JPH05168471A - Method for culturing adhesive cell and apparatus for its culturing - Google Patents

Abstract

PURPOSE:To set and control culture conditions by monitoring the state of a cell peeling from or sticking to a culture carrier with the absorbance of a culture solution. CONSTITUTION:The absorbance of a culture solution in a culture vessel is measured with an absorbance measurement sensor 3 to monitor the state of a cell peeling from and sticking to the culture carrier. A variation in absorbance with the lapse of time is used as an index to control the peeling from and sticking to the culture carrier of the cell.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for culturing adherent cells using a culture carrier.

[0002]

2. Description of the Related Art As a large-scale culturing method for adherent cells, for example, adherent animal cells or adherent plant cells, a culture carrier, which is a substance for adhering cells, is suspended in a culture medium and the cells are adhered to the culture carrier. A carrier culture method for proliferating is proposed,
It has already been put to practical use. An example of such a general culturing method and culturing apparatus is "Microcarrier Cell Culture-Principles and Method-" (Pharmacia Fine Chemical Company).
-principles & method- '', published in 1987), this carrier culture method, when cells grow until they fill the entire carrier, the cells are detached from the carrier and a new cell suspension is prepared. And then attach it to a new carrier. By performing this operation, the cells are passaged and the culture is scaled up. A proteolytic enzyme such as trypsin is usually used to detach the cells from the carrier.

A more specific operation of detaching cells from the carrier will be described below. 1) First, the carrier in which the cells are growing is separated from the medium, the carrier in which the cells are growing is washed with an appropriate buffer, and the washing solution is discarded. 2) Add the enzyme solution and stir. This causes the cells to detach from the carrier and float. 3) When the cells are detached, stop the enzyme treatment. In this case, excessive enzyme treatment damages the cells and reduces the adherence of the cells to the carrier and the viability. On the contrary, when the enzyme treatment is insufficient, the cells remaining on the carrier are wasted. .. That is, in order to carry out the carrier culture method efficiently, it is necessary to completely remove the cells from the carrier without damaging the cells.

Regarding the detachment of the cells from the carrier, as to the above-mentioned operation 1), a method of improving the composition of the buffer solution to reduce the damage of cells is described in JP-A-62-44176. There is. Regarding the operation of 3), there is a method of reducing cell damage using an immobilized enzyme inhibitor.
171183 published. However, regarding the operation 2), no particular proposal has been made so far, as will be described later.

The point of this operation 2) is to quickly grasp the state that all the cells have detached from the carrier and to operate promptly.
3) is in place. However, in this regard, in general, preliminary experiments are conducted to empirically set the time for enzyme treatment. That is, sampling is carried out several times during the enzyme treatment and microscopic observation is carried out to grasp the time required for cells to detach from the carrier. The time required for the cells to detach from the carrier is related to the adhesive force of the cells to the carrier. The stronger the adhesive force of the cells, the longer the time required for detachment, and the weaker the adhesive force, the shorter the time. The culture condition setting based on this experience is also used in the operation of attaching cells to the carrier.

[0006]

Therefore, in the prior art, it was necessary to carry out a preliminary experiment for each cell. Furthermore, even if it is the same cell, the physiological state of the cell is constantly changing, and therefore it can be said that the reliability of the culture condition setting set from experience is considerably low. Therefore, the purpose of the present invention depends on the preliminary experiment by the sampling of the culture solution and the microscopic observation, which are used in the conventional method, and the experience of the experimenter in order to efficiently culture the adherent cells by the carrier method. It is an object of the present invention to provide a method for monitoring the state of detachment of cells from a carrier and adhesion to the carrier, setting and controlling optimal culture conditions, and an apparatus for carrying out the method.

