JP2017038556A - Method for culturing cells and culture kit for cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for culturing cells and a cell culture kit, which are capable of stably producing more cells.SOLUTION: According to the present invention, the method comprises the steps of: preparing a scaffold 300 having a first functional molecule 100 including a first molecular structure 1 capable of capturing a lipid bilayer membrane structure and a second molecular structure 2 capable of self-organizing; contacting a cell 5 having a lipid bilayer membrane structure with the scaffold 300 in a physiologically active environment for cell culture to allow the cell 5 to adhere the scaffold 300 via the first molecular structure 1; maintaining the physiologically active environment for cell culture to allow the cell 5 to proliferate on the scaffold 300; and separating the proliferated cells 5 from the scaffold 300.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a cell culture method and a cell culture kit.

<Conventional cell culture scaffold and mass culture technology>
Petri dishes, flasks, roller bottles, follow fibers, alginic acid gels, microcarriers, and the like have been developed as conventionally used cell culture scaffolds. For suspension cells, products using roller bottles and similar bioreactors have been developed, and there are reports on automation systems, etc., which are also used for mass production of suspension IPS cells and the like that have recently attracted attention It is like that. Some adherent cells use a system in which the adhesive surfaces are stacked. In addition, products using three-dimensional higher order structures or microcarriers are known, but have not been generalized.

<Adherent cell culture using microcarriers>
In recent years, microcarriers having various shapes (particulate carrier, porous carrier, etc.) have been developed.
In addition, in order to adhere cells to a carrier, fibronectin, vitronectin, laminin, etc. and cells such as RGD (S) (one-letter code of peptide; arginine-glycine-asparagine- (serine)) peptide as an adhesion sequence peptide of these proteins Several functional microcarriers in which an adhesion factor is added to a culture solution or these are coated on a microcarrier in advance have been commercialized. Examples in which these proteins or RGD peptides are immobilized by coating or chemical bonding have also been reported (see Patent Document 1, Patent Document 2, and Non-Patent Document 1). An example in which a temperature-responsive gel is coated has also been reported (see Patent Document 3).

Special table 2014-533309 gazette JP 2006-136212 A JP 2004-236553 A

Ruoslahti, E .; (1983). Proc. Natl. Acad. Sci. USA 80, 1224-1227.

When the functional microcarrier described above is applied to large-scale culture, a larger adhesion area allows more cells to be cultured, so that a three-dimensional space can be used effectively and particle-shaped micros with excellent operability. A carrier is selected.
However, at present, the existing functional microcarriers have not established stable methods and products that can build a mass production system. The reason for this is that the existing functional microcarrier is a mechanism that adheres cells only by the interaction between the cell surface and the surface of the microcarrier, and therefore maintains the cell adhesion state in a fluid culture environment. The inability to do so is considered to be influencing. Alternatively, since the cell adhesion state is weakly stretched by external force, it is considered that the cell cannot survive in a fluid environment and the cell survival rate is low.
Accordingly, the present invention has been made in view of such circumstances, and an object thereof is to provide a cell culture method and a cell culture kit that can stably produce more cells.

  In order to solve the above problems, a cell culture method according to one embodiment of the present invention includes a first molecular structure capable of capturing a lipid bilayer structure and a second molecular structure that self-assembles. A step of preparing a scaffold having a first functional molecule, a cell having the lipid bilayer structure, and the scaffold are contacted in a physiologically active environment for cell culture, and the first molecular structure is Attaching the cells to the scaffold, maintaining the physiologically active environment for cell culture, growing the cells on the scaffold, and separating the grown cells from the scaffold It is characterized by having.

  According to one embodiment of the present invention, more cells can be stably produced.

It is a schematic diagram which shows each structural example of the 1st functional molecule | numerator 100 and the 2nd functional molecule | numerator 200 which concern on this embodiment. It is a schematic diagram which shows each structural example of the 1st scaffold 300 and the 2nd scaffold 400 which concern on this embodiment. It is a schematic diagram which shows each structural example of the 1st composite body 500 and the 2nd composite body 600 which concern on this embodiment. It is sectional drawing which shows typically the process of culture | cultivation of the cell 5 which concerns on this embodiment. It is a top view which shows typically the molecular structure of the 2nd molecular structure 2 which concerns on this embodiment. It is a top view which shows typically the self-organization mode of the 2nd molecular structure 2 which concerns on this embodiment.

