A plating method to improve hERG-HEK293 cell stability during patch-clamp experiments
By using matrix gel coating and optimized cell suspension preparation methods, combined with the use of DMEM high-glucose medium and heparin and FBS, the stability problem of hERG-HEK293 cells in patch-clamp experiments was solved, achieving stable cell adhesion and long-term recording.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN HEYAN BIOMEDICAL TECH CO LTD
- Filing Date
- 2023-03-30
- Publication Date
- 2026-05-26
AI Technical Summary
In existing technologies, hERG-HEK293 cells exhibit poor stability in patch-clamp experiments, are easily washed away by perfusion, and have short recording times.
Cell slides were coated with matrix gel and cultured in combination with DMEM high-glucose medium, heparin, and FBS to optimize cell suspension preparation and culture conditions, including adjustments to incubation time and centrifugation parameters.
It improved cell stability and adhesion, extended recording time, increased the success rate of sealing and membrane rupture, and reduced the amount of cells used.
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Figure BDA0004153229720000091
Abstract
Description
Technical Field
[0001] This invention relates to the field of patch-clamp cell culture technology, and in particular to a plating method for improving the stability of hERG-HEK293 cells during patch-clamp experiments. Background Technology
[0002] Patch-clamp assays are considered the gold standard for studying cellular ion channels and can be used for screening ion channel-related drugs. This technique involves placing the tip of a microelectrode (typically <5 μm in diameter) onto the cell membrane surface using a micromanipulation device, then gently lifting the cell membrane with negative pressure to form a relatively closed region (i.e., sealing). Because the material is primarily non-conductive silicon dioxide, a resistance of up to 10 ohms is generated in the tip region. 9 The high impedance above Ω creates an electrical protective shield effect, making Na... + K + Ca 2+ Plasma cannot pass through other areas; it can only enter and exit through a localized membrane region inside the tube, thus enabling the recording of Na. + K + Ca 2+ The ultra-weak current generated when ions that play an important role in maintaining cell physiology flow through the cell membrane.
[0003] The invention patent with patent number CN111849915A discloses a culture method to improve the stability of hERG-HEK293 cells during patch clamp experiments. However, the cells do not adhere firmly to the wall and are easily washed away by perfusion, resulting in a short recording time. Summary of the Invention
[0004] In view of this, the present invention proposes a plating method to improve the stability of hERG-HEK293 cells during patch-clamp experiments. This method results in strong cell adhesion and longer recording time.
[0005] The technical solution of this invention is achieved as follows: This invention provides a plating method for improving the stability of hERG-HEK293 cells during patch-clamp experiments, comprising the following steps:
[0006] S1, Place the cell spreader in a culture dish, cover the cell spreader with matrix gel for 10-30 minutes, and then remove the coating solution.
[0007] S2, add hERG-HEK293 cell suspension to the cell slide prepared by matrix gel in step S1, place it in an incubator and let it stand for 5-20 minutes, then add culture medium to cover the glass slide for cell culture;
[0008] S3. Remove the cell slide from the culture dish and place it in a culture dish containing extracellular fluid. Then, remove the slide and place it in a chamber filled with extracellular fluid for patch-clamp detection.
[0009] Based on the above technical solutions, preferably, the culture medium in step S2 is DMEM high-glucose culture medium.
[0010] Based on the above technical solutions, preferably, the cell culture time in step S2 is 15-20 hours.
[0011] Based on the above technical solutions, the preferred method for preparing the hERG-HEK293 cell suspension in step S2 includes the following steps:
[0012] S21. Take a culture dish containing hERG-HEK293 cells, remove the culture medium and wash once with PBS, then add enzyme solution and incubate at 30-37℃ for 1-2 min. When the cells detach from the bottom of the culture dish, add culture medium to obtain a cell suspension.
