Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

Summary

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

Abstract

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

Introduction

Owing to its efficient and less labor-intensive experimental process, label-free cell-based technology has witnessed rapid growth over the past decade for analytical as well as screening purposes such as in the aspect of proteomics^1,2, drug delivery³, etc.^4,5 Compared with traditional biochemical methods aimed at cell analysis, label-free real-time cell assay with the prototype developed by Giaever and coworkers previously⁶ is based on the principle of recording electric signal changes on the surface of cell-attached microchips, which permit a continuous measurement of cell growth or migration in a quantitative manner. Following this strategy, a real-time cell electronic sensing (RT-CES) system using electric impedance-based detection principle was introduced^7,8 and more recently a commercial real-time cell analyzer (RTCA) was launched for laboratory research⁹.

The commercial real-time cell analyzer mainly reads out the cells' evoked signals of electric impedance, which result from the physiological changes of incubated cells including cell proliferation, migration, viability, morphology, and adherence on the surface of microchips^10,11. Such electric signals are further converted by the analyzer into a dimensionless parameter named the Cell Index (CI) to assess the cell status. The change of the impedance of microchips mainly reflects the local ionic environment of covered cells at the electrode/solution interface. Therefore, the analytical performance of the cell analyzer relies heavily on the core sensing unit, the disposable microchips (i.e., so-called electronic plates, e.g., 96/16/8-well), which are made of arrayed gold microelectrodes lithographically printed at the bottom of incubation wells. The gold microelectrodes assemble in a circle-on-line format (Figure 1) and cover most of the surface area of the incubation wells, which allow for dynamic and sensitive detection of attached cells^3,12,13,14. The CI will increase in the case of more surface coverage of cells on the chip, and decrease when cells are exposed to a toxicant resulting in apoptosis. Although the real-time cell analyzer has been frequently used to determine cytotoxicity¹¹ and neurotoxicity¹⁵ and provide more kinetic information than classical endpoints method, the disposable electronic plates are the costliest consumable.

Until now, there have been no available methods for the regeneration of the electronic plate, which is probably due to the fact that harsh regeneration conditions, such as piranha solution or acetic acid are involved^16,17,18, which may alter the electric status of gold microchips. Therefore, a mild and efficient method to remove the adherent cells and other substances from the surface of gold chips will be desirable for the electronic plate regeneration process. We have recently developed a protocol aimed at the regeneration of disposable electronic plates using non-corrosive reagents and the regenerated chips were characterized by electrochemical as well as optical methods¹⁹. By using readily available and moderate laboratory reagents including trypsin and ethanol, we have established a general method to regenerate the commercial electronic plate without adverse effects, which is successfully applied to regenerate the two main types of electronic plate (both 16 and L8) used for RTCA.

Protocol

NOTE: In general, the regeneration process includes trypsin digestion and rinsing step with ethanol and water. The digestion time changes according to the number of cells used, and the type and number of cells used may differ depending on the experimental purposes. It is advised to check the regenerated microchips using optical and electrochemical methods to optimize the regeneration conditions. During the experiment, soluble and insoluble chemicals may be involved, and here these two typical cases of the regeneration procedures are detailed.

1. Preparation of Regeneration Solutions

NOTE: Prepare and handle all the solutions under sterile conditions.

1. Prepare fresh 0.25% (g/v) trypsin solution. Trypsin can be purchased or freshly prepared as follows.
   1. Dissolve 0.25 g of trypsin in 100 mL of pH 7.4 phosphate-buffered saline (PBS).
   2. Stir the solution until the trypsin is fully dissolved at 4 ^oC. Stir the solution at low speed taking care to avoid any bubble formation.
   3. Filter the solution with a 0.22 µm filter in the biosafety hood.
2. Prepare 75% ethanol using absolute ethanol. Pour 75 mL absolute ethanol into a volumetric flask and add deionized water until the volume reaches 100 mL. Perform further sterilization if necessary.

2. Incubation and Proliferation of A549 Cells in Electronic Plates

NOTE: Electronic plate types 16 and L8 are both made of arrayed gold microelectrodes lithographically printed at the bottom of incubation wells. Electronic plate 16 has 16 wells while electronic plate L8 has 8 wells. The wells of electronic plate 16 are round while the wells of electronic plate L8 are rounded rectangles. The volume of each electronic plate 8 well is about 830 µL and each electronic plate 16 well is about 270 µL.

