven fabric as the support 1 and can grow on it. .

  When the cells are detached from the culture surface by passage or the like, the thin film 2 containing the cell membrane protein and the pH / temperature-responsive polymer 3 as shown on the right side of FIG. The binding at the cell binding site is eliminated, and the iPS cells 5 and the feeder cells 4 float in the culture medium. 3 (a) shows the structure of the thin film 2 containing the pH / temperature-responsive polymer in which the pH response site, the temperature response site, and the cell binding site are all bonded. FIG. As shown, the same effect can be obtained even with the thin film 6 including a pH / temperature-responsive polymer composed of a pH-responsive site and a temperature-responsive site and a pH / temperature-responsive polymer having a structure in which the cell binding sites are independent. be able to.

  As described above, in the culture vessel using the separation and recovery membrane of the present invention, a structure change occurs in a short time by adopting a structure having a pH response site in addition to a temperature response site on its surface. Cells can be detached quickly. Next, each element of a thin film including a support, which is a constituent element of the separation / recovery membrane of the cell culture container in the present invention, and a hydrophobic polymer site such as pH / temperature-responsive polymer will be described.

<Support>
The support 1 provided in the separation / recovery membrane fixes a hydrophobic polymer portion such as a pH / temperature-responsive polymer 3. The support 1 may be of any type, but in particular, it is preferable that the hydrophobic polymer portion such as the pH / temperature-responsive polymer 3 can be chemically fixed. Specifically, the support is preferably configured to include at least one of a non-woven cloth, a polymer material, and a glass material. By using such a material, surface treatment for fixing pH / temperature-responsive polymer sites such as pH / temperature-responsive polymer is particularly easy, and when pressure is applied for surface treatment There is an advantage of excellent durability.

  Examples of the polymer material include polystyrene, polymethyl methacrylate, surface-modified agarose gel, and the like. However, the support 1 does not need to be composed of these materials as long as the pH / temperature-responsive polymer portion can be fixed. For example, a metal material such as a simple metal or a metal oxide may be used. it can. The shape of the support surface may be any shape as long as the pH / temperature-responsive polymer portion can be fixed. For example, the shape of the support surface can be flat. May have a pillar structure, a spherical structure, a porous shape, or the like.

<Thin film>
The thin film 2 is composed of a hydrophobic polymer portion composed of a pH / temperature-responsive polymer portion, a cell binding portion, more preferably a hydrophilic portion, and is fixed to the support 1 described above. This thin film can be referred to as a pH / temperature-responsive cell adsorption / desorption polymer.

(Hydrophobic polymer part)
The hydrophobic polymer portion is composed of the above-described pH / temperature-responsive polymer. As described above, the structure of the hydrophobic polymer site fixed to the support 1 changes depending on pH and temperature. By such a structural change, the cell binding site bound to the hydrophobic polymer site can be buried in the hydrophilic site described later, and the target cells can be easily detached from the separation / recovery membrane. The specific structure of the hydrophobic polymer portion is as shown in the example above, and is not particularly limited as long as the structure changes depending on pH and temperature. However, the hydrophobic polymer portion preferably has a helical structure at least in part. By using a hydrophobic polymer site having such a structure, the hydrogen bond that controls the helical structure can be cleaved by pH and temperature, and the structure can be easily changed.

  Further, the hydrophobic polymer portion may be fixed to the support 1 by any method. For example, methods such as chemical bonding (covalent bonding, etc.) and physical adsorption (adsorption using antigen-antibody reaction, etc.) can be mentioned.

  Preferable specific examples of the hydrophobic polymer site include polyamino acids. A polyamino acid is formed by polymerizing two or more amino acids. At this time, the amino acid is usually polymerized in a straight chain. Polyamino acids have a helical structure due to the occurrence of hydrogen bonds within the molecule. Therefore, the use of a polyamino acid has an advantage that the structural change is easily caused. In addition, since polyamino acids are usually linear, it is easy to control the orientation of the polymer sites. Therefore, the hydrophobic part can be easily fixed at a desired position on the support. Furthermore, there is an advantage that it becomes easy to bind a cell binding site described later to a polymer site.

  The polyamino acid may be composed of only one amino acid (homopolymerization), or two or more amino acids may be composed of any ratio and combination (copolymerization). The amino acid constituting the polyamino acid is not particularly limited, and examples thereof include glutamic acid, aspartic acid, asparagine, lysine, glutamine, cysteine, alanine, leucine and the like. The number average molecular weight of the polyamino acid is not particularly limited, but is usually 5 kDa or more, preferably 10 kDa or more, more preferably 50 kDa or more. By setting it as this range, it becomes easy to fix the polyamino acid by the support.

(Cell binding site)
The cell binding site can be adsorbed to the target cell when brought into contact with the liquid to be treated containing the target cell. Therefore, the cell binding site may be appropriately determined and used according to the target cell to be separated and collected. For example, gelatin is commonly used as a cell binding substance for fixing feeder cells to a culture vessel.

