JP2009095291A - Method of and device for monitoring cytotoxicity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of and a device for easily monitoring cytotoxicity at a low cost, enabling cytotoxicity to be measured, in real time. <P>SOLUTION: The device of monitoring cytotoxicity comprises a means for supplying a culture liquid and a culture liquid containing a test drug, while switching them, to a container 6 in which culture cells will be sealed; means 7, 9 and 10 for measuring the absorption of light having a specified wavelength with lapse of time by irradiating the culture liquid or the culture liquid containing the test drug flowing out from the container 6 by perfusing the culture cells with the light; and a means 11 for carrying out information handling of the measured change of the absorption of the light that has the specified wavelength. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method and apparatus for monitoring cell damage when a test drug is given to cultured cells.

  Knowing whether a drug has cytotoxic side effects is essential in developing a drug. This requires measuring cell damage. In addition, it is necessary to measure cell damage in order to elucidate the mechanism of cell damage caused by such drugs and to develop a suppression method. For this reason, conventionally, a drug to be tested is given to cultured cells such as mammals to measure cell damage.

Non-Patent Document 1 discloses that cell damage is measured by quantifying enzyme activity or mitochondrial activity in a cell to which a test drug is given using a specific reagent. Specifically, in each hole of a measuring instrument called a microplate having 96 holes, cells are cultured with a culture solution, and after the cells have sufficiently grown, a drug to be tested for toxicity is added. After a certain time, an MTT solution as a reagent is added, and the amount of the added MTT changed to formazan by an enzyme present in intracellular mitochondria is measured. This measurement is performed by measuring the absorbance with a microplate reader because formazan absorbs light having a wavelength of 570 nm.
In addition, Patent Document 1 discloses that in order to accurately quantify cell damage, the detection of the viability of each cell is detected and the cell damage is measured based on the number of live cells and dead cells. . Specifically, the cells to be adhered are cultured on the surface of a membrane pattern such as collagen to which the cells are adhered, and a drug to be tested whose toxicity is to be measured is added. Next, the cells are stained with a specific staining solution that varies in color after staining depending on whether the cells are alive or dead. After staining, cell viability on the membrane pattern is determined for each cell by a microscope, and cell damage is measured by viability.
Green LM, Reade JL, and Ware CF, (1984) .J. Immunol.Methods, 70 (2) 257-268.Rapid colorimetric assay for cellviability: application to the quantitation of cytotoxic and growth inhibitorylymphokines. JP-A-10-327854

However, in the measurement of Non-Patent Document 1, only one measurement at a certain time can be performed for one hole of the microplate. Therefore, in order to see a long-term change over time, a culture test must be performed using a large number of holes, and measurement with samples in different holes must be performed intermittently, for example, at intervals of 10 minutes. Even when only detecting the presence or absence of cell damage without requiring such changes over time, it is not possible to know when cell damage caused by the drug under test occurs, so it is possible to know the appropriate measurement timing. Have difficulty. Moreover, it is necessary to use a special reagent for measuring cell damage.
From these things, much labor and cost are required. Nevertheless, only intermittent measurements can be made, and in one measurement, it takes a considerable amount of time from the start of measurement until results are obtained. It was not clear whether the data was related to cell damage, and real-time measurement could not be performed.

  Moreover, in the measurement of patent document 1, a cell damage can be measured with the exact data regarding the life and death of a cell. However, also in this method, as in the case of Non-Patent Document 1, only one-time measurement at a certain time can be performed, and much labor and cost are required. It is the same as that. For this reason, it was impossible to measure in real time. Furthermore, if the cells did not die, it was not possible to judge cell damage.

In order to solve the above-mentioned problems, the present invention has been made through extensive research, and as a result, when a cultured cell is chemically or physically damaged, a substance that absorbs ultraviolet light having a specific wavelength is transferred from the cell to the culture solution. Based on finding that it is released. And based on such new knowledge, the method and apparatus which can measure a cell damage in real time have been completed.
That is, an object of the present invention is to provide a cell damage monitoring method and apparatus capable of measuring cell damage in real time while being simple and low-cost.