In recent years, the regenerative medicine industry with iPS cells etc. as a pillar has become full-scale in terms of legal regulations due to the implementation of the Pharmaceuticals and Medical Devices Act in recent years, and a more stable, efficient and simple cell culture method is desired. ing. In particular, iPS stem cells are mostly adherent cells, but there are currently no products or systems suitable for production. The culture method of the present invention is a method suitable for mass culture of such adherent cells and / or cell masses.
Hereinafter, a cell culture method and a cell culture kit according to embodiments of the present invention will be described respectively.

<Cell culture method>
〔Overview〕
A scaffold according to an embodiment of the present invention (hereinafter referred to as this embodiment) includes a first functional molecule and an arbitrary carrier. Moreover, the scaffold according to the present embodiment may have a second functional molecule in addition to the first functional molecule. In the present embodiment, a scaffold that has the first functional molecule and an arbitrary carrier and does not have the second functional molecule is referred to as a “first scaffold”. Further, a scaffold having the first functional molecule, the second functional molecule, and an arbitrary carrier is referred to as a “second scaffold”.

  Here, the first functional molecule includes a first molecular structure capable of capturing a lipid bilayer structure, a second molecular structure that self-assembles, a second molecular structure, and a carrier. A linker molecule for linking with each other. The second functional molecule includes a second molecular structure that self-assembles and a linker molecule that connects the second molecular structure and the carrier, and can capture the lipid bilayer structure. The first molecular structure is not included.

The first scaffold can be formed by self-organizing only the first functional molecule on the carrier. The second scaffold can be formed by self-organizing the first functional molecule and the second functional molecule on the carrier.
The above-mentioned scaffold is brought into contact with a suspension containing cells and / or cell masses (hereinafter simply referred to as cells) in a physiologically active environment for cell culture, so that the cells maintain a high density and original form. A composite can be formed that adheres with. Here, the cells include at least one kind of adherent cells, for example. Moreover, a cell contains at least 1 or more types of animal origin cells, for example. In the following description, a complex in which cells are adhered to a first scaffold is referred to as a “first complex”, and a complex in which cells are adhered to a second scaffold is referred to as a “second complex”.

In the present embodiment, the first complex or the second complex is retained for a certain period of time in a physiological environment for cell culture, so that the surface area can be effectively used on the scaffold as much as possible, and a form close to a living body. The cells can be cultured. In addition, after this culture, cells can be separated and collected from the scaffold.
Next, the present embodiment will be described with reference to the drawings.

〔Constitution〕
FIG. 1A is a schematic diagram illustrating a configuration example of the first functional molecule 100 according to the present embodiment. As shown in FIG. 1 (a), the first functional molecule 100 recognizes the hydrophilic polar head and the hydrophobic portion of the phospholipid constituting the lipid bilayer structure of the cell and recognizes the lipid bilayer. A first molecular structure 1 that can be captured in a form that penetrates into the multilayer structure, a second molecular structure 2 that can be self-assembled by interaction between molecules, and an arbitrary carrier 4 A linker molecule 3 capable of binding to The first molecular structure 1 includes a first portion 11 that captures (eg, binds or adsorbs) a hydrophilic polar head, and a second portion that captures (eg, binds or adsorbs) a hydrophobic portion. 12 and so on.

  FIG. 1B is a schematic diagram illustrating a configuration example of the second functional molecule 200 according to the present embodiment. As shown in FIG. 1 (b), the second functional molecule 200 is bonded to the second molecular structure 2 that can be self-assembled by the interaction between the molecules and the arbitrary carrier 4. And a linker molecule 3 capable of The difference between the second functional molecule 200 and the first functional molecule 100 is only the presence or absence of the first molecular structure 1, and the second functional molecule 200 includes the first molecular structure 1. No.

  FIG. 2A is a schematic diagram illustrating a configuration example of the first scaffold 300. As shown in FIG. 2A, the first scaffold 300 includes the first functional molecule 100 and the carrier 4. The first functional molecule 100 is bound / adsorbed to the carrier via the linker molecule 3 that is a component of the first functional molecule 100. Further, in the first scaffold 300, the first functional molecule 100 can be self-assembled by the interaction of the second molecular structure 2 that is a constituent element (see the arrow in FIG. 2A). And a high-density structure of the first functional molecule 100 can be formed.

  FIG. 2B is a schematic diagram illustrating a configuration example of the second scaffold 400. As shown in FIG. 2 (b), the second scaffold 400 includes the first functional molecule 100, the second functional molecule 200, and the carrier 4. The first functional molecule 100 is bonded / adsorbed to the carrier 4 via the linker molecule 3 which is a constituent element. Similarly, the second functional molecule 200 is also bonded / adsorbed to the carrier 4 via the linker molecule 3 that is a constituent element thereof.