  The cell binding site is preferably bound to a hydrophobic polymer site. The cell binding site and the hydrophobic polymer site may be directly bonded, but are preferably bonded via a linker. By binding the cell binding site to the hydrophobic polymer site via a linker, the cell binding site is more reliably projected outward from the surface of the separation and recovery membrane, that is, exposed to the outer surface. Can do. As a result, when the separation / recovery membrane is brought into contact with the liquid to be treated containing the target cell, the cell binding site can be more easily adsorbed to the target cell.

  The linker is preferably biotin-avidin. By using biotin-avidin, the cell binding site and the hydrophobic polymer site can be bound more easily. Specifically, the cell binding site and the polymer can be easily bound via avidin by biotinylating the cell binding site and biotinylating the polymer site.

  The specific binding position of the cell binding site in the hydrophobic polymer site is not particularly limited. However, although details will be described later, from the viewpoint of more reliably exposing the cell binding site to the outer surface of the separation and recovery membrane, it is preferable that the cell binding site is bonded to the end of the polymer constituting the polymer site. Specifically, for example, when the polymer constituting the polymer portion is linear, it is a linear end, and when the polymer is network-like, it is a network end. By binding the cell binding site to these positions, the cell binding site can be more reliably exposed to the outer surface of the separation and recovery membrane.

  As described above, the cell binding site is exposed on the outer surface of the separation and recovery membrane. Therefore, when the liquid to be treated is brought into contact with the separation and recovery membrane, the target cell can be specifically bound to the cell binding site and recovered. The method for exposing the cell binding site to the outer surface is not particularly limited. For example, as described above, the cell binding site is adsorbed to the hydrophobic polymer site via the linker, so that the cell binding site is exposed to the outer surface. Can be exposed to. In addition, the position (height) of the portion other than the cell binding site is relatively lower than the position (height) of the cell binding site, so that it is exposed to the outer surface without using a linker. Can do. These methods for exposing to the outer surface are only examples, and any method may be used as long as the cell binding site is exposed to the outer surface of the separation / recovery membrane. It may be.

  As described above, the non-specific adsorption portion on the surface of the separation / recovery membrane can be reduced by binding the cell binding site to the polymer site while the cell binding site is exposed on the outer surface. That is, since the cell binding site is arranged so as to cover the polymer site, the area of the non-specific adsorption portion derived from the polymer site can be reduced.

  In general, cells tend to cause non-specific adsorption to hydrophobic parts, so if there is a hydrophobic part on the surface of the separation and recovery membrane, cells other than target cells tend to non-specifically adsorb to the hydrophobic part. . However, in this embodiment, since the cell binding site is exposed to the outer surface and bound to the polymer site, the target cell can be specifically adsorbed by the cell binding site. Moreover, since the area of the hydrophobic portion is reduced as described above, nonspecific adsorption of cells other than the target cells to the separation and recovery membrane can be suppressed.

(Hydrophilic site)
The hydrophilic portion is a portion that is exposed on the outer surface of the separation / recovery membrane and contacts the liquid to be processed when it is contacted (for example, impregnated) with the liquid to be processed containing target cells. By providing a hydrophilic site so as to be exposed on the outer surface in a site other than the hydrophobic polymer site and the cell binding site, nonspecific adsorption of hydrophobic cells can be further suppressed.

  Examples of such a hydrophilic site include those having a hydrophilic functional group, specifically, a hydroxyl group, a carboxyl group, or the like. More specific examples of the hydrophilic portion include ethylene glycol and its polymer (polyethylene glycol), molecules having a phospholipid structure, phosphoric acid and its polymer (polyphosphoric acid), and the like. Among these, from the viewpoint of good hydrophilicity and ease of binding to a hydrophobic polymer site, the hydrophilic site preferably contains polyethylene glycol obtained by polymerizing two or more ethylene glycols.

  Further, the hydrophilic part may be provided independently of the hydrophobic polymer part, but from the viewpoint of further promoting the structural change of the hydrophobic part, the hydrophilic part is bound to the hydrophobic polymer part. It is preferable. That is, the hydrophilic part is preferably integrated with the hydrophobic polymer part. Since the hydrophilic part is bonded to the hydrophobic polymer part, the structural change of the hydrophobic polymer part accompanying the temperature change and pH change can be further promoted. However, it is not necessary that all the hydrophilic sites provided in the separation and recovery membrane are bonded to the hydrophobic polymer site, and the temperature responsiveness of the hydrophobic polymer site is controlled by appropriately binding an alkyl group or the like. be able to.

  The bonding position of the hydrophilic part to the hydrophobic polymer part is not particularly limited. However, when the hydrophobic polymer moiety is linear, it is preferable that the hydrophilic moiety is bonded to the side chain of the hydrophobic polymer moiety. Thereby, a hydrophilic part can be easily arrange | positioned in parts other than a hydrophobic polymer part in a separation collection membrane. Therefore, the hydrophilic part can be more reliably exposed on the outer surface of the separation / recovery membrane.

  As described above, the hydrophobic polymer moiety is preferably a polyamino acid, and the hydrophilic moiety preferably includes polyethylene glycol. Moreover, it is preferable that the hydrophilic part is bonded to the hydrophobic polymer part. Examples of suitable materials for the hydrophobic polymer and the hydrophilic portion satisfying these are compounds represented by the following formula (1).