The cell damage monitoring method according to the present invention is characterized in that a culture solution containing a drug to be tested is applied to cultured cells, and the absorption of light of a specific wavelength is measured over time for the subsequent culture solution.
According to the present invention, a culture solution containing a test drug to be detected for the presence or absence or the degree of cytotoxicity is given to cultured cells. Then, since the light absorption of the specific wavelength by the culture medium changes with the occurrence of the cell damage, the occurrence or degree of the cell damage is monitored in real time by measuring the absorption of the light of the specific wavelength over time. be able to.

In addition, the method for monitoring cell damage according to the present invention continuously supplies a culture medium containing a test drug to cultured cells, and continuously absorbs light of a specific wavelength over time for a culture liquid that continuously flows out. And measuring.
According to the present invention, the supply of the culture solution containing the test drug to the cultured cells, the outflow of the culture solution from the cultured cells, and the measurement of light absorption at a specific wavelength are all performed continuously. Therefore, if conditions such as the flow rate are set first, the measurement can be easily performed without any trouble. And when light absorption of a specific wavelength by a culture solution changes with generation | occurrence | production of cell damage, generation | occurrence | production or the extent of cell damage can be monitored in real time.

  In the present invention, when the specific wavelength is about 260 nm or about 285 nm, cell damage can be monitored by light absorption at these wavelengths.

In addition, the method for monitoring cell damage according to the present invention supplies a culture solution containing a test drug to the cultured cells, measures the absorption of light of a specific wavelength over time for the flowing culture solution, and A comparison test is performed simultaneously, in which a culture solution containing is supplied to a culture cell different from the culture cells, and the flowing culture solution is measured over time for the absorption of light of the specific wavelength.
According to the present invention, a culture solution containing a test drug to be detected for the presence or absence of cytotoxicity or its degree is supplied to the cultured cells. Then, since the light absorption of a specific wavelength by the culture medium changes with the occurrence of cell damage, by measuring the absorption of light of a specific wavelength over time for the culture liquid that perfuses and flows out of the cultured cells, The occurrence or extent of cell damage can be monitored in real time. At the same time, a comparative test can be simultaneously performed on cultured cells to be compared with respect to the occurrence of cell damage. Since both tests can be measured in real time, it is possible to obtain the result of a comparative test that coincides with time.

In addition, the method for monitoring cell damage according to the present invention supplies a culture solution containing a test drug to the cultured cells, measures the absorption of light of a specific wavelength over time for the flowing culture solution, and Supply a culture solution containing a test drug that does not contain or different from the test drug to the cultured cells, and simultaneously perform a comparative test that measures the absorption of light of the specific wavelength over time for the flowing culture solution It is characterized by.
According to the present invention, a culture solution containing a test drug to be detected for the presence or absence of cytotoxicity or its degree is supplied to the cultured cells. Then, since the light absorption of a specific wavelength by the culture medium changes with the occurrence of cell damage, by measuring the absorption of light of a specific wavelength over time for the culture liquid that perfuses and flows out of the cultured cells, The occurrence or extent of cell damage can be monitored in real time. At the same time, a comparative test can be simultaneously performed for a drug to be compared or no drug to be tested regarding the occurrence of cell damage. Since both tests can be measured in real time, it is possible to obtain the result of a comparative test that coincides with time.

Further, the cell damage monitoring apparatus according to the present invention comprises a means for switching between a culture solution and a culture solution containing a test drug and supplying the culture cell to a container in which the culture cell is sealed, and perfusing the culture cell from the container. Means for measuring the absorption of light of a specific wavelength over time by irradiating the culture medium containing the flowing out culture medium or drug to be tested, and means for processing information on changes in the measured light absorption of the specific wavelength It is characterized by including.
According to the present invention, the normal culture solution and the culture solution containing the drug to be tested for detecting the presence or absence of cytotoxicity are switched to either one and supplied to the container in which the cultured cells are enclosed. it can. Then, in the container, the measurement means irradiates light to the culture solution containing the culture solution or the drug to be tested that perfuses the cultured cells and flows out of the vessel, and measures the absorption of light of a specific wavelength over time. . Since the light absorption at a specific wavelength by the culture medium changes with the occurrence of cell damage, the occurrence or degree of cell damage can be known in real time from the measurement result processed by the information processing means.

