JPH10108666A - Hollow fiber type incubator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hollow fiber type incubator having scarce deviation of culture media even in a large scale incubator. SOLUTION: In this hollow fiber type incubator provided with a header forming a disk like inner space at least on one end of the incubator body, the interior surface of the header is composed of a straight part 7 of the incubator body and the nozzle straight part 8 joining continuously at radius part 9, this incubator satisfies the formula, S<1/2> /20<d or V<1/3> <d, (D-d)/8<=R<=(D-d)/2 or D/10<=H, when [where, D(mm) is an inside diameter of the header of the incubator body side, d(mm) is a minimum inside diameter of the nozzle of the header, R(mm) is a radius of curvature of curved part of the header, H(mm) is a height of the disk like inner space, S(cm<2> ) is a membrane surface area of the out side of the hollow fiber of the hollow fiber type incubator, V(ml) is the delivery volume of the hollow fiber].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the culture of cells. More specifically, the present invention relates to a hollow fiber type incubator suitable for mass production of animal cells for producing useful substances.

[0002]

2. Description of the Related Art A hollow fiber type incubator has been developed by Knazek et al. (Knazek et. Al. 1972.S.) for the purpose of culturing animal cells in an environment close to a living body.
science 178: 65-67). In the hollow fiber type incubator, the material exchange between the culture solution flowing inside the hollow fiber and the cells growing on the outside of the hollow fiber through the wall surface of the hollow fiber is continuously and effectively performed. In addition, the hollow fiber type incubator secures a large number of surfaces to which cells indispensable for cell growth adhere to cells as a hollow fiber outer membrane surface in a limited volume. Therefore, if a hollow fiber type incubator is used, animal cells can be cultured at a high density of 1 to 5 × 10 ⁸ cells / ml. Usually, the reached density of animal cells is only 10 ⁶ cells / ml in static culture using a Petri dish or a flask, and is only 10 ⁷ cells / ml in perfusion culture or the like, which has been carefully studied. High-density cultivation greatly contributes to downsizing and labor-saving of the culture system.

[0003] Therefore, the hollow fiber type incubator has been considered to be a very effective means for mass-culturing animal cells for the purpose of industrial production of useful substances. However, mass culture of animal cells using a hollow fiber type incubator has not yet been sufficiently commercialized. The reason is that the hollow fiber type incubator inherently has a major drawback. The disadvantage is that in a large hollow fiber type incubator, the culture environment becomes uneven at various points in the incubator body. In order to use the hollow fiber type incubator effectively, it is necessary to homogenize the entire inside of the incubator body to an optimal environment for culture.

[0004] A hollow fiber type incubator usually has a form in which a hollow fiber bundle is housed in a cylindrical container, and has a structure called a header at both ends of the cylinder. The culture solution is supplied from one end of the nozzle of the header (the inlet side of the culture solution), and is discharged to the nozzle of the other header (the outlet side of the culture solution). Non-uniformity of the culture environment occurs in two directions: a longitudinal direction of the incubator (a direction from the culture solution inlet side to the culture solution outlet side) and a circumferential direction (a direction from the center of the culture vessel to the circumference of the culture vessel). Regarding the former,
It appears in the form of a concentration gradient. That is, oxygen,
The concentration of substances consumed by cells, such as glucose and amino acids, is higher on the inlet side of the culture solution and lowers on the outlet side. In addition, the concentrations of waste products and secretions discharged from cells such as carbon dioxide, lactic acid, and ammonia are lower on the inlet side of the culture solution and higher on the outlet side. This non-uniformity in the longitudinal direction of the incubator was considered to be a very large problem for efficient culture, but can be easily solved by appropriately reversing the flow direction of the culture solution during the culture period. there were. On the other hand, the non-uniformity of the culture environment in the circumferential direction is such that the culture solution flows quickly through the hollow fibers located at the center of the hollow fiber bundle housed in the incubator body, and slower through the hollow fibers located at the periphery. This is due to a so-called one-sided flow. In order to reduce the one-sided flow, it is necessary to optimize the structure of the hollow fiber type incubator. However, the header of the conventional hollow fiber incubator has been designed in accordance with a hollow fiber type blood processing device (a hemodialyzer, a plasma separator, an artificial lung, etc.). Alternatively, a header of a hollow fiber type blood processing apparatus has been diverted to a hollow fiber type incubator. In the case of a hollow fiber type blood processing device, the liquid flowing inside is blood or plasma having a relatively high viscosity containing blood cell components that are vulnerable to physical stress. Therefore, many headers are designed not to reduce one-sided flow in the container body, but to reduce stagnation of liquid flow in the header with the aim of reducing coagulation and residual blood in the header. (Japanese Unexamined Patent Publication No. Hei 4-30522)
9). In addition, since the flow rate of the liquid flowing inside the processing device such as the hollow fiber type blood is 50 to 200 ml / min, the header is designed to reduce the one-sided flow in the processing device main body under the conditions of 50 to 200 ml / min. Design (Japanese Unexamined Patent Publication No. 8-572)
66). However, in the case of the hollow fiber type incubator, the liquid flowing inside is a culture liquid, and does not include components that are vulnerable to physical stress such as blood cells. Further, it is an almost perfect aqueous solution and has a lower viscosity than blood or plasma. Furthermore, since the flow rate of the culture solution is almost proportional to the number of cells to be cultured, when culturing a large number of cells in a large hollow fiber type incubator, 200 times
ml / min or more, and 1000 ml / min or more depending on the type of cells. One-sided flow generated inside the hollow fiber type incubator is
There is almost no problem with small ones used at the laboratory level. However, it is not negligible for large-scale products used on a scaled-up industrial level.
Regardless of how the cultivation conditions are examined, the cultivation performance per unit area of the hollow fiber membrane (the number of maintained cells and physical production) in the small-sized hollow fiber type incubator is extremely low, It was a disappointing result that we could not scale up as expected.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a large hollow fiber type incubator having extremely high culture performance. Specifically, it is an object of the present invention to provide a hollow fiber type incubator having a small one-sided flow in the incubator body even in a large incubator.

