JP4924415B2 - Apoptosis detection method and apparatus - Google Patents

Description

  The present invention relates to an apoptosis detection method and an apoptosis detection apparatus that can be applied, for example, when culturing animal cells that produce substances used as main raw materials such as pharmaceuticals.

  Drugs including antibody drugs contain substances produced by cells as the main component. Since such a substance is secreted and produced by animal cells, for example, it can be obtained by culturing animal cells and separating and purifying the target substance secreted in the culture solution. In the process of culturing cells, the cells proliferate by repeating division, but if the culture environment deteriorates, the number of cells that cause cell death increases, and if the deterioration of the environment continues, all cells eventually die. Factors that deteriorate the culture environment include mechanical crushing by agitation, nutrient depletion, ammonia secreted by cells, and accumulation of lactic acid.

  Cell death is classified into apoptosis and necrosis. Apoptosis is characterized by changes in the nuclear structure and accompanying cell shrinkage. At the early stage of apoptosis, cell surface microvilli disappear, cells shrink and chromatin loses the normal retinal structure, and around the nuclear membrane. Aggregation and nuclear fragmentation occurs. Thereafter, protrusions appear on the surface of the cell (blebbing), and the cell membrane structure is broken to form apoptotic bodies. On the other hand, in necrosis, first of all, organelles such as mitochondria are enlarged, and the cells gradually expand. Finally, osmotic pressure cannot be controlled and cell lysis occurs and ruptures.

  There are various methods for detecting apoptosis. The main detection methods are listed below. The DAPI (4 ′, 6-diamidino-2-phenylindole) staining method is a method for detecting chromatin aggregation associated with apoptosis. The cells are fixed, stained with the fluorescent dye DAPI that specifically binds to DNA, and observed with a fluorescence microscope. In living cells, a chromatin DNA network structure is observed, and in apoptotic cells, large and small spherical chromatin aggregates are observed. As another method, there is a method of detecting a DNA fragment because chromatin DNA is cleaved in an oligonucleosome unit with apoptosis. Methods for detecting this DNA fragment include agarose gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay, and SubG1 assay. In agarose gel electrophoresis, DNA is extracted from cells and electrophoresed by agarose gel electrophoresis. Apoptotic cells are detected as an integral multiple of 180 to 200 bp. In the TUNEL assay, biotin-labeled dUTP is bound to DNA ends using terminal deoxynucleotidyl transferase (TdT) by labeling the ends of DNA cleaved with apoptosis, and FITC (fluorescein isothiocyanate) -avidin The complex is bound and observed with a fluorescence microscope or measured by flow cytometry (FACS). In the SubG1 assay, DNA fragments are washed out of the cell, then the DNA is stained with PI (propidium iodide) and measured by FACS. Apoptotic cells are cells with less DNA than live G1 cells (SubG1 cells) Identify as There is also a method for detecting cell membrane changes due to apoptosis. Phosphorous components are distributed unevenly in the inner and outer membranes of normal cell membranes, for example, sphingomyelin and phosphatidylcholine are localized in the outer membrane, while phosphatidylserine (PS) and phosphatidylethanolamine are It exists mainly in the inner membrane. Early in the apoptotic process, phosphatidylserine and phosphatidylethanolamine in the inner membrane migrate to the cell surface and are exposed. Since annexin V is a protein that binds to phosphatidylserine in the presence of calcium, annexin V labeled with FITC or the like is bound to phosphatidylserine exposed on the cell surface and measured by FACS to detect apoptotic cells. There is also a method for measuring signal transduction when apoptosis occurs. Detection is performed by immunohistochemical staining or enzyme immunoassay (ELISA) using antibodies against various caspase cross-sections cleaved during the activation process.

  It is also known that cytokeratin 18 is cleaved by caspase at an early event of apoptosis. It is known that a caspase degradation product of cytokeratin 18 produced along with apoptosis exists in a wide range in the protoplasm of cells (Non-patent Document 1). As an antibody that specifically binds to a caspase degradation product of cytokeratin 18, an M30 antibody (M30 cytodes (trade name)) is commercially available. This M30 antibody is capable of detecting a caspase degradation product of cytokeratin 18 in the protoplasm, and is used as a means for determining the initial stage of apoptosis. Cytokeratin 18 is known to exist specifically in epithelial cells (Non-patent Document 2).

