JP4173357B2 - Devices for tissue culture, protein extraction and protein concentration - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for performing on-line a series of operations of tissue culture, protein extraction, and protein concentration on a microchip.
In addition, the tissue culture in this invention is a tissue culture in a broad sense, and includes both tissue piece culture and cell culture.
[0002]
[Prior art]
Conventional tissue culture, medium exchange, and protein extraction operations always require centrifugation. For example, the cells contained in the culture dish and the old medium are transferred together to a centrifuge tube, centrifuged with a centrifuge, the cells are spun down, and the old medium in the supernatant is removed by decantation. Next, a new medium is added to the cells sinking to the bottom of the centrifuge tube, suspended, and transferred to a new dish or the like. Similarly, centrifugation is also required for protein extraction after culture. Therefore, the operation is complicated and time-consuming, and at the same time, it is actually impossible to process a plurality of samples simultaneously.
On the other hand, as a means for rapidly analyzing a minute amount of protein, microchip electrophoresis has recently attracted attention (see, for example, Non-Patent Document 1, Non-Patent Document 2, and Patent Document 1). Under the present circumstances as described above, development of a system for performing on-chip, on-line operations such as protein expression, extraction, and concentration is unavoidable.
[0003]
[Non-Patent Document 1]
Yao, S. et al. 1999, Proc. Natl. Acad. Sci. USA 96, 5372-537?
[Non-Patent Document 2]
Bousse, L. et al. 2001, Anal. Chem. 73, 1207-1212
[Patent Document 1]
Japanese Patent Application No. 2002-315027
[0004]
[Problems to be solved by the invention]
In the present invention, tissue culture, medium exchange, and protein extraction can be performed on-chip and online without complicated centrifugation, and further, in combination with this, protein concentration and separation can also be performed on-chip and online. The goal is to establish on-chip, on-line, high-throughput protein expression analysis systems.
[0005]
[Means for Solving the Problems]
The present invention has a function of a microchip having a plurality of wells composed of one or a plurality of wells, and a sieve capable of separating a cultured cell (or cultured tissue) and a medium, and each is loaded into the well. The present invention relates to a device for performing tissue culture, protein extraction, and protein concentration online, in combination with one or more membrane inserts that can be easily detachable.
[0006]
The present invention also relates to an on-chip, on-line tissue culture, protein extraction and protein concentration method characterized by using the above-mentioned device.
[0007]
Furthermore, the present invention relates to an on-chip, online tissue culture, protein extraction and protein concentration method characterized by comprising the following series of operations.
(1) having a sieve function capable of separating a cultured cell (or cultured tissue) and a medium in a first row of wells on a microchip provided with one or a plurality of wells; and One or more membrane inserts each having a size that can be loaded into the well and easily detachable are loaded, and a culture medium and cells (or tissues) are placed therein to perform culture.
(2) Next, the membrane insert is taken out and transferred to the second row of wells, and further cultured in a new medium (if necessary, the membrane insert is further transferred to the third row of wells, and the medium is changed again. Incubate).
(3) After culturing, the membrane insert is taken out, and this is a well for performing protein extraction operation from cells (or tissues) (well in the third row. However, if the medium is exchanged twice, the fourth row After adding the protein extraction reagent to dissolve the cells (or tissues) and extracting the protein, the membrane insert is removed leaving the extract.
(4) The protein extract obtained in (3) is transferred to a well filled with a protein-concentrating reagent (well in the fourth row; however, if the medium is changed twice, the well in the fifth row). Then, the concentrated protein is transferred to a well in which the concentrated protein is stored (well in the fifth row. However, if the medium is exchanged twice, the well in the sixth row).
[0008]
Furthermore, the present invention has a function of (1) one or more microchips having a plurality of rows of wells and (2) a sieve capable of separating cultured cells (or cultured tissues) and culture media. And one or more membrane inserts each of which can be loaded into the well and can be easily attached and detached, (3) medium, (4) protein extraction reagent, and (5) protein concentration reagent. And a kit comprising:
[0009]
DETAILED DESCRIPTION OF THE INVENTION
An example of a microchip according to the present invention is shown in FIG. 1 for a plurality of microchips provided with a plurality of rows of wells.
In FIG. 1, 1, 2, 3, 4, 5, 6,... Represent 1 column, 2 represents the second column, 3 represents the third column, and so on. In addition, 1a, 1b, 1c,..., 1j, 2a, 2b, 2c,..., 2j, 3a, 3b, 3c,. Reference numeral 10 denotes a microchip substrate.
In the case where there are a plurality of membrane inserts according to the present invention, it is desirable that they are connected to each other so that they can be simultaneously loaded into a plurality of wells and taken out at the same time. It becomes like 2. Here, 12 represents a connecting hand.
