JP2001259389A - Porous structure for bioreactor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a prous structure for a bioreactor having excellent affinity for the surface of a cell membrane. SOLUTION: In the porous structure for a bioreactor, a phosphorychloline analogous structure is provided on at least a part of the surface of the porous structure.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a porous structure suitable for application to a bioreactor.

[0002]

2. Description of the Related Art In recent years, so-called bioreactors, in which useful cells and enzymes are immobilized on a porous structure, have been applied to the production of physiologically active substances, replacement of some organ functions, wastewater treatment, and the like. Widely used for mass production and processing in factories.

[0003] Many types of porous materials for bioreactors having different materials, shapes, surface properties, and the like depending on the type, size, and characteristics of useful cells or enzymes to be immobilized or cultured have been developed. Representative examples include organic polymer porous membranes usually used as microfiltration membranes, ultrafiltration membranes, and dialysis membranes, sponges, particulate organic porous structures, and inorganic porous materials such as ceramics and glass. Examples of the structure include a hollow fiber membrane, a flat membrane, a sponge, and a porous particle.

[0004] Materials used include organic substances such as cellulose or cellulose derivatives, natural or naturally derived polymers such as chitin, chitosan and dextran;
Examples include polyethylene, polypropylene, polyester, polyacrylonitrile, acrylic acid derivatives, synthetic organic polymers such as polyvinyl alcohol, polysulfone, and polyurethane, and inorganic polymers such as silica and alumina.

[0005]

The conventional porous structure for a bioreactor has a problem in that the affinity between the porous structure and the surface of the cell membrane is low particularly when the purpose is to fix and grow cells. Therefore, at the time of cell attachment and growth, the survival time of the cells is reduced as the culture time is prolonged, and it is difficult to grow cells at high density and in large quantities. In addition, when a bioreactor is used for cell culture, when the cells are collected after the culture is completed, the use of proteolytic enzymes may significantly damage the cultured cells, and the recovery rate of viable cells is reduced. There is a problem. That is, an object of the present invention is to provide a porous structure for a bioreactor having excellent affinity with the cell membrane surface.

[0006]

The gist of the present invention is that a porous structure for a bioreactor has a structure similar to a phospholipid component constituting a cell membrane on a surface of a porous structure for a bioreactor in order to obtain excellent affinity with the cell membrane surface. It is to introduce.

That is, the present invention is as follows. A porous structure for a bioreactor, wherein at least a part of the surface of the porous structure has a structure represented by the following general formula [1].

Embedded image (In the general formula [1], R ¹ and R ² are the same or different and each have an alkyl group having 1 to 20 carbon atoms,
2 represents an aryl group or an aralkyl group having 7 to 20 carbon atoms. )

[0008] A porous structure for a bioreactor, wherein at least a part of the surface of the porous structure has a structure represented by the following general formula [2].

Embedded image (In the general formula [2], R ¹ and R ² are the same or different and each represent an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 12 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms; R ³ represents an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 12 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, or the following group [3].

Embedded image (A is at least one group selected from the group consisting of an oxyethylene group, an oxypropylene group, an oxybutylene group, an oxypentamethylene group, and an oxyhexamethylene group, and two or more may be mixed; When A is composed of one kind of group, n is an integer of 1 to 30. When A is composed of two or more kinds of groups, n is
Is an average value and represents a number from 1 to 30. R ⁴ represents an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 12 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms. ))

A porous structure for a bioreactor as described above or above, wherein a diol component having a structure represented by the following general formula [4] is used as a part of the raw material.

Embedded image (In the above general formula [4], R ¹ , R ² and R ³ each have the same meaning as defined in the general formula [2],
R ^5, R ⁶ are the same or different, represent an alkylene group having 1 to 10 carbon atoms, m represents an integer of 1 to 10, R ⁷
Represents a hydrogen atom, an alkyl group having 1 to 20 carbon atoms,
Represents an aryl group having 12 or an aralkyl group having 7 to 20 carbon atoms. The above-mentioned porous structure for a bioreactor, comprising a polyurethane or a polyurethane urea obtained by using a diol having a structure represented by the general formula [4] as at least a part of a diol component. The porous structure for a bioreactor according to any one of the above-mentioned or the above, wherein the porous structure is in the form of a hollow fiber membrane, a flat membrane, or a sponge.