Further, when a plurality of lines including each means are provided, a plurality of tests including various comparative tests can be performed simultaneously.
In addition, a tank for storing a culture solution and a culture solution containing a drug to be tested is further provided, and when these are shared by a plurality of lines, when performing a plurality of tests at the same time, The apparatus can be simplified by sharing the culture medium bath containing the drug under test.

  According to the present invention, it is possible to provide a cell damage monitoring method and apparatus capable of measuring cell damage in real time while being simple and low-cost.

Hereinafter, a cell damage monitoring method and apparatus according to an embodiment of the present invention will be described with reference to the accompanying drawings. In the description, the same reference numerals are used for the same elements, and redundant descriptions are omitted.
FIG. 1 is an example of a cell damage monitoring apparatus 1 for carrying out a cell damage monitoring method according to an embodiment of the present invention. Contains a culture bath 2 that contains a normal culture solution suitable for the cultured cells to be tested, and a culture solution that contains the drug to be tested for the presence or absence of cytotoxicity and the level of toxicity. The tested drug-containing culture solution tank 3 is arranged. In the flow path from these tanks to the experimental cell enclosure 6, a switching valve 4 and a pump 5 are installed, and the normal culture solution and the test solution-containing culture solution are switched, and either one is tested. It can be supplied to a cell enclosure.
The experimental cell enclosure 6 has an inlet through which the culture solution in the culture solution tank 2 or the test solution-containing culture solution in the test solution-containing culture solution tank 3 flows, and a flow from which the culture solution perfused with the cultured cells flows out. An outlet is provided approximately on the opposite side of the container. The container is provided with a support object suitable for the cells to be encapsulated, for example, a porous solid such as hydroxyapatite or the like having cell adhesion, and fine particles, and the cells to be tested are supported there. Means for keeping the temperature in the container at a predetermined constant value is also provided.

  The downstream pipe line connected to the outlet of the experimental cell enclosure 6 is provided with a light measuring unit 7 having a window that transmits ultraviolet light and visible light. The window of the light measuring unit 7 is irradiated with light from the light source 9. The light source 9 is, for example, a xenon lamp, and irradiates light having a range from ultraviolet light to visible light including wavelengths of about 260 nm and about 285 nm. A light detector 10 is provided for measuring the light intensity of a predetermined wavelength of about 260 nm or about 285 nm, which is irradiated from the light source 9 to the light measurement unit 7 and transmitted through the culture solution in the light measurement unit 7. The photodetector 10 is, for example, a photomultiplier tube PMT. A drainage tank 8 is provided downstream of the light measurement unit 7.

  In addition, an information processing / control device 11 for recording and analyzing the measurement result of the photodetector 10 and controlling the switching valve 4 and the pump 5 is provided. This is composed of, for example, a computer, and from the light intensity of a specific wavelength detected by the light detector 10, a change with time in the degree of light absorption of the wavelength by the culture solution is continuously obtained, recorded, and necessary. Display accordingly. Furthermore, when the experiment is started, the switching valve 4 is connected to the normal culture medium tank 2 side to operate the pump, and after a predetermined time, the switching valve 4 is switched to the test substance-containing culture liquid tank 3 side, a photodetector. When it is determined from the data from 10 that the absorption of light of a specific wavelength has reached a predetermined threshold, control is performed such as switching the switching valve 4 again, or stopping the pump operation and terminating the experiment. May be.