In formula (1), n and x are each independently an integer of 2 or more.

  Next, the configuration and operation of the separation / recovery membrane and the cell culture container according to the first embodiment will be described. That is, in this example, a separation / recovery membrane in which a support is coated with a thin film containing a pH / temperature-responsive polymer having a temperature-responsive site and a pH-responsive site, and the hydrophilicity and hydrophobicity change depending on the temperature and pH, and It is an Example of the cell culture container using this.

  1 is a perspective view of a separation and recovery membrane of Example 1. FIG. When the separation / recovery membrane 10 is observed from a macro viewpoint, a thin film 2 containing a pH / temperature-responsive polymer is formed on the surface of the support 1. As described above, the cell culture container itself may be the support 1. The thin film 2 includes a hydrophobic polymer portion, a cell binding portion, and a hydrophilic portion that are composed of the pH / temperature-responsive polymer portion described in the previous principle.

  FIG. 4 is a diagram for explaining the operation of the separation and recovery membrane 10 of Example 1. FIG. 4A shows a state before heating and a high pH, and FIG. 4B shows a state after heating. It is a figure which shows the separation collection membrane 10 of the state of low pH. In the present embodiment, the structure of the hydrophobic polymer portion is changed or destroyed by heating the separation and recovery membrane 10 to a low pH. As shown in FIG. 4 (a), in the present embodiment, the thin film 2 is bonded to a helical hydrophobic polymer portion 2a whose structure changes with temperature and pH, and to the hydrophobic polymer portion 2a. A cell binding site 2b and a hydrophilic site 2c bonded to the hydrophobic polymer site 2a are included. A cell binding site 2b is bonded to each of the three hydrophobic polymer sites 2a shown in FIG. Further, on the surface of the support 1, hydrophobic polymer portions 2 a and hydrophilic portions 2 c are alternately arranged. That is, the surface of the support 1 is covered with the hydrophilic portion 2c except for the portion of the hydrophobic polymer portion 2a covered with the cell binding portion 2b. The position of the cell binding site 2 b is relatively higher than the position of the hydrophilic site 2 c with respect to the support 1.

  Then, in the separation / recovery membrane 10 of this example, as shown in (a) of the figure, after the target cells 20 are adsorbed to the cell binding site 2b, the separation / recovery membrane 10 is heated to a low pH. The structure of the hydrophobic polymer site 2a changes, and the target cells 20 adsorbed on the cell binding site 2b are detached as shown in FIG. In the illustrated example, for convenience, one target cell 20 is adsorbed to one cell binding site 2b. However, one target cell 20 may be adsorbed to two or more target cell sites 2b. Good.

  In the configuration of the present embodiment, an operation when the state of FIG. 4A changes to the state of FIG. 4B will be described. The helical structure is maintained in the hydrophobic polymer part 2a before heating at a high pH. Further, the target cell 20 is adsorbed to the cell binding site 2b. When the separation / recovery membrane 10 is heated to a low pH in this state, the helical structure of the hydrophobic polymer portion 2a is destroyed and changed to a random structure by the heat energy, and the cell binding portion 2b exposed on the outer surface is changed. Embedded in the hydrophilic part 2c. As a result, the adsorption of the cell binding site 2b and the target cell 20 is blocked by the steric hindrance of the hydrophobic polymer site 2a changed to a random structure, as shown in FIG. Furthermore, when the cell binding site 2b is buried in the hydrophilic site 2c, the target cell 20 is placed on the support 1 covered with the hydrophilic site 2c. If it does so, the target cell 20 which is hydrophobic will be in the state which can be collect | recovered easily from the hydrophilic region 2c.

  The temperature at which the state shown in FIG. 4A changes to the state shown in FIG. 4B can be controlled by, for example, the structure of the hydrophobic polymer portion 2a. For example, when the hydrophobic polymer portion 2a has a long helical structure, the number of hydrogen bonds increases, so that the thermal energy required to change the structure by cutting them is also increased. Therefore, the temperature for changing the structure also tends to increase. On the other hand, when the included helical structure is short, the number of hydrogen bonds is also small. Therefore, the temperature for changing the structure tends to be low. Furthermore, when the hydrophilic part 2c has couple | bonded with the hydrophobic polymer part 2a, it is controllable also by the structure of the hydrophilic part 2c.

  In the description of the first embodiment of the separation and recovery membrane using the pH / temperature-responsive polymer, the spiral structure changes to a random structure at a predetermined temperature or higher and lower than or equal to pH. The structure is a random structure above a predetermined pH, but may be a spiral structure above a predetermined temperature. In this case, structural changes occur due to cooling and high pH, or cooling and low pH, and the target cells 20 can be recovered. Further, the spiral structure may be changed to a random structure at a predetermined temperature or higher and pH or higher.