1. A549 cell culture
   1. Culture A549 cells in Roswell Park Memorial Institute-1640 medium (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-streptomycin in the cell culture dish.
   2. Passage the cells when the cell density reaches ~75% then wash the adherent cell layer with 1 × PBS and trypsinize with 0.25% trypsin solution for 1 min at 37 ^oC. Centrifuge the cells at 179 × g for 5 min to remove trypsin. Resuspend the cells in 10 mL medium for further culture or other experiments.
      NOTE: The solution of cells resuspended in the medium is here designated as the cell suspension buffer.
   3. Take out 10 µL and count the cell number using a hemocytometer. Use the RPMI-1640 medium to further dilute the cell suspension buffer to prepare 30,000-80,000 cells/mL cell suspension buffer for the experiments.
2. Proliferation/cytotoxicity experiment and data acquisition
   1. Add 50 µL (150 µL for electronic plate L8 ) cell medium to each incubation well of the electronic plate 16 and leave it for 30 min in the biosafety hood for equilibration. Manually insert the electronic plate 16 in the real-time cell analyzer in the CO₂ incubator at 37 ^oC.
   2. Launch the real-time cell analyzer operation program on the computer. On its default experiment pattern setup page, select “Experiment model” and name the running assay.
   3. On its Layout page, select the corresponding wells that are included in the experiment and input the information like cell type, number and drug names in the edit boxes. On its Schedule page, add the experimental steps, then select the running time (e.g., 12, 24, or 48 h) of each step and interval of real-time data acquisition.
      Note: Here, the interval of step 1 and 2 are 1 min and 15 min afterwards.
   4. Save the file and click “Start” button to measure the background impedance of the media on its Cell Index page; the resulting data will be automatically subtracted. On its Well Graph page, all well curves can be visited. On its Plot page, add wells and the graph of time-cell index will be showed.
   5. For the cell proliferation experiment, firstly click the “Pause” button to pause the program . Then take out the electronic plate and add 100 µL A549 cell suspension buffer per well at room temperature. Leave the plate for 30 min in the biosafety hood to let the cells settle to the bottom of incubation wells.
   6.  Manually insert the electronic plate in the real-time cell analyzer station. Click the “Start” button to continue the experiment.
      Note: On the Plot page, the analyzer continuously records Cell Index values or curves throughout the experiment.
   7. When the experiment is complete, enter the Plot page and open the experiment file. Then select all the tested incubation wells and the resulting Cell Index curves that can be averaged or normalized for the last time before adding pharmaceutical drugs or changing medium to reduce variations between assays. On the Data Analysis page, EC50 or IC50 can be calculated

3. Regeneration of Electronic Plates

NOTE: For each rinsing step, the solution in the electronic plate was mixed thoroughly (5-10 times) using a pipette.

