JPH0225748A - Method of measuring number of cultured cells and ultrasonic oscillator used therein - Google Patents

Abstract

PURPOSE:To easily and rapidly measure the number of cultured cells by sticking a cell-adhesive dye to the cultured cells, dissolving the dye and crushing the cells by using ultrasonic wave, then measuring the absorbancy. CONSTITUTION:After the cells are cultured in a microtiter plate, a soln. prepd. by dissolving the cell-adhesive dye into the physiological salt soln. of a phosphoric acid buffer is added to the medium. The tip of the ultrasonic oscillator mounted with a plate guide formed of rigid rubber, etc., is inserted into the medium in the microtiter plate and the ultrasonic waves are oscillated upon lapse of the prescribed time. The cells are crushed and the dye is completely dissolved in this way, by which the medium contg. no insoluble matter is obtd. The absorbancy thereof is, therefore, measured and the number of cells is easily counted.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for simply and quickly measuring the number of cells proliferated by culture, and an ultrasonic oscillator used therefor.

Saramai Ibaraki Conventionally, methods for measuring the number of proliferating cells obtained through culture include: 1) Directly measuring the cell number with a hemocytometer or Coulter counter, and 2) Measuring changes in cell DNA synthesis due to proliferation ('H). - A method of measuring thymidine incorporation is known.

The above method (■) includes 1) direct counting of the number of cells, 2) counting of the number of cells in which the formation of spindle fibers is inhibited by colchicine during cell division and a chromosomal image appears, and 3) (3H) - There is a method of identifying and counting cells with advanced DNA synthesis by autoradiography using thymidine or the like.

On the other hand, as for method ① above, 4) culturing in a test tube [
5) A method to incorporate 3H]-thymidine and collect trichloroacetic acid insoluble matter on a glass filter, and 5) a method to culture in a microtiter plate and collect cells that incorporate 3H]-thymidine on a glass filter using a cell harvester. be.

Among these methods, method 1) or 5) above is currently the most popular method, but l)
This method allows cells to be grown on a relatively large scale (2~1o11!!
) has the disadvantage that it must be cultured in 5)
This method must be carried out in a controlled radioisotope laboratory, and therefore requires knowledge of radiobiology and technical proficiency.

Furthermore, in 1983, Mossmann developed an MTT assay using dye degradation products from animal cells as an indicator as a cell proliferation measurement method (Journal of Immunological Methods, 65.55-63.1983).

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
is cleaved by enzymes in the mitochondria of living cells,
Produces blue-purple MTT formazan.

The amount of MTT formazan produced is correlated with the number of viable cells, and cell proliferation at the time of addition of MTT can be measured by dissolving it with isopropanol and measuring the absorbance. Therefore, according to this method, cell proliferation can be measured simply and with good reproducibility using a small amount (50 to 200 μl) of a culture system without using radioactive substances.

However, after dissolving MTT formazan with MTT Afusei,
There is a problem in that if left for a long time, serum proteins will precipitate, so colorimetric determination must be carried out within one hour.

Furthermore, Denizot et al.
reported an improved method of Afsei (Journal of Immunological Methods, Deaf, 271-277, 1986), but in this improved method, after culturing in a serum-containing medium, the medium is changed to a serum-free medium when MTT is added. There is a problem in that the operation is complicated because it is replaced by .

Furthermore, after adding isopropanol, it is necessary to repeat the viping and piping several times in order to dissolve the MTT formazan. However, since the piping operation requires a long time, there is a problem in that serum proteins may precipitate when a large number of specimens are processed.

In addition to the MTT assay, Coomassie brilliant blue (Analytical Biochemistry, 140,417-423.1) is also used as a dye that adheres to cultured cells.
984) or Crystal Violet (Journal of Immunology, Month 37. (6).

1878-1884.1986) has also been reported. However, even with cell proliferation measurement methods using these dyes, the dye must be solubilized in a solvent within a limited time, and if the solubilization is insufficient, errors may occur in the measured values. There are drawbacks.