  In addition, the separation / recovery membrane of the present embodiment is not limited to the illustrated contents and the contents described above. For example, as shown in FIG. 5 (a), it is not necessary that the cell binding sites 2b are bound to all the hydrophobic polymer sites 2a, and the cell binding sites 2b only to some of the hydrophobic polymer sites 2a. The separation / recovery membrane 10A may be combined. By doing in this way, even when the number of target cells contained in the liquid to be treated is small, the target cells can be efficiently collected.

  Further, as shown in FIG. 5B, in addition to the hydrophilic portion 2c bonded to the hydrophobic polymer portion 2a, the support 1 is exposed between the adjacent hydrophilic portions 2c. The separation / recovery membrane 10B may be provided with another hydrophilic portion 2d at the portion. By doing in this way, nonspecific adsorption | suction can be prevented more reliably.

<Method for producing separation and recovery membrane>
Next, a method for manufacturing the separation / recovery membrane of this example will be described. The production method described below describes the case where one kind of lower critical solution temperature (LCST) type antibody is used. However, the same procedure should be followed when a plurality of types of LCST type antibodies are immobilized on a separation / recovery membrane. Can do. Although the manufacturing method of the separation / recovery membrane of the present embodiment is not particularly limited, for example, it can be manufactured by the following method. In the following description, as an example of a method for producing a separation / recovery membrane, a case where a separation / recovery membrane in which a hydrophilic part is further bonded to a hydrophobic polymer part composed of a pH / temperature-responsive polymer part is described. This will be described with reference to FIG.

  First, the hydrophobic polymer part 2a to which the hydrophilic part 2c is bonded is prepared by an arbitrary method, and is fixed to the surface of the support 1 by, for example, spin coating, dip method, vapor deposition method or the like. As a fixing method, a chemical bond in which the hydrophobic polymer portion 2a itself is not eliminated by a reversible reaction is preferable. Specifically, for example, amide bond, ester bond, silanol bond, maleimide, thioester bond, chemical bond by Diels-Alder reaction, chemical bond by click reaction, or gold-thiol bond, imine bond, etc. depending on the application .

  Here, when the hydrophobic polymer part 2a is fixed, the hydrophilic part 2c may or may not be fixed to the support 1. However, if the hydrophobic polymer part 2a is fixed to the support 1, the hydrophilic part 2c bonded to the side chain can be sufficiently arranged on the surface of the support 1 without fixing. Thereafter, the hydrophobic polymer portion 2a that has not been fixed is removed with a good solvent.

  Next, the cell binding site 2b is bonded to the end of the fixed hydrophobic polymer site 2a by chemical bonding. At this time, the cell binding site 2b is not necessarily bound to the end of the hydrophobic polymer site 2a. Therefore, as long as the cell binding site 2b is exposed on the outer surface of the cell separation membrane 10, it may be bound to any part of the hydrophobic polymer site 2a. By doing so, it is possible to manufacture a separation and recovery membrane in which the hydrophilic portion 2c is exposed on the surface.

  The hydrophobic polymer part 2a is bonded to the support 1 by dispersing the hydrophobic polymer part 2a in a solvent as necessary, for example, spin coating, spray coating, dip coating, roll coating, bead coating, etc. You may apply | coat to the surface of the support body 1 by these various methods. Also by such a method, the hydrophobic polymer part 2a can be arranged on the support 1.

<Application of separation and recovery membrane>
Next, specific uses of the separation and recovery membrane of the present embodiment will be described with reference to the drawings. The separation / recovery membrane of the present embodiment can be applied to various uses in addition to the culture vessel, and is suitable for, for example, a biodevice such as a cell culture sheet, a cell culture apparatus, and the like. Hereinafter, these two examples will be described as suitable uses of the separation and recovery membrane of this embodiment.

  FIG. 6 is a top view of a culture sheet 50 configured using the separation and recovery membrane of this example. The cell culture sheet 50 of this example has the same configuration as the separation and recovery membrane 10. The cell culture sheet 50 includes a support body 1 having polystyrene having a thickness of 0.5 μm as a main component, and a plurality of columnar fine protrusions 2A that are erected on the thin film and have polystyrene as a main component. The protrusion 2A has a cylindrical shape, and has a diameter of 500 nm and a height of 1 μm. Each protrusion 2A is arranged with a period (pitch) of 1 μm. Further, in the culture sheet 50 of this example, the protrusion 2A is first formed in a columnar shape, and then the protrusion 2A is removed in a cross shape to form a cross-shaped gap 51.

  The protrusion 2A can be formed by the following method. That is, first, a base projection having the above-described shape made of polystyrene is formed on the support 1 by an imprint method. The surface of the base is subjected to surface oxidation treatment using an ultraviolet light / ozone generator. Finally, a culture sheet provided with protrusions 2A capable of adsorbing target cells by binding a hydrophobic polymer member or the like to the base subjected to the surface oxidation treatment as shown in FIG. 50 is obtained. The culture sheet 50 is impregnated with the culture solution in a glass petri dish or the like in which the culture solution is placed, and the target cells 20 are cultured on the culture sheet 50.