J. Cell. Biol. 2000 Mar 20; 148 (6): 1239-54. Biomed. Pharmacother. 2005 Oct; 59 Suppl 2: S359-62.

  By the way, in any apoptosis detection method as described above, a step of crushing the cell itself to extract intracellular components or fixing the cell and treating with a staining solution is required. In particular, the apoptosis test method using the M30 antibody was applied to a tissue sample such as a paraffin-embedded tissue section fixed according to a conventional method.

  On the other hand, in the cell culture using a culture tank, if the above-described method is applied when examining apoptosis of cells, the cells extracted from the medium are disrupted or stained by a measuring device such as FACS or electrophoresis device. It was necessary to perform processing and the like, and very complicated processing was required. Therefore, in view of the above-described circumstances, the present invention provides an apoptosis detection method and an apoptosis detection device that measure the state of apoptosis in a culture tank by measuring a culture solution without requiring treatment of the cells themselves. With the goal.

  The method for detecting apoptosis according to the present invention that has achieved the above-described object includes a step of detecting a caspase degradation product of cytokeratin 18 contained in a culture medium in which animal cells are cultured, and the amount of the caspase degradation product in the culture medium is used as an indicator. Thus, the apoptosis of the animal cell is measured. On the other hand, an apoptosis detection apparatus according to the present invention includes a reaction unit that supplies a medium in which animal cells are cultured, and a detection unit that detects a caspase degradation product of cytokeratin 18 contained in the medium supplied to the reaction unit. The caspase degradation product contained in the culture medium supplied to the reaction section is measured by the detection means, and the apoptosis of the animal cell is detected using the amount of the caspase degradation product as an index.

  The apoptosis detection method and detection apparatus according to the present invention do not require various treatments by aseptically removing animal cells in culture. Here, the caspase degradation product is preferably detected by M30 antibody. Animal cells can be Chinese hamster ovary cells and / or hybridoma CRL-1606 cells.

  The apoptosis detection method and detection apparatus according to the present invention are based on the novel finding that a caspase degradation product of cytokeratin 18 is secreted into an extracellular medium at an early stage of apoptosis of animal cells. Therefore, the detection of the caspase degradation product is not limited at all, and any method and apparatus can be applied as long as the caspase degradation product contained in the medium can be detected. As an example, a caspase degradation product contained in a medium can be detected by using an ELISA method.

  In the apoptosis detection method according to the present invention, a caspase degradation product contained in the medium may be detected in the microchannel. For example, an antibody that specifically binds to a caspase degradation product (for example, M30 antibody) is bound to the inner wall of the microchannel, and the caspase degradation product contained in the medium can be detected by applying the ELISA method. .

  According to the apoptosis detection method and the detection apparatus according to the present invention, it is possible to detect the apoptosis of animal cells by a very simple process without requiring a complicated cell pretreatment before analysis, which has been conventionally required. In addition, according to the method and apparatus for detecting apoptosis according to the present invention, since it does not require complicated cell pretreatment before analysis, which has been necessary in the past, a process that has conventionally taken several hours for cell pretreatment, etc. This can be omitted, and the time for detecting apoptosis can be greatly shortened.

  Hereinafter, an apoptosis detection method and an apoptosis detection device according to the present invention will be described in detail with reference to the drawings. The usage of the apoptosis detection apparatus according to the present invention is not limited in any way, and examples include apoptosis monitoring in a culture tank, apoptosis monitoring during tissue culture, and apoptosis analysis of a stored solution of cultured cells.

  In the apoptosis detection method and apoptosis detection device according to the present invention, a caspase degradation product of cytokeratin 18 contained in a medium in which animal cells are cultured is detected. This is based on the novel finding that a caspase degradation product of cytokeratin 18 is secreted into the extracellular medium in the early stage of apoptosis of animal cells. Therefore, the detection of the caspase degradation product of cytokeratin 18 is not limited at all, and any method and apparatus can be applied as long as it is a means capable of detecting the caspase degradation product contained in the medium.