A method for monitoring cell damage using the apparatus shown in the example of FIG. 1 will be described. First, in a state where the switching valve 4 is in communication with the culture solution tank 2 side, the pump 5 is operated to perfuse the culture solution into the experimental cell enclosure 6 in which predetermined experimental cells are enclosed. Here, the cells may be perfused for a sufficient period of time to culture and proliferate, but even when such a long period of time is not applied, the cells are stabilized with a normal culture solution, At that time, the time required to obtain the light absorbance of the specific wavelength light of the culture solution discharged from the experimental cell enclosure 6 as basic data is supplied from the culture solution tank 2.
In the light measurement unit 7, the light emitted from the light source 9 such as a xenon lamp passes through the culture solution discharged by perfusing the cells in the experimental cell enclosure 6, and the light having a specific wavelength is detected by the photodetector 10. Intensity is detected. Specific wavelengths include 260 nm or the vicinity thereof, 285 nm or the vicinity thereof, or both. The culture solution that has passed through the light measuring unit 7 is discharged to the drainage tank 8.

Next, the switching valve 4 is switched so as to communicate with the test drug-containing culture solution tank 3 containing the culture solution containing the test drug to be tested for toxicity to cells. Then, the test drug-containing culture solution is perfused with respect to the cells in the experimental cell enclosure 6. The photodetector 10 continuously detects the light intensity of a specific wavelength with respect to the culture medium discharged from the experimental cell enclosure 7. In the information processing / control device 11, the light absorbance of the culture solution is recorded over time based on the data of the light intensity of the specific wavelength from the photodetector 10.
When the cells in the experimental cell enclosure 6 are damaged due to the toxicity of the drug to be tested, light absorption at a specific wavelength, particularly light absorption at wavelengths of 260 nm and 285 nm, increases by the culture medium perfused and discharged from the cells. . Here, especially the change in the absorbance of light at 260 nm appears prominently. This is because nucleotides and their polymer nucleic acids leak out from damaged cells, thereby absorbing light of this wavelength. It is thought that there is. Regarding the wavelength of 285 nm, it is considered that the peptide or protein that is an amino acid or a polymer thereof leaks from the damaged cell and absorbs light of that wavelength.

  If a threshold value when the degree of absorption of light of a specific wavelength increases is determined in advance, when the information processing / control device 11 detects that the threshold value has been reached, the pump 5 is stopped and the experiment is terminated. It is also possible to observe the subsequent change by switching the switching valve 4 so as to perfuse a normal culture solution.

In this example, the light absorption of the culture solution after being discharged from the experimental cell enclosure 6 was measured in the light measurement unit 7 provided downstream. Absorption of light may be measured by providing a reservoir for the perfused culture medium and making it transparent to allow light to pass therethrough. In this case, the light absorption is measured continuously, but the supply and discharge of the culture solution do not necessarily have to be performed continuously, so that the amount of the culture solution and the test drug can be saved. In this example, in the information processing / control device 11, the light absorption is obtained from the light intensity detected by the light detector 10, but the transmitted light intensity and the degree of light absorption appear in an inverse relationship. Therefore, when monitoring, the detected light intensity may be directly adopted as a numerical value related to light absorption.
In addition, the information processing / control device 11 automatically processes the detection result of the light detector 10 and controls the switching valve 4 and the pump 5. However, if such automation is not necessary, May be performed as appropriate.

Next, another example in the present embodiment will be described with reference to FIG. In this example, the cell damage monitoring device 1 has two devices described in FIG. 1 installed in parallel, and among them, the culture solution tank 2, the test solution-containing culture solution tank 3, and the drainage tank 8 are shared. Is equivalent to These are shown as a flow path (A) and a flow path (B) in the figure.
When such an apparatus is used, various comparative tests can be performed. As described in the apparatus of FIG. 1, in this embodiment, since it is possible to observe the occurrence of cell damage in real time, as shown in FIG. 2, real-time observation is performed in each flow path arranged in parallel. It is possible to perform simultaneous observation in two flow paths, and is greatly different from a conventional batch type cell damage testing apparatus.

