JPH0644857B2 - Cell culture device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is intended to grow microorganisms, particularly filamentous fungi such as molds and actinomycetes, or animal cells, plant cells, etc., to produce various antibiotics, various enzymes, proteins, The present invention relates to a cell culture device suitable for a cell culture method for producing useful substances such as polysaccharides, physiologically active substances, animal and plant hormones.

(Prior Art) Conventionally, a liquid culture method and a solid culture method have been used for culturing filamentous fungi, etc., and a liquid or solid culture method is selected depending on the type of bacteria and the type of target product.

In the case of the solid culture method, natural products such as rice and bran are used as a medium, and the culture is mainly performed by static culture. In recent years, as an alternative to the solid culture method using a natural product as a medium, a method of impregnating a sponge-like substance with a culture solution and performing static culture has also been reported.

The present applicant has proposed a novel cell culture method in Japanese Patent Application No. 61-106393. This cell culture method is used for synthetic polymeric materials such as urethane foam, foamed composite polymeric materials, or porous materials such as natural sponge such as sponge, and synthetic fibers such as holyester. , Macromolecules such as cotton, natural fibers and other thread-like, lace-like, cloth-like, napped, etc., are impregnated with the culture solution, cells are inoculated, and then the polymer By moving the porous object by shaking, shaking, mixing, etc., to enhance the contact between the cells and the culture solution and air (oxygen), and to form the cells as a uniform biofilm in the surface layer of the porous object. , A method of increasing the growth rate of cells, increasing intracellular activities of various enzymes, promoting extracellular secretory action, and increasing productivity of a target product. It is attracting attention from the academic field.

(Problems to be Solved by the Invention) In the case of the solid culture method, since the composition of the medium components varies depending on the lot of the solid natural medium, the amount of cells to be propagated or the yield of the desired product may vary greatly, or Since the product is accumulated in the solid medium, it has a drawback that the separation / purification becomes complicated. In the method of impregnating the sponge-like object with the culture solution and statically culturing, the target product accumulates in the liquid medium, so separation and purification is easy, but contact between the bacterial cells and the medium and air is insufficient. In addition, there is a problem in that the efficiency of the movement of the substance is poor because it is mainly due to diffusion, and the growth of the cells is non-uniform, so that the proliferation rate is reduced and the productivity of the target product is suppressed.

The invention described in the above-mentioned Japanese Patent Application is a novel cell culturing method, which is different from the above-mentioned conventional method, in which a polymeric porous material is uniformly impregnated with a liquid medium, cells are inoculated, and the polymeric porous material is shaken and shaken. By increasing the contact between the cells and the culture solution and air (oxygen)
Although it has a feature that it is possible to increase the growth rate of cells in the surface layer of the porous polymer object and increase the production of the target product, there is a problem that there is no apparatus suitable for this. That is, the object of the present invention is to further utilize the characteristics of the invention described in the above-mentioned Japanese Patent Application No. 1 to 5, by continuously kneading and culturing a polymeric porous body containing cells and a culture solution, and continuously producing cells and objects. It is an object of the present invention to provide a culture device that separates a culture solution containing a product, and collects a polymer porous body containing cells and the culture solution, respectively.

(Means for Solving the Problems) The above-mentioned problems are caused by the cell culture apparatus of the present invention, that is, the introduction of a polymeric porous body having a strength that does not shatter into small pieces only by being compressed to reduce the volume. A porous body containing portion having a mouth at one end and an outlet for discharging the polymer porous body at the other end, and cells and a culture solution which are located in the porous body containing portion and are to be proliferated. A cell culture device body having a screw kneading blade that moves from the input port to the discharge port while kneading and mixing the polymer porous object, and squeezing the polymer porous object discharged from the discharge port. This is solved by a cell culture device provided with a squeezing device for squeezing out the liquid impregnated into the polymer porous body.

(Operation) A culture apparatus for inoculating cells to be proliferated in a conventional solid culture method, in particular, a solid culture method, which is found in a conventional solid culture method, and allowing the solid medium to stand for culturing, and the present invention. The difference from a culture device that impregnates a molecular porous material with a culture solution, inoculates cells to be proliferated, and continuously cultivates while moving is that the polymer porous material is moved to impregnate the culture solution. Is uniformly dispersed in the porous body, and at the same time, the surface layer of the polymer porous body can be constantly brought into contact with air oxygen, and by positively promoting the mass transfer of oxygen and culture medium to cells, Means are provided for continuously growing cells as a uniform biofilm within the surface of the porous body. That is, a polymeric porous body containing a culture solution and cells to be proliferated is continuously or batchwise charged from one end of the porous body storage part, and a screw provided in the porous body storage part The kneading blades appropriately knead and mix the individual polymeric porous objects to promote oxygen transfer to the cells by keeping them in contact with the injected gas whose temperature and humidity are adjusted, as well as the fermentation heat and carbon dioxide gas generated during the culture process. It can be eliminated and the appropriate culture temperature is maintained to promote cell growth. The setting of the cell culture period in the porous object housing part is performed by setting the pitch of the screw kneading blade, the number of pitches and the rotation speed of the screw kneading shaft appropriately, and controlling it to achieve a high value for the length of the porous object housing part. A predetermined culture period can be given by defining the moving speed of the molecular porous object, and new polymeric porous objects containing cells and culture solution are continuously added according to the moving amount of the polymeric porous object. By doing so, continuous culture can be performed.