  Further, in the second scaffold 400, the first functional molecule 100 and the second functional molecule 200 interact with the second molecular structure 2 that is a component thereof (the arrow in FIG. 2B). And a high-density structure in which the first functional molecule 100 and the second functional molecule 200 are mixed can be formed. For example, the first functional molecule 100 and the second functional molecule 200 can form a high-density structure arranged so as to be adjacent to each other on the carrier 4.

  FIG. 3A is a schematic diagram illustrating a configuration example of the first complex 500. As shown in FIG. 3A, the first complex 500 includes a first scaffold 300 and cells 5 attached to the first scaffold 300. In the first complex 500, the cell 5 adheres to the first scaffold 300 via the first molecular structure 1 that is a component of the first scaffold 300. The cell 5 has a hydrophilic polar head (an example of a hydrophilic part) 51 and a hydrophobic part 52 of a phospholipid constituting a lipid bilayer structure. The first portion 11 of the first molecular structure 1 recognizes and captures the hydrophilic polar head 51 of the cell 5. Further, the second portion 12 of the first molecular structure 1 recognizes and captures the hydrophobic portion 52 of the cell 5.

FIG. 3B is a schematic diagram illustrating a configuration example of the second complex 600. As shown in FIG. 3 (b), the second complex 600 includes the second scaffold 400 and the cells 5 attached to the second scaffold 400. Similar to the first complex 500, in the second complex 600, the cells 5 adhere to the first scaffold 300 via the first molecular structure 1 that is a component thereof.
FIG. 4 is a cross-sectional view schematically showing the process of culturing the cell 5 according to this embodiment. Note that an enlarged view of a portion surrounded by a broken line in FIG. 4 corresponds to the above-described FIG. 3A (or FIG. 3B). As shown in FIG. 4, the cell culture method according to the present embodiment has a smaller adhesion area per cell and changes the shape of the cell as compared with the case where the cell adheres onto the existing functional microcarrier. The cells can adhere at a high density in a state close to that of the living body (In vivo). For this reason, a cell culture can be performed more effectively utilizing the scaffold area.

〔Concrete example〕
Next, the present embodiment will be described with a specific example.
(1) First and second functional molecules and synthesis method thereof First, the first functional molecule 100 and the second functional molecule 200 will be described.
The first functional molecule 100 includes the following three molecular structures 1 to 3.
A first molecular structure 1 capable of binding cells by recognizing both the hydrophilic polar head and the hydrophobic part of the lipid bilayer membrane
Second molecular structure 2 that can self-assemble
A molecular structure capable of binding to any carrier (ie linker molecule 3)

The second functional molecule 200 includes the second molecular structure 2 and the linker molecule 3.
As the first functional molecule 100 and the second functional molecule 200, for example, a polypeptide having a high degree of design freedom and high biocompatibility can be used. However, it is not limited to this as long as the same function can be obtained.

  The first molecular structure 1 has a polypeptide composed of natural or artificial amino acids and at least one of alkanes having 6 to 18 carbon chains. For example, the first structure 1 uses a polypeptide having a high degree of design freedom and high biocompatibility. Examples of the portion (first portion 11) that captures the hydrophilic polar head 51 of the lipid bilayer structure include acidic amino acids (glutamic acid (E), aspartic acid (D)) or basic amino acids (lysine ( K), arginine (R), and histidine (H) can be used in combination with one or more amino acids and repeated 1 to 10 residues, for example, lysine (K) 9 residues (Hereinafter, it may be referred to as a hydrophilic polar head recognition sequence.) Here, the residue means one unit of amino acid constituting the peptide.

  For the recognition of the hydrophobic part of the lipid bilayer structure, non-polar amino acids (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F ), Tryptophan (W), proline (P)), and one or more amino acids consisting of the group consisting of 1 to 10 residues can be used. As an example, there is an amino acid sequence composed of 5 residues of alanine (A) and 1 or 2 residues of tryptophan (W) (hereinafter also referred to as a hydrophobic portion recognition sequence).

The second molecular structure 2 only needs to have self-organization ability and biocompatibility. Further, the second molecular structure 2 may have a molecular structure capable of forming a monomolecular film. The second molecular structure 2 may be a most general alkane derivative having 6 or more chains, and may be a polypeptide like the first molecular structure 1.
FIG. 5 is a plan view schematically showing the molecular structure of the second molecular structure 2. As an example, the polypeptide is the same as the first molecular structure 1, and as shown in FIG.
It consists of 4 residues of glutamic acid (E), which is an acidic amino acid, 4 residues of lysine (K), which is a basic amino acid, 5 residues of leucine (L), which is a nonpolar amino acid, and 2 residues of alanine (A). Forms an alpha helix as the next structure.