As an example of a comparative test using the apparatus shown in FIG. 2, monitoring of cell damage of different cells for the same drug to be tested will be described. In this case, different cells to be used for the experiment are accommodated in the experimental cell enclosures 6A and 6B, respectively. Then, the same experiment as described in FIG. 1 is performed in each of the flow paths (A) and (B). FIG. 3 shows an example of a time chart showing which tanks 2 and 3 communicate with the switching valves 4A and 4B in that case. As shown in FIG. 3, the switching of the switching valves 4A and 4B in the flow paths (A) and (B) is the same.
First, the switching valves 4A and 4B communicate with the culture solution tank 2, and a normal culture solution is supplied to the experimental cell enclosures 6A and 6B. Next, at t1 after elapse of a predetermined time, the switching valve 4A, 4B is communicated with the test-substance-containing culture medium 3 so that the same test-substance-containing culture medium is contained in the experimental cell enclosures 6A, 6B. Supplied. During this time, light absorption is detected by the photodetectors 10A and 10B in both flow paths. Therefore, if the degree of light absorption in one of the flow paths or the flow path where the cell damage appears lately exceeds a predetermined threshold value n after elapse of time t2 as shown in FIG. 5, the time t2 is used as a reference. At a time point t3 or t2 after a predetermined time, the switching valves 4A and 4B may be switched to the culture solution tank 2 again, and the subsequent progress may be observed, or the pumps 5A and 5B are stopped and the experiment is performed. You may end.

Next, an example will be described in which a comparison test is performed with and without the drug to be tested on the same cell using the apparatus of FIG. The same cells are accommodated in the experimental cell enclosures 6A and 6B. Then, as shown in FIG. 4, in the flow path (A), the switching of the switching valve 4A similar to that described in the time chart of FIG. 3 is performed. On the other hand, in the flow path (B), the switching valve 4B always communicates with the culture solution tank 2 and does not communicate with the drug-containing culture solution tank 3. Therefore, the cells in the experimental cell enclosure 6B in the flow path (B) are not given the drug to be tested and can be compared with the cells in the flow path (A) given the drug to be tested. .
Further, in the apparatus of FIG. 2, if the drug-containing culture fluid tank 3 is not provided in common but separately provided in the flow paths (A) and (B), different drugs to be tested can be applied to the same cell. Needless to say, you can experiment.

In the embodiment described with reference to FIGS. 1 to 5, the light measuring units 7, 7 </ b> A, and 7 </ b> B take extra effort by measuring the absorption of light of a specific wavelength for the discharged culture solution. It is possible to know the occurrence of cell damage in real time.
In addition, the fact that the light absorption appears greatly indicates that the degree of cell damage is large, so that the magnitude of toxicity of the test drug can also be known.
In addition, there is no need to use special chemicals to detect the occurrence of cell damage, and the occurrence and extent of cell damage can be monitored simply by measuring the light absorption at a specific wavelength for the fluid perfused with cultured cells. can do.
Furthermore, the occurrence of cell damage can be detected even when the cells do not die.

[Example 1] In this example, in order to clarify the usefulness of the present invention, the apparatus shown in FIG. The experiment was conducted using a culture solution HBSS having a low osmotic pressure.
A cultured cell line RGM1 derived from rat gastric mucosa cells is cultured on the surface of hydroxyapatite granules (diameter 0.2 mm), and these granules are packed in a cylindrical plastic experimental cell enclosure 6 having a diameter of 5 mm and a length of 10 mm. And packed. In the culture solution tank 2, the culture solution HBSS is stored, the switching valve 4 is communicated with the culture solution tank 2, and the pump 5 is operated so that the culture solution is supplied to the culture cells in the experimental cell enclosure 6. HBSS was perfused at a flow rate of 0.2 ml / min.
The culture solution discharged from the experimental cell enclosure 6 after perfusion was measured in the light measurement unit 7. The light source 9 is irradiated with light having a wavelength of 260 nm, 285 nm, 445 nm, and 500 nm using a 100 W xenon lamp, and the light intensity that is transmitted is detected by the photodetector 10, so that the amount of light absorbed by the culture solution can be increased. Continuous measurement was performed.
After confirming that the cultured cells were in a stable state, the switching valve 4 was switched to the drug solution-containing culture bath 3 side. The test solution-containing culture solution tank 3 stores 50% of a low osmotic pressure culture solution HBSS diluted with distilled water. After the switching valve 4 is switched, the low osmotic pressure HBSS causes the same as described above. Cultured cells were perfused at a flow rate.
The continuous measurement result after switching to 50% HBSS was shown in FIG. 6 for the amount of light absorbed by each wavelength.