In the method and apparatus for detecting apoptosis according to the present invention, it is preferable to use an antibody that specifically binds to a caspase degradation product of cytokeratin 18. Specifically, the caspase degradation product of cytokeratin 18 is not particularly limited, but an M30 antibody (trade name: M30 cytodes, manufactured by Roche) can be used. The caspase degradation product of cytokeratin 18 to which this M30 antibody specifically binds may be referred to as M30.
When detecting the degradation product using an antibody that specifically binds to a caspase degradation product of cytokeratin 18, an ELISA method can be used as an example. More specifically, the detection is performed using a sandwich ELISA method using the individual. The detection of apoptosis is not limited to M30, and any substance secreted with apoptosis can be detected. In addition, a caspase degradation product of cytokeratin 18 can be detected using sandwich ELISA for detection of M30. The detection method is not limited to the ELISA method, and a method such as Western blotting may be used.

  Animal cells cultured in a liquid medium are not particularly limited, and may be epithelial cells that are known to accumulate the above-mentioned caspase degradation products in the protoplasm in the early stages of apoptosis. Other animal cells for which such knowledge is unknown may be used. For example, in the test method and test apparatus for apoptosis according to the present invention, Chinese hamster ovary cells and hybridomas producing specific antibodies can be used as animal cells to be cultured. The knowledge that a caspase degradation product of cytokeratin 18 is detected in the medium during apoptosis in these cell lines is a novel finding.

  As an example of the apoptosis detection apparatus, an apparatus using the microchannel 5 as a reaction field can be exemplified as shown in FIGS. Since the amount of caspase degradation product of cytokeratin 18 contained in the culture solution is very small, the detection time using the microchannel as a reaction field can greatly reduce the detection time. However, if the detection time is not a problem, a detection apparatus to which an ELISA method using a plate is applied can be used.

  As shown in FIGS. 1 and 2, in the apoptosis detection device 13, a chip 6 to which a primary antibody (anti-cytokeratin 18 antibody; M5 antibody) is bound is installed in the microchannel 5. As shown in detail in FIG. 2, the chip 6 includes an upper member in which a liquid feeding inlet 17 for supplying a liquid to the microchannel 5 and a liquid feeding outlet 18 for discharging the liquid from the microchannel 5 are formed. And a lower member in which the micro flow path 5 is formed on one main surface, and a structure in which the one main surface is opposed to each other.

  When detecting apoptosis with the apoptosis detection device 13, first, the culture solution that has passed through the filter 2 so as not to mix cells from the culture tank 1 is fed into the microchannel 5 using the pump 3. When cell death is apoptotic, a caspase degradation product (M30) of cytokeratin 18 is present in the culture medium, and this M30 binds to the primary antibody on the inner wall of the flow path. Next, the M30 antibody to which biotin is bound is fed using the microsyringe 4 and the pump 3. After feeding the washing solution, streptavidin combined with alkaline phosphatase is sent. After the washing solution is fed, NBT substrate (nitro-blue tetrazolium chloride substrate) is fed, and the absorbance at 405 nm is measured by the absorptiometer 7. The absorptiometer 7 includes a detector 8 for detecting absorbance, a cell 9 for feeding a solution that has passed through the microchannel 5, a wavelength filter 10 for passing light having a desired wavelength (405 nm), and The light source 11 is configured.

  As described above, M30 contained in the culture medium can be measured, and apoptosis in animal cells can be detected. Information indicating the concentration of M30 in the medium measured by the apoptosis detection device or the stage of apoptosis can be recorded in the recording device 14. The analysis device 15 analyzes the information indicating the M30 concentration in the culture medium or the stage of apoptosis recorded in the recording device 14, and performs analysis so as to obtain appropriate culture conditions. Specifically, the control value is calculated with respect to the culture temperature condition of the culture tank 1, the rotation speed condition of the stirring blade, the concentration condition of the medium component, the pH condition of the culture solution, etc. The control device 16 controls the culture in the culture tank 1 by outputting the control value calculated by the analysis device 15. Specifically, the control device 16 controls the temperature control device of the culture tank 1 in accordance with the culture temperature condition of the culture tank 1 output from the analysis device 15, and in the case of stirring culture, The driving device to be driven is controlled, the medium component supply device is controlled according to the concentration condition of the medium component, and the pH adjustment solution supply device is controlled according to the pH condition of the culture solution. Thereby, culture can be performed in an environment suitable for animal cells, and productivity is improved. The data stored in the recording device 14 can also be used as one index for quality control.