Furthermore, in recent years, various measurement methods and devices that directly use microwell plates have been developed and commercially available, and it has been desired to develop a cell number counting method that can be easily incorporated into these devices.

The present invention was made for the purpose of solving the problems seen in the conventional technology on ridges. An object of the present invention is to provide a measurement method and an ultrasonic oscillator used therein.

The present invention will be explained in detail below.

The method according to the present invention involves dispersing and dissolving a dye attached to a cell or a dye decomposition product obtained by reacting a dye with a cell using an ultrasonic oscillator, and measuring its absorbance. It is characterized by

In other words, the method for measuring the number of cultured cells according to the present invention uses an ultrasonic oscillator to crush the proliferating cells and instantly solubilize the dye in a cell proliferation activity measurement method using a dye. It is possible to understand the power of being able to do something.

The dye used in the present invention is one that can attach to cells,
or those that can produce dye decomposition products by cells, such dyes include 3-(45-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (referred to as MTT), Crystal Bioleft, Examples include Coomassie brilliant blue.

In the present invention, the above dye is attached to proliferating cells of any animal or plant that can grow, or a dye is reacted with the cells to produce a dye decomposition product. In this case, the dye is used after being dissolved in phosphate buffered saline (PBS).

The above cell culture is performed using RPMI 16 for animal cells.
Use conventional media such as 40 medium, Dulbecco's modified Eagle's medium, minimum essential medium (MEM), etc., and add 1 to 20% (wt) animal serum such as fetal bovine serum or human AB type serum to these media. , preferably 5-10% (wt
) or a serum-free medium supplemented with growth factors. On the other hand, plant cells are cultured using a medium in which in addition to inorganic elements, organic substances such as vitamins are added to sugars as a carbon source and energy source, and plant growth regulators are also added.

Incidentally, when measuring the growth activity of microorganisms, it is preferable to culture them using a natural liquid medium or a synthetic liquid medium containing a natural product such as meat juice, wort, or serum as a main component.

In the present invention, cultured cells to which dyes have been attached as described above, or cells which have been decomposed by reacting with dyes,
Ultrasonic waves are applied to crush the cells and dissolve the dye.

The ultrasonic oscillator used here to apply ultrasonic waves may be a commercially available hand-held ultrasonic homogenizer, and the tips for cell disruption may be one with a conical or cylindrical tip, or one with an interval of 9 mm. 8 to 1
It is characterized by having 2, or 48 in total, 8 vertical and 6 horizontal at 9 m+w intervals, or 96 in total, 8 vertical and 12 horizontal at 9 mm intervals.

The above-mentioned cell disruption chip has a diameter of 41 mm or less, preferably 1 mm
mm to 31 and the length is 7m1II or more, preferably 301
It is preferable to use titanium or stainless steel with a diameter of 111 to 50 mm. Also, a height of 30 IIll11 to 1 on one side or both sides along the row of chips.
It is preferable to have a guide made of hard rubber, rubber or silicone coated (or covered) plastic, stainless steel, acrylic resin, or vinyl chloride resin with a diameter of 0.00 mm and a thickness of 11 to 5 nV.

The attached Figure 1 shows an example of the shape of the chip, in which fat indicates one chip, (bl indicates 8 chips, (C1 indicates 12 chips, and (dl) indicates 1 chip. 96 pieces (8 x 12) to fit a 96-well plate
A connected chip is shown.

When an ultrasonic oscillator configured as described above is used to apply ultrasound to cultured cells with dye attached or cultured cells reacted with dye, the cells can be disrupted in an extremely short period of time. At the same time, the dye can be completely dissolved, making it possible to suppress the precipitation of serum proteins seen in conventional methods.

Next, a method of applying ultrasonic waves using the above-mentioned ultrasonic oscillator will be explained.

After culturing the cells in a microtiter plate, a solution of a dye dissolved in phosphate buffered saline (PBS) is added to the culture solution and left to stand for a predetermined period of time. Insert at a distance of at least 211111% from the bottom of the well, preferably 3m+ or more, to avoid damaging the bottom of the plate, and at least 3mm or more, preferably 4ff1m or more deeper than the culture solution surface to prevent the culture solution from scattering from the well. It is desirable to do so.