It can be seen that a large amount of light-absorbing substances leaked from the cells with the occurrence of cell damage. Leakage patterns are different at each wavelength. The light-absorbing substance with a wavelength of 260 nm is a nucleotide and its polymer nucleic acid, and the light-absorbing substance with a wavelength of 285 nm is an amino acid and its peptide or protein as a candidate. A light absorbing substance having a wavelength of 445 nm is presumed to be flavins. As shown in FIG. 6, the change in absorption of light having a wavelength of 260 nm appears most prominently, and 285 nm follows.
Thus, it can be seen that cell damage due to low osmotic pressure stress can be detected by an increase in light absorption in the vicinity of a specific wavelength, for example, 260 nm, 285 nm, and 445 nm, with respect to the culture medium perfused with cells.

[Example 2] Using the same apparatus and cultured cells as in Example 1, an experiment was conducted using an oxidant that is supposed to cause cell damage due to oxidation of membrane lipids in the cell membrane as a test drug. 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is an oxidant, was used as a test solution-containing culture solution. A culture solution HBSS containing AAPH at a concentration of 0.1 mM was stored in the test solution-containing culture solution tank 3, and the cultured cells in the experimental cell enclosure 6 were perfused at the same flow rate as in Example 1. The measurement results of the light absorption of the culture solution after perfusion for each wavelength of light immediately after the perfusion of the culture solution containing AAPH are as shown in FIG.
This revealed that substances that absorb light of wavelengths of 260 nm and 285 nm leak from the cells due to cell damage caused by oxidation of membrane lipids by AAPH. The leakage pattern was different at both wavelengths, and the 260 nm absorbing substance was released rapidly immediately after AAPH administration, but the 285 nm absorbing substance continued to leak for a while after administration, and sudden leakage occurred about 60 minutes later. This spill acceleration disappeared after 110 minutes and the subsequent spill rate was maintained at a high level.
It is clear that multiple substances that absorb 260nm and 285nm separately leaked out of the cell due to the oxidation of membrane lipids by AAPH because the time-dependent change pattern of the outflow rate of the absorbing substance at two wavelengths. It became. In addition, the outflow amount of the substance having an absorption characteristic at 445 nm was small compared with the low osmotic pressure treatment shown in FIG. From these results, it was shown that cell damage due to lipid oxidative stress in the cell membrane can be detected by measuring the light absorption amount of the cell culture solution at wavelengths near 260 nm and 285 nm.
Thus, also in this example, it can be seen that cell damage can be detected by an increase in light absorption at specific wavelengths, for example, 260 nm and 285 nm, with respect to the culture medium perfused with cells.

"Confirmation test of Example 1 and Example 2" When the cells were stimulated by the low osmotic pressure and the treatment with the lipid oxidant according to Examples 1 and 2, the ultraviolet light absorbing substance outflow phenomenon seen in Figs. In order to confirm the relationship between the occurrence of cell damage and the time course of cell damage, the influence of each stimulus on the function of mitochondria and cell membrane was examined by a conventional technique for measuring the degree of damage.
Regarding the function of mitochondria, the activity of mitochondrial dehydrogenase was measured using a reagent kit Tetra Color ONE (Seikagaku Corporation). Regarding the cell membrane function, the activity of lactate dehydrogenase leaking out of the cell due to the semipermeable loss of the cell membrane was measured using a commercially available drug CytoTox96 (Promega).