FIG. 3 shows a case where a plurality of membrane inserts connected according to the present invention are loaded into the first well of a microchip having a plurality of wells according to the present invention. It becomes like this.
[0010]
Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings, in which a plurality of microchips having a plurality of rows of wells and a plurality of membrane inserts are used.
[0011]
First, as shown in FIG. 2, a membrane insert 11 consisting of a membrane filter only at the bottom, each of which is connected by a connecting hand 12 so that a plurality of wells can be simultaneously loaded and taken out simultaneously, is shown in FIG. Are loaded into the wells in the first row on the substrate 10 of the microchip, and the medium and cells (or tissue pieces) are placed therein and cultured. It is essential that the membrane insert used here has a sieve function that can separate cultured cells (or cultured tissues) and culture media. The bottom part is made of a membrane filter (of course, the whole membrane insert may be made of a membrane filter). The pore size of the membrane filter is usually 10 μm or less, preferably 0.02 to 10 μm. In addition, the material of the membrane filter includes cellulose derivatives such as polycarbonate, polypropylene, cellulose acetate and cellulose nitrate, and derivatives of bacterial cellulose, polyester, tetrafluoroethylene, nylon 6, nylon 6,6, and Kevlar. ^TM (Polyamide, du Pont), Anopore membrane ^TM (Anopore ^TM , Whatman Scientific), Low Prodyne (Pole Corporation), Biodyne ^TM (Pole Corporation) etc. may be used.
In the case of a membrane insert having a membrane filter only at the bottom, examples of materials other than the bottom include polycarbonate, polypropylene (PP), polypropylene copolymer (PPCO), polystyrene (PS), polyethylene (PE), and high density. Polyethylene (HDPE), low density polyethylene (LDPE), crosslinked high density polyethylene (XLPE), polyester, poly (ethylene terephthalate) (PET), polyether, silicon, polyolefin, polysulfone (PSF), acetal (ACL), polyethylene terephthalate Polymer (PETG), polymethyl methacrylate (PMMA), polyvinylidene difluoride (PVDF), tetrafluoroethylene (TFE), polyamide, fluorinated ethylene propylene (FE) ), Therma Knox (TMX), perm Knox ^TM Although various materials such as (PMX), aluminum, quartz glass, borosilicate glass, soda glass and the like can be used, the same material as the membrane filter is usually used.
The material of the connecting hand 12 is not particularly limited, but normally, the same material as the membrane insert is used, and a plurality of membrane inserts are integrally formed with the connecting hands connected. It is common.
Different specimens (cells or tissues) may be placed in a plurality of wells each loaded with a membrane insert, or the same specimen (cells or tissues) may be placed therein.
In the former case, a plurality of specimens (cells or tissues) can be processed simultaneously. In the latter case, reproducibility can be seen.
[0012]
After culturing for a certain period of time, the membrane insert is taken out in a connected state, loaded into the second row of wells, the medium is changed, and the culture is further continued.
When moving the membrane insert, the membrane filter passes the medium but does not pass the cells (or tissues), so only the cells (or tissues) move to the wells in the second row together with the membrane insert, and the old medium is in one row. It remains in the eyes. A new medium may be placed in the well in the second row in advance, or may be added to the membrane insert after the membrane insert is moved.
When removing the membrane insert from the well after tissue culture, the pore size of the membrane filter is 2 μm or less, or the cell density is 2 × 10. ⁶ When cell / well or more or the well size is 3 mm or less, the medium may not pass through the membrane filter due to clogging of the surface tension or the pore size of the membrane filter. In that case, pressure filtration may be appropriately performed using, for example, a syringe, a pipette, a pump, or a multi-channel pipette capable of simultaneously pressurizing a plurality of pipes as a pressurizer.
In the case of syringe pressurization, pipette pressurization, multichannel pipette pressurization, etc., it is naturally desirable that these pressurizers themselves can be loaded into the membrane insert. In the case of pump pressurization, it is desirable that the tip of the tube can be loaded into the membrane insert.
For reference, examples of pressurization using a syringe and pressurization using a multichannel pipette are shown in FIG. 4A shows an example of pressurization with a syringe, and FIG. 4B shows an example of pressurization with a multichannel pipette.
[0013]
After culturing, as in the first culturing, when the membrane insert is connected, it is removed from the second row of wells and moved to the third row of wells. Only move to the third row of wells with the membrane insert. If necessary, the medium may be exchanged again and the culture may be continued. Here, the case where the culture cells (or cultured tissues) are washed in the third row of wells with one medium exchange will be described. . Washing of cultured cells (or tissue) is usually free of FBS (fetal bovine serum), 10-20 mM HEPES-containing PPMI-1640 medium, FBS-free, HEPES-containing HamF-12 medium, FBS-free HEPES-containing minimum essential medium Eagle (MEM), FBS-free, HEPES-containing basal medium Eagle (BME), FBS-free, HEPES-containing 199 medium, FBS-free, HEPES-containing Iscove's, etc. , Using a buffer solution such as a phosphate buffer, a HEPES buffer, a tricine buffer, a Tris buffer, a borate buffer, or a physiological saline.