[0013] S22, use a pipette to gently agitate the cell suspension to separate the aggregated cells, then transfer the cell suspension to a centrifuge tube and centrifuge to collect the cells;
[0014] S23, after centrifugation, the cells were resuspended in culture medium to obtain hERG-HEK293 cell suspension.
[0015] Based on the above technical solutions, preferably, the enzyme solution in step S21 is an EDTA pancreatic enzyme solution.
[0016] Based on the above technical solutions, preferably, the mass fraction of pancreatic enzyme in the EDTA pancreatic enzyme solution is 0.2%-0.3%.
[0017] Based on the above technical solutions, preferably, the culture medium described in steps S21 and S23 is a DMEM high-glucose culture medium containing 5%-10% FBS and 3-5 U / mL heparin.
[0018] Based on the above technical solutions, preferably, the cell density in the culture dish in step S21 is 5 × 10⁻⁶. 5 -7×10 5 pcs / cm 2 .
[0019] Based on the above technical solutions, preferably, the culture medium used in steps S2, S21 and S23 is a culture medium preheated at 35-37℃.
[0020] Based on the above technical solutions, preferably, the centrifugation conditions in step S22 are 800-1000 rpm for 5-8 minutes.
[0021] The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments, as described in this invention, has the following advantages over existing technologies:
[0022] (1) The present invention uses matrix gel to coat the slides, which increases cell stability, makes the cells adhere firmly to the wall, and is not easily washed away by perfusion, thus increasing the membrane breakage rate and extending the cell recording time.
[0023] (2) Heparin can protect cell membrane morphology and reduce cell damage; FBS can increase cell growth and help cells adhere to the wall; the combination of heparin and FBS improves cell stability and survival rate and increases the success rate of cell sealing and membrane rupture. Detailed Implementation
[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0025] The DMEM high-glucose medium and matrix gel used in this invention were both purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. The DMEM high-glucose medium was model 41401ES76, and the matrix gel was... Matrix LDEV-Free matrix adhesive, model number 40183ES08.
[0026] Example 1
[0027] A plating method for improving the stability of hERG-HEK293 cells during patch-clamp assays includes the following steps:
[0028] S1. Place four 9mm diameter cell spread sheets in a 6cm diameter culture dish, cover the cell spread sheets with 70μL of matrix gel and coat for 10min. After completion, remove the coating solution.
[0029] S2, Take a culture dish containing hERG-HEK293 cells, with a cell density of 5 × 10⁶ cells / mL. 5 pcs / cm 2 Remove the old culture medium and wash once with PBS buffer. Then add 1 mL of 0.2% EDTA trypsin solution and incubate at 30°C for 1 min. When the cells detach from the bottom of the culture dish, add 3 mL of DMEM high glucose medium containing 5% FBS and 3 U / mL heparin, which has been preheated at 35°C, to obtain the cell suspension.
[0030] S3. Use a pipette to gently pipette the cell suspension to separate the aggregated cells, then transfer the cell suspension to a 15 mL centrifuge tube, and then centrifuge at 800 rpm for 5 min to collect the cells.
[0031] S4, after centrifugation, the cells were resuspended in 2 mL of DMEM high glucose medium containing 5% FBS and 3 U / mL heparin preheated at 35°C to obtain hERG-HEK293 cell suspension;
[0032] S5, take 50 μL of hERG-HEK293 cell suspension and drop it onto the cell slide prepared by the matrix gel in step S1. Place it in an incubator and let it stand for 5 min. Then add DMEM high glucose medium preheated at 35℃ to cover the slide and incubate for 15 h.
[0033] S6. After the culture is completed, remove the cell slide from the culture dish and place it in a culture dish containing extracellular fluid. Then, remove the slide and place it in a chamber filled with extracellular fluid for patch-clamp detection.
[0034] Example 2
[0035] A plating method for improving the stability of hERG-HEK293 cells during patch-clamp assays includes the following steps:
[0036] S1. Place four 9mm diameter cell spread sheets in a 6cm diameter culture dish, cover the cell spread sheets with 70μL of matrix gel for 15min, and then remove the coating solution.