Since mitochondria have the function of synthesizing ATP by oxygen respiration and are an important organ for cell survival, the influence on the level of mitochondrial function is important in the investigation of the degree of cytotoxicity. Mitochondrial function depends on the activity of dehydrogenases located within mitochondria. Therefore, changes in the activity of mitochondrial endogenous dehydrogenase over time were examined in RGM1 cultured cells exposed to a hypotonic solution (50% HBSS) or a membrane lipid oxidant (AAPH).
The degree of damage in untreated cells was set to 0%, the degree of damage in cells killed by treatment with distilled water for 20 minutes was set to 100%, and the measured values after stress treatment at various times were normalized. The results are shown in FIG.
In both treatments, the measured values showed similar changes, and both enzyme activities decreased to almost half after 30 minutes of treatment. This indicates that mitochondrial function was impaired by the applied stress stimulus. However, mitochondrial activity was not completely lost even after 2 hours from the start of treatment, and these stress stimuli did not kill all RGM1 cells because both treatments were maintained at low levels. I found that there was no.
Although the weakening of mitochondrial activity in FIG. 8 reflects cell damage due to applied stress, the life / death information of the damaged cell is indeterminate.

Therefore, in order to determine whether the treated cells were alive or dead, the LDH activity in the culture solution was measured. In living cells, biological substances such as proteins, nucleic acids, and sugars are trapped in the cells due to the semi-permeability of the cell membrane. If the cell dies, the polymer membrane leaks out of the cell due to the damage of the cell membrane. The ratio of dead cells reflected in the ratio of semipermeable disruption of the cell membrane can be evaluated by the amount of enzyme activity of LDH, which is a kind of leaked protein. FIG. 9 shows the results of measuring the LDH activity in the culture medium after treating RGM1 cells with 50% HBSS or 0.1 mM AAPH for various times. The measurement value was normalized by setting the measurement value in a condition not containing cells to 0% and the degree of damage in dead cells treated with distilled water for 20 minutes as 100%. The culture solution LDH amount of untreated cells was seen as weak as 1.6 to 1.8% of the total intracellular amount.
The LDH activity of the stimulated cells increased little by little depending on the treatment time, but the leaked LDH activity was about 6.8% for the diluted culture and about 4.9% for the lipid oxidant even after 2 hours treatment. Was a trace amount. These stress stimuli do not lead to devastating death in most cells, although the percentage of cells that die even after 2 hours of stress exposure is low at about 5%. It turned out to be a level.

Based on the results of FIGS. 8 and 9, the effluents of FIGS. 6 and 7 were considered.
First, substances having absorption at 285 nm are amino acids tyrosine and tryptophan and proteins containing them. Of these, if the protein leaks out of the cell, the amount is reflected in the amount of LDH contained in the protein leaked into the culture solution. The LDH amount observed in FIG. 9 increased gently in a time-dependent manner immediately after the start of treatment with both stress stimuli, indicating that the amount of protein efflux gradually and uniformly increased.
On the other hand, the outflow amount of the 285 nm absorbing substance changed in a pattern different from the extracellular LDH amount. With 50% HBSS stimulation, the 285 nm absorbing material increased the efflux rate from around 40 minutes following a slight increase in efflux immediately after treatment.
Even with AAPH stimulation, the amount of spillage started to flow out at a high rate from about 80 minutes following a slight increase in spillage after the start of treatment. The outflow velocity acceleration under both conditions disappeared after a while and then stabilized at a high outflow rate level. As described above, the change pattern of extracellular efflux of 285 nm absorbing substance and protein represented by LDH does not match. Therefore, most of the 285 nm absorbing substance leaked by stress stimulation is not a protein but an amino acid. I guess it would be.