[0006]

As a result of intensive studies to achieve the above object, the present inventor has found that the above object can be achieved by using a header having a specific structure even in a large incubator. Invented the invention. That is, the present invention relates to a hollow fiber type incubator, in which a hollow fiber bundle provided with a header forming a disk-shaped internal space at at least one end of a main body of the incubator in which a bundle of hollow fibers is housed in a cylindrical container. In the type incubator, the inner surface of the header has a straight portion of the incubator body side and a nozzle straight portion continuous at a round portion, and the inner diameter of the header on the incubator body side is D (mm). The minimum inner diameter of the header nozzle is d (mm), and the radius of curvature of the radius portion of the header is R (m
m), the height of the disk-shaped internal space of the header is H (m
m), the hollow fiber outer culture area of the hollow fiber type incubator is S (cm).
² ) When the outer volume of the hollow fiber is V (ml), S ≧ 500
0 or V ≧ 60, and S ^1/2 / 20 <d or V ^1/3
<D, and (D−d) / 8 ≦ R ≦ (D−d) / 2 or D
/ 10 ≦ H, which is a hollow fiber type incubator. FIG. 1 shows an example of a hollow fiber type incubator according to the present invention, in which an incubator body 4 containing headers 1 and 2, a bundle of hollow fibers 3, and both ends of the hollow fiber are adhered and held inside a container body. It consists of bonding parts 5 and 6. Nozzles 1a, 2a for introducing or discharging the culture solution are provided in headers 1 and 2, respectively.
However, the incubator body 4 is provided with addition ports 4a and 4b for adding or collecting cells or adding or collecting a culture solution or the like. As the material of the incubator body and header,
Usually used, polypropylene, polycarbonate,
Polymethacrylate, styrene-butadiene copolymer and the like are also used in the present invention.