[0037] S2, Take a culture dish containing hERG-HEK293 cells, with a cell density of 6 × 10⁶ cells / mL. 5 pcs / cm 2 Remove the old culture medium and wash once with PBS buffer. Then add 1 mL of 0.25% EDTA trypsin solution and incubate at 35°C for 1.5 min. When the cells detach from the bottom of the culture dish, add 3 mL of DMEM high glucose medium containing 6% FBS and 4 U / mL heparin, which has been preheated at 37°C, to obtain the cell suspension.
[0038] S3. Use a pipette to gently pipette the cell suspension to separate the aggregated cells, then transfer the cell suspension to a 15mL centrifuge tube, and then centrifuge at 900rpm for 6min to collect the cells.
[0039] S4, after centrifugation, the cells were resuspended in 2 mL of DMEM high glucose medium containing 6% FBS and 4 U / mL heparin preheated at 37°C to obtain hERG-HEK293 cell suspension;
[0040] S5, take 50 μL of hERG-HEK293 cell suspension and drop it onto the cell slide prepared by the matrix gel in step S1. Place it in an incubator and let it stand for 8 min. Then add DMEM high glucose medium preheated at 37℃ to cover the slide and incubate for 18 h.
[0041] S6. After the culture is completed, remove the cell slide from the culture dish and place it in a culture dish containing extracellular fluid. Then, remove the slide and place it in a chamber filled with extracellular fluid for patch-clamp detection.
[0042] Example 3
[0043] A plating method for improving the stability of hERG-HEK293 cells during patch-clamp assays includes the following steps:
[0044] S1. Place four 9mm diameter cell spread sheets in a 6cm diameter culture dish, cover the cell spread sheets with 70μL of matrix gel for 20min, and then remove the coating solution.
[0045] S2, Take a culture dish containing hERG-HEK293 cells, with a cell density of 7 × 10⁻⁶ cells. 5 pcs / cm 2 Remove the old culture medium and wash once with PBS buffer. Then add 1 mL of 0.3% EDTA trypsin solution and incubate at 37°C for 2 min. When the cells detach from the bottom of the culture dish, add 3 mL of DMEM high glucose medium containing 10% FBS and 4 U / mL heparin, which has been preheated at 37°C, to obtain the cell suspension.
[0046] S3, use a pipette to gently pipette the cell suspension to separate the aggregated cells, then transfer the cell suspension to a 15mL centrifuge tube, and then centrifuge at 1000rpm for 7min to collect the cells;
[0047] S4, after centrifugation, the cells were resuspended in 2 mL of DMEM high glucose medium containing 8% FBS and 3 U / mL heparin preheated at 37°C to obtain hERG-HEK293 cell suspension;
[0048] S5, take 50 μL of hERG-HEK293 cell suspension and drop it onto the cell slide prepared by the matrix gel in step S1. Place it in an incubator and let it stand for 15 min. Then add DMEM high glucose medium preheated at 37℃ to cover the slide and incubate for 18 h.
[0049] S6. After the culture is completed, remove the cell slide from the culture dish and place it in a culture dish containing extracellular fluid. Then, remove the slide and place it in a chamber filled with extracellular fluid for patch-clamp detection.
[0050] Example 4
[0051] A plating method for improving the stability of hERG-HEK293 cells during patch-clamp assays includes the following steps:
[0052] S1. Place four 9mm diameter cell spread sheets in a 6cm diameter culture dish, cover the cell spread sheets with 70μL of matrix gel for 25min, and then remove the coating solution.
[0053] S2, Take a culture dish containing hERG-HEK293 cells, with a cell density of 5 × 10⁶ cells / mL. 5 pcs / cm 2 Remove the old culture medium and wash once with PBS buffer. Then add 1 mL of 0.2% EDTA trypsin solution and incubate at 30-37℃ for 2 min. When the cells detach from the bottom of the culture dish, add 3 mL of DMEM high glucose medium containing 10% FBS and 5 U / mL heparin, which has been preheated at 37℃, to obtain the cell suspension.