  In addition, after the measurement of M30 in the culture solution is completed, the protein other than the primary antibody can be washed out to the waste liquid tank 12 by sending an acidic solution or an alkaline solution to the microchannel 5. By flowing the washing solution and sending the blocking solution, it becomes possible to measure apoptosis again with the same chip 6.

  The chip 6 to which the primary antibody is bound has an amino group treatment on the inner wall of the flow path. As shown in Fig. 3, the glutaraldehyde solution is first sent to activate the amino group (a1), and after washing with pure water, the M5 antibody is sent (a2), thereby allowing the M5 antibody to flow through the inner wall of the channel. Can be covalently bound to (a3). Drying can be prevented by feeding a blocking liquid after washing and closing the liquid feeding inlet 17 and the liquid feeding outlet 18. Further, the chip 6 can be stored at 4 ° C. by a temperature adjusting device (not shown). If it is installed in the apoptosis detection device 13 before the culture tank 1 is operated, the chip 6 can be reused, so that it is not necessary to replace the chip 6 until the operation is completed. The method of covalently binding the antibody to the microchannel 5 may be binding using a carboxyl group as shown in FIGS. 3 (b1) to (b3). In addition, a bond using a covalent bond is a desirable method when the chip 6 is reused, but a bond using a hydrophobic bond may be used when there is no need for reuse.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.

[Example 1] Expression confirmation of cytokeratin 18 in Chinese hamster ovary cells (CHO) and hybridoma cells (experimental method)
In this example, mouse mouse hybridoma CRL-1606 cells (purchased from American Type Culture Collection) and Chinese hamster ovary cells (CHO-S cells; purchased from Invitrogen) were used. CRL-1606 cells are floating cells that secrete anti-fibronectin antibodies. For culture, a medium in which Fetal bovine serum (FBS) was added to Iscove's Modified Dulbecco's Medium (IMDM medium) to a concentration of 5% was used. . Hereinafter referred to as serum medium. CHO-S cells are cells that have been genetically modified from adherent CHO-K1 cells to form floating cells, and are often used in recombinant protein production. In the culture of CHO-S cells, CD-CHO medium (manufactured by Invitrogen) was used. For these two types of cells, the presence of cytokeratin 18, which is characteristic of epithelial cells, was confirmed.

First, CHO-S cells are shown. CHO-S cells were cultured in an incubator (37 ° C., 5% CO ₂ ,> 95% humidity) using CD-CHO medium. The cultured cells were dispensed into microtubes so that the number of cells became 2 × 10 ⁵ cells, centrifuged at 5000 rpm for 5 minutes with a centrifuge, and the supernatant was removed to recover the cells. A 4% formaldehyde solution was added to the collected cells and allowed to stand for 15 minutes. Centrifugation was performed at 5000 rpm for 5 minutes to remove the formaldehyde solution, PBS solution was added for washing, and centrifugation was performed at 5000 rpm for 5 minutes to remove the PBS solution. 0.5% TritonX-100 solution was added and allowed to stand at room temperature for 5 minutes. Centrifugation was performed at 5000 rpm for 5 minutes to remove the 0.5% Triton X-100 solution, a PBS solution was added for washing, and centrifugation was performed at 5000 rpm for 5 minutes to remove the PBS solution. A 3% BSA solution was added and allowed to stand at room temperature for 1 hour. After removing the BSA solution by centrifugation, anti-cytokeratin 18 antibody (M5) was incubated at 37 ° C. for 1 hour.

