JPS61152282A - Cell culture method - Google Patents

Abstract

PURPOSE:To determine the cell proliferation combatibility, accurately, by irradiating the cell culture liquid in a transparent vessel with visible light, and calculating the pH from the absorption change of the visible light. CONSTITUTION:A culture liquid in a transparent vessel such as mcroplate, dish, culture bottle, etc. is irradiated with visible light, and the pH of the liquid is calculated from the transmission spectrum or reflection spectrum of the light. When the calculated pH is out of the range suitable for the cell culture, the culture liquid is exchanged. Phenol red added to the culture liquid at a low concentration has absorption peaks near 430-440nm and 560nm, and the absorption peak of 430-440 increases and that of 560nm decreases according to the decrease of pH within the pH range of 6.8-7.6.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for culturing cells, and more particularly to a method for culturing cells in which a culture medium is replaced by tracking pr-r of the culture medium. It is.

[Prior Art 1] Cells are cultured in a culture solution containing a large amount of nutrients, but the pH of the culture solution changes during culture due to consumption and metabolism of the nutrients in the culture solution.

When the nutrients in the culture solution are consumed, cell proliferation becomes slow, and when the pH of the culture solution changes due to metabolism, the pH falls outside of the range suitable for cell growth, making it difficult for cells to proliferate. Therefore, during culture, if these problems arise, the culture medium is replaced.

The timing of replacing the culture solution is determined mainly by visually observing the color change due to I) TI of phenol red contained in the culture solution.

[Problem to be solved by the invention 1: Estimating I)H of a culture solution by visually judging the color change of phenol red in the culture solution 1-The conventional technology as described above
It is very ambiguous, and the cell solution may be left in a state unsuitable for cell growth, or it may be left outside the culture environment for a long time because it takes time to make a decision.
In severe cases (J cells could die).

[Structure 1 of the Invention The present invention was made to solve the above-mentioned problems. irradiation and the resulting transmission spectrum also ('reflection spectrum)
- Calculate the pIf from the obtained I).If the pIf is out of the range suitable for culturing the cells, the culture medium should be replaced.

Phenol red (Phenol red) is usually included in the culture solution at a dilute concentration that does not harm the cells, but at such a dilute concentration (JPhenol red ( ”430-44 in the visible light range
(It has absorption peaks near lnm iq and 560nm i=j, and has an isosbestic point at 480nm.) In the pH range of 68 to 76, which is the pH range in which cells can grow,
As p][ decreases, the absorption peak near 430 to 440 nm increases, and the absorption peak near 560 nm (1. Depends on the ratio of nearby absorption.
The throat rests on a single curve. Therefore, the pH of the culture solution can be determined from the ratio of these two peaks.

Mike [l play 1., dissono 3. A certain amount of absorption and scattering occurs in containers such as culture bottles (1) due to dirt, etc., and absorption and scattering occurs from the cells themselves growing on the walls of the container. Measuring absorption changes
(550nm)
pIf can be calculated using the curve in Figure 1 by subtracting the absorption at a wavelength where phenol red has almost no absorption such as L) from the two peaks and taking the ratio of L).

[Effect of the invention 1 The suitability of the culture solution for cell expansion 1 can be quickly determined by exchanging the culture solution (and culturing) according to the method of the present invention/7; Since compatibility can be detected accurately, cell expansion can now be carried out stably.

[Example 1 96-hole microphone [1 play!・Mouse myeloma cells and culture medium (Tal\Soko MEM medium, r) I-16, 8
, phenol red 15/ρ) was cultured for 21-1 days in an inguacter with CO, and then measured using a microplate luminometer. The absorbance was 3 degrees. When the absorbance at 650 nm is subtracted from the absorbance at 440 nm and the absorbance at 560 nm, and the ratio of each value is taken, the result is 4.64. Converting the value to p l (in Figure 1) results in 658.Actually, the pH is p I - (result I measured with a sensor) H is 661, showing good agreement. pH 6, Since 58 was not suitable for cell proliferation, the culture medium was replaced.

Furthermore, prI was measured every 2 days, and if the pH was inappropriate, the culture medium was replaced and cultured for 2a. As a result, cell iJ:
, 1: <proliferated.

[Brief explanation of the drawing]

Figure 1 shows the IJ for each pl-f of phenol red solution.
This is a curve representing the ratio of absorbance at 430 nm and absorbance at 560 nm. Patent applicant: Sumitomo Electric Industries, Ltd. Agent: Patent attorney: Aobai Ao and 2 others Figure 1H

Claims (2)

[Claims] (1) When culturing cells in a transparent device, the pH is calculated from the change in absorption of visible light by the culture solution, and the culture solution is replaced when the pH becomes inappropriate for cell growth. How to culture cells. (2) The culture method according to claim 1, wherein the change in absorption of visible light by the culture solution is an absorption change due to phenol red contained in the culture solution.