The structure represented by the general formula [1] (hereinafter referred to as a phosphorylcholine-like structure) is similar to a phospholipid (phosphatidylcholine) forming a biological membrane.
A polymer material having this structure on the surface has a high affinity for cells. For this reason, when a phosphorylcholine-like structure is introduced onto the surface of a porous structure for a bioreactor, a high affinity between the porous structure and immobilized or proliferating cells is developed. It was found that when the cells were collected from the structure, damage to the cells was reduced.

[0011] The phosphorylcholine-like structure may cover the entire surface of the porous structure, but a high affinity with cells can be obtained even if it is present only on a part of the surface of the porous structure. This is considered to be because phospholipids on the cell membrane surface selectively adsorb to phosphorylcholine-like portions on the porous material surface.

The phosphorylcholine-like structure has the general formula [1]
In the above, the structure represented by the general formula [2] has a phosphorylcholine-like structure in the bioreactor even if it is hydrolyzed due to the presence of a phosphate ester bond which is easily hydrolyzed at the terminal. It is preferable because R ³ in the general formula [2] is represented by the general formula [3] because the compound has a hydrophilic group at the terminal, so that the affinity with cells is further increased. preferable. Also,
It is particularly preferable to use a diol component having a structure represented by the general formula [4] as a raw material for a porous structure, since a phosphorylcholine-like structure is easily expressed on the surface and a hydrophilic group is present on the outermost surface of the material.

The method of using a diol component having the structure represented by the general formula [4] as a raw material for a porous structure is not limited at all, but a diol having a phosphorylcholine-like structure represented by the general formula [4] is used. Is used at least as a part of the diol component, the polyurethane or polyurethane urea has an excellent hydrophilic / hydrophobic balance,
It is preferable to have a phosphorylcholine-like structure in the side chain, so that the phosphorylcholine-like structure can be effectively expressed on the material surface.

The porous structure is generally used in the form of a hollow fiber membrane, a flat membrane, a sponge, or the like, and has a preferable shape. The porous structure is suitable for cells to be immobilized or cultured, and is optimal for use as a bioreactor. The shape is not particularly limited as long as the shape is appropriate.

The method of introducing the phosphorylcholine-like structure into the porous structure is not particularly limited, and a method of physically or chemically immobilizing phosphorylcholine on the surface of the porous structure after molding, such as a porous method Using a functional group or a charge on the surface of the structure to chemically bond a phosphorylcholine-like structure, or to coat a substance having a phosphorylcholine-like structure on the surface, or to use a substance having a phosphorylcholine-like structure in the molecule. A method of forming a porous structure by using the same, and a method of mixing a material having a phosphorylcholine-like structure in a molecule into a raw material at the time of forming the porous structure.

Hereinafter, embodiments of the present invention will be described with reference to examples.

EXAMPLES Production of urethanes

Embedded image Diol represented by the above formula [5] (hereinafter abbreviated as CDO) 5
0.00g, polytetramethylene glycol (average molecular weight 1
320) (hereinafter abbreviated as PTMG) 56.23 g and N-methyl-2-pyrrolidone (hereinafter abbreviated as NMP) 1000 ml were dissolved and heated at 100 ° C. on an oil bath. Hexamethylene diisocyanate (HDI)
33.87 g was slowly added after sufficiently replacing the inside of the reactor with argon gas. After the addition, the mixture was stirred at 100 ° C. for 2 hours, 123.28 g of 4-methylenebis (cyclohexyl isocyanate) (hereinafter abbreviated as HMDI) was slowly added, and the mixture was stirred at 100 ° C. for 7 hours after the addition. Slowly add 38.26 g of butanediol and add 12
Stirred for hours. After the reaction, this reaction mixture was poured into 5000 ml of a 50% aqueous acetone solution. The resulting precipitate was separated by filtration, washed again with acetone, and the resulting precipitate was recovered and dried under reduced pressure to obtain 241.31 g of polymer A. The weight average molecular weight of the obtained polymer A was 112,000.