FIG. 6 is a plan view schematically showing the self-assembly mode of the second molecular structure 2. As shown in FIG. 6, the self-assembly mode of the second molecular structure 2 includes electrostatic interaction between molecules by acidic and basic amino acids (that is, charged amino acids), and intermolecular interactions by nonpolar amino acids. Can be self-organized uniformly by the hydrophobic interaction.
The linker molecule 3 only needs to be able to bind to the support 4. For example, if the support 4 is gold, it may have a thiol group or a disulfide group. If the support 4 is silica, a linker that can be bound by silane coupling. It only has to have molecules. In addition, when a functional group is modified in advance by surface treatment regardless of the material of the support 4, it is only necessary to have a linker molecule that can bind to the functional group.

The carrier 4 may be a solid phase or gel / sol body composed of organic and inorganic molecules, and the size and shape are not particularly limited, and any flat plate, three-dimensional object (particles, etc.), film, etc. may be used. be able to.
Next, the outline of the synthesis method will be described by taking as an example the case where the first functional molecule 100 and the second functional molecule 200 are polypeptides.

The method for synthesizing the first functional molecule 100 and the second functional molecule 200 may be synthesized by any of a liquid phase method and a solid phase method, which are general peptide synthesis methods. The following example is a synthesis example of the first functional molecule 100 and the second functional molecule 200 protein conjugate modified with a protein (for example, biotin), and can be synthesized in the following five steps. .
(Step 1) This step is a step of preparing a solid support. The solid support has a linker (L) having an amino group on the resin (R). Here, the solid support may not have a linker when the amino group is directly present in the resin. The solid support can be prepared by modifying a linker having an amino group on the resin. The preparation method may be a generally known method. For example, a resin such as polystyrene can be used as the resin for the solid carrier. As the linker, a commonly used linker may be used.

(Step 2) Next, amino acids necessary for the synthesis of the target first functional molecule and second functional molecule are prepared. Examples of these amino protecting groups include a tert-butoxycarbonyl group (Boc group) and a 9-fluorenylmethyloxycarbonyl group (Fmoc group). A known method may be used to introduce the protecting group.
(Step 3) This step is a step of binding the various amino acids prepared in Step 2 to the solid support prepared in Step 1 above. First, the first amino acid residue whose amino group is protected is bound to the amino group of the solid support. The amino group of the first amino acid residue is then deprotected and the next amino acid residue is attached. By repeating this deprotection of the amino group and the bonding of the monomer, the intended first functional molecule and second functional molecule having a solid support and an amino protecting group are obtained. Known methods can be used for amino group deprotection and amino acid binding / extension.

(Step 4) The resin (R) and linker of the first functional molecule and the second functional molecule obtained in the above step 3 are removed, and the first functional molecule and the second functional molecule having an amino protecting group are removed. Obtain functional molecules.
(Step 5) Deprotecting the amino protecting group (P) of the first functional molecule and the second functional molecule, and linker molecules at the respective ends of the first functional molecule and the second functional molecule Are combined. A known method can be used to bond the linker molecule.
Through the above steps 1 to 5, the first functional molecule 100 and the second functional molecule 200 can be obtained.
Another example of the method for synthesizing the first functional molecule 100 and the second functional molecule 200 is described below.

  In the above steps 1 and 2, the amino group of the solid support is replaced with a carboxyl group, the carboxyl group of the constituent amino acid is protected, and the constituent amino group and the carboxyl group of the solid support are first bonded. Then, the protected carboxyl group is deprotected, and in the same manner as in Step 3, the deprotection of the carboxyl group and the coupling of the amino group of the appropriate monomer are repeated, thereby including the first functionality containing the solid support and the protecting group. A molecule and a second functional molecule can be obtained. Thereafter, a linker molecule is introduced in the same procedure as in Steps 4 and 5 above. As still another example, constituent amino acids may be sequentially bound using a linker molecule as an end point.

(2) Cell Culture Method Next, a cell culture method will be described.
The cell culture method according to the present embodiment includes the following four steps 1 to 4.
Step 1: Forming a scaffold containing a self-assembled assembly of the first functional molecule 100 Step 2: Contacting the suspension with a suspension containing cells in a physiologically active environment for cell culture, the first function Step 5 for adhering cells 5 onto the scaffold via the first molecular structure 1 of the sex molecule 100: Step 4 for maintaining cells in a physiologically active environment for cell culture and allowing the cells to grow on the scaffold Step 4: Steps for separating cells are described below.

