JPS6158582A - Continuous circulation cell culture apparatus - Google Patents

Abstract

PURPOSE:To carry out the efficient culture of cerebral cells, etc. in the state of high dissolved oxygen content, preventing the foaming to the culture liquid, by absorbing oxygen to culture liquid in a pressure chamber, and circulating the oxygen-containing culture liquid to the culture tank. CONSTITUTION:A pressure chamber 3 to add oxygen to a culture liquid under high pressure is attached to a culture chamber 2, and culture liquid is circulated with a circulation pump 4 through the circulation line 6. The solenoid valves 13 and 14 are in open state in the circulation of the culture liquid. When the dissolved oxygen content of the culture liquid is decreased below a specific level, the controlling apparatus 10 operated by the signal transmitted from the sensor units 8 and 9 detecting the oxygen content, the leak valve 12 and the solenoid valves 13, 14 are closed, and the valve 11 is opened to supply oxygen from the tank 5 to the pressure chamber 3. The supplied oxygen is dissolved in the culture liquid under the pressure of about 2-5atm. After the absorption of oxygen, the leak valve 12 is opened to return the pressure chamber to atmospheric pressure, and the solenoid valves 13 and 14 are opened to start the circulation of the culture liquid having high dissolved oxygen content while removing the bubbles with the filter 15.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a continuous perfusion culture device 1 for culturing various animal cells.

Special cells such as brain cells and hepatic parenchymal cells show an extreme decrease in cell number and functional decline in culture column 1'1 with a low oxygen concentration. jffl common cultivation! The dissolved oxygen concentration of the culture solution in device f was limited to approximately 1 to 3 ppm.

It has been revealed that moistening is highly effective in culturing the aforementioned animal cells at higher dissolved oxygen concentrations. Conventionally, a method of bubbling oxygen or air into a culture solution under normal pressure has been proposed as a method for increasing the dissolved oxygen concentration. In this case, the dissolved oxygen concentration is considerably improved, but it has the disadvantage that homing occurs in the culture solution. like this. The former bubbles are caused by the denaturation of proteins contained in serum, etc. of the culture solution, and are an obstacle when culturing cells. In the latter case, during bubbling, oxygen is not completely dissolved and 0 remains. However, since it is impossible to measure the dissolved oxygen concentration, etc., it is difficult to grasp the data and monitor the concentration. Therefore, when culturing animal cells, the method of increasing dissolved oxygen concentration by bubbling is
Particularly when this system is used as an artificial organ, it has extremely serious drawbacks.

] In view of the above, as a result of intensive research, the present inventors introduced a pressurization chamber to increase the dissolved oxygen concentration into the culture apparatus, filled this pressurization chamber with oxygen, and pressurized it (2 to 5a1. +n), oxygen is well dissolved in the culture medium, and this pressurized culture medium containing high oxygen concentration can maintain a sufficiently high dissolved oxygen concentration in the culture medium even after the pressure is reduced to normal pressure. The present invention was developed based on the discovery that homing produces only a small amount of air bubbles in the culture solution and these air bubbles can be easily removed using a filter or the like.

Hereinafter, a first embodiment of the present invention will be described with reference to FIG.
In detail, the culture tank (2) uses hollow fibers and/or fibers (8) and (9) as carriers for the cultured cells in order to maintain the function of the cultured cells, especially the parenchymal cells, at a temperature of the culture solution.
Various sensors for measuring pi-1, dissolved oxygen concentration, carbon dioxide concentration, gluten concentration, etc. are installed inside.

This sensor unit (8M) is connected to the controller (10) and is configured to control each device of this culture device.

(5) is an oxygen tank. This oxygen tank (5) is
It is connected to the pressurizing chamber (3) by a connecting pipe (7). Furthermore, an electrically controlled opening/closing valve (11) is provided at the connection point between the communication pipe (7) and the pressurizing chamber (3),
This opening/closing valve (11) is connected to the fan 10-le unit (
10). (12) is a leak valve, (
+:1)(+4) are solenoid valves, and their opening/closing control is all performed by the control/roll unit) (In)]21). .

(16) is a stirrer bar which is rotated by a stirrer (not shown) placed in the 1ζ section of the pressurizing chamber (3) L-):
j In order to stir the M solution, the pressurizing chamber 11 (which is placed in
5) to supply the culture solution from which air bubbles have been removed to the culture tank (2).