  A method for using the culture sheet 50, that is, a specific method for culturing cells will be described with reference to FIG. FIG. 7 is a cross-sectional view of the culture sheet 50 of this example. The cell 20 is adsorbed and fixed to the cell binding site on the surface of each protrusion 2A. In the illustrated example, a state in which one cell 20 is adsorbed to the cell binding sites on the surface of the plurality of protrusions 2A is shown. Then, the cells 20 are cultured by supplying a culture solution such as a medium and nutrients in a state where the cells 20 are adsorbed in this way. In addition, although it does not restrict | limit especially as the cell 20 which can be cultured, For example, they are cells (tissue), such as skin, bone, and blood.

  As shown in FIG. 7, the support 1 and the cell 20 are separated by a gap 51. Therefore, a culture solution and oxygen for culturing the cell 20 are supplied from both the upper side and the lower side of the cell 20. Thereby, the cell 20 can be cultured efficiently. Further, for example, carbon dioxide, which is a waste product discharged with the culture of the cell 20, is efficiently discharged from the upper side and the lower side of the cell 20.

  By using such a culture sheet 50, the cell 20 is supported only by the upper surface of the protrusion 2A. Therefore, the contact area of the cells 20 conventionally cultured on the bottom surface of the glass petri dish can be reduced. Accordingly, it is possible to greatly reduce cell damage to the cells 20 when the cells 20 are peeled off. Thereby, the fixation rate to the transplant site | part at the time of transplanting the cell 20 can be raised. In addition, as shown in FIG. 7, the culture solution can easily flow through the entire cell 20 through the gap below the cell 20 formed by the protrusion 2A. As a result, it is possible to efficiently supply nutrients to the cells 20 and discharge waste products of the cells 20, and to suppress the killing of the cells 20 in the culture that has conventionally occurred.

  The protrusion 2A may be a polymer material that has been subjected to a hydrophilic treatment by plasma treatment or the like. As the polymer material, it is preferable to select a material having little influence on the cells (tissue) to be cultured. Specifically, polystyrene is used, but the material is not limited thereto. For example, as the polymer material, polymethyl methacrylate, polylactic acid and the like are also preferable.

  In addition to the above-described biodevices, examples of biodevices include those generally called μTAS as a medical / diagnostic tool. Specific examples include those whose surface is finely processed and those used for detection and synthesis in the medical and chemical fields.

  Subsequently, as Example 2, an example of a cell culture apparatus using the separation and recovery membrane of Example 1 and a cell separation and recovery method will be described. This cell culture apparatus separates and collects target cells contained in the liquid to be treated. That is, this example is a cell culture provided with a separation and recovery membrane comprising a support and a thin film containing a pH / temperature-responsive polymer that is fixed to the support and changes in hydrophilicity and hydrophobicity depending on temperature and pH. A container, an impregnating device for impregnating a separation / recovery membrane of the cell container into a treatment liquid containing cells, a heating / cooling device for heating or cooling the separation / recovery membrane, and a liquid feeding device for feeding an acidic solution to the culture solution A cell recovery device that recovers the cells adsorbed on the separation / recovery membrane, and the cell recovery device quickly recovers the cells by changing the temperature and pH of the separation / recovery membrane using a heating / cooling device and a liquid feeding device. It is an Example of the cell culture apparatus which enables it to do.

  Further, the present example is a method for separating and recovering cells cultured in a cell culture device, the cell culture device having a support, a cell fixed site, a cell binding site, and being hydrophilic by temperature and pH. A cell culture vessel having a separation / recovery membrane composed of a pH / temperature-responsive polymer that changes pH and hydrophobicity, an impregnation apparatus for impregnating the separation / recovery membrane into a treatment liquid containing cells, and separation / recovery A separation / recovery membrane contacting step in which the separation / recovery membrane is brought into contact with the treatment liquid containing the cells by the impregnation device, and the cells are adsorbed to the cell binding site; After the recovery membrane contact step, the pH / temperature change step for changing the pH and temperature of the separation / recovery membrane, and the membrane treatment liquid is applied to the surface of the separation / recovery membrane whose pH and temperature are changed in the pH / temperature change step. Supply and supply The membrane treatment solution was collected by cell harvester, an embodiment of a separation and recovery method comprising: cell recovery step of recovering the cells adsorbed to the cell binding domain, a. Hereinafter, a specific apparatus configuration of the cell culture apparatus of the present embodiment and a cell separation and recovery method using the cell culture apparatus will be described.

  FIG. 8 is a block diagram of a configuration example of a cell culture device configured using the separation and recovery membrane described above. The cell culture apparatus 100 heats or cools the culture container 102a provided with the separation / recovery film 10, the impregnation apparatus 102b for impregnating and contacting the separation / recovery film 10 with a liquid to be treated containing target cells, and the separation / recovery film 10. A heating / cooling device 104, a cell recovery device 105 that recovers target cells adsorbed on the separation / recovery membrane 10, and a liquid supply device 106 that supplies an acidic solution are provided.