(Process 1)
Step 1 is a step of forming the first scaffold 300 or the second scaffold 400. In this step, the first functional molecule 100 alone (or the first functional molecule 100 and the second functional molecule 200) is brought into contact with the carrier 4 in a state in which the first functional molecule 100 and the second functional molecule 200 are dissolved in the buffer. The scaffold 300 (or the second scaffold 400) can be formed. This step needs to be performed in a reaction vessel in which the first functional molecule 100 or the like is not adsorbed.

  The buffer can be freely selected depending on the linker molecule and the binding mode of the support 4 and the first functional molecule 100 or the second functional molecule 200. For example, if the first functional molecule and the second functional molecule are polypeptides, the conditions may be such that the self-assembly and the linker molecule-carrier binding reaction proceed. The number of molecules necessary for the reaction is not limited as long as a uniform self-assembled aggregate can be formed, and can be freely selected according to the size and surface area of the support 4.

  In the case of forming the second scaffold 400, the amount ratio between the first functional molecule 100 and the second functional molecule 200 (that is, the number of molecules of the first functional molecule 100 and the second functionality). The ratio of the molecule 200 to the number of molecules) can also be freely selected as necessary. This ratio may be in the range of 1000: 1 to 1:10, preferably in the range of 100: 1 to 1: 9, more preferably in the range of 10: 1 to 1: 5. Regarding the reaction time, it is sufficient if a uniform self-assembled aggregate can be formed, and it can be freely selected according to the size and surface area of the support 4. An example is as described in the examples.

(Process 2)
Step 2 is a step of forming the first composite 500 (or the second composite 600). In this step, the first complex 500 (or the second scaffold 400 or the second scaffold 400) is brought into contact with a cell as a sample in a physiological environment for cell culture. A composite 600) is formed. This step needs to be performed in a reaction vessel in which the first complex 500 (or the second complex 600), a sample cell, and the like are not adsorbed.
Regarding the physiological environment for cell culture, it can be freely selected according to the type of cell. For example, if the cells are commercially available, media and culture conditions recommended by the vendor may be selected.

Regarding the volume ratio of the first scaffold 300 (or the second scaffold 400) and the cell in contact, the volume of the cell culture environment, the size of the first scaffold 300 (or the second scaffold 400). , Shape, or cell type, property, etc., can be selected freely. For example, the first scaffold 300 (or the second scaffold 400) that can secure the area to which all of the cells can adhere is sufficient, and preferably the area that can adhere all the necessary cells after culture. The first scaffold 300 (or the second scaffold 400) of the first scaffold 300 (or the second scaffold 400) is more preferable, and more preferably, the first scaffold 300 (or the second scaffold sufficiently exceeds the area where all necessary cells can adhere after culture). Scaffold 400). An example is as described in the examples.
Regarding the reaction time, it is only necessary that a sufficient amount of cells as a sample adhere to the first scaffold 300 (or the second scaffold 400), and the reaction time can be freely set as necessary. An example is as described in the examples.

(Process 3)
Step 3 is a step in which the first complex 500 (or the second complex 600) is held in a physiological environment for cell culture for a certain period of time, and the sample cells are cultured and amplified. Similar to step 2, this step needs to be performed in a reaction vessel in which the first scaffold 300 and the sample cells are not adsorbed. Moreover, the same container as the process 2 may be used and you may transfer to another container as needed.
With respect to the physiological environment for cell culture, the same conditions as in step 2 can be applied. The reaction time can be freely set according to the type of cell and the required amount. For example, as long as the culture is performed on a container such as an ordinary petri dish, it is sufficient that the concentration does not become 100% confluent. An example is as described in the examples.

(Process 4)
Step 4 is a step of separating the cells amplified after the culture and the first scaffold 300 (or the second scaffold 400) after the step 3. In this step, it is necessary to carry out the reaction in a reaction vessel in which the first scaffold 300 and the sample cells are not adsorbed as in step 2. Moreover, the same container as the process 2 may be used and you may transfer to another container as needed.

  With respect to the separation method, the cells are detached from the first scaffold 300 (or the second scaffold 400) by treatment with an enzyme such as trypsin or actase known as a general method, and then the first scaffold. This can be achieved by removing only 300 (or the second scaffold 400) or only the cells from the container. For example, when the carrier 4 constituting the first scaffold 300 (or the second scaffold 400) is a magnetic body, a solution in which cells are suspended and the first scaffold 300 (or the first scaffold 300 (or the first scaffold 300) are formed using a magnet. 2 scaffolds 400). As another method, when the carrier 4 constituting the first scaffold 300 (or the second scaffold 400) is a magnetic body, the carrier 4 is fixed with a magnet while being centrifuged or hydrodynamically. By applying force, the cells may be separated from the first scaffold 300 (or the second scaffold 400) without using a special reagent.