When harvesting the target product, cells and culture liquid are continuously separated and collected by squeezing the culture liquid by squeezing the polymer porous material holding the culture liquid containing the proliferated cells and the target product with a squeezing device. can do.

(Effects of the Invention) According to the present invention, cells are inoculated into a polymer porous body such as urethane foam impregnated with a liquid medium, and the porous body is appropriately kneaded and cultured by a screw kneading blade. Unlike the solid-state culture method described above, cells can be cultured without rate-limiting the oxygen and medium mass transfer processes to the cells, increasing the cell growth rate, increasing the intracellular activity of various enzymes, and secreting them outside the cells. The action can also be promoted, the production activity of the target product can be enhanced, and the polymer porous body can be continuously or semi-batchly moved in the cell culture device,
Since continuous squeeze separation can be performed, the production efficiency of the target product can be increased.

As a result, 1) it is possible to easily set culture conditions suitable for the cell growth characteristics of various cells by using the cell culture device of the present invention,
In particular, it makes it possible to continuously culture the solid culture method by batch operation found in the conventional method, and it is possible to continuously separate and recover the target product and cells, which gives a great effect to industrial production. be able to.

2) By improving the mass transfer process, a large amount of cells can be grown in a small amount of medium, a large amount of various industrially useful enzymes can be secreted, the productivity of useful target products can be increased, and a high concentration of The target product can be continuously separated and collected directly from the culture solution,
It is possible to improve the efficiency of the entire production process. That is, the productivity of useful substances such as various antibiotics, various enzymes, proteins, polysaccharides, physiologically active substances, animal and plant hormones, etc. by various cells can be increased, and continuous, large-scale production, separation and recovery can be easily performed. It can be said that it is a functionally integrated bioreactor.

Embodiments Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a diagram showing the configuration and flow sheet of a preferred embodiment of the culture process by the cell culture device of the present invention.

The cell culture device body D basically includes a porous object storage portion D5 and a screw kneading blade D2. Porous object storage section D
In FIG. 5, a polymeric porous material is aseptically impregnated with the culture solution, and a predetermined amount is supplied from the porous material supply tank A to continuously or batch-impregnate the culture solution through the supply rotary valve A1 and the inlet D3. A porous body is provided, and also
Cells are supplied in parallel when a macromolecular porous body containing a culture solution is supplied from a cell supply tank B for aseptically inoculating cells to be grown into a porous body via a supply valve B1 and an input port D3. , Blower C1 to air conditioner C and inlet D
Pressurized air whose temperature and humidity are adjusted is continuously supplied via 3. The supplied porous material containing cells and the culture solution is appropriately kneaded and mixed by the screw kneading blade D2 driven by the variable speed motor D1 in the porous material vertical accommodating portion D5 to promote the culture. The polymer porous body after the cells have grown or the target product has been produced is transferred from the outlet D4 into the conical pressing device E. Then, this polymer porous body is provided with a variable speed motor E1 in the conical pressing device E.
The culture solution and cells are squeezed and separated by a squeeze screw blade E2 driven by
In addition, the dehydrated polymer porous body containing cells is separately collected in the porous body recovery tank G. The pressure in the cell culture device body D and the conical squeezing device E is controlled by the pressure control exhaust nozzle H having a pressure control valve provided at the outlet of the cell culture device body D and the pressure control exhaust nozzle F1 provided in the culture solution recovery tank F. Is maintained at a higher pressure than outside the device. When the target product is an intracellular product, washing water is injected through a sterile washing water supply pipe I arranged at the outlet of the cell culture device body D to wash and squeeze and dehydrate the cells, and then the polymeric porous object. At the same time, it can be recovered in the porous object recovery tank G.

FIG. 2 is a detailed side view of a preferred embodiment of the cell culture device of the present invention, and FIG. 3 is a side view of the embodiment when the cell culture device is used while being tilted.