[0007]

[Means for Solving the Problems] In order to achieve the above object, the inventors of the present invention conducted diligent experiments and studies on changes in absorbance of adherent cells, particularly adherent animal cells, in culture by a carrier method. In the culture of adherent cells by the carrier method, the absorbance of the culture solution does not change when the cells are attached to the carrier, but the cells and the carrier are suspended separately in the culture solution. Attention was paid to the phenomenon that the absorbance of the culture solution increases in proportion to the amount of floating cells at the time, that is, when the cells are detached from the carrier or when the cells are not attached to the carrier. Furthermore, focusing on this phenomenon,
By measuring the absorbance in the culture medium, the amount of cells floating without adhering to the carrier in the culture medium can be regarded as the turbidity of the culture medium, that is, the high turbidity means that It means that the cells are floating without being attached to the carrier, and conversely, the low turbidity means that the cells are attached to the carrier, and therefore by measuring the absorbance of the culture solution, Attention was paid to the fact that it becomes possible to monitor the state of cells in the culture solution.

[0008] Further, the change in the state of cells in the culture medium was perceived as a change in the absorbance of the culture medium. For example, it is assumed that the change in the absorbance with the passage of time is as shown by the solid line in FIG. The horizontal axis of the graph represents time and the vertical axis represents absorbance. In FIG. 1, the absorbance increases linearly between times t ₁ and t ₃ , and in the case of such a change, cells are gradually detached from the carrier. And
From time t ₃ to t ₄ , the increase in absorbance gradually slows down, the increase in absorbance stops at time t ₄ , and then becomes constant. This means that the amount of detached cells gradually decreased from time t ₃ to t ₄ , and the detachment of cells ended at time t ₄ . As described above, by measuring the absorbance in the culture medium, the state of the cells can be monitored without the troublesome operations such as sampling of the culture medium and microscopic observation.

Furthermore, paying attention to the fact that the amount of change in the measured absorbance with time is calculated and this amount of change serves as an index for controlling the culture conditions, and when the cells are detached from the carrier by the enzyme treatment, the enzyme treatment When the absorbance of the culture solution inside is measured, and if the change amount of the absorbance with time corresponds to the preset condition, it is judged that the peeling has been completed and it is reasonable to carry out the enzyme treatment stop operation. did.

That is, we first found that the change in the absorbance of the culture medium during cell detachment is typically as shown by the solid line graph in FIG. The horizontal axis of the graph in FIG. 1 represents time and the left vertical axis represents absorbance. First, during the period when cells are actively detached from the start of enzyme treatment (from time t ₁
t ₃ ), the absorbance increases linearly. Then, the increase gradually becomes dull (time t ₃ to t ₄ ) and shows a constant value after the time when all cells are detached (time t ₄ to t ₅ ).

The change in the absorbance can be summarized as follows by focusing on the amount of change in the absorbance per unit time. The amount of change in this absorbance (the amount of change is now ΔD) is
Shown as the slope of the graph. At times t ₁ to t ₃ at which cells are actively detached, ΔD is a positive number. Then, ΔD gradually decreases from time t ₃ to t ₄ , and becomes almost 0 from time t ₄ when all cells are detached. In this way, the progress of cell detachment can be known from the changes in absorbance and ΔD. In particular, if ΔD is used, the progress of cell detachment can be grasped most easily.

In the detachment of cells, the most important point is how many cells, especially living cells, can be obtained without waste, and this depends on when the enzyme treatment is completed. For example, in FIG.
If the treatment is completed at time t ₂ , the enzyme treatment is insufficient and many cells remain on the carrier, and at time t ₅ , the enzyme treatment is excessive and the cells are damaged.

We have further found that, in the detachment operation of cells by enzyme treatment, the amount of viable cells detached during enzyme treatment is typically shown as the dotted line graph in FIG. 1 (see FIG. 1). The vertical axis on the right shows the number of viable cells). From time t ₁ to t ₃ , the number of viable cells increased linearly as well as the absorbance. Then, it became the highest at time t ₃ when the increase in the absorbance became dull, and gradually decreased after time t ₃ . In the number of viable cells decreased time t ₃ or later, at time t ₃ from the time t _4, at the same time as the separation of cells, the cells were detached initially is enzymatically damaged, continue to die, the amount of the latter It is thought that this is because there are more people. As a result of actual measurement, at time t ₄ when all cells were detached, a large number of viable cells had already died, and living cells were also considerably damaged.