  Next, the 260 nm wavelength absorbing material was considered. This substance is a candidate for nucleotides and RNA or DNA nucleic acids that are macromolecules based on the absorption wavelength. In order for nucleic acids, which are macromolecules, to leak out of the cell, physical destruction of the cell membrane is essential as in the case of proteins. Nucleic acid efflux from cells should show the same pattern of change as protein efflux, and thus the amount of nucleic acid efflux is expected to increase slowly and evenly, similar to the culture LDH activity of FIG. However, the outflow amount of the 260 nm absorbing material temporarily increased with a maximum peak in the vicinity of 30 minutes in the 50% HBSS treatment, and showed an increase in the vicinity of 45 minutes in the AAPH treatment. Since such a temporary increase pattern does not coincide with the change pattern of extracellular LDH activity, the main 260 nm absorption substance is not a RNA or DNA of a high molecular nucleic acid but a nucleotide monomer of its constituent unit. Guessed.

  From the above, by measuring the amount of absorption of ultraviolet light at wavelengths of 260 nm and 285 nm in the culture solution of cultured cells, cell damage induced by stress stimulation can be detected and weak cell damage that does not lead to cell death can be detected. It was shown that it could be detected in stages. Moreover, it was speculated that the ultraviolet light-absorbing substance leaking from the cells is a low-molecular substance such as an amino acid or a nucleotide.

It is a block diagram which shows an example of the monitoring apparatus of the cell disorder in embodiment of this invention. It is a block diagram which shows the other example of the monitoring apparatus of the cell disorder in embodiment of this invention. It is a figure which shows an example of the control regarding the apparatus of FIG. 2 in embodiment of this invention. It is a figure which shows the other example of the control regarding the apparatus of FIG. 2 in embodiment of this invention. It is explanatory drawing of t2 regarding FIG. 3 or FIG. 4 in embodiment of this invention. It is a figure which shows the result of Example 1 in embodiment of this invention. It is a figure which shows the result of Example 2 in embodiment of this invention. It is a figure which shows the result of the confirmation test of Example 1,2. It is a figure which shows the result of the confirmation test of Example 1,2.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Cell damage monitoring apparatus, 2 ... Culture solution tank, 3 ... Test solution containing culture solution tank, 4 ... Switching valve, 5 ... Pump, 6 ... Experimental cell enclosure, 7 ... Light measuring part, 8 ... Drain Tank 9 Light source 10 Light detector 11 Information processing / control device

Claims (8)

  A method for monitoring cell damage, comprising: providing a culture solution containing a test drug to cultured cells, and measuring the absorption of light of a specific wavelength over time for the subsequent culture solution.   A cell damage monitor characterized by continuously supplying a culture solution containing a test drug to cultured cells and continuously measuring the absorption of light of a specific wavelength over time for the continuously flowing culture solution. Method.   The method for monitoring cell damage according to claim 1 or 2, wherein the specific wavelength is about 260 nm or about 285 nm.   Supply the culture medium containing the test drug to the cultured cells, measure the absorption of light of a specific wavelength over time for the flowing culture liquid, and transfer the culture liquid containing the test drug to a cultured cell different from the cultured cells. A method for monitoring cell damage, comprising simultaneously performing a comparative test for measuring the absorption of light of the specific wavelength over time for the culture solution supplied and discharged.   Supply the culture solution containing the test drug to the cultured cells, measure the absorption of light of a specific wavelength over time for the culture solution flowing out, and do not include the test drug or different from the test drug A method for monitoring cell damage, wherein a culture solution containing a drug is supplied to cultured cells, and a comparative test for measuring the absorption of light of the specific wavelength over time is simultaneously performed on the flowing culture solution. Means for switching between the culture solution and the culture solution containing the drug to be tested, and supplying the vessel to which the cultured cells are enclosed;
Means for irradiating light to the culture solution containing the drug to be tested or the test solution to be perfused from the container by perfusing the cultured cells, and measuring the absorption of light of a specific wavelength over time;
And a means for processing information on a change in light absorption measured at a specific wavelength.   The cell damage monitoring apparatus according to claim 6, wherein a plurality of lines including the respective means are provided. The cell damage monitoring apparatus according to claim 7, further comprising a tank for storing the culture solution and a culture solution containing the drug to be tested, which are shared by the plurality of lines. .

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