The cell culture device body 1 has a polymeric porous body 8a containing cells and a culture solution at one end, and an inlet 3a into which sterile pressurized air whose temperature and humidity have been adjusted is injected, and a height after cell culture. Porous substance accommodating portion 1a for culturing cells having an outlet 3b for discharging a molecular porous substance, and an outlet 3 at the lower end thereof
Introducing tube 1b connected to b for discharging a polymeric porous object 8b containing cells after culturing, a hollow provided with an inlet nozzle 1c for injecting a pressure-controlled exhaust nozzle 1d and cell washing water to this introducing tube 1b. It consists of a cylindrical shape. The polymer porous body 8a containing the cells and the culture solution injected into the porous body containing portion 1a is kneaded with a screw supported by bearings 1e, 1f provided at one end of the porous body containing portion 1a and the introduction tube 1b. Screw kneading blade 2 arranged on shaft 2
When a is rotated, it is appropriately kneaded and mixed, uniformly contacts the air injected from the input port 3a, promotes cell growth, and is moved to the downstream of the porous object housing portion 1a. The screw kneading blade 2a has a polymer porous body 8a,
It is possible to facilitate the flow of the injected gas by providing the perforated blades 2b with openings smaller than the size of 8b to form perforated blades. Also,
A stirring plate 2d may be provided between the screw kneading blades 2a to further enhance the mixing of the polymer porous body. By pouring the polymer porous body 8a containing the new cells and the culture liquid in the same amount as the moving amount by which the polymer porous body is moved by the screw kneading blade 2a from the charging port 3a in a continuous or batch operation, It is possible to continuously grow cells in the porous object storage portion 1a. The cell culturing period is defined by the rotation speed of the screw kneading shaft 2, the pitch speed given by the kneading blade pitch 2c between the screw kneading blades 2a, and the length of the porous object accommodating portion 1a. Therefore, in the case of cells having a short culture period, the kneading blade pitch 2c can be increased, the rotation speed of the screw kneading shaft 2 can be increased, or the length of the porous object accommodating portion 1a can be shortened. Conversely, in the case of cells having a relatively long culturing period, the kneading blade pitch 2c is made small, the rotation speed of the screw kneading shaft 2 is slowed, or the length of the porous object accommodating portion 1a is lengthened to make the culturing period longer. Can be lengthened. Furthermore, when culturing cells having a long culturing period, by arranging the inlet 3a side of the porous object accommodating portion 1a with a low inclination, the self-weight of the polymeric porous object 8a is utilized to make the porous object 8a. The screw kneading blade 2a by moving the screw kneading blade 2a in a direction opposite to the direction in which the screw kneading blade 2a moves.
It is possible to discharge only the input amount into the discharge port 3b introduction pipe 1b of the porous object accommodating portion 1a by allowing the polymer porous substance 8a injected from the input port 3a to be retained between a and pushed out from the injection port side. . In the culture period in this case, the polymeric porous objects 8a and 8b held in the porous object housing portion 1a are used.
It is regulated from the amount and the input amount. Further, the rotation speed of the screw kneading shaft 2 is temporarily increased, and a part of the polymer porous body 8b containing the cells after the culturing, preferably, the holding capacity of the introduction body 1b and the porous body 8b in the conical squeezing apparatus 4. A semi-batch culture can be carried out by discharging a polymer porous body 8a containing the same amount of new cells and a culture solution as the above through a batch operation from the charging port 3a (see FIG. 3). . Further, by stacking the porous object storage portion 1a and connecting the U-shaped porous object introduction tube 1b, the length of the porous object storage portion 1a can be increased to prolong the culture period ( Drawing omitted). A constant temperature jacket 7 is provided around the porous object storage portion 1a, and by supplying constant temperature water from the lower inlet 7a to the upper outlet 7b, the inside of the porous object storage portion 1a is maintained at a constant temperature. The culture is promoted at the optimum temperature.

At the time of harvesting the target product, the porous object 8b containing the cells and the target product after the culturing period is introduced into the conical pressing device 4 connected to the introduction pipe 1b, and the cells and the culture solution are squeezed and separated. It

The conical squeezing device 4 is a squeezing device having a tapered overall shape, which is arranged on a compression screw shaft 5 supported by bearings 4e and 4d provided on a porous object introduction opening and a lower end porous object discharge pipe 4c. It comprises a screw blade 5a, a conical perforated tube 4a having a perforation 4b arranged so as to be in contact with the tip of the compression screw blade 5a, and a culture solution recovery section 6 provided on the outer circumference of the conical perforated tube 4a. The diameter of the screw blade of the squeezing screw blade 5 becomes smaller according to the transport direction of the polymer porous object, and the squeezing screw blade 5 is fitted in the conical porous tube 4a. The space between the pitches of the squeezing screw blades 5a becomes smaller toward the lower end, and the volume of the introduced polymer porous body 8b is squeezed and reduced toward the lower end, and the impregnated culture solution is a conical perforated tube. It is squeezed out from the perforations 4b provided in 4a, gathers in the culture solution recovery section 6, and is discharged from the culture solution discharge nozzle 6a. The squeezed and dehydrated polymer porous body 8c containing cells is discharged from a discharge pipe 4c provided at the lower end. If the target product is an intracellular product, the introduction tube 1b
Aseptic wash water is continuously or semi-continuously injected from the injection nozzle 1c provided in the above to squeeze and dehydrate the wash water and the culture solution while washing the cells, and the polymer porous body 8c containing the dehydrated cells is placed at the lower end. It can be continuously or semi-continuously separated and collected from the discharge pipe 4c. The amount of the polymeric porous object 8b that is pressed and dehydrated by the conical pressing device 4 is determined by the pressing screw shaft 5
It can be adjusted by changing the rotation speed.