  The anti-cytokeratin 18 antibody was removed by centrifugation, and after washing twice with a washing solution, a FITC-labeled anti-IgG antibody was added and incubated at 37 ° C. for 1 hour. After washing with the washing solution, the fluorescence image of the cells was observed with a fluorescence microscope (excitation light 490 nm, fluorescence 520 nm or more). The results are shown in FIG. As shown in FIG. 4, in CHO-S cells, fluorescence was observed with anti-cytokeratin 18 antibody (upper left in the figure), whereas no fluorescence was observed when anti-cytokeratin 18 antibody was removed. (Upper right in the figure). From this, it was confirmed that cytokeratin 18 was expressed in CHO-S cells and hybridoma cells.

  For CRL-1606 cells, since the cells themselves secrete IgG, it is necessary to adopt a method that does not use a FITC-labeled anti-IgG antibody as a secondary antibody, and directly with a fluorescently labeled (FITC) anti-cytokeratin 18 antibody The staining method was used. The cells were treated in the same manner as in the case of the above CHO-S cells. After blocking, the anti-cytokeratin 18 antibody was incubated at 37 ° C. for 1 hour, washed, and then observed with a fluorescence microscope (excitation light 490 nm , Fluorescence 520 nm or more). The results are shown in FIG. As shown in FIG. 5, it can be seen that CRL-1606 cells are stained with the anti-cytokeratin 18 antibody (upper in the figure).

  From the above, it was confirmed that cytokeratin 18 was expressed intracellularly in CHO-S cells and CRL-1606 cells. The finding that cytokeratin 18, which was supposed to be specifically expressed in epithelial cells, is expressed in these CHO-S cells and CRL-1606 cells was a novel finding.

[Example 2] Confirmation of M30 by apoptosis induction using cell disruption solution (experimental method)
When Asp-Glu-Val-Asp-CHO, a caspase 3 inhibitor, is added to cultured cells (CHO-S cells or CRL-1606 cells), staurosporine, an apoptosis inducer, is added as a control. When sulfoxide (DMSO) was added (because staurosporine is dissolved in DMSO), each was cultured for 4 hours in an environment of 37 ° C. and 5% CO ₂ . Each cultured cell was separated into a cell and a culture solution by centrifugation (800 rpm, 5 minutes), and then a 0.5% NP-40 solution was added to the cell precipitate and disrupted and lysed by vortexing. Under each culture condition, M30 was detected using the following ELISA method from the cell disruption solution and the culture solution from which the cells had been removed.

  Anti-cytokeratin 18 antibody (M5 antibody; Roche Diagnostics) was bound to a 96-well plate. Next, after blocking with skim milk, the cell disruption solution and culture solution prepared above were added, and incubation was performed at 37 ° C. for 1 hour. After washing three times, a biotin-labeled M30 antibody solution (Roche Diagnostics) was added and incubated at 37 ° C. for 1 hour. After three washes, HRP-labeled streptavidin was added and incubated at 37 ° C. for 1 hour. After three washes, a TMB substrate solution was added and incubated at room temperature for 20 minutes, after which sulfuric acid was added and the absorbance at 450 nm was measured.

  The results are shown in FIG. Fig. 6 (a) shows the results of measuring the absorbance of the cell disruption solution in CHO-S cells, and Fig. 6 (b) shows the results of measuring the absorbance of the culture solution in CHO-S cells, Fig. 6 (c). Fig. 6 shows the results of measuring the absorbance of the cell disruption solution in CRL-1606 cells, and Fig. 6 (b) shows the results of measuring the absorbance of the culture solution in CRL-1606 cells. As shown in FIGS. 6 (a) and 6 (b), in CHO-S cells, the absorbance decreased in the presence of the caspase 3 inhibitor both in the cell and in the culture solution, and the absorbance increased by the apoptosis inducer. This shows that the change in absorbance by this method is due to apoptosis. Similar results were also obtained in CRL-1606 cells (FIGS. 6C and 6D), indicating that apoptosis can be detected by this method.