To do this, as shown in Figure 2, for example, by using a plate-shaped guide made of hard rubber attached to the tip of the oscillator, the tip can be placed on the microtiter plate without touching the bottom of the plate. can be inserted evenly into the interior.

The plate-shaped safety guide used here has a thickness of 0.5+ms or more, preferably 111m to 5Illll, and a width of 1105u.
Above, preferably 15IIIl to 130III11, and a height of 30mm or more is desirable. In addition, the material of the guide should be made of hard rubber or 0.1 on+ thick so as not to damage the adhesive part with the microtiter plate.
As mentioned above, it is preferably made of hard rubber of 11m to 20au++ or plastic coated with silicone on the adhesive surface, stainless steel, acrylic resin, vinyl chloride resin, or the like.

In Fig. 2, fat shows the front and side (horizontal) sides of the plate-shaped guide, (b) shows the plate-shaped guide attached to a model with 8 chips, and (C) shows the model with 96 chips. The state in which it is attached is shown. In addition, (dl indicates a state in which a chip equipped with a plate-shaped guide is inserted into a microtiter plate.

After inserting the ultrasonic oscillator into the culture solution in the microtiter plate as described above, ultrasonic waves are generated, and the oscillation time is sufficient for 2 seconds to 10 seconds, preferably about 5 seconds.

In addition, by applying ultrasonic waves to the culture solution by oscillating for 2 seconds or more, no insoluble matter is observed.

That is, according to the present invention, by applying ultrasound to cultured cells with dyes attached or reacted with them using an ultrasonic oscillator, the cells are crushed and the dyes are completely dissolved, leaving virtually no insoluble matter. Since a culture solution free of cellulose can be obtained, the number of cells can be easily counted by measuring its absorbance. Therefore, according to the present invention, the cell proliferation activity of a large number of specimens can be measured.

In addition, the ultrasonic oscillator used in the present invention is equipped with a tip for disrupting cells at its tip, and a safety guide is attached to the tip, so that it does not damage the bottom of the microtiter plate containing cultured cells. I don't have to worry about getting it on.

EXAMPLES The present invention will be specifically described below with reference to Examples.

Example 1 This example shows the relationship between the number of cells in the culture medium and the absorbance obtained by MTT assay using three wavelength measurement, and the time required for the measurement using a human cultured cell line.

As the human cultured cell line, the B lymphoblastoid cell line Wl-L2 obtained from Dai-Nippon Pharmaceutical Co., Ltd. was used, and the cell line was 1 to 16
The cells were cultured in a 96-well microtiter plate (Falcon 3072) at a temperature of 37° C. in an atmosphere containing 5% carbon dioxide gas and 100% humidity so that the number of cells/well was 100%. For the MTT assay, the cell culture medium was
Add μl of MTT solution to a 96-well microtiter plate (Falcon 3072) and incubate for a predetermined period of time, then add 10 μl of an MTT solution prepared by dissolving MTT in phosphate buffered saline (PBS) to a concentration of 5 tag/-β. Add, 37
The sample was left standing for 4 to 6 hours in an atmosphere containing 5% carbon dioxide gas and 100% humidity at a temperature of .degree. 150 μl to dissolve MTT formazan generated by MTT cleavage.
When hydrochloric acid-ituburobanol was added and pipetting was performed immediately using Pipetman (Gilman);
- carried out using an ultrasonic oscillator with a book tip. Furthermore, a safety guide made of acrylic resin coated with hard rubber was attached to the ultrasonic oscillator.

The continuous wavelength was set to a main wavelength of 577 nm and a sub wavelength of 620 nm, and MTT assay measurements were performed using a medium containing no cells as a control.