When culturing cells with a relatively short culture period,
As shown in FIG. 4, the screw kneading shaft 2 can be coaxially arranged with the screw kneading blade 2a and the pressing screw blade 5a to integrate the cell culture device body 1 and the conical pressing device. In this case, the cell culturing period is defined by the rotation speed of the screw kneading blade 2, the pitch speed given by the kneading blade pitch 2c, and the length of the porous object storage portion 1a. In addition, a pressure regulating exhaust nozzle 1d and a wash water injection nozzle 1c are arranged above the introduction portion to the conical pressing device.

The polymer porous body used in the present invention is sufficient if it has a strength such that only the volume is reduced by being squeezed and is not finely crushed, and it may be a material such as urethane foam, but is not particularly limited. . The size of the porous polymer object is 2.5 mm on a side or the diameter.
20 mm is preferable.

In each of the above-mentioned examples, as the squeezing device for squeezing the culture solution impregnating the polymer porous body, a conical squeezing device consisting of a conical porous tube and a tapered squeezing screw blade was used. It goes without saying that it is possible to use another squeezing device in which the cylindrical rolls are arranged so as to face each other and the porous body is passed between the rolls to squeeze it.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration and a flow sheet of a preferred embodiment of the culture process by the cell culture device of the present invention, FIG. 2 is a detailed side view of the preferred embodiment of the cell culture device of the present invention, and FIG. FIG. 2 is a side view of the implementation side when the cell culture device of FIG. 2 is used in a tilted state, and FIG. 4 is a detailed side view of another embodiment of the cell culture device of the present invention. A ... Porous material supply tank B ... Cell supply tank C ... Air conditioner, C1 ... Blower D ... Cell culture device body D1 ... Variable speed motor D2 ... Screw kneading blade D3 ... Input port D4 ... … Discharge port D5 …… Porous substance storage unit E …… Cone type squeezing device E1 …… Variable speed motor E2 …… Squeeze screw blade F …… Culture solution recovery tank F1 …… Pressure adjusting exhaust nozzle G …… Porous substance recovery Tank H ... Pressure-regulating exhaust nozzle I ... Wash water injection nozzle 1 ... Cell culture device body 1a ... Porous object storage part 1b ... Introduction pipe 1c ... Wash water injection nozzle 1d ... Pressure-regulating exhaust nozzle 2 ...... Screw kneading shaft 2a ...... Screw kneading blade 2b ...... Perforation 2c …… Kneading blade pitch 2d …… Stirring plate 3a …… Inlet port 3b …… Discharge port 4 …… Cone squeezing device 4a …… Cone type Perforated tube 4b ... many Holes 4c ... Discharge pipe 5 ... Compressing screw shaft 5a ... Compressing screw blades 6 ... Culture solution collecting section 7 ... Constant temperature jacket 8a ... Polymer cells containing culture medium and new cells 8b. Polymeric porous material 8c ... Cell-containing polymeric porous material after compression dehydration

Claims (4)

[Claims] 1. An ejector having at one end an inlet for introducing a polymer porous body having a strength such that the volume is reduced by being squeezed and is not crushed finely, and the other end is for discharging the polymer porous body. A porous object containing portion having an outlet, and the polymer porous object containing cells and a culture solution located in the porous object containing portion to be proliferated and kneading and mixing the mixture from the input port to the outlet. A cell culturing apparatus main body having a screw kneading blade that moves up to, and a squeezing apparatus that squeezes the polymer porous body discharged from the outlet to squeeze the liquid impregnated into the polymer porous body. Cell culture device. 2. The squeezing device is located in a conical perforated tube and is fitted into the perforated tube. The squeezing screw is of a taper type as a whole in which the diameter of the blade is reduced according to the transfer direction of the polymeric porous object. The cell culture device according to claim (1), which is a conical pressing device including a wing. 3. The screw kneading blade according to claim 1, wherein the screw kneading blade is a porous blade having an opening smaller than the size of the polymeric porous body, and a stirring plate is provided between the blades. The cell culture device according to the item. 4. The method according to claim 1, wherein the porous object storage portion is arranged so as to be inclined so that the input port side thereof becomes lower.
The cell culture device according to any one of (1) to (3).