[0007] The hollow fiber referred to in the present invention is a hollow fiber membrane having material permeability, and as a material, generally used polyethylene, polypropylene, cellulose, and the like.
Regenerated cellulose, cellulose acetate, polysulfone, polyacrylonitrile, polystyrene and the like are used. Either a hydrophobic material or a hydrophilic material can be used.
The membrane itself may be used, or a membrane coated with a substance having hydrophilicity, cell affinity, antithrombotic property and the like may be used. Further, a film whose surface has been modified by graft polymerization or chemical bonding may be used. The area of the outer membrane of the hollow fiber referred to in the present invention is a portion effective for material exchange of the hollow fiber accommodated in the container body of the hollow fiber type incubator, that is, between the adhesive portions on both sides (the length of this portion is effective). (Referred to as length) of the outer membrane area of the hollow fiber, and is given by the following equation. (Hollow fiber outer membrane area) = (Hollow fiber outer diameter) x π x (Hollow fiber effective length) x (Number of hollow fibers) The hollow fiber outer volume referred to in the present invention means the volume of the incubator body excluding the hollow fiber portion. Is the volume of the so-called ECS (ex
Tra-capillary space). When the main body is cylindrical, it is given by the following equation. (Hollow fiber outer volume) = (Inner radius of main body) ² x π x (effective length of hollow fiber)-(hollow fiber outer radius) ² x π x (effective length of hollow fiber)
× (the number of hollow fibers) The present invention is directed to a large-sized incubator having S ≧ 5000 or V ≧ 60. The cells to be cultured in the hollow fiber type incubator according to the present invention are mainly animal cells, and include both adherence and floating. Examples include Hela (cells derived from human cervical cancer), Balb3T3 (cells derived from mouse embryo), L
929 (cells derived from mouse synthetic tissues), BGM (cells derived from African green monkey kidney), Vero (cells derived from African green monkey kidney), BHK (cells derived from neonatal Syrian hamster kidney), CHO (cells derived from Chinese hamster ovary), and genes , And hybridomas. Also, not limited to cell lines, hepatocytes,
Primary culture cells such as pancreatic cells, blood stem cells, progenitor cells, lymphocytes, blood cells such as T cells and B cells are also included. These include cells in which a gene has been introduced into these cells, cells that have been activated by the application of an appropriate humoral factor, and the like. Further, the hollow fiber type incubator according to the present invention is effective for culturing any animal cells, but is particularly suitable for culturing animal cells for producing useful substances. Examples of this include the production of pharmaceuticals, diagnostics, and the like by animal cells into which genes have been introduced, the production of vaccine viruses by animal cells that have been infected with viruses, and the production of antibodies by hybridomas.

The structure of the header of the hollow fiber type incubator according to the present invention is such that the straight portion 7 on the inner side of the incubator and the nozzle straight portion 8 on the inner surface of the header in FIG. When the inside diameter of the incubator body side opening 10 is D, the minimum inside diameter of the nozzle 11 is d, the radius of curvature of the round portion 9 is R, and the height of the disc-shaped internal space is H, d:
R and H have characteristics. If the value of d is small, the flow of the culture solution is biased toward the center when the culture solution is flowed at a high flow rate, and the flow rate in the peripheral portion is reduced. Therefore, d is S ≧ 500
0 or in a large incubator with V ≧ 60, S ^1/2/2
It is necessary that 0 <d. When d takes a value smaller than this, it is not preferable because one-sided flow of the culture solution occurs inside the incubator at a high flow rate. In addition, adopting a structure conforming to the standard, such as a sanitary ferrule-shaped nozzle, facilitates the incorporation of the hollow fiber type incubator into the culture system, and facilitates maintenance and maintenance of sterility during the culture period. It is effective from the point of view. When the value of R is small, when the culture solution flows at a high flow rate, the culture solution is not evenly distributed to the hollow fiber bundles. Therefore, R is S
≧ 5000 or in a large incubator with V ≧ 60,
(D−d) / 8 ≦ R ≦ (D−d) / 2, preferably
It is necessary that (D−d) /3.5≦R≦ (D−d) / 2. It is not preferable that R takes a value less than this range because a single flow of the culture solution occurs inside the incubator at a high flow rate. If the value of R exceeds this range, stagnation of the liquid flow in the header is likely to occur, so the upper limit is preferably (D−d) / 2. Also,
Even if R <(D−d) / 8, if H satisfies D / 10 ≦ H, more preferably D / 4.54 ≦ H,
The same effect as in the case of (D−d) / 8 ≦ R ≦ (D−d) / 2 can be obtained. The hollow fiber type incubator of the present invention is large, and hardly generates a one-sided flow in the main body of the incubator even when the flow rate of the culture solution is high. Therefore, it is possible to maintain cells at high density throughout the incubator.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to embodiments.