After washing, remove the membrane insert from the third row of wells in a connected state, and move to the fourth row of wells. The washing solution will remain in the third row, and only the cells (or tissues) will become the membrane insert. Move together to the well in the fourth row. A protein extraction reagent is put in this, a cell (or tissue) is dissolved, and protein is extracted. After extraction, if the membrane insert is connected and removed from the fourth row of wells, the residue of cells (or tissues) insoluble in the extract is removed together with the membrane insert, and only the protein extract is in four rows. It remains in the eyes. In addition, although washing | cleaning operation of a cultured cell (or cultured tissue) is not essential, it is desirable to perform it.
Even when the membrane insert is taken out from the well after the culture medium is exchanged or washed, the culture medium or the washing solution may not pass through the membrane filter in the same manner as in the removal after the culture. In that case, pressure filtration may be performed using a pressurizer in the same manner as the removal after the culture. The pressurizer used in this case may be exactly the same as that used for removal after the culture.
[0014]
The process then proceeds to protein concentration.
The wells in the fifth row are filled with a protein concentration reagent in advance.
The 4th to 5th rows and the 5th to 6th rows are connected by the microchannel 8, the sample in the 4th row is transferred to the 5th row by pressurization, and the 5th row is filled. The protein is concentrated by transferring it to the sixth sample reservoir (well in which the concentrated protein is stored) through the agent.
The pressure during pressurization is usually 10 to 100 kPa / mm ³ , Preferably 30-80 kPa / mm ³ , More preferably 50-70 kPa / mm ³ The pressing time is usually 0.1 to 5 seconds, preferably 0.5 to 2 seconds, and more preferably about 1 second.
The pressurizer used at this time may be the same as the pressurizer used if needed when taking out the membrane insert after the culture, or after taking out the membrane insert after medium replacement or washing.
In this case, however, the pressurizer is used by loading it in one or more wells.
[0015]
Further, although not shown in FIG. 1, if a microchannel for microchip electrophoresis for subjecting the concentrated protein to microchip electrophoresis is incorporated on the same microchip in advance (for electrophoresis) After that, the concentrated protein can be transferred to the microchannel of the microchip electrophoresis prepared on the same microchip, and then the separation can be continued (note that (For example, see Non-Patent Document 1 and Non-Patent Document 2 for the ordinary microchannel of microchip electrophoresis).
[0016]
The separation operation is performed by feeding the concentrated protein to another microchip electrophoresis microchannel prepared separately (connected to another microchip electrophoresis microchannel prepared separately). May be.
In this case, that is, when the concentrated protein is subjected to electrophoresis on another microchip for microchip electrophoresis, a flow path (microchannel) is formed from the well in which the concentrated protein is stored to the outside of the microchip. A channel provided with a channel may be used. The transfer of the concentrated protein from the well in which these are stored to the sample reservoir on the microchip for microchip electrophoresis is usually performed by pressurization. The pressurizer used at this time is exactly the same as described above.
Of course, when the separation operation of the protein on the microchip is not considered, it is not necessary to provide a flow path (microchannel) toward the outside of the microchip in the well in which the concentrated protein is stored.
[0017]
As microchannels for microchip electrophoresis, in addition to those used in ordinary electrophoresis, those for two-dimensional electrophoresis and those for nanochannel electrophoresis are naturally The invention can be applied to the device of the invention, or can be used in combination with the device of the invention. For example, the microchannel of the special two-dimensional electrophoresis method (Patent Document 1) previously filed by the present inventors is also applicable. Or can be used.
In the case of using a plurality of membrane inserts connected to a microchip having a plurality of wells according to the present invention and a plurality of membrane inserts in this way, in a multi-channel manner, from protein expression to extraction and concentration. A series of operations up to the separation operation can be performed by combining this with microchip electrophoresis.
[0018]
Examples of the material of the microchip used in the present invention include the same materials as used in microchip electrophoresis. More specifically, for example, quartz glass, borosilicate glass, soda glass, polymethyl methacrylate, Examples include polycarbonate and dimethylsiloxane. Among them, preferred are glass or polymethylmethacrylate, which has less sample adsorption and can be easily chipped.
As the size of the microchip, for example, those having a length of 10 to 120 mm, a width of 10 to 120 mm, and a thickness of 600 to 5000 μm are usually used as in the case of the microchip electrophoresis. .
The size of the well provided in the microchip is usually about 1.5 to 10 mm in diameter and about 500 μm to 12 mm in depth.