Next, the external sensor unit (8H9) senses that the dissolved oxygen concentration of the continuous perfusion cell culture device of the present invention has become low, and the oxygen tank (5) is transferred from the pressurized chamber ( 3) is filled with oxygen and the inside of the pressurized chamber is 2-5a1. It is pressurized to about m. Next, rotate the stirrer bar (16). This action causes oxygen to dissolve into the culture solution from both sides of the culture solution. After dissolving enough oxygen, open and close the valve (
11) and open the leak valve (12) to reduce the pressure in the pressurized chamber to normal pressure. After pressure reduction, solenoid valve (+3) (+4
) is opened and the culture solution is continuously refluxed into the culture tank (2) again.

Since the bubbles generated in the pressurized chamber at this time are few, they are removed by the bubble removal filter (15).

Next, a second embodiment of the continuous perfusion cell culture apparatus of the present invention will be described in detail with reference to FIG. 1, and its explanation will be omitted. (
+7) (+8) is a planter pump. (19)
The beam server tank (20) is a pre-tank. The plunger pump (17) sends the culture solution in the pressurized chamber (3) to the reservoir tank (+9), so the plunger pump (17) sends the culture solution in the tank (20) to the pressurized chamber (3). 1 (alternative structure), each of which is connected to a controller 10 (10).

The operation of this embodiment is the same as that of the first embodiment, in which the pre-tank (20), the reservoir tank (+!1), and the system are the same as those of the first embodiment.
3) While (14) is blocked, the beam server tank (1
The culture solution stored in 9) is supplied to the culture tank (2). When the dissolved oxygen concentration reaches a predetermined value, the plunger pumps (+7) (18) start operating, causing reflux and supplying the culture solution to the reservoir tank (19) as well.

Next, another embodiment of the pressurizing chamber of the continuous perfusion cell culture device of the present invention will be described in detail with reference to FIG. did. (21)
is a hollow fiber and is connected to a communication pipe (7) connected to an oxygen tank (5) = 4-. The tips of the hollow fibers (21) are closed. Note that a Teflon tube, a Boretex tube, etc. can also be used instead of the hollow fiber.

-1- The operation of the pressurizing chamber will be explained. When the dissolved oxygen concentration of the culture medium becomes low, the leak valve (12), solenoid valve (13) (+4) is activated by the control of the fan unit (10).
When the opening/closing valve (11) is closed, the external oxygen is removed from the hollow fiber (2).
1) and is filled into the pressurized chamber (3). As a result, oxygen is dissolved out from within the hollow fibers and dissolved in the culture solution. At the same time, the pressurizing chamber is also pressurized. Therefore, dissolution proceeds also from the negative side of the culture solution. During this action, the stirrer bar (1
6) also rotates. The actions of reducing the pressure and ◇・refluxing after sufficiently dissolving oxygen are the same as in the first embodiment, so their explanation will be omitted.

It is preferable to configure it so that it takes

Furthermore, the device of the present invention can be applied to culturing cells of various organs and producing substitute artificial organs. In addition to maintaining a high dissolved oxygen concentration in the culture medium, the characteristics of this case include parenchymal cells and collagen on the outside of the hollow fibers in the culture tank.

Extracellular matrices such as glycoproteins and non-parenchymal cells such as cytoblasts coexist, allowing the original organ functions to be substituted for a long period of time. The point is that it has a unit that separates.

As is clear from the explanation in (1) below, the continuous perfusion cell culture device of the present invention allows the cultivation of facultative cells, renal parenchymal cells, hepatic parenchymal cells, etc., which are suitable for cultured cells with a high dissolved oxygen concentration in the culture medium. When applied to parenchymal cells, the action of the bar tank (19) makes it possible to constantly flow the culture solution in a certain chamber into the culture tank without interruption. Furthermore, from a practical standpoint, it is advantageous to add blood, serum, etc. to the reservoir tank.

In addition, in other embodiments of the pressurized chamber, the contact area between oxygen and the culture medium is large, that is, oxygen is dissolved from all surfaces of the hollow fibers, so the dissolution rate is fast and the amount of oxygen consumed is small. The sufficient effect is j(1).

Next, an experimental example of the influence of oxygen in culturing hepatic parenchymal cells using the continuous perfusion cell culture device of the present invention and its application to a substitute artificial organ will be explained.