<Culture kit>
Next, the cell culture kit according to the present embodiment will be described.
The cell culture kit according to the present embodiment is, for example, the first scaffold 300 or the second scaffold 400 described above. The culture kit includes the first functional molecule 100 (or the first functional molecule 100 and the second functional molecule 200) and the carrier 4. In addition to the above, the culture kit may include a container or the like as necessary. When the culture kit includes the first functional molecule 100 and the second functional molecule 200, the quantity ratio (the number of molecules of the first functional molecule 100 and the number of molecules of the second functional molecule 200) Ratio) may be in the range of 1000: 1 to 1:10, preferably in the range of 100: 1 to 1: 9, more preferably in the range of 10: 1 to 1: 5.

The carrier 4 included in the culture kit may be a solid phase or gel / sol body composed of organic and inorganic molecules, and the size and shape are not particularly limited. A flat plate, a three-dimensional object (particles, etc.), a film Any form may be used. Moreover, it is good also as a magnetic body in consideration of operativity, automation, etc. as needed. For example, by using an inorganic particle-like magnetic material, the container and volume can be freely selected, and a large amount of cells can be cultured by a simpler operation.
The container used in the present embodiment is preferably processed so that cells and materials as samples are not adsorbed inside the container. In another embodiment, a container may be used as the carrier 4.

<Effect of this embodiment>
This embodiment has the following effects (1) to (3).
(1) Cell 5 is cultured using a scaffold (first scaffold 300 or second scaffold 300) having a function of capturing a lipid bilayer structure and including a functional molecule that self-assembles. To do. The cells can be cultured in a state where the cells are adhered to the scaffold (hereinafter referred to as a cell adhesion state), and the cells are proliferated by the interaction between the cells. For this reason, compared with the mechanism which adhere | attaches only by the interaction between the conventional cell surface-microcarrier surface, the adhesive force of a scaffold and a cell is large, and it is strong against external force. Therefore, for example, cells can be cultured with a high survival rate even in a fluid environment. In addition, since the cells are cultured in a cell-adhered state, not by extension, the cells can be cultured at high density. Therefore, more cells can be stably produced.

(2) When the second scaffold 400 is used, the adhesion area per cell is controlled by adjusting the quantitative ratio between the first functional molecule 100 and the second functional molecule. Can do. Thereby, for example, the adhesion area per cell can be reduced, and more cells can be efficiently cultured. Excellent operability and efficient cell culture.

(3) Each of the first functional molecule 100 and the second functional molecule 200 has a second molecular structure 2. Between adjacent second molecular structures 2, electrostatic interaction between molecules and hydrophobic interaction between molecules work. Thereby, the first functional molecule 100 and the second functional molecule 200 can be self-assembled uniformly.
EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited to the following Example.

<Example 1: Synthesis of first functional molecule and second functional molecule>
For the first functional molecule and the second functional molecule, a polypeptide composed of amino acids was used. Cysteine having a thiol group was introduced as a linker molecule. The synthesis was carried out by the FMOC solid phase method in which the amino group was protected with an FMOC group. A commercially available resin for peptide synthesis was used as the solid phase. A detailed procedure will be described below.

[Binding of C-terminal amino acid residue to polystyrene solid phase]
(1) About 150 mg (1 × 10 ⁻⁴ mol) of peptide synthesis resin is put into a synthesis column, and it is swollen while being held with 4 mL of N-methylpyrrolidone (NMP) for 5 min (min) and washed. did. Further, washing was performed about 3 times using 2 mL of NMP.

(2) In order to deprotect the Fmoc group from the terminal amino group of the synthesis resin, 2 ml of piperidine (Pperidine: PPD) / NMP (1/4, vol / vol) solution (basic deprotection solution) was injected into the column. And held for 1 min. Similarly, 2 ml of basic deprotection solution was used for 15 minutes, and the deprotection was completed by shaking and stirring every 3 minutes.

(3) In order to remove the Fmoc group in the column and the basic deprotection solution, the column was washed 5 times with 2 ml of NMP.
(4) The basic deprotection solution and washing waste liquid of (1) and (3) were collected in a 20 ml volumetric flask.