When it is desired to use a well whose depth is deeper than the thickness of the microchip, for example, a plate having an appropriate thickness made of the same material as that of the substrate is attached to the portion where the well is to be provided on the microchip substrate. Then, a well hole is cut out in this portion, and then a film is attached to the bottom surface of the microchip substrate to close the hole.
[0019]
Examples of cells used in the present invention include T cell lymphoma Jurkat cells, promyelocytic leukemia HL-60 cells, cervical cancer HeLa cells, inertial myeloid leukemia K-562 cells, various bacterial cells, various yeast cells and the like. All of the cells used in normal cell culture can be mentioned.
Examples of the tissue piece used in the present invention include tissue pieces of organs such as lung, heart, brain, uterus and the like, which are normal, cancer-derived or lesion-derived.
Examples of the medium used in the present invention include RPMI-1640 + FBS (10%), basal medium eagle (BME) + FBS (10%), minimum essential medium eagle (MEM) + FBS (10%), HamF-12 medium + FBS. (10%) 199 medium + FBS (10%), Iscove's medium + FBS (10%), etc. In normal tissue culture, all the mediums that are used as suitable for each cell or tissue piece combination Can be mentioned.
[0020]
As a protein extraction reagent for lysing cells and extracting proteins, for example, for mammalian cells, CelLytic ^TM M (Sigma), M-PER ^TM (Pierce) CelLytic for bacterial cells ^TM B, CelLytic ^TM BII (Sigma), for yeast cells, CelLytic ^TM Y (Sigma) etc. are mentioned.
In the case of tissue piece culture instead of cell culture, for example, CelLytic ^TM MT (Sigma) and T-PER ^TM (Pierce) and the like.
Ionic surfactants 0.5 to 1.0% Triton X-100 and Nonidet P-40 (Cosmo Bio) are usually used as protein extraction reagents, but these are homogenized using a pencil mixer or the like. Doing so increases the effect.
Examples of the protein concentrating reagent include gel filtration carriers such as Sephadex, biogel, and agarose gel, and, for example, C having a particle size of 5 to 105 μm. ₄ Or C ₁₈ Examples of the product name include Honda Pack and Honda Pack HC. ₁₈ , Prep C ₁₈ (Waters) and ZipTip (Millipore).
Gel Absorbant (trade name: Spectra Gel Absorbant (SPECTRUM)), dialysis tube, cotton cellulose dialysis membrane, regenerated cellulose dialysis membrane, cellulose ester dialysis membrane, etc. can also be used, and the trade name is Cellu-Sep T. Examples include Tubular Membrane (Membrane Filtration Products), Spectra Biotech Membrane (SPECTRUM), Spectra / Pore CE Dialysis Tube (SPECTRUM).
[0021]
【Example】
EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.
[0022]
Example 1
(1) A microchip (material: polymethyl methacrylate) as shown in FIG. 1 having a well of 10 mm × 6 rows, having a diameter of 3 mm and a depth of 5000 μm, 80 mm × 75 mm and a thickness of 5000 μm, and a pore diameter of 8 μm A 10-piece membrane insert as shown in FIG. 2 having a polycarbonate membrane filter at the bottom (other parts are also polycarbonate).
(2) As cells to be cultured, Jurkat cells (Jalkat cells) (New Nippon Pharmaceutical) are 2 × 10 ⁶ Each cell / well was used, and these were cultured in the membrane insert in the first row of wells loaded with the connected membrane insert.
(3) RPMI-1640 medium (Sigma) -10% fetal calf serum (Equitech-Bio, Inc, Ingram, TX, USA) was used as the medium.
(4) As described above, the medium was exchanged by taking out the membrane insert from the well in the first row while being connected to the membrane insert, and loading the well in the second row.
(5) Washing | cleaning of the cultured cell was performed by the method mentioned above using a PPMI-1640 culture medium (Sigma) -10mM HEPES.
(5) Protein extraction uses CelLytic as an extraction reagent ^TM -M (Sigma) was used for the method described above.
(6) Protein Concentration uses Honda Pack Filler C as a protein concentration reagent. ₁₈ (Waters) 20 mg was used by the method described above.
The transfer to the sample reservoir (the well in which the concentrated protein is stored) through the well filled with the protein concentration reagent of the protein extract (that is, the protein concentration operation of the protein extract) is performed by syringe pressurization. It went by.
(7) Separation is performed by a two-dimensional electrophoresis method described in Patent Document 1 using a Hitachi microchip (i-chip 12) and a Hitachi microchip electrophoresis apparatus (cosmo-i SV1200, manufactured by Hitachi Electronics Engineering). It was.
(Agilent protein chip kit) was used as the fluorescent reagent and buffer.
The concentrated protein was transferred to the sample reservoir of the electrophoresis microchip by syringe pressurization.
(8) The obtained electropherogram is shown in FIG.