  In addition to these, the culture apparatus 100 also has a washing solution 100b for washing cells non-specifically adsorbed on the separation and recovery membrane, a medium reagent 100c for culturing target cells, and a cold storage for storing the medium reagent 100a. And a liquid supply system 101 that supplies the cleaning liquid 100b and the culture medium reagent 100c to the temperature-controlled room 102. Furthermore, the culture apparatus 100 is provided with a temperature-controlled room 102, and a separation / recovery membrane 10 is impregnated with a culture solution containing target cells in a culture vessel 102a in the temperature-controlled room 102. The atmosphere in the culture vessel 102 a is controlled by the gas exchange device 103.

  A method for separating and recovering cells using the cell culture apparatus 100 having the above configuration will be described. In the following description, cells are collected after being adsorbed on a separation / recovery membrane and cultured, but may be collected immediately after adsorption without being cultured.

  First, the to-be-processed liquid containing a target cell is supplied to the culture container 102a. Thereafter, the impregnation apparatus 102b performs a separation / recovery membrane contact step in which the separation / recovery membrane 10 is impregnated and brought into contact with the liquid to be treated containing the target cells to adsorb the target cells in the liquid to be treated to the separation / recovery membrane. Thereafter, the separation / recovery membrane 10 is washed with the washing solution 100b to wash away nonspecifically adsorbed cells, and the specifically adsorbed target cells are cultured. By doing in this way, a target cell can be cultured efficiently in the temperature-controlled room 102.

  Subsequently, the impregnation apparatus 102b takes out the impregnated separation / recovery membrane 10 from the liquid to be treated, adds a carbonic acid solution to the culture solution by the liquid feeding device 106 that feeds the acidic solution, and the culture in which the separation / recovery membrane 10 contacts. After the pH of the liquid is lowered to a predetermined pH, a heating / cooling step is performed in which the separation / recovery membrane 10 is cooled by the heating / cooling device 104. Thereby, the structure of the hydrophobic polymer portion 2a of the separation / recovery membrane 10 changes, and the target cells adsorbed on the culture surface, that is, the cultured target cells can be easily detached. Then, the cell recovery device 105 supplies the cell recovery liquid 105a to the surface of the separation / recovery membrane 10 in this state. Thereby, the cultured target cells are peeled off from the surface of the separation / recovery membrane 10. The detached target cells are collected by the cell collection device 105.

  According to the method of the culture device using the separation and recovery membrane of Example 2 described in detail above, damage to cells due to detachment that has conventionally occurred when using a normal culture medium or a conventional culture sheet, or at the time of recovery It becomes possible to suppress the use of chemicals. Thereby, the fall of the collection rate of a target cell can be suppressed.

  In the above example, the target cells are detached by cooling, but the target cells are detached by heating by changing the structure of the hydrophobic polymer portion 2a such as pH / temperature-responsive polymer. You may do it. In this case, the separation / recovery membrane 10 may be controlled to be heated instead of cooling by the heating / cooling device 104. Similarly, in the above example, the target cells are detached by changing the pH to a low pH. However, the device 106 that changes the structure of the hydrophobic polymer site 2a and the like and sends an acidic solution is used as an alkaline solution. In this case, the target cells may be detached with a high pH.

  As described above, in the description of this example, the case where one type of LCST type antibody is used has been described. However, when there are a plurality of LCST type antibodies having different phase transitions, the culture temperature is changed as described above. Just do it.

  Next, the separation / recovery membrane in each of the examples described above will be described and the evaluation of cell detachment will be described with specific examples.

<Preparation of PEG-modified polylysine>
First, 1-ethyl-3-carbodiimide (WSC), N-hydroxysuccinimide (NHS), and carboxylic acid-terminated polyethylene glycol were dissolved in pure water, and this solution was cooled to 4 ° C. and stirred while polylysine (various Molecular weight mixed). And it stirred at normal temperature for 2 hours, and obtained the PEG modification polylysine which polyethyleneglycol (hydrophilic part) couple | bonded with the side chain of polylysine (hydrophobic polymer part).

<Immobilization of polylysine to the support>
The obtained polylysine was each covalently bonded according to the following procedure on a resin plate (polystyrene support) whose surface was treated with an amino group. 1-ethyl-3-carbodiimide (WSC) and N-hydroxysuccinimide (NHS) were added to a 1 mol% polylysine aqueous solution, and each plate was immersed. Then, it was made to react at room temperature for 2 hours. As a result, a support to which polylysine was bound was obtained. Thereafter, each plate was washed three times with pure water.

<Binding of gelatin to polylysine>
The obtained three kinds of plates to which polylysine was bound were immersed in 2% glutaraldehyde diluted with a carbonate buffer and allowed to stand at 37 ° C. for 2 hours. Each plate after the reaction was washed three times with a phosphate buffer (PBS). Then, a gelatin solution having a concentration of 0.1 wt% (using a PBS buffer solution having a pH of 7.4) was prepared, and each plate was immersed in the gelatin solution to bind gelatin to polylysine as described above.

<MEF cell detachment test 1>
Mouse embryo fibroblast primary culture cells (MEF cells) were seeded in a culture vessel coated with a pH-temperature responsive polymer. The MEF cells were prepared according to the following procedure.