As a polymer, 18% by weight of polyether sulfone (Sumitomo Chemical Co., Ltd., Sumika Excel 4800P),
63.82% of NMP as a solvent, 16% by weight of triethylene glycol as a non-solvent, and 0.18% of polymer A in 1% by weight of polyvinylpyrrolidone K90 as a hydrophilizing agent were stirred and dissolved at 50 ° C. for 3 hours. After leaving still for 1 hour, the undissolved material was removed with a sintering filter to obtain a spinning stock solution. Slit outer diameter 300 μm, slit inner diameter 200 μ
m, a spinning stock solution was discharged from an outer slit of a tube-in-orifice type nozzle having an inner liquid discharge hole diameter of 100 μm, and an inner liquid of 30 wt% of water, 60 wt% of NMP, and 10 wt% of triethylene glycol was discharged from an inner liquid discharge hole. The spinning solution discharged from the nozzle passed through a 30 cm aerial traveling section and was led to a coagulation bath immediately below the nozzle. The coagulation liquid is water 60wt%, NMP3
2 wt%, triethylene glycol 8 wt%, temperature is 6
It was 0 ° C. The solidified hollow fiber membrane was washed with water, dried, and wound up with a winder. Winding speed is 15m / min
Met. The inner diameter of the obtained hollow fiber membrane was 200 μm, the outer diameter was 280 μm, and the film thickness was 40 μm. Approximately 10,000 hollow fiber membranes are bundled and filled into a case, and both end faces are adhered so that the ends of the hollow fiber are open, and the membrane area is 1.5 m ^2.
Produced a hollow fiber membrane type bioreactor. Proximal tubular cells of Yorkshire pigs from 4 to 6 weeks of age were seeded in the prepared bioreactor, connected to the bioreactor system and cultured. After 14 days, when the number of cultured cells was counted, 1.1 × 10 ¹⁰ cells were present.

Comparative Example A hollow fiber membrane-type bioreactor having a membrane area of 1.5 m ² was prepared by the same operation except that the polymer A was not added during the production of the hollow fiber membrane, and cell culture was performed by the same operation. When the number of cultured cells was counted, 2.3 × 10 ⁹ cells were present.

[0019]

As is clear from the examples and comparative examples, the porous structure for a bioreactor of the present invention has a phosphorylcholine structure on the surface of the porous structure, thereby increasing the affinity for cells and improving the bioactivity. As a reactor, high-density, large-scale processing is possible in cell immobilization and culture.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C08G 18/50 C08G 18/50 B C12N 11/08 C12N 11/08 G F-term (Reference) 4B033 NA01 NA16 NB02 NB34 ND01 ND07 4D006 GA06 GA07 GA13 HA01 MA01 MA03 MA28 MA31 MA33 MC09 MC09X MC52 MC53 MC53X MC88 NA10 NA17 NA63 NA75 PC67 4J034 BA01 BA02 BA03 CA01 CA04 CB02 CB03 CC01 CD01 CD07 DA01 DB03 DB04 DG01 HC02 DG02 HC03 DG02 DG02 HC03 HC46 HC52 HC64 HC66 HC67 HC71 MA03 MA04 MA22 MA24 MA26 QC07 RA02

Claims (5)

[Claims] Claims: 1. At least a part of the surface of a porous structure,
A porous structure for a bioreactor, having a structure represented by the following general formula [1]. Embedded image (In the general formula [1], R ¹ and R ² are the same or different and each have an alkyl group having 1 to 20 carbon atoms,
2 represents an aryl group or an aralkyl group having 7 to 20 carbon atoms. ) 2. At least a part of the surface of the porous structure,
A porous structure for a bioreactor, having a structure represented by the following general formula [2]. Embedded image (In the general formula [2], R ¹ and R ² are the same or different and each represent an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 12 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms; R ³ represents an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 12 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, or the following group [3]. (A is at least one group selected from the group consisting of an oxyethylene group, an oxypropylene group, an oxybutylene group, an oxypentamethylene group, and an oxyhexamethylene group, and two or more may be mixed; When A is composed of one kind of group, n is an integer of 1 to 30. When A is composed of two or more kinds of groups, n is
Is an average value and represents a number from 1 to 30. R ⁴ represents an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 12 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms. )) 3. A diol component having a structure represented by the following general formula [4] is used as a part of the raw material.
The porous structure for a bioreactor according to the above. Embedded image (In the above general formula [4], R ¹ , R ² and R ³ each have the same meaning as defined in the general formula [2],
R ^5, R ⁶ are the same or different, represent an alkylene group having 1 to 10 carbon atoms, m represents an integer of 1 to 10, R ⁷
Represents a hydrogen atom, an alkyl group having 1 to 20 carbon atoms,
Represents an aryl group having 12 or an aralkyl group having 7 to 20 carbon atoms. ) 4. The porous structure for a bioreactor according to claim 3, comprising a polyurethane or polyurethane urea obtained by using a diol having a structure represented by the general formula [4] as at least a part of a diol component. 5. The bioreactor porous structure according to claim 1, wherein the porous structure is a hollow fiber membrane, a flat membrane, or a sponge.