JPH02109970A - Ph control of culture solution and system therefor - Google Patents

Abstract

PURPOSE:To provide the title system so designed that a pH indicator is added to a culture solution and light intensities corresponding to respective two different wavelengths are measured to calculate a pH value to make a pH control, thereby enabling pH calculation to be made in a stabilized manner. CONSTITUTION:A specified culture solution 2 is put into a culture tank 1 and stained with phenol red. The aimed pH value is inputted into an operating equipment 12 by an assigned pH-inputting means 13. When the culture solution 2 is subjected to color change from red to yellow with culture advancement, the culture solution is introduced through a pump 4 into a cell 5, and respective light intensities at 558nm and 433nm are measured with a light-receiving device 9, thus measuring a pH value. This value and the aimed value are compared by an operating equipment 12 to determine the amount to be controlled, thereby controlling the pH value through a CO2 gas cylinder 15. Therefore, a pH monitor is replaced by a photo-electric sensor to enable the equipment to be stably used for a long period.

Description

Detailed Description of the Invention (6) Industrial Application Field The present invention provides a high-density cell culture device in which the pH of the culture medium is
1-Relating to control methods and devices (Naka) Prior art When trying to culture animal cells at high density, maintaining an optimal culture environment becomes a problem. The environment necessary for culturing animal cells in vitro is the environment in which the cells existed in vivo. In other words, high-density culture is possible by making conditions such as nutrients -9He dissolved oxygen, temperature, and removal of waste products as close as possible to the in-vivo environment.

Here, conventional pH control of the culture solution involves dipping an autoclavable glass metal sensor into the culture solution to measure the DH value, and then injecting an alkaline or acidic solution into the culture solution. ) C (H gas was injected to control the pH.) (c) Problems to be solved by the invention In the above pH control, although the Galanu electrode type sensor can be autoclaved, deterioration due to autoclaving is unavoidable. In addition, since the sensor involves a chemical reaction, there was a problem that the indicated value would deviate due to changes over time.In addition, if the indicated value deviates by -degrees, it will not be possible to calibrate it while cells are being cultured, and it will not be possible to calibrate it in the optimal environment. could not be cultured.

An object of the present invention is to solve the above problems by controlling the pH of a culture solution without using a glass-electric sensor.

B) Means for Solving the Problems The present invention solves the above problems by adding a pH indicator to the culture solution and measuring the light intensity of two different wavelengths of the culture solution to calculate 9pH1l. pH4l! Pour acid or alkaline solution or gas into the culture solution
In addition, an apparatus for carrying out the method includes a culture tank to contain a culture solution containing a pH indicator, a means for irradiating the culture solution with light, and a device for carrying out the method. a light-receiving unit that receives reflected or transmitted light from two different wavelength ranges; and means for calculating pHi based on the intensity of the received light.

Based on the pH 1 liter, acid or alkaline solution or ■
It is equipped with control means for fully controlling the flow rates of the two gases.

In addition, examples of the above-mentioned pH indicators include phenol red and bromocresol barb.

The two different wavelength ranges are 2.For example, when using pheno-red as a pH indicator, use the red wavelength around 55Bnrn and the yellow wavelength around 433nm. pH
Calculate.・That pHi? I k Momotoni Cultivation! ! Liquid I
) H control 1vtl-Execute (f) Embodiment An embodiment of the present invention will be described based on the drawings.

FIG. 1 relates to an apparatus for carrying out the method according to the present invention. 1 is a culture tank in which a culture solution 2 is stored.

Dissolved oxygen sensor 1. A temperature sensor is attached (not shown)
, the culture medium 11 and liquid 21 are set to optimal environmental conditions. A pH indicator, Pheno-μ Red, is added to the culture solution 2.03 is a culture solution circulation channel in which the culture solution is pumped by a peristaltic pump 4.
Liquid 2 is sucked up from culture tank 1, passed through measurement station 5, and transferred to culture tank 1.
Return to.

Measurement set/L15 consists of Gax's set ρ.9. A white reflector plate 6 with a reflective effect is placed on the back surface to reflect the transmitted light from the light emitting means (light emitting diode or lamp) 7.

In addition, a light receiving element 9 is disposed at a position facing the reflecting plate 6 via a fiber 8, and receives light of different wavelengths transmitted through the culture solution 2. Here, Fi ~ ri 8 is * 555 ""
It selectively transmits nearby red light and yellow light near 4,330 m. In this wavelength range, phenol red, which is a pH indicator, exhibits a yellow color in an acidic region and a red color in an alkaline region. Therefore, if another pH indicator is used, the filter used will also be different. In addition, pi-It! used by the light receiving element! A filter is not necessary if it is sensitive only to different wavelengths corresponding to the indicator.

10.11 shows the amplifier and ADD conversion, respectively, and 12 shows the light intensity ratio J pH received by each light receiving element 9.
014, which is an arithmetic device that calculates the H control amount, calculates C based on the control amount calculated by the arithmetic device 12.
This is a control means for controlling the amount of gas supplied from the O2 gas pump 15 into the culture tank 1.

Next, the operation of the device according to the present invention will be explained.

First, a predetermined culture vII solution is placed in the culture tank 1 and stained with phenol red. The medium II solution is initially slightly alkaline and shows a dark red color.

In addition, the specified pH input means 13 is inputted into the control target pHk calculation device 2.

As the culture progresses, the culture medium 2 changes color (Generally, when cells reach a high density, they accumulate in the medium as a result of cell metabolism 1 (acid concentration increases). The pH drops significantly, causing the medium to change color to yellow. ), the culture solution 2 is introduced into the measurement cell 5 using the pump 4, the intensity of light at 558 nm and 433 nm is measured, and *pHt'' is calculated.

The measured pH is compared with the control target pHf calculation device 12 to determine a control amount, and CO2 gas corresponding to the control amount is sent from the gas pump 15 to the culture tank 1 to control the pH.

1・-@Nursing tank 3-W1 circulation path 2-Culture solution 5-Measurement section The same effect can be achieved by feeding an alkali.

Alternatively, without providing a means for circulating the culture solution, a reflector may be placed in the culture solution in the culture tank to directly irradiate the culture tank with light, measure the light intensity, and calculate the pH.

(G) Effect The present invention replaces the chemical sensor with a photo-electric sensor for pH monitoring, so it can be used stably for a very long time, and since the sensor is external to the autoclave, it will not be affected in any way. There is an advantage.

[Brief explanation of drawings]

Claims (1)

[Claims] 1. Calculate the pH value by adding a pH indicator to the culture solution and measuring the light intensity of two different wavelengths of the culture solution, and
Add acid or alkaline solution or C to the culture medium based on the H value.
A method for controlling the pH of a culture solution, which comprises controlling the pH by flowing O_2 gas. 2. A culture tank that contains a culture solution containing a pH indicator;
a means for irradiating light to a culture solution; a light receiving section for receiving reflected or transmitted light from the culture solution in two different wavelength ranges; a means for calculating a pH value based on the intensity of the received light;
A pH control device for a culture solution, comprising a control means for controlling the flow rate of acid or alkaline solution or CO_2 gas based on the H value.