From the above, the optimum time of enzyme treatment for obtaining the most live cells is time t ₃ ,
It can be seen that the enzyme treatment can be stopped at time t ₃ . This time t ₃ is the time when the amount of change ΔD in absorbance starts to decrease, as described above. Therefore, during enzyme treatment,
The absorbance of the culture solution is measured, and the amount of change in absorbance per unit time is calculated. The optimum enzyme treatment time that maximizes the number of viable cells obtained is taken as the time when the amount of change in absorbance starts to decrease, and the enzyme treatment may be stopped at this point.

The optimum time of the enzyme treatment shown as time t _{3 in} FIG. 1 varies depending on the cell type and the state of the same cell. When the cell adhesion is strong, the slope of the graph of FIG. 1 becomes smaller, the time t ₃ becomes longer, and when the cell adhesion is weak, it becomes shorter. However, in any case, the optimum time for the enzyme treatment is regarded as the time when the amount of change in absorbance starts to decrease.

Therefore, according to the present invention, the type of cell,
Regardless of the state of the cells, cell exfoliation can always be performed under optimum conditions. INDUSTRIAL APPLICABILITY The present invention can be applied not only to the operation of exfoliating cells from a carrier, but also to the control of the attachment state of cells to a carrier. That is, as in the case of the detachment of cells described above, the adhered state of cells can be controlled using the amount of change in the absorbance of the culture solution as an index.

In the attachment of cells to the carrier, the number of cells floating in the culture medium decreases as the cells attach to the carrier, and the absorbance of the culture medium decreases for the same reason as in the case of detachment. Usually, the main control factor involved in the adhesion of cells to the carrier is agitation, and when attaching cells to a carrier, 1/4 to 1/2 of the agitation speed that is optimal for cell growth.
Stir continuously at the speed of. In some cases, intermittent stirring may be used in which the operations of standing and stirring are repeated if necessary. Therefore, in the present invention, by using the change of the absorbance of the culture medium as an index, it becomes possible to control the stirring and adjust the attachment of the cells on the carrier to an optimum state.

By the way, the principle itself of determining the density or concentration of the suspension by measuring the absorbance of the suspension is known,
It is generally known as a turbidity sensor and is also used for microbial culture and animal cell culture. The present invention is one in which the principle is applied to the culture of adherent animal cells, but in the present invention, it is desired to measure the absorbance of the culture solution more accurately, and therefore, the measurement result of the absorbance. It is preferable to measure the absorbance at a small wavelength or if there is no absorption of the medium or the carrier itself, which is the background of the absorbance measurement, such that is within the range of 0 to 1.

The graph of FIG. 2 shows the results of measuring the absorbances of the five kinds of solutions a to e at wavelengths of 300 nm to 900 nm. Solid line a is a mixed solution of cells, carrier and medium, solid line b
Indicates a buffer solution in which cells and carriers are mixed, a solid line c indicates a buffer solution in which carriers to which cells are attached are mixed, a solid line d indicates a buffer solution in which carriers are mixed, and a solid line e indicates a measurement result of a medium. As can be seen from FIG. 2, in the graphs other than d, the absorbance changes sharply at the boundary of 600 nm. Then, the absorbance at 600 nm to 900 nm shows a substantially constant value, the absorption of the medium was hardly seen, and the absorbance was in the range of about 0 to 1. From this, it can be seen that in the present invention, it is more desirable to measure the absorbance at a wavelength in the range of 600 nm to 900 nm.

As a method of measuring the absorbance, the measurement may be carried out by installing it inside the culture tank, or by installing the sensor outside the culture tank and circulating the culture solution.

[0021]

EXAMPLES The present invention will be described in more detail with reference to the case of culturing adherent animal cells as adherent cells. FIG. 5 shows an example of a suitable culture apparatus for carrying out the culture method of the present invention. In the figure, reference numeral 1 is a culture tank, inside which a stirring means 2 and a sensor 3 for measuring absorbance are installed. As these, conventionally known ones can be appropriately used. 4 is an enzyme solution storage tank for cell detachment, 5
Is a storage tank for the solution for washing the carrier, and 6 is a storage tank for the enzyme treatment stop solution, which is filled with each conventionally known solution. One tube is opened in each of the storage layers 4 to 6, and the other end of the tube is opened to the culture tank 1. Each tube has one
The individual pumps 7, 8 and 9 are incorporated. Further, the culture tank 1 is opened with a pipe 13 for transferring and returning the solution to another culture tank or storage tank not shown in the figure, and a pipe 14 for discharging the solution in the culture tank 1. .. Tube 13 and tube 1
Pumps 15 and 16 are incorporated in the pump 4, respectively.