In FIG. 5, channel numbers 1 to 5 are control samples, and Jurkat cells are cultured at 37 ° C. (1 to 5 are the same sample). Channel numbers 6 to 10 are samples subjected to heat shock at 51 ° C. for 30 minutes (6 to 10 are the same sample).
The signal marked with a triangle in the figure is a marker protein, and the lower is a peak of 7 kDa, and the higher is a peak of 18 kDa. An increase in peak intensity of 5-10 giving a heat shock is observed.
As is clear from FIG. 5, 1 to 5 and 6 to 10 show almost the same results, respectively, and it can be seen that the method of the present invention has very good reproducibility.
In this example, medium replacement, cell washing, recovery, protein extraction and protein concentration were performed by the online system of the present invention, and further separation was performed by microchip electrophoresis according to the method described in Patent Document 1. The time required for separation was only 10 minutes including all steps.
Thus, a significant reduction in analysis time has been achieved with the present invention.
[0023]
Comparative Example 1
The Jurkat cells cultured at 37 ° C. were collected by conventional cell recovery methods (medium exchange and cell washing) and protein extraction methods, and analyzed by conventional two-dimensional gel electrophoresis. The analysis results are shown in FIG.
The conventional cell recovery method and protein extraction method are as follows. Jurkat cells 2 × 10 ⁶ cells were cultured in a 100 mm culture dish, medium A: RPMI-1640 containing 2 mM glutamine (Sigma), 0.005% gentamicin (Sigma), 10% fetal calf serum (Equitech-Bio, Inc, Ingram, TX, USA) was used for culture. Cells after growth 2 × 10 ⁶ 10 mL of the culture solution containing cells was collected with a pipette into a 15 mL centrifuge tube, and centrifuged at 170 × g for 5 minutes. The supernatant after centrifugation was removed by decantation, and another new medium, medium B: medium composition RPMI-1640 (manufactured by Nissui Corp.), 0.005% gentamicin, 2 mM glutamine, 10 mM HEPES (Sigma) 10 mL of the suspension was added, and the cells were suspended with a pipette, washed, and centrifuged again at 170 × g for 5 minutes to pellet the cells. 10 mL of medium B was added to the cell pellet, the cells were suspended with a pipette, transferred to a 100 mm culture dish, and cultured at 37 ° C. for 30 minutes. Cells containing the culture broth after culturing were collected with a pipette into a 15 mL centrifuge tube, centrifuged again at 170 × g for 5 minutes, the cells were pelleted, and the supernatant was removed by decantation. To the pellet, 10 mL of 10 mM phosphate buffer (Sigma) was added as a washing buffer, suspended with a pipette, centrifuged again at 170 × g for 5 minutes, the supernatant was removed, and the pellet was recovered. Further, 2 mL of 1% Nordide P-40 (manufactured by Cosmo Bio) and 1 mM EDTA (pH 7.4) were added to the pellet to homogenize the cells. The cell lysate was centrifuged at 20000 × g for 10 minutes, and the supernatant was used as a protein mixture sample as a sample for two-dimensional gel electrophoresis.
[0024]
The conventional two-dimensional gel electrophoresis is as follows. Two-dimensional gel electrophoresis was performed by a modified method of O ′ Farrell (O ′ Farrell, PH, J. Biol. Chem. 1975, 250, 4007-4021). Polyacrylamide capillary gel (4% T, 5.4% C, 2% Amphrine pH 3.5-10; manufactured by Amersham Pharmacia Biotech) 62 mm long, 1.2 mm diameter, isoelectric focusing (hereinafter referred to as 1-dimensional electrophoresis) , IEF). Load a sample containing 50 micrograms of protein onto an IEF gel and gradually increase the voltage at 16 ° C (15 minutes at 200V, 15 minutes at 400V, 30 minutes at 800V, 288 minutes at 2500V) Electrophoresis and SDS polyacrylamide gel electrophoresis were carried out at 85 mA (W) × 60 mm (H) × 1 mm (T) slab gel (15% T, 2.67% C) at 15 mA per plate. After completion of the second-dimensional electrophoresis, staining was performed using 0.5% CBB (Coomassie Brilliant Blue R-250; manufactured by Nippon Biorat Laboratories). The pH gradient in IEF was determined by measuring the pH extracted from each 5 mm of the IEF gel with a micro pH meter (pH Boy P-2; manufactured by Shin dengen).
[0025]
Spot I, spot IIa, and spot IIb in FIG. 6 are marker proteins. Each spot is an amino acid sequencer and shaving spot I is thymosin beta 4 (7 kDa), stathmin (Mw 18.0 kDa, pI 5.9), phosphorylated stathmine (Mw 18.5 kDa, pI 5.6).