(5) 0.134 g (3 × 10 ⁻⁴ mol) of Fmoc-Cys (Trt) -OH, benzotriazolyltetramethyluronium hexafluorophosphine (2- (1-H-Benzotriazole-1-yl) -1,1,3,3-Tetramethyluronium Hexafluorophosphate (HBTU) 0.114 g (3 × 10 ⁻⁴ mol), Hydroxybenzotriazole (1-Hydroxybenzene Monohydrate: HOBt · H ₂ O (0.046) ^10-4 mol), 2 ml of NMP, and 0.11 ml of diisopropylethylamine (DIEA) were added to the column and held for 24 min. The mixture was vibrated and stirred every 3 minutes.
(6) In order to remove the remaining reagent in the column, it was washed 5 times with 2 ml of NMP.

[Elongation of peptide chain]
On the basis of the C-terminal amino acid residue bound on the solid phase in the previous section, amino acids were sequentially bound according to the designed amino acid sequence, and the peptide chain was elongated on the solid phase. The procedure is as follows. The amino acid sequences are shown in Table 1.
(1) In order to deprotect the Fmoc group from the α-amino group of the amino acid bonded to the resin for synthesis, 2 ml of a basic deprotection solvent was injected and held for 1 min.

(2) Similarly, 2 ml of basic deprotection solvent was injected and held for 15 minutes, and the mixture was shaken and stirred every 3 minutes, and washed 5 times with 2 ml of NMP to remove the remaining reagent.
(3) According to the designed amino acid sequence, Fmoc-Ala-OH (0.094 mg, 3 × 10 ⁻⁴ mol), Fmoc-Lys (Boc) —OH (0.141 g, 3 × 10 ⁻⁴ mol), Fmoc-Trp —OH (0.128 g, 3 × 10 ⁻⁴ mol), Fmoc-Glu (tBu) —OH (0.127 g, 3 × 10 ⁻⁴ mol), Fmoc-Tyr (tBu) —OH (0.187 g, 3 × 10 ⁻⁴ mol), Fmoc-Leu-OH (0.125 g, 3 × 10 ⁻⁴ mol) and HBTU, HOBt · H ₂ O, DIEA, and NMP described above are added to the column and stirred every 5 min. Hold for 60 min.

(4) In order to remove the remaining reagent in the column, washing was performed 5 times with 2 ml of NMP.
(5) The peptide chain was elongated according to the amino acid sequence designed by repeating the steps (1) to (4).
(6) The last amino acid deprotection solution and the washing waste liquid were collected in a 20 ml volumetric flask.
(7) The extract was washed 5 times with 2 ml of chloroform and dried under reduced pressure.

[Deprotection of amino acid side chain functional groups and excision from the solid phase]
The side chain functional group was deprotected and cleaved from the solid phase to purify it for actual use in the analysis. When using the synthesized peptide as a free peptide, the deprotection and excision steps were performed according to the following procedure.
(1) The synthesized peptide-modified polystyrene particles were put into a synthesis column. Separately from the column, 5 ml of diethyl ether was put into a 10 ml ground glass and stored refrigerated.

(2) 1.85 ml of trifluoroacetic acid (Trifluoroacetic acid: TFA), 0.05 ml of triisopropylsilane (0.1 ml) and 0.1 ml of pure water are added to the column of (1), and the first 1 h (hours) Vibrated every 10 min and stirred, and then held for 4 h.

(3) After holding, the excised solution of (2) was put into the refrigerated diethyl ether of (1) (ether precipitation). The resulting solution and the precipitate were divided into 1.5 ml tubes.
(4) After the divided precipitate and solution were stirred (vibration, ultrasonic treatment), the precipitate and the solution were separated by centrifugation at 2000 rpm for 5 minutes.

(5) The supernatant was removed so as not to remove the precipitate in the tube, about 1 ml of diethyl ether was added again and the mixture was stirred and washed, and then centrifuged again at 2000 rpm for 5 minutes to remove the supernatant.
(6) The obtained precipitate was dissolved in H ₂ O / acetonitrile (1/1, vol / vol), purified by HPLC, and the molecular weight was confirmed by MALDI-TOF-MS.

<Example 2: Production of first scaffold or second scaffold>
The production of the first scaffold or the second scaffold will be described. For the first functional molecule or the second functional molecule, the polypeptide synthesized in Example 1 was used, and as the scaffolding material, gold-coated magnetic silica particles (custom product manufactured by Micromond) were used. 50 mg of gold-coated magnetic silica particles and the first functional molecule or the second functional molecule were mixed in an EtOH / MeOH (1/1, vol / vol) solution with a tube rotator for 1 hour under the conditions shown in Table 2. The magnetic silica particles were collected using a magnet. Table 2 shows the quantitative ratio (ratio of the number of molecules) of the first functional molecule and the second functional molecule in the scaffold.