Further, the culture device comprises a control unit 10 and a display unit 1.
1 and a setting unit 12, the absorbance measured by the sensor 3 is processed by the control unit 10. Control unit 1
At 0, according to the set value set by the setting unit 12,
ON / OFF of each pump 7, 8, 9, 13, 14
And the stirring speed of the stirrer 2 is determined. The display unit 11 displays the absorbance processed by the control unit 10 as a numerical value or a graph. The display unit 10 is not always essential, but by providing the display unit, the operation of the culture device can be reliably performed.

The operation of peeling cells from the carrier by the enzyme treatment using the above apparatus will be described. First, the stirrer 2 is stopped and the carrier to which the cells are attached is allowed to settle.
When everything has settled, the pump 16 is operated and the medium is discharged from the pipe 14. Next, the pump 8 is operated to inject the carrier cleaning solution into the culture tank 1. After the stirrer 2 is operated and stirred for several times, the stirrer 2 is stopped, the carrier is allowed to settle, and the pump 1 is again used.
6 is operated to discharge the cleaning liquid from the pipe 14. This washing operation is performed twice or three times. After washing, the pump 7 is operated to inject the enzyme solution into the culture tank 1, and the stirrer 2 is operated to start detachment of cells. From the time when stirring is started, the absorbance is measured by the sensor 3 for measuring absorbance.

It is the easiest and preferable method to install the sensor inside, but when the sensor is installed outside the culture tank, as shown in FIG. A pipe for circulating the culture liquid, such as taking out and returning a certain amount of the culture liquid, is provided, and a sensor is incorporated in the middle of this pipe. The measured absorbance data is the control unit 1
0, it is processed according to the flow chart shown in FIG. That is, the measured absorbance D (step 1) is compared with the previously measured absorbance, and the change amount Δ per unit time
D is calculated (step 2). Next, ΔD is compared with the previously calculated value (step 3). If ΔD has decreased as a result of the comparison, it is determined that the detachment of the cells has been completed, and the enzyme treatment stop operation (step 4) is performed. That is, the pump 9
And the enzyme treatment stop solution is injected into the culture tank 1. Thereby, the enzymatic treatment is stopped. If ΔD is constant or increases, it is determined that the detachment of cells has not been completed, and the process returns to step 1.

The condition for deciding the termination of the enzyme treatment may be one measured value of ΔD as described above, and it is stopped when the value of ΔD is decreased a plurality of times (for example, 3 times). May be determined. Further, the calculation results of several times may be averaged to set the value as ΔD, and the size of Δt may be changed appropriately. In any case, they should be empirically determined to have an optimum value according to the culture conditions such as the type of cell and the type of carrier.

Next, the operation of the culture apparatus shown in FIG. 5 for the case of attaching cells to the carrier will be described. A mixed solution of the carrier and the medium is poured into the culture tank 1, the cell suspension is inoculated, and the mixture is stirred by the stirrer 2 at a stirring speed R ₁ . Simultaneously with the start of stirring, the absorbance is measured by the sensor 3 at regular intervals. Absorbance data measured by the sensor 3 is sent to the control unit 10, and the flow chart shown in FIG.
The stirrer 2 is controlled according to the chart. That is, the absorbance measured by the sensor 3 (step 1) is compared with the previously measured absorbance (step 2). As a result, if the absorbance has decreased, the stirring speed R ₁ is maintained (step 3). When there is no change in the absorbance or when the absorbance is increased, the stirring speed R is decreased at a constant ratio r (step 4).