Experimental Example 1 - Effect of oxygen on hepatic parenchymal cell culture i, Isolation of parenchymal cells from IIF1M and culture Genase Type was changed to cell dispersion (manufactured by Wako Pure Chemical Industries, Ltd.), calf serum was changed to fetal bovine serum (manufactured by GTBCO), The culture medium was DE (manufactured by Hinaga Pharmaceutical Co., Ltd.) and RPMT 164. (1((Hibiki IBCO
The conventional collagenase reflux method was used except for the addition of (manufactured by Manufacturer Co., Ltd.). (Nakamura et al. Betsuma Protein Nucleic Acid Enzyme No. 24 19
I31) 2. Cultured liver parenchymal cells due to differences in gas phase components in the culture tank were heated to 1/-F in an incubator with C, C, and C as gas phase components.
Culture was carried out in a conventional manner by fixing 02 to 5% and changing the N2 ratio to 17.02. (Nakamura et al. Betsuma Protein Nucleic Acid Enzyme No. 241'981) Then, the adhesion rate of hepatic parenchymal cells to the plate after culture and the survival rate of hepatic parenchymal cells based on the number of culture ports were determined. Regarding the adhesion □ rate, use the Hira! plate after 10 years of culture. It was determined by toluidine blue staining according to the method of , et al. (Gann I 984.).

The survival rate was determined from the number of cells that were removed from the culture plate by trypsin treatment, treated with trypan blue, and not stained.

The results are shown in FIGS. 4 and 5. FIG. 4 shows the adhesion rate of hepatic parenchymal cells to the plate, where the vertical axis is the adhesion rate (%) and the horizontal axis is the concentration of oxygen in the gas phase in the culture tank (%). Figure 5 shows the survival rate, the vertical axis is the survival rate (%) of hepatic parenchymal cells, and the horizontal axis is the time after culture (B).Graphs 1, 2, 3, 4
The concentration of oxygen in the gas phase in the culture tank is 0.20 and 5, respectively.
This is a graph under the condition of 0.70(%).

3. Survival rate associated with changes in dissolved oxygen concentration in the culture solution The hepatocytes separated and cultured in 1 above were placed in the culture tank part of the continuous perfusion cell device of the present invention (1,5-1, OX, 10” cells/
A petri dish was placed in which the cells were adhered to a size of cm2, and the survival rate was examined by varying the concentration of dissolved oxygen in the culture solution. The survival rate was measured in the same manner as in 2 above.

Further, the amount of albumin was measured by the BCG method after sufficiently disrupting the cells with a sonicater, and the GOT activity was measured by the POP method using a fraction obtained in the same manner and culture solution 1.

The results are shown in FIG. The vertical axis in the figure is the survival rate (%),
The horizontal axis represents the time (days) after culture in graphs 1, 2.3, 4. S
, 6, the dissolved oxygen concentration in the culture solution is 0.1.2, 5. 'L
It is a graph under the condition of 10.15 (ppm).

= 8-4, Results: 5% CO2, 9S% air for a long time as conditions for culture.
However, the #ll cells in living organisms differ greatly in the environment in which they exist, and at the same time, they also differ depending on the cell. It has also been qualitatively reported that, without optimizing the conditions, for example, Mtblast cells grow well under considerably low oxygen concentration conditions.The results of this new oxygen amount are shown in Figure 4. , as shown in Figure 5: 50%02 where the gas phase oxygen concentration in the culture tank is higher than the normal culture conditions (20%02)
The adhesion rate to the plate and the survival rate are the highest at 70%.
After 02, the effect decreased slightly. On the other hand, all cells do not have the same requirement for oxygen, such as the cyanoblast cells of Kinzan rice, which survive only in hypoxic conditions. -Quencher of heavyt oxygen-
Vitamin E (Ct-tocopl+
erol) + N-pro-pylgal lat, e
We investigated the effect on the survival rate of liver cells and 111 Yamaki Cotton Hagi cells under hyperoxic conditions by adding ISI grna. The effect was 1- and 5 was -4.

From FIGS. 4 and 5, it is clear that the culture condition fl of hepatic parenchymal cells is too low with normal oxygen levels. Furthermore, as shown in Figure 6, the continuous perfusion cell culture device of the present invention has been used to precisely control the dissolved oxygen concentration to 1 (as shown in Figure 6). Oxygen concentration 5-15ppm
was necessary for cell survival, and 7-10 ppm was optimal. It also showed a remarkable effect on liver function M [2 ppm oxygen], although albumin synthesis decreased to 5-10% of the initial level after 7 days of culture.
In terms of ppm, it showed a value of 80% or more. Also, the G of the cell
OT is maintained at almost 100% at 7-10 pl-1 m, but decreases to 10-20% at 2ρ1111+. GOT in the culture solution showed a significantly lower value at 7-10 ppm than at 2 ppm.