1. Case 1: For cytotoxicity evaluation of anti -cancer drug
   1. Seed A549 cells on the electronic plate as described in section 2.
   2. Dissolve the anti-cancer drug doxorubicin hydrochloride (DOX) in the cell medium first. Slowly add DOX to the cells (final concentration of DOX is 30 µg/mL) using a pipette. Record the CI as described in section 2.
      NOTE: The electronic plate used was regenerated immediately after the experiment was complete. In this case, as DOX is a soluble chemical, the regeneration of the electronic plate following the protocol is relatively easy to handle and no additional steps are needed.
   3. Regeneration
      1. Take out the electronic plate containing cells from the real-time cell analyzer station. Place the used electronic plate in a sterile biosafety hood at room temperature and mix culture medium thoroughly using a pipette. Pipette out all the medium.
      2. Rinse the electronic plates with 200 µL deionized water for 3 times at room temperature.
      3. Digest the remaining cells on the electronic plates with 0.25% freshly prepared trypsin for 1-2 h (200 µL/well) in an incubator at 37 ^oC. When the digestion is completed, mix the incubation solution 5-10 times using a pipette and pipette out all the solution in the biosafety hood at room temperature.
      4. Rinse the electronic plates with 200 µL ethanol and 200 µL water, respectively, as follows: deionized water 2 times, 100% ethanol 2 times; 75% ethanol 2 times; deionized water 2 times. Invert the electronic plate on a sterile gauze or tissue paper to decant the remaining water at room temperature.
      5. Ultraviolet sterilize the regenerated electronic plate for 1-2 h in the biosafety hood at room temperature.
         NOTE: Double the volume of all solutions for the regeneration of electronic plate L8.
2. Case 2: drug delivery using mesoporous material.
   1. Use rod-like mesoporous silica materials with an average pore size of 5.6 nm as drug delivery matrices, as previously reported²⁰.
      NOTE: Here, the electronic plate was employed to monitor the drug-release effect on cells.
      1. Add mesoporous materials into the incubation solution of each well of the electronic plates. As silica materials are not soluble and precipitate in the electronic plate, rinse the plate as follows:
   2. Perform steps 3.1.3.1-3.1.3.3 as described for Case 1.
   3. Rinse the microchips with 200 µL deionized water once in the biosafety hood at room temperature.
   4. Rinse the microchips with 200 µL absolute ethanol 2 times at room temperature.
   5. Rinse the microchips with 200 µL 75% ethanol once at room temperature.
   6. Add 250 µL of 75% ethanol in each well and seal the electronic plate with the sealing film at room temperature.
   7. Spin at 114 x g for 2 min and empty the electronic plate. Invert the electronic plate on a sterile gauze or tissue paper to decant the remaining water at room temperature.
   8. Repeat step 3.2.5-3.2.7 once.
   9. Ultraviolet sterilize the regenerated electronic plate for 1-2 h in the biosafety hood at room temperature.
      NOTE: Double the volume of all solution when rinsing L8 electronic plates.

4. Assessment of Regeneration Effect

1. Optical characteristics of regenerated electronic plate surface
   1. Use a steel spoon to carefully disassemble the bottom part of the incubation well of fresh and regenerated electronic plate that contains arrayed gold microelectrodes. Wearing a plastic glove, carefully wipe out the microchip-containing part from the electronic plate using the steel spoon.
   2. Place both parts at the center of the glass microscope slides and collect the resulting Raman spectra by Raman Microscope equipped with a CCD detector²¹.
      NOTE: The spectrometer was equipped with a 633 nm laser and the spectra was recorded at an interval of 30 s laser exposure at a wavelength in the range of 500-3500 cm^-1. The optical images were obtained by a Raman Microscope using a 10x eye lens and 50x objective. Figure 1A and Figure 1C were also obtained using the installed Raman Microscope apparatus.
   3. To evaluate the viability of attached cells after anti-cancer drug uptake on the fresh and regenerated electronic plates, use a confocal laser scanning microscope to monitor the spontaneous fluorescence of DOX molecules absorbed by A549.
      1. To record the spontaneous fluorescence of absorbed DOX by the cells (fresh and regenerated electronic plates with A549 cells), use a 63X oil objective and 485 nm as the excitation wavelength.
2. Electrochemical behaviors of regenerated electrodes
   NOTE: As manual dissembling of a commercial electronic plate is difficult and not ideal for the assessment of surface electric status as checked, the regeneration efficiency of the protocol was evaluated by using normal gold/glassy carbon electrode (GCE) as a test platform, on which electrochemical impedance spectroscopy (EIS) was performed. The diameters of the Au electrode and GCE both are 3 mm. Electrochemical data were obtained by an electrochemical analyzer with a three-electrode system and the impedance measurements were carried out using an alternating current (AC) signal of 10 mV amplitude at a wide frequency range of 100 kHz to 0.01 Hz.
   1. Before the measurement, polish the Au electrode and GCE to a mirror-like surface with an alumina slurry on a polishing cloth, followed by sonication in water to remove any particles.
   2. Activate the Au electrode in 0.5 M H₂SO₄. Obtain EIS data of the bare Au electrode and GCE in 10 mM KCl electrolyte solution containing 1mM K₃[Fe(CN)₆] as described²².
   3. Prepare stock A549 cell solution according to 2.1.2 and 2.1.3. Drop 10 µL cell suspension on the surface of Au electrode and GCE. Incubate Au electrode and GCE modified with cells in a 37 ^oC incubator for 3 h. Obtain EIS data of the Au electrode and GCE modified with cells in 10 mM KCl electrolyte solution containing 1mM K₃[Fe(CN)₆].
   4. Regenerate the electrodes using the protocol and measure EIS.
      1. Immerse the Au electrode and GCE modified with cells in 0.25% trypsin for 0.5-2 h. Rinse the Au electrode and GCE softly with water 4 times. Rinse the electrodes softly with absolute ethanol 4 times. Immerge Au electrode and GCE in 75% ethanol for ~5 min. Rinse the chips softly with 75% ethanol 4 times.
      2. Obtain EIS data of regenerated Au electrode and GCE in 10 mM KCl electrolyte solution containing 1mM K₃[Fe(CN)₆].