EXAMPLE A 6600 polyethylene hollow fiber coated with a hydrophilic material (inner diameter 340 μm, film thickness 50 μm) was housed in a cylindrical polycarbonate container having an inner diameter of 52 mm at both ends, and both ends of the hollow fiber were placed inside the container with urethane. By maintaining the adhesion, an incubator body having an effective length of the hollow fiber of 220 mm, a hollow fiber outer membrane area of 2.0 m ² , and a hollow fiber outer volume of 246 ml was produced. Headers were arranged at both ends to obtain a hollow fiber type incubator, and a flow visualization test was performed. The flow visualization test is as follows. Tubes of an appropriate length were attached to header nozzles at both ends of the incubator, and a 1.5% transparent agar solution kept at 60 ° C. was flowed at a predetermined flow rate. Next, switch to an agar solution appropriately colored with ink or the like,
It flowed at a predetermined flow rate. When the colored agar solution had flowed to 1/3 of the incubator main body, the pump was stopped, and the entire incubator was quickly cooled by, for example, immersing in cold water. After the agar solution was completely solidified, the incubator was cut in a longitudinal direction into two parts along a plane including the axis passing through the center of the cylinder, and the length of the colored agar (penetration length) of the cross section was measured. It was expressed as a ratio of the penetration depth of the peripheral portion to the penetration length of the central portion (hereinafter, referred to as a peripheral penetration length ratio (%)).

[0010]

Example 1 At both ends of the hollow fiber type incubator body described above, D was 52 (mm) and d was 8 (m) with the header shown in FIG.
m), R was set to 12 (mm), and H was set to 5 (mm), and a flow visualization test was performed. Flow rate 500ml
/ Min, the penetration ratio at the periphery was 79.6%.

[0011]

Example 2 At both ends of the hollow fiber type incubator body described above, D was 52 (mm) and d was 8 (m) with the header shown in FIG.
m), R was set to 21 (mm) and H was set to 5 (mm), and a flow visualization test was performed. Flow rate 500ml
At / min, the peripheral penetration ratio was 103.0%, and almost no one-sided flow occurred. In addition, flow velocity 1
At 500 ml / min, it was 90.0%, and almost no one-sided flow occurred.

[0012]

Example 3 At both ends of the hollow fiber type incubator body described above, D was 52 (mm) and d was 8 (m) with the header shown in FIG.
m), R was set to 5 (mm), and H was set to 12 (mm), and a flow visualization test was performed. Flow rate 500ml
/ Min was 80.1%.

[0013]

Comparative Example 1 D was 52 (mm) and d was 8 (m) at both ends of the hollow fiber type incubator body described above with the header shown in FIG.
m), R was set to 5 (mm), and H was set to 5 (mm), and a flow visualization test was performed. Flow rate 500ml /
In minutes, the amount was 66.9%, and the one-sided flow was remarkable. Table 1 shows the flow test results of Examples 1 to 3 and Comparative Example 1.
Table 2 summarizes the calculation results of each parameter. The criterion was x when the peripheral flow rate ratio was less than 70%, ％ when 70% or more and less than 90%, and ◎ when 90% or more.

[0014]

[Table 2]

[0015]

When the hollow fiber type incubator of the present invention is used, even when a culture solution is flowed at a high flow rate in a large-sized incubator, almost no one-sided flow occurs in the incubator body. Therefore, uniform culture conditions can be provided for the cells maintained at the center of the incubator and the cells maintained at the periphery of the incubator, and the entire incubator can be effectively used even in a large incubator. Therefore, the scale-up of high-density cultivation of animal cells by the hollow fiber type incubator can be achieved, and efficient mass cultivation of animal cells has become possible.

[Brief description of the drawings]

FIG. 1 is a schematic view showing an example of a hollow fiber type incubator of the present invention.

FIG. 2 is a schematic sectional view showing an example of a header used in the hollow fiber type incubator of the present invention.

[Explanation of symbols]

 1, 2 Header 1a, 2a Nozzle 3 Hollow fiber 4 Incubator main body 4a, 4b Addition port 5, 6 Adhesive part 7 Container main body side straight part 8 Nozzle straight part 9 Round part 10 Incubator main body side opening 11 Nozzle

Claims (1)

[Claims] 1. A hollow fiber type incubator, wherein at least one end of an incubator main body in which a hollow fiber bundle is housed in a cylindrical container is provided with a header forming a disc-shaped internal space. In the type incubator, the inner surface of the header has a straight portion of the incubator body side and a nozzle straight portion continuous at a round portion, and the inner diameter of the header on the incubator body side is D (mm). The minimum inner diameter of the header nozzle is d (mm), and the radius of curvature of the radius portion of the header is R (m
m), the height of the disk-shaped internal space of the header is H (m
m), the hollow fiber outer culture area of the hollow fiber type incubator is S (cm).
² ) When the outer volume of the hollow fiber is V (ml), S ≧ 500
0 or V ≧ 60, and S ^1/2 / 20 <d or V ^1/3
<D, and (D−d) / 8 ≦ R ≦ (D−d) / 2 or D
/ 10 ≦ H, a hollow fiber type incubator.