FIG. 3 shows the relationship between the number of cells and the absorbance of the MTT assay using three wavelength measurements. As seen in the figure, as a result of the three-wavelength measurement, the difference between the number of cells and the absorbance (AS77Abzo) showed good linearity, and the correlation coefficient was 0.992. No difference was observed between dissolution by pipetting and dissolution using an ultrasonic oscillator, but the time required for dissolving MTT formazan was approximately 16 minutes for dissolving 20 wells using pipetting, whereas the time required for dissolving MTT formazan was extremely long. Approximately 2 when using a sonic oscillator
shortened to minutes.

Example 2 In this example, human cultured cell line H0323 (Ohashi II, et al.,
Cell Biology Intern Report, ■.

77.1986. ) was cultured and subjected to MTT assay. To dissolve MTT formazan, pipetting using Pipetman (Gilson) and 8 as shown in Figure 2fb)
This was done using an ultrasonic oscillator with multiple tips.

A stainless steel safety cover coated with hard rubber was attached to the ultrasonic oscillator.

The measurement wavelength was the same as in Example 1.

As a result, as in Example 1, the difference in cell number and absorbance (A,
Although a good linear relationship was observed for MTT formazan, the time required for dissolving MTT formazan was approximately 70 minutes using pipetting to dissolve 96 wells, whereas the time required for dissolving MTT formazan was approximately 70 minutes using 8-tip ultrasonics. The oscillator completed the test in 1 minute and 30 seconds.

Example 3 This example describes human-human hybridoma 9 P-13-
2 (Nimoto et al., Agricultural Biological Chemistry, 2217.1986), the cells were cultured in 20 96-well microtiter plates, and MTT assay was performed. To dissolve MTT formazan, pipetting using a Pipetman (Gilson) and the procedure shown in Figure 2 (
The experiment was conducted using an ultrasonic oscillator having 96 chips shown in C1.

A hard rubber safety guide was attached to the ultrasonic oscillator. The measurement wavelength was the same as in Example 1.

As a result, during the pipetting operation, protein precipitated from the second plate, making measurement impossible. In contrast, when using an ultrasonic oscillator, 20 plates can be
The treatment was completed in minutes, and no protein precipitation was observed. Furthermore, when using an ultrasonic oscillator, a good linear relationship was observed between cell number and absorbance value for all plates.

[Brief explanation of the drawing]

FIG. 1 shows the chips attached to the ultrasonic oscillator according to the present invention, fat indicates one chip, (e) indicates 8 chips, (e) indicates 12 chips, and FDL indicates 48 chips. The formation states of 96 consecutive chips are shown. Figure 2 shows an example of a plate-shaped guide attached to a chip, where (a) shows the front and side views of the guide, and (bl)
indicates the state in which guides are attached to 8 series of chips, fcl indicates the state in which guides are attached to 96 series of chips, and (d+ indicates the state in which one chip with a guide attached is inserted into the microtiter plate. Fig. 3 shows the relationship between the absorbance of MTT Afsei and the number of cells measured by three wavelength measurements.

Claims (5)

[Claims] (1) A cell-adhesive dye is attached to or reacted with cells grown by culture, and then ultrasonic waves are applied to dissolve the dye and disrupt the cells, and then the absorbance is measured. How to measure the number of cultured cells. (2) The method for measuring the number of cultured cells according to claim 1, wherein the cell-adhesive dye is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. (3) Use 1 cell disruption chip, 8 chips at 9 mm intervals, 12 chips, or 8 chips vertically and 6 chips horizontally at 9 mm intervals.
48 pieces in total or 9 pieces in total, 8 pieces vertically and 12 pieces horizontally at 9mm intervals.
An ultrasonic oscillator consisting of six connected units. (4) The ultrasonic oscillator tip has a diameter of 4 mm or less and a length of 7 mm.
The ultrasonic oscillator according to claim 3, which has a diameter of mm or more. (5) A guide made of hard rubber or rubber with a height of 30 mm to 100 mm and a thickness of 1 mm to 5 mm, or made of silicone-coated plastic, stainless steel, acrylic resin, or A claim comprising a guide made of polyvinyl chloride (
3) or the ultrasonic oscillator described in (4).