Identification was performed as follows. After the separation was completed, the two-dimensional gel electrophoresis was transferred to polyvinylidene fluoride (Immobilon P; manufactured by Milipore) membrane using a semi-dry plotter (KS-8460; manufactured by Marysol) at 10 mM CAPS buffer pH 11, 150 mA for 2 hours. . The peptide on the membrane was subjected to a gas phase protein sequencer (PPSQ-21; manufactured by Shimadzu Corporation). The homology search of peptide amino acid sequencers was carried out using the Internet homology search program MP search (http://www.dna.affrc.go.jp/htdocs/homology/homology.html).
[0026]
Comparative Example 2
Jurkat cells 2 × 10 ⁶ After the cells were subjected to heat shock at 51 ° C. for 30 minutes on a microchip, the protein was recovered by the conventional cell recovery method (medium replacement and cell washing) described in Comparative Example 1 and the protein extraction method. Measurement analysis was performed by the conventional two-dimensional gel electrophoresis method described in 1. The analysis results are shown in FIG. Increases in the expression levels of the proteins in spot I and spot IIb were observed due to heat shock.
The time required for analysis according to the conventional method performed in Comparative Example 1 and Comparative Example 2 was 1 hour in the operations from cell culture medium exchange, cell washing, recovery, and protein extraction, and the total time including 2D electrophoresis and analysis. The time required was 24 hours.
[0027]
Comparative Example 3
The sample of Comparative Example 1 is recovered by the conventional cell recovery method and protein extraction method described in Comparative Example 1, and separated by an existing protein analysis method using an microchip [Agilent 2100 bioanalyzer (manufactured by Agilent Technology)]. And measurement analysis. A microchip kit (protein 200 Plus; manufactured by Caliper Technology) was used as the electrophoresis buffer and the microchip. The electrophoresis conditions were in accordance with the manual attached to the Agilent 2100 bioanalyzer. The measurement analysis results are shown in FIG. Since each peak of 7 kDa and 18 kDa overlaps with a molecular weight marker or system peak, Spot I, Spot IIa and Spot IIb could not be measured by this Agilent system.
[0028]
Comparative Example 4
FIG. 9 shows the results of measurement and analysis of the sample of Comparative Example 2 collected by the conventional tissue culture method and protein extraction method described in Comparative Example 1 and measured and analyzed by the conventional microchip electrophoresis method described in Comparative Example 3. Similar to the experimental results of Comparative Example 3, since the respective peaks of 7 kDa and 18 kDa overlap with the molecular weight marker or system peak, Spot I, Spot IIa and Spot IIb could not be measured by this Agilent system.
[0029]
Comparative Example 5
The sample of Comparative Example 1 was recovered by the conventional cell recovery method and protein extraction method described in Comparative Example 1, and separated and analyzed by Hitachi Microchip (i-chip 12) and Hitachi Microchip Electrophoresis Device (cosmo). -i SV1200 (manufactured by Hitachi Electronics Engineering Co., Ltd.) was performed by the two-dimensional electrophoresis method described in Patent Document 1. The measurement analysis results are shown in FIG. In the figure, I (7 kDa), IIa and IIb (18 kDa) are marker proteins.
[0030]
Comparative Example 6
The sample of Comparative Example 2 was recovered by the conventional cell recovery method and protein extraction method described in Comparative Example 1, and separation and measurement analysis were performed using Hitachi Microchip (i-chip 12) and Hitachi Microchip Electrophoresis Device (cosmo). -i SV1200 (manufactured by Hitachi Electronics Engineering Co., Ltd.) was performed by the two-dimensional electrophoresis method described in Patent Document 1. The measurement analysis results are shown in FIG. In the figure, I (7 kDa), IIa and IIb (18 kDa) are marker proteins. Marker protein was detected by heat shock.
[0031]
In Comparative Examples 5 and 6 in which proteins were recovered by the conventional cell recovery method and protein extraction method, and separation and measurement analysis were performed in the same manner as in Example 1, when compared with FIG. It can be seen that almost equivalent results are obtained. From this, it can be seen that the medium exchange on the microchip, cell washing, and protein extraction operation according to the present invention are not significantly different from the existing cell recovery method and protein extraction method and the obtained results themselves. . Therefore, the method according to the present invention can be replaced with an existing method, and by replacing the existing method with the method according to the present invention, the operability can be simplified and the cell processing time can be shortened. I understand. Incidentally, the time required for the cell washing, medium exchange, and protein extraction steps by the conventional method is 1 hour, whereas in the method according to the present invention, 10 minutes or less (10 minutes including the separation step) as described above. Met.