Next, as a result of reducing R in step 4, it is determined whether R is 0 or negative (step 5), and if it is, stirring is stopped (step 6). If this is not the case, the absorbance is further measured and compared (steps 7 and 8), and R is decreased at the ratio r until a decrease in the absorbance is observed. When a decrease in absorbance is observed in step 8, this time, conversely, the stirring speed is set to a constant ratio r and R =
Increase until R ₁ (steps 9, 10, 11,
12). When the absorbance has not decreased, the process returns to step 8.

Next, an example in which cell exfoliation is actually performed using the apparatus shown in FIG. 5 will be described.

[Example 1] Cytodex2 (Pharmacia, Pharmacia) was used as a culture carrier, and ERDF medium (Kyokuto Pharmaceutical Co., Ltd.) was used.
Fetal calf serum (Gibco, Gibco) was added to 10% to culture mouse-derived cells. After culturing until the cells completely filled the carrier, the cells were detached with a trypsin solution (Mochida Pharmaceutical Co., Ltd.). The absorbance of the culture solution during cell detachment was measured. The change in absorbance is shown in the graph of f in FIG. As you can see from the graph, 5 minutes after starting the enzyme treatment 30
The absorbance increased linearly until about 2 seconds, then gradually increased, and became almost constant at about 7 minutes and 30 seconds. No cells remained on the carrier at 7 minutes and 30 seconds when the absorbance value became constant. In addition, the survival rate of the cells at this time is 87%
However, it decreased to 82% after 10 minutes. When measured at 5 minutes and 30 seconds when the increase in absorbance became slow, the survival rate was as high as 99%, and the recovery rate was 95% with the cell yield when all the cells were detached as 100%.

Thus, the cell viability was highest when the rate of increase in absorbance decreased, and decreased thereafter. Therefore, it was confirmed that in order to obtain the maximum yield of living cells, the enzyme treatment should be stopped at the time when the rate of increase in absorbance decreases.

Example 2 As a culture carrier, Dormacell2.6 (Pfeifer & Lagen, Pfeifer & La)
ngen Dormagen) in ERDF medium (Kyokuto Pharmaceutical Co., Ltd.)
Fetal bovine serum (Gibco, Gibco) was added to adjust the concentration to 100%, and hamster-derived cells were cultured. After culturing until the cells completely filled the carrier, the cells were detached with a trypsin solution (Mochida Pharmaceutical Co., Ltd.). The absorbance of the culture solution during cell detachment was measured. The change in absorbance is shown in the graph of g in FIG. The absorbance increased linearly from the start of the enzyme treatment to about 8 minutes, then gradually increased, and became almost constant after 11 minutes and 30 seconds. 11 minutes when the absorbance value becomes constant
At 30 seconds, no cells remained on the carrier. Also,
The cell viability at this time was 82%, but after 15 minutes it was 76%.
It had fallen to%. The survival rate was 98% at 8 minutes when the increase in absorbance was slow, and the recovery rate was 90% when the cell yield when all the cells were detached was 100%.

In this example, as in Example 1, the cell survival rate reached the maximum when the rate of increase in absorbance decreased, and decreased thereafter. Then, in order to obtain the maximum yield of viable cells, it was confirmed that the enzyme treatment should be stopped at the time when the rate of increase in absorbance decreased.

[0031]

EFFECTS OF THE INVENTION According to the present invention, the troublesome operations such as sampling of the culture solution and microscopic observation are not required,
Optimum culture conditions can be set and controlled by monitoring the state of cells detached from the carrier and adhered to the carrier. Further, since the culture solution is not sampled, there is an effect that the risk of contamination with various bacteria is reduced.

[Brief description of drawings]

FIG. 1 is a diagram showing a relationship between an enzyme treatment time, an absorbance of a culture solution, and the number of living cells detached in a cell detachment operation.

FIG. 2 is a diagram showing patterns of absorbance of various culture solutions according to measurement wavelengths.

FIG. 3 is a flow chart of a cell detachment operation.

FIG. 4 is a flow chart of cell attachment operation.

FIG. 5 is an explanatory view of an embodiment of a culture device for carrying out the present invention.

FIG. 6 is an explanatory view showing a culture tank having another configuration.