[0054] S3, use a pipette to gently pipette the cell suspension to separate the aggregated cells, then transfer the cell suspension to a 15mL centrifuge tube, and then centrifuge at 800-1000rpm for 6min to collect the cells;
[0055] S4, after centrifugation, the cells were resuspended in 2 mL of DMEM high-glucose medium containing 5% FBS and 5 U / mL heparin preheated at 37°C to obtain hERG-HEK293 cell suspension;
[0056] S5, take 50 μL of hERG-HEK293 cell suspension and drop it onto the cell slide prepared by matrix gel in step S1. Place it in an incubator and let it stand for 10 min. Then add DMEM high glucose medium preheated at 37℃ to cover the slide and incubate for 18 h.
[0057] S6. After the culture is completed, remove the cell slide from the culture dish and place it in a culture dish containing extracellular fluid. Then, remove the slide and place it in a chamber filled with extracellular fluid for patch-clamp detection.
[0058] Example 5
[0059] A plating method for improving the stability of hERG-HEK293 cells during patch-clamp assays includes the following steps:
[0060] S1. Place four 9mm diameter cell spread sheets in a 6cm diameter culture dish, cover the cell spread sheets with 70μL of matrix gel for 30min, and then remove the coating solution.
[0061] S2, Take a culture dish containing hERG-HEK293 cells, with a cell density of 7 × 10⁻⁶ cells. 5 pcs / cm 2 Remove the old culture medium and wash once with PBS buffer. Then add 1 mL of 0.25% EDTA trypsin solution and incubate at 37°C for 1 min. When the cells detach from the bottom of the culture dish, add 3 mL of DMEM high glucose medium containing 5% FBS and 4 U / mL heparin, which has been preheated at 37°C, to obtain the cell suspension.
[0062] S3, use a pipette to gently pipette the cell suspension to separate the aggregated cells, then transfer the cell suspension to a 15mL centrifuge tube, and then centrifuge at 1000rpm for 8min to collect the cells;
[0063] S4, after centrifugation, the cells were resuspended in 2 mL of DMEM high glucose medium containing 8% FBS and 5 U / mL heparin preheated at 37°C to obtain hERG-HEK293 cell suspension;
[0064] S5, take 50 μL of hERG-HEK293 cell suspension and drop it onto the cell slide prepared by the matrix gel in step S1. Place it in an incubator and let it stand for 20 min. Then add DMEM high glucose medium preheated at 37℃ to cover the slide and incubate for 20 h.
[0065] S6. After the culture is completed, remove the cell slide from the culture dish and place it in a culture dish containing extracellular fluid. Then, remove the slide and place it in a chamber filled with extracellular fluid for patch-clamp detection.
[0066] Comparative Example 1
[0067] The difference between Comparative Example 1 and Example 1 is that: in step S1, the cell spreader is not coated with matrix gel, and in step S5, the cell suspension is spread into the cell culture dish with the cell spreader, and the detection is started after 5 minutes.
[0068] Comparative Example 2
[0069] The difference between Comparative Example 2 and Example 1 is that the culture medium does not contain heparin.
[0070] Comparative Example 3
[0071] The difference between Comparative Example 3 and Example 1 is that the culture medium in steps S4 and S5 does not contain FBS.
[0072] Comparative Example 4
[0073] The difference between Comparative Example 4 and Example 1 is that the culture medium does not contain heparin and FBS.
[0074] Comparative Example 5
[0075] The difference between Comparative Example 5 and Example 1 is that in step S5, the hERG-HEK293 cell suspension was dropped onto the cell slide prepared with matrix gel, placed in an incubator and left to stand for 5 minutes, and then DMEM high-glucose medium preheated at 35°C was added to cover the slide and cultured for 10 hours.