Remove MEF cells that have been frozen in the vial from the liquid nitrogen tank, warm them in a 37 ° C water bath for 90 seconds, remove them from the water bath in a semi-dissolved state, move them into the safety cabinet, and then move the cells in the vial. The entire amount was transferred to a centrifuge tube by decantation, and after adding MEF medium (added bovine serum to DMEM medium to a final concentration of 10%) heated to 37 ° C., it was centrifuged at 170 × g for 5 minutes (room temperature). The medium supernatant was removed with an aspirator so as not to aspirate the cell pellet in the safety cabinet, 1 mL of MEF medium was added to the centrifuge tube and pipetted several times to break the pellet, and 9 ml of medium was added to make 10 ml. The number of cells was counted, and MEF cells were seeded in the culture vessel of the present invention at 5 × 10 3 cells / cm 2. The seeded culture vessel was placed in a 37 ° C., 5% CO 2 incubator and cultured for 3 days, and then the temperature and pH conditions were changed and a cell detachment test was performed.

  FIG. 9 shows the state of MEF cell attachment when the temperature and pH conditions are changed. As a control experiment, the results of culturing MEF cells in a culture vessel coated with gelatin, which is a cell adhesion factor, are also shown. At pH 7.2 and 37 ° C., the MEF cells adhered to the culture vessel in both the pH-temperature-responsive polymer coating and the gelatin coating, and the cultured cells were not affected. When the culture container was lowered to 4 ° C. while maintaining the pH of 7.2, the cells in the pH-temperature-responsive polymer-coated culture container were finally detached after 3 hours. The cells in the collagen-coated culture vessel had no change in the attachment of MEF cells. When carbonic acid was added to the culture solution, the pH was lowered to 6.5, and the temperature of the culture vessel was lowered to 4 ° C., the cells in the pH-temperature-responsive polymer-coated culture vessel were detached within 5 minutes. The cells in the collagen-coated culture vessel had no change in the attachment of MEF cells. Thus, in the pH-temperature-responsive polymer-coated culture vessel, the cells can be detached by lowering the temperature, and the pH is adjusted to a pH that does not affect the cell culture (in the above, pH 6.5). Thus, it was confirmed that detachment of cells can be performed quickly.

<Peeling test of iPS cell colony>
iPS cells were cultured according to the following procedure. First, reagents were prepared for culturing iPS cells. The composition of each reagent is as follows.
・ PBS (-) solution: 80g NaCl, 2g KCl, 29g Na ₂ HPO ₄・ 12H ₂ O, 2g KH2PO4
・ Centrifuge medium: 400 mL KO-DMEM / F12 medium, 0.925 mL 2-mercaptoethanol, 4 mL NEAA, 4 mL L-glutamine (200 mM), 5 mL PSA, 100 mL KSR
・ Culture medium: FGF-2 added to the above-mentioned centrifuge medium
Since iPS cells are cultured on feeder cells, iPS cells were seeded on the MEF cells seeded in Example 1. The sowing method is as follows. The iPS cell culture medium cultured by the usual culture method (collagen-coated culture vessel) was removed by aspiration, 2 mL 0.1% (w / v) dispase solution was added, and the mixture was reacted at 37 ° C. for 3-10 min. The dispase solution was removed by suction, 5 mL of iPS (−) medium was added, and the cell was removed with a cell scraper. Transfer the cell suspension to a 15 mL tube for centrifugation, add 5 mL of iPS (-) medium, precipitate the cells by centrifugation (300 rpm, 2 min, room temperature), and remove the supernatant. IPS (−) medium was added and the cells were lightly suspended. On the other hand, MEF cells were seeded in a pH-temperature-responsive polymer-coated culture vessel the day before, and the MEF cells cultured in a 37 ° C., 5% CO 2 incubator were removed from the incubator, and the suspension of iPS cells as described above was used in the present invention. The cells were seeded in a pH-temperature-responsive polymer-coated culture vessel, and cultured in a 37 ° C., 5% CO 2 incubator for 5 days. Thereafter, the detachment test of iPS cells was performed by changing the temperature and pH conditions.

  FIGS. 10 and 11 show photographs showing the state of attachment of iPS cell colonies when the temperature and pH conditions are changed. As a control experiment, the results of culturing iPS cells in a culture vessel coated with gelatin, which is a cell adhesion factor, are also shown. At pH 7.2 and 37 ° C., both the pH-temperature-responsive polymer coating and the gelatin coating adhered iPS cells to the culture vessel, and the cultured cells were not affected. When the culture vessel was lowered to 4 ° C. while maintaining the pH of 7.2, the cells in the pH-temperature-responsive polymer-coated culture vessel were finally detached after 3 hours as in the case of MEF cells. As shown in FIG. 10, the cells in the collagen-coated culture container had no change in iPS cell attachment. When carbonic acid is added to the culture solution, the pH is lowered to 6.5, and the temperature of the culture vessel is further lowered to 4 ° C., the iPS cells in the pH-temperature-responsive polymer-coated culture vessel are within 5 minutes as shown in FIG. It peeled. The cells in the collagen-coated culture vessel did not change iPS cell attachment. In this way, in the pH / temperature-responsive polymer-coated culture vessel, cells can be detached by lowering the temperature, and the pH is adjusted to a pH that does not affect the cell culture (in the above, pH 6.5). Thus, it was confirmed that detachment of cells can be performed quickly.