[0032]
【The invention's effect】
According to the present invention, medium replacement using a centrifuge becomes unnecessary in the field of tissue culture, and the culture operation can be simplified. Further, concentration can be performed on the microchip by incorporating a concentration reagent on the microchip. Furthermore, by incorporating a microchannel for microchip electrophoresis into this, or by connecting it to a microchannel of another microchip electrophoresis prepared separately, extraction and concentration from a series of protein expression The separation can be performed on the microchip. Furthermore, by simultaneously producing multiple channels on a microchip, protein expression analysis can be performed simultaneously on multiple samples, and high throughput of proteome analysis can be achieved.
According to the present invention, not only protein expression analysis and function analysis can be easily and rapidly performed in this way, but also for medical diagnosis, laborious centrifugation operation can be omitted, and analysis can be brought online. In addition, since the cell (or tissue) and protein do not touch many instruments and the adsorption of the protein can be minimized, the effect is extremely great.
[Brief description of the drawings]
FIG. 1 is an example of a microchip according to the present invention.
[Fig. 2] Fig. 2 is an example of a membrane insert according to the present invention in which a plurality are connected and only a bottom part is constituted by a membrane filter.
FIG. 3 is a cross-sectional view of a microchip according to the present invention in which a plurality of connected micro inserts having a membrane filter formed only at the bottom are provided with a plurality of wells in a plurality of rows according to the present invention; It is the figure which looked at the case where it loads to the well of the 1st row from right above.
FIG. 4 is an example of a pressurization method used in the present invention, in which (a) is an example of pressurization using a syringe, and (b) is an example of pressurization using a multichannel pipette.
5 is an electropherogram obtained in Example 1. FIG.
FIG. 6 shows the results obtained by measuring the sample obtained by the conventional cell recovery method and protein extraction method obtained in Comparative Example 1 by the conventional two-dimensional electrophoresis method.
FIG. 7 shows the results obtained by measuring the sample obtained by the conventional cell recovery method and protein extraction method obtained in Comparative Example 2 by the conventional two-dimensional electrophoresis method.
FIG. 8 shows the results obtained by measuring a sample obtained by the conventional cell recovery method and protein extraction method obtained in Comparative Example 3 by a microchip electrophoresis method using an Agilent system.
FIG. 9 shows the results obtained by measuring the sample obtained by the conventional cell recovery method and protein extraction method obtained in Comparative Example 4 by microchip electrophoresis using an Agilent system.
FIG. 10 shows the results obtained by measuring the samples obtained by the conventional cell recovery method and protein extraction method obtained in Comparative Example 5 by the same microchip electrophoresis method as in Example 1.
FIG. 11 shows the results obtained by measuring the samples obtained by the conventional cell recovery method and protein extraction method obtained in Comparative Example 6 by the same microchip electrophoresis method as in Example 1.
[Explanation of symbols]
1a, 1b, ... 2a, 2b, ... 6j well
8 channel (micro channel)
10 Microchip substrate
11 Membrane inserts with multiple connections
12 Connecting hands

Claims (30)

One or a plurality of microchips provided with a plurality of rows of wells and a function of a sieve capable of separating cultured cells (or cultured tissues) and a culture medium, and each of which can be loaded into the wells A device for performing tissue culture, protein extraction, and protein concentration on-line, in combination with one or more membrane inserts that can be easily attached and detached. The device according to claim 1, comprising a plurality of microchips provided with a plurality of rows of wells and a plurality of membrane inserts. 3. The device of claim 2, wherein a plurality of membrane inserts are connected to each other so that they can be loaded and removed simultaneously from a plurality of wells. A microchip having at least two rows of wells for tissue culture and medium exchange, a well for performing protein extraction from cells, and at least two rows of wells for performing protein concentration The device according to claim 1, wherein the device is a chip. The device according to claim 4, wherein the microchip further comprises a well for washing cultured cells (or cultured tissues) after tissue culture. The device according to claim 4 or 5, wherein one of the wells for performing the protein concentration operation is a well filled with a concentration reagent, and the other is a well in which the concentrated protein is stored. The device according to claim 6, wherein a protein concentration reagent for performing a protein concentration operation is incorporated in the well. In order to transfer the extracted solution after the protein extraction operation through the well filled with the protein concentration reagent for performing the protein concentration operation to the well in which the concentrated protein is stored, a flow path is formed between the wells. The device according to claim 6 or 7 connected by (microchannel). Further, in order to subject the concentrated protein to electrophoresis on a separately prepared microchip for microchip electrophoresis, a flow path (microchannel) extends from the well in which the concentrated protein is stored to the outside of the microchip. ) Is provided. The device according to claim 8. The device according to claim 8, further comprising a microchannel for microchip electrophoresis incorporated in the microchip in order to subject the concentrated protein to microchip electrophoresis on the same microchip. The device according to claim 9 or 10, further comprising a pressurizer that can be loaded into one or more membrane inserts and / or one or more wells to pressurize them. The device according to any one of claims 1 to 11, wherein the membrane insert comprises a membrane filter at least at the bottom thereof. The device according to claim 12, wherein the membrane filter has a pore diameter of 10 μm or less. The device according to claim 1, wherein the microchip has a length of 10 to 120 mm, a width of 10 to 120 mm, and a thickness of 600 to 5000 μm. The device according to claim 1, wherein the well provided on the microchip has a diameter of 1.5 to 10 mm and a depth of 500 μm to 12 mm. An on-chip, online tissue culture, protein extraction, and protein concentration method using the device according to claim 1. An on-chip, on-line tissue culture, protein extraction and protein concentration method comprising the following series of operations.