[0076] The voltage stimulation protocol for whole-cell patch-clamp recording of hERG potassium currents is as follows: After whole-cell sealing, the cell membrane voltage is clamped at -80 mV. The clamping voltage is depolarized from -80 mV to -50 mV and maintained for 0.5 s (as a leakage current detection), then stepped to 30 mV and maintained for 2.5 s, and then rapidly restored to -50 mV and maintained for 4 s to excite the tail current of the hERG channel. Data is collected every 16 s to observe the effect of the drug on the hERG tail current. A 0.5 s stimulation at -50 mV is used as a leakage current detection.
[0077] Patch-clamp tests were performed on samples prepared according to the above voltage stimulation protocol for the examples and comparative examples, and the results are as follows:
[0078] Table 1 Test Results
[0079]
[0080] As shown in Table 1, the use of matrix gel coating resulted in good cell stability and a high success rate of sealing and membrane rupture; the cells adhered firmly to the cell wall and were not easily washed away by perfusion, allowing for longer recording time; and because the cells were not easily washed away by perfusion, the amount of cells used was reduced, saving on the amount of cell samples.
[0081] Comparative Examples 2-5 show that heparin, FBS, and the length of culture time all affect cell stability, sealing and membrane perforation rate, and recording time. This verifies that the combination of heparin and FBS can improve cell stability and viability, and increase the success rate of cell sealing and membrane perforation.
[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A plating method for improving the stability of hERG-HEK293 cells during patch-clamp experiments, characterized in that: Includes the following steps: S1, Place the cell spreader in a culture dish, cover the cell spreader with matrix gel for 10-30 minutes, and then remove the coating solution. S2, add hERG-HEK293 cell suspension to the cell slide prepared by matrix gel in step S1, place it in an incubator and let it stand for 5-20 minutes, then add culture medium to cover the glass slide for cell culture; The preparation method of the hERG-HEK293 cell suspension includes the following steps: S21. Take a culture dish containing hERG-HEK293 cells, remove the culture medium and wash once with PBS, then add enzyme solution and incubate at 30-37℃ for 1-2 min. When the cells detach from the bottom of the culture dish, add culture medium to obtain a cell suspension. S22, use a pipette to gently agitate the cell suspension to separate the aggregated cells, then transfer the cell suspension to a centrifuge tube and centrifuge to collect the cells; S23, after centrifugation, the cells were resuspended in culture medium to obtain hERG-HEK293 cell suspension; The culture medium described in steps S21 and S23 is a DMEM high-glucose medium containing 5%-10% FBS and 3-5 U / mL heparin. S3. Remove the cell slide from the culture dish and place it in a culture dish containing extracellular fluid. Then, remove the slide and place it in a chamber filled with extracellular fluid for patch-clamp detection.
2. The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments as described in claim 1, characterized in that: The culture medium described in step S2 is DMEM high-glucose medium.
3. The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments as described in claim 1, characterized in that: The cell culture time in step S2 is 15-20 hours.
4. The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments as described in claim 1, characterized in that: The enzyme solution mentioned in step S21 is an EDTA pancreatic enzyme solution.
5. The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments as described in claim 4, characterized in that: The mass fraction of pancreatic enzyme in the EDTA pancreatic enzyme solution is 0.2%-0.3%.
6. The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments as described in claim 1, characterized in that: In step S21, the cell density in the culture dish is 5 × 10⁻⁶. 5 -7×10 5 pcs / cm 2 .
7. The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments as described in claim 1, characterized in that: The culture medium used in steps S2, S21 and S23 is a preheated culture medium at 35-37℃.
8. The plating method for improving hERG-HEK293 cell stability during patch-clamp experiments as described in claim 1, characterized in that: The centrifugation conditions for step S22 are 800-1000 rpm for 5-8 min.