<Example 3: Production of first complex or second complex and cell culture>
For preparation of the first complex or the second complex and cell culture, two types of samples, human-derived lung cancer cells MCF-7 and human neonatal dermal fibroblasts (NHDF), are used as scaffolds. As, the 1st scaffold produced in Example 2 or the 2nd scaffold was used. As for the physiological environment for culture, MCF-7 is D-MEM (10 vol% fetal bovine serum, 1 vol% NEAA, 1 vol% penicillin / streptomycin), NHDF is D-MEM (10 vol% fetal bovine serum, 1 vol% penicillin). / Streptomycin) medium was used, and the container was repeatedly used 3 times using a 50 mL capacity polystyrene tube as the container. In addition, as a representative example of an existing functional microcarrier for comparison, the test was performed using Global Eucalyptic Microcarrier: GEM ^™ beads manufactured by Global cell solution. After culturing for evaluation were compared by counting the number of living cells in Life Technologies Inc. Cell Counter (CountessII ^TM) using trypan blue solution. The detailed procedure is as follows.

(1) 40 mL of the medium was added to the container, and the cells were suspended at 1 × 10 ⁵ cells / mL.
(2) Add 50 mg of the first complex or the second complex (GEM ^TM beads) into the container and stir well by pipetting.
(3) Incubate for 5 days at 37 ° C. in a CO ₂ incubator.
The results of Example 3 are shown in Table 3.

  As shown in Table 3, when the first scaffold and the second scaffold were used, better results were obtained with respect to the number of cells and the survival rate than when the existing functional microcarrier was used under any conditions. It was. In the case of MCF-7, it was shown that the maximum number of cells was 1.4 times that of existing functional microcarriers and the survival rate (%) was 1.2 times higher. In the case of NHDF, it was shown that the maximum cell number was 1.5 times and the survival rate (%) was 1.2 times higher. Also, the standard deviations were all small and stable.

DESCRIPTION OF SYMBOLS 1 1st molecular structure 2 2nd molecular structure 3 Linker molecule 4 Carrier 5 Cell 11 1st part 12 2nd part 51 Hydrophilic polar head 52 Hydrophobic part 100 1st functional molecule 200 Second functional molecule 300 First scaffold 400 Second scaffold 500 First complex 600 Second complex

Claims (12)

Providing a scaffold having a first functional molecule comprising a first molecular structure capable of capturing a lipid bilayer structure and a second molecular structure that self-assembles;
Contacting the cell having the lipid bilayer structure with the scaffold under a physiologically active environment for cell culture, and adhering the cell onto the scaffold via the first molecular structure;
Maintaining the physiologically active environment for cell culture and growing the cells on the scaffold;
Separating the proliferated cells from the scaffold, and a method for culturing cells. The scaffold further comprises a carrier;
The cell culture method according to claim 1, wherein the first functional molecule includes a linker molecule that links the second molecular structure to the carrier. The scaffold is
The cell culture according to claim 2, further comprising a second functional molecule containing the second molecular structure and the linker molecule, and not containing the first molecular structure. Method.   The cell culture according to claim 3, wherein the ratio of the number of molecules of the first functional molecule to the number of molecules of the second functional molecule is in the range of 1000: 1 to 1:10. Method.   The cell culture according to claim 3, wherein the ratio of the number of molecules of the first functional molecule to the number of molecules of the second functional molecule is in the range of 100: 1 to 1: 9. Method.   The cell culture according to claim 3, wherein the ratio of the number of molecules of the first functional molecule to the number of molecules of the second functional molecule is in the range of 10: 1 to 1: 5. Method. The first molecular structure includes a first part that binds or adsorbs to the hydrophilic part of the lipid bilayer structure, and a second part that binds or adsorbs to the hydrophobic part of the lipid bilayer structure. And having a portion,
The cell culture method according to any one of claims 1 to 6, wherein the second molecular structure has a molecular structure capable of forming a monomolecular film.   The first molecular structure includes a polypeptide composed of natural or artificial amino acids and at least one of alkanes having 6 to 18 carbon chains, according to claim 7. Cell culture method.   The method for culturing cells according to any one of claims 1 to 8, wherein the cells contain at least one kind of adherent cells.   The cell culture method according to any one of claims 1 to 9, wherein the cell contains at least one kind of animal-derived cells. A first functional molecule;
A carrier that carries the first functional molecule, and the first functional molecule comprises:
A first molecular structure capable of capturing a lipid bilayer structure;
A second molecular structure that self-assembles;
A cell culture kit comprising: a linker molecule that links the second molecular structure to the carrier.   The cell culture according to claim 11, further comprising a second functional molecule that includes the second molecular structure and the linker molecule, and does not include the first molecular structure. kit.

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