This made it possible to mildly increase the dissolved oxygen concentration in the culture solution by 1" without losing the function of the factor.

The experiment was carried out in the same manner as in Example 1 of the Cold Experiment: Application to a Substitute Artificial Liver System, Isolation and Cultivation of Parenchymal Cells from the Liver.

2. A line from IIFl foolishness! A primary culture of fibroblasts isolated from +1 blast cells was used.

3. Preparation of model organ model Ty+tel collagen (Koken, atelocollagen derived from cowhide 7) is dissolved in a pl+3 hydrochloric acid or acetic acid solution to a concentration of 0.2%.

After thorough stirring, add highly concentrated phosphoric acid bt+f fer (pH 7
, 4) or neutralize with a 10-fold concentrated cell culture medium. Type J, ■ in the colladen solution kept at 4 °C.
Collagen (BRL, Hoechstji + Han).

Extracellular 7 Trix components such as fibronectin (manufactured by Sigtna) and laminin (manufactured by 7 Nakoshi) were added at 0.01-0.
0 (101%) and stir slowly.Also, use amniotic membrane as the extracellular matrix (SD platform crudely extracted components were better in terms of cell adhesion and functional TF, retention, etc.). Next, mix hepatic parenchymal cells and fibroblasts with this solution and make it homogeneous.
Fill the outside of the hollow fiber and dissolve in water in a constant temperature water bath at 37°C.
It will gel in about 10 minutes. Next, DE culture solution containing 10% FBS is quickly filled into the -I+- hollow fiber to start cell culture.

Another method is to coat the outside of the hollow fiber with 0.2% T.
yr + el colladen, (1, (11 - 0,0001%
Type IV t V: 7 After gelling a solution of laden, fibronectin, and laminin at 37°C, lyophilize it to form a porous matrix. Culture is started by filling the gel with the germ cells.

4.Culture conditions For culturing, a continuous perfusion cell culture apparatus shown in FIG. 1 is used.

At this time, the opening degree of the culture solution was 37±0.2° C., and the culture solution reflux rate was 0.3 mN/cells outside the hollow fiber, especially parenchymal cells, survived well in the culture at a high oxygen concentration level. In addition, serum-free medium DM (R, Enat, el, at.

Proc, Na1.1. /\cad, S c,
i, \'o1.811'lil '84) was significantly superior to the serum-containing medium. In addition, the expression of hepatic ability was excellent when the cells were returned to a normal medium containing serum).

Furthermore, when the System 12 system containing the hollow fibers described above was used in rats whose whole liver had been removed, they survived for several days. 20 minutes until the perfusion solution approaches 100% serum for the purpose of cell adaptation to serum.
% and 50% serum for one day each, the survival rate and functional expression of the cells in the gel were good, and if the adaptation to internal concentration changes is taken into account, it can be used as a substitute artificial organ.

[Brief explanation of the drawing]

Part 1l: Partial sectional view showing the first embodiment of the present invention. Fig. 2: Partial sectional view showing the second embodiment of the invention. Fig. 3: Other embodiments of the pressurizing chamber of the present invention. Figure 4 is a sectional view of the main part showing the adhesion rate of hepatic parenchymal cells to the plate in the experimental example of the present invention (
%) and oxygen in the gas phase of the culture tank (%) Figures 5 and 6N: Viability rate (%) of hepatic parenchymal cells in experimental examples of the present invention
) and the time (days) after culture (1) Continuous reflux culture device, (2) Culture tank. (3)...pressure chamber, (4)...reflux pump. (5)...Oxygen tank, (6)...Recirculation pipe. Patent Applicant: Advance Development Research Institute, Inc., 1
&le (%) Figure 1 to 5 After culture, B% simple (8) Figure 6

Claims (1)

[Claims] (1) In order to always maintain a high concentration of dissolved oxygen in the culture solution supplied to the culture tank, there is a pressurized chamber, an oxygen tank connected to this pressurized chamber, a culture tank, and a culture tank in which the culture solution is supplied to the culture tank. 1. A continuous reflux cell culture device comprising: a reflux pipe for refluxing the blood to the pressurized chamber; and a reflux pump.