FIG. 7 shows two examples of the present invention in detaching cells.

[Explanation of symbols]

1 ... Culture tank, 2 ... Stirrer, 3 ... Absorbance measurement sensor, 4 ...
Enzyme solution storage tank for cell detachment, 5 ... Carrier cleaning solution storage tank, 6 ... Enzyme treatment stop solution storage tank, 7-9 ... Pump, 1
0 ... Control unit, 11 ... Display unit, 12 ... Setting unit, 13 to 14
… Piping, 15-16… Pumps

Claims (14)

[Claims] 1. A method in which adherent cells are adhered to a culture carrier for culturing, and then the adherent cells are detached from the culture carrier by an enzyme treatment for culturing, and the cells are determined from the absorbance value of a culture solution. 2. A method for culturing adherent cells, which comprises monitoring the state of detachment from the culture carrier or adherence to the culture carrier. 2. The method for culturing adherent cells according to claim 1, wherein the absorbance is the amount of change in absorbance with the passage of time. 3. The method for culturing adherent cells according to claim 1, wherein the wavelength for measuring the absorbance is a wavelength in the range of approximately 600 nm to 900 nm. 4. The method for culturing adherent cells according to claim 1, wherein the absorbance of the culture solution in the tank is measured by a sensor installed in the culture tank. 5. The adhesion according to claim 1, wherein the culture solution is extracted from the culture tank and the absorbance of the culture solution in the tank is measured by a sensor installed outside the tank. Method for culturing sex cells. 6. The enzyme treatment stopping operation is performed when the value of the amount of change in the absorbance of the culture medium reaches a value that satisfies a preset condition. A method for culturing adherent cells. 7. The method for culturing adherent cells according to claim 6, wherein the preset condition is that the amount of change begins to decrease. 8. A culture carrier and a culture solution are injected into a culture vessel of an apparatus for culturing animal cells, and a suspension of adherent cells is inoculated to adhere the adherent cells to the culture carrier for culturing. In the method, the method for culturing adherent cells is characterized in that the adhered cells are controlled by using the amount of change in the absorbance of the liquid in the culture tank as an index. 9. The culture vessel of the apparatus for culturing animal cells has a stirring means capable of controlling a stirring speed, and the stirring condition of the stirring means is changed according to the change amount of the absorbance. The method for culturing adherent cells according to claim 8. 10. The method for culturing adherent cells according to claim 1, wherein the adherent cells are adherent animal cells. 11. A culture tank capable of encapsulating a carrier for culturing adherent cells, a means for measuring the absorbance of a liquid in the culture tank, and an enzyme solution having a function of exfoliating cells adhered to the carrier in the culture tank. An apparatus for culturing adherent cells, comprising: a supply means for supplying, and an adjusting means for stopping the supply operation of the enzyme solution of the supplying means based on information from the means for measuring the absorbance. 12. A culture carrier and a culture medium are injected, and a suspension of adherent cells is further inoculated to adhere the adherent cells to the culture carrier, and then the adherent cells are removed from the culture carrier by enzyme treatment. Is a culture device for peeling and culturing, at least one culture tank, means for washing the culture carrier, and an enzyme solution for peeling the adherent cells that have grown by adhering to the culture carrier. Means for injecting into the culture tank, means for injecting a solution containing an inhibitor of the enzyme solution into the culture tank, and means for injecting the liquid in the culture tank into another culture tank or another tank, Means for injecting the liquid in the other culture tank or the other tank into the culture tank, means for discharging the liquid in the culture tank, a sensor for measuring the absorbance of the liquid in the culture tank, the Calculate the amount of change in absorbance over time Means, from the calculation result of the amount of change with time of the absorbance, the detachment of the adherent cells from the culture carrier, and the adhesion of the adherent cells to the culture carrier is performed efficiently, An apparatus for culturing adherent cells, which comprises a means for controlling operating conditions. 13. The apparatus for culturing adherent cells according to claim 11, further comprising means for displaying the measurement result of the absorbance and the calculation result of the amount of change in the absorbance with time. 14. The device for culturing adherent cells according to claim 11, wherein the adherent cells are adherent animal cells.