[Example 3] Detection of M30 in culture (experimental method)
CRL-1606 cells were fed and cultured in a 1 L culture tank. The culture conditions were 37 ° C. and 60% saturated dissolved oxygen concentration. Once every 12 hours, 5 mL of a culture solution containing cells aseptically was sampled, and 1 mL of the culture was counted using ViCell (Beckman Coulter) for total cell count and viable cell count. In the remaining culture solution, cells were precipitated by a centrifuge (800 rpm, 5 minutes), and the supernatant was stored frozen. After completion of the culture, M30 in the culture broth was detected using the method described in Example 2.

  FIG. 7 shows a graph of the time course of M30 in the culture solution in the 1 L culture tank and the time course of the total number of cells and the number of living cells. As shown in FIG. 7, it can be seen that the amount of M30 increases as the number of living cells decreases, that is, the number of dead cells increases. It became clear that apoptosis can be detected from the culture medium by using this apoptosis detection method.

It is a schematic block diagram which shows typically the apoptosis detection apparatus to which this invention is applied. It is a schematic block diagram which shows a microchannel chip | tip typically. It is the schematic which shows the covalent bond method to the flow path of an antibody. It is a figure which shows the expression confirmation by the immunohistochemical staining of the Chinese hamster ovary cell (CHO cell) in an Example. It is a figure which shows the expression confirmation by immunohistochemical staining of the hybridoma cell (CRL-1606 cell) in an Example. It is a figure which shows the result of the light absorbency in this apoptosis detection method in the extracellular and intracellular of each Chinese hamster ovary cell (CHO cell) and hybridoma cell (CRL-1606 cell) in an Example. It is the figure which showed the detection of M30 in the culture solution in the batch culture of the CRL-1606 cell in an Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Culture tank, 2 ... Filter, 3 ... Pump, 4 ... Syringe, 5 ... Micro flow path, 6 ... Chip, 7 ... Absorbance meter, 8 ... Detector, 9 ... Cell, 10 ... Wavelength filter, 11 ... Light source, 12 ... Waste liquid tank, 13 ... Apoptosis detection device, 14 ... Recording device, 15 ... Analysis device, 16 ... Control device, 17 ... Liquid feed inlet, 18 ... Liquid feed outlet

Claims (12)

  A step of detecting a caspase degradation product of cytokeratin 18 to which M30 antibody specifically binds, which is contained in a culture medium in which animal cells are removed by a filter, wherein the caspase degradation in the culture medium is detected. A method for detecting apoptosis, comprising measuring apoptosis of the animal cell using the amount of the substance as an index. The method for detecting apoptosis according to claim 1, wherein the caspase degradation product is detected with an M30 antibody.   The method for detecting apoptosis according to claim 1, wherein the animal cells are Chinese hamster ovary cells and / or hybridoma CRL-1606 cells.   The method for detecting apoptosis according to claim 1, wherein an ELISA method is used to detect the caspase degradation product.   2. The method for detecting apoptosis according to claim 1, wherein a microchannel is used as a reaction field when detecting a caspase degradation product contained in the medium.   2. The method for detecting apoptosis according to claim 1, wherein an ELISA method is used for detection of the caspase degradation product, and a primary antibody used in measurement by the ELISA method is covalently bound to the wall surface of the microchannel. A reaction part that supplies a culture medium in which animal cells are removed by filtering, and a caspase degradation of cytokeratin 18 that specifically binds to M30 antibody contained in the culture medium supplied to the reaction part. Detecting means for detecting an object,
An apoptosis detection apparatus, wherein the detection unit measures caspase degradation products contained in the medium supplied to the reaction unit and detects apoptosis of the animal cells using the amount of the caspase degradation products as an index.   The apoptosis detection apparatus according to claim 7, wherein the detection means is an M30 antibody.   The apoptosis detection apparatus according to claim 7, wherein the animal cells are Chinese hamster ovary cells and / or hybridoma CRL-1606 cells.   8. The apoptosis detection apparatus according to claim 7, wherein the detection means applies an ELISA method.   The apoptosis detecting apparatus according to claim 7, wherein the reaction unit includes a microchannel.   8. The apoptosis detection apparatus according to claim 7, wherein the detection means is applied with an ELISA method, and a primary antibody used for measurement by the ELISA method is covalently bound to a wall surface of a microchannel. .

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