  In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail for better understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

DESCRIPTION OF SYMBOLS 1 Support body 2 and 6 Thin film 2a Hydrophobic polymer part 2b Cell binding part 2c Hydrophilic part 3 pH and temperature-responsive polymer 4 Feeder cell 5 iPS cell 10 Separation collection membrane 50 Culture sheet 51 Crevice 100 Cell culture device 100a Cold storage 100b Washing solution 100c Medium reagent 101 Liquid supply system 102 Temperature-controlled room 102a Culture vessel 102b Impregnation device 103 Gas exchange device 104 Heating / cooling device 105 Cell recovery device 105a Cell recovery solution 106 Liquid supply device

Claims (15)

It has a temperature recovery site and a pH response site, and has a separation and recovery membrane in which a support is coated with a thin film containing a pH / temperature response polymer whose hydrophilicity and hydrophobicity change depending on the temperature and pH.
A cell culture container characterized by the above. The cell culture container according to claim 1,
The pH / temperature-responsive polymer has a helical structure at least in part.
A cell culture container characterized by the above. The cell culture container according to claim 1,
The thin film has a hydrophilic portion bonded to the pH / temperature-responsive polymer.
A cell culture container characterized by the above. The cell culture container according to claim 1,
The pH / temperature-responsive polymer contains a polyamino acid,
A cell culture container characterized by the above. The cell culture container according to claim 3,
The hydrophilic portion contains polyethylene glycol,
A cell culture container characterized by the above. The cell culture container according to claim 3,
The pH / temperature responsive polymer contains a cell binding site,
A cell culture container characterized by the above. The cell culture container according to claim 1,
The support includes at least one of a polymer material and a glass material.
A cell culture container characterized by the above. The cell culture container according to claim 6,
The position of the cell binding site is relatively higher than the position of the hydrophilic site with respect to the support.
A cell culture container characterized by the above. The cell culture container according to claim 6,
The cell binding site is a cell adhesion factor or part of a cell adhesion factor;
A cell culture container characterized by the above. A cell culture vessel comprising a support and a separation and recovery membrane fixed to the support and comprising a thin film containing a pH / temperature-responsive polymer that changes in hydrophilicity and hydrophobicity depending on temperature and pH;
An impregnation apparatus for impregnating the separation / recovery membrane of the cell container into a liquid to be treated containing cells;
A heating and cooling device for heating or cooling the separation and recovery membrane;
A liquid feeder for feeding an acidic solution to the culture solution;
A cell recovery device for recovering cells adsorbed on the separation and recovery membrane,
By changing the temperature and pH of the separation and recovery membrane by the heating and cooling device and the liquid feeding device, the cell recovery device recovers the cells.
A cell culture device. The cell culture device according to claim 10,
The pH / temperature-responsive polymer includes a cell-binding site capable of adsorbing the cells when contacted with a treatment liquid containing the cells,
The thin film has a hydrophilic portion bonded to the pH / temperature-responsive polymer.
A cell culture device. The cell culture device according to claim 11,
The position of the cell binding site is relatively higher than the position of the hydrophilic site with respect to the support.
A cell culture device. A method for separating and collecting cells cultured in a cell culture device,
The cell culture device comprises:
A cell having a separation and recovery membrane fixed to the support and having a cell binding site and a thin film containing a pH / temperature-responsive polymer that changes in hydrophilicity and hydrophobicity depending on temperature and pH A culture vessel, an impregnation device for impregnating the separation / recovery membrane in a treatment liquid containing cells, and a cell recovery device for recovering cells adsorbed on the separation / recovery membrane,
A separation / recovery membrane contacting step in which the separation / recovery membrane is brought into contact with a liquid to be treated containing the cells by the impregnation device, and the cells are adsorbed to the cell binding site;
After the separation / recovery membrane contact step, a pH / temperature change step for changing the pH and temperature of the separation / recovery membrane;
In the pH / temperature changing step, a membrane treatment solution is supplied to the surface of the separation and recovery membrane whose pH and temperature are changed, and the supplied membrane treatment solution is recovered by the cell recovery device, and the cell binding site A cell recovery step of recovering the cells adsorbed on
A separation and recovery method characterized by the above. The separation and recovery method according to claim 13,
The thin film has a hydrophilic portion bonded to the pH / temperature-responsive polymer,
The position of the cell binding site is relatively higher than the position of the hydrophilic site with respect to the support.
A separation and recovery method characterized by the above. The separation and recovery method according to claim 13,
The cell culture device further includes a heating / cooling device for heating or cooling the separation / recovery membrane, and a liquid feeding device for feeding an acidic solution to the culture solution,
In the pH and temperature changing step, the pH and temperature of the separation and recovery membrane are changed by the liquid feeding device and the heating and cooling device,
A separation and recovery method characterized by the above.

Images