(1) having a sieve function capable of separating a cultured cell (or cultured tissue) and a medium in a first row of wells on a microchip provided with one or a plurality of wells; and One or more membrane inserts each having a size that can be loaded into the well and easily detachable are loaded, and a culture medium and cells (or tissues) are placed therein to perform culture.
(2) Next, the membrane insert is taken out and transferred to the second row of wells, and further cultured in a new medium (if necessary, the membrane insert is further transferred to the third row of wells, and the medium is changed again. Incubate).
(3) After culturing, the membrane insert is taken out, and this is a well for performing protein extraction operation from cells (or tissues) (well in the third row. However, if the medium is exchanged twice, the fourth row After adding the protein extraction reagent to dissolve the cells (or tissues) and extracting the protein, the membrane insert is removed leaving the extract.
(4) Transfer the protein extract obtained in (3) to a well filled with a protein-concentrating reagent (well in the fourth row; however, if the medium has been changed twice, the well in the fifth row) The protein is concentrated, and the concentrated protein is transferred to a well in which the concentrated protein is stored (well in the fifth row. However, if the medium is changed twice, the well in the sixth row). 18. When removing a membrane insert from a well after tissue culture or / and after exchanging the medium, if necessary, the membrane insert is loaded with a pressurizer, filtered after pressurizing the medium, and then removed from the well. the method of. Wells containing the protein extract (3rd row wells; if the medium was changed twice, 4th row wells) and wells filled with protein-concentrating reagent (4th row wells; medium) The well in the fifth row when the exchange is performed twice and the well in which the concentrated protein is stored (the well in the fifth row. However, the well in the sixth row when the medium is exchanged twice) Using a microchip with a channel (microchannel) connected between them, the protein extract is passed through a well filled with a protein-concentrating reagent by pressurization and transferred to a well containing the concentrated protein The method according to claim 17 or 18. After tissue culture, the membrane insert is transferred to a well for washing cultured cells (or cultured tissue) (well in the third row. However, if the medium is changed twice, the well in the fourth row) After washing the cells (or cultured tissue), the membrane insert is taken out from the well, and this is a well for performing the protein extraction operation (well in the fourth row. However, if the medium is changed twice, the fifth row To the well of the 5th row and 6th row (however, if the medium was changed twice, the 6th and 7th rows were added). Concentration operation is performed using the wells in the row [in this case, however, between the wells in the fourth row and the fifth row and between the wells in the fifth row and the sixth row (medium exchange) Is performed twice, the fifth row of wells and the sixth row of wells Using the microchip during and between the sixth column well and seventh column of wells) is connected with the channel (microchannels). The method of claim 19. 21. When removing the membrane insert from the well after washing the cultured cells (or cultured tissue), if necessary, the membrane insert is loaded with a pressurizer, the washing solution is filtered by pressurization, and then removed from the well. The method described. The method according to any one of claims 17 to 21, wherein a plurality of microchips provided with a plurality of rows of wells and a plurality of membrane inserts are used. 23. The method of claim 22, wherein the plurality of membrane inserts are membrane inserts that are connected to each other so that they can be loaded and removed simultaneously from a plurality of wells. The method according to any one of claims 17 to 23, wherein the membrane insert comprises a membrane filter at least at the bottom thereof. The method according to any one of claims 17 to 24, wherein the microchip to be used has a length of 10 to 120 mm, a width of 10 to 120 mm, and a thickness of 600 to 5000 µm. The method according to any one of claims 17 to 25, wherein the well provided on the microchip to be used has a diameter of 1.5 to 10 mm and a depth of 500 to 12 mm. (1) One to a plurality of microchips provided with a plurality of rows of wells, and (2) a sieve function capable of separating a cultured cell (or cultured tissue) and a culture medium, and each of the wells A kit comprising one or a plurality of membrane inserts that can be easily loaded and removed, (3) a medium, (4) a protein extraction reagent, and (5) a protein concentration reagent. 28. The kit according to claim 27, further comprising a washing solution for cultured cells (or cultured tissue). 29. The kit according to claim 28, further comprising a pressurizer that can be loaded into one or more membrane inserts and / or one or more wells to pressurize them. 30. The kit of claim 29, wherein the pressurizer is a syringe, pipette, pump, or multichannel pipette.

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