Results

Surface properties of gold microchips: The regeneration procedures used in this protocol were outlined in Figure 1. Figure 2 shows the microscopic surface pictures of fresh and regenerated electronic plates by the optical microscope. As shown in Figure 2A and Figure 2C, microscopic observations indicated that there was virtually no diffe...

Discussion

We summarized several available methods^{17,23,24,25,26} for regenerating microchips in Table 1. Basically, these methods involved relatively harsh experimental conditions to achieve the complete regeneration of chips because of the presence of strong molecule-molecule interactions such as that of the immune complex used for SPR chips. However, t...

Materials

Name	Company	Catalog Number	Comments
Rat: Sprague-Dawley	Charles River	Cat# 400
mouse anti-rat CD11b monoclonal (clone OX42)	Bio-Rad	Cat# MCA275R	Panning: 1:1,000; Staining: 1:500
Goat polycolonal anti-Iba1	Abcam	Cat# AB5076	Staining: 1:500
Rabbit polyclonal anti-Ki67	Abcam	Cat# AB15580	Staining: 1:500
Alexa Fluor Donkey anti-mouse 594	Invitrogen	Cat# 11055	Staining: 1:500
Alexa Fluor Donkey anti-goat 488	Invitrogen	Cat# A-21203	Staining: 1:500
Alexa Fluor Donkey anti-Rabbit 647	Invitrogen	Cat# A-31573	Staining: 1:500
Triton-X (detergent in ICC staining)	Thermo Fisher	Cat# 28313
Heparan sulfate	Galen Laboratory Supplies	Cat# GAG-HS01
Heparin	Sigma	Cat# M3149
Peptone from milk solids	Sigma	Cat# P6838
TGF-β2	Peprotech	Cat# 100-35B
Murine IL-34	R&D Systems	Cat# 5195-ML/CF
Ovine wool cholesterol	Avanti Polar Lipids	Cat# 700000P
Collagen IV	Corning	Cat# 354233
Oleic acid	Cayman Chemicals	Cat# 90260
11(Z)Eicosadienoic (Gondoic) Acid	Cayman Chemicals	Cat# 20606
Calcein AM dye	Invitrogen	Cat# C3100MP
Ethidium homodimer-1	Invitrogen	Cat# E1169
DNaseI	Worthington	Cat# DPRFS
Percoll PLUS	GE Healthcare	Cat# 17-5445-02
Trypsin	Sigma	Cat# T9935
DMEM/F12	Gibco	Cat# 21041-02
Penicillin/ Streptomycin	Gibco	Cat# 15140-122
Glutamine	Gibco	Cat# 25030-081
N-acetyl cysteine	Sigma	Cat# A9165
Insulin	Sigma	Cat# 16634
Apo-transferrin	Sigma	Cat# T1147
Sodium selenite	Sigma	Cat# S-5261
DMEM (high glucose)	Gibco	Cat# 11960-044
Dapi Fluoromount-G	Southern Biotech	Cat# 0100-20
Poly-D-Lysine	Sigma	Cat# A-003-E
Primaria Plates	VWR	Cat# 62406-456
Stock reagents	reconstitution	Concentration used	Storage
Apo-transferrin	10 mg/mL in PBS	1:100	-20°C
N-acetyl cysteine	5 mg/mL in H2O	1:1,000	-20°C
Sodium selenite	2.5 mg/mL in H2O	1:25,000	-20°C
Collagen IV	200 μg/mL in PBS	1:100	-80°C
TGF-b2	20 μg/mL in PBS	1:10,000	-20°C
IL-34	100 μg/mL in PBS	1:1,000	-80°C
Ovine wool cholesterol	1.5 mg/mL in 100% ethanol	1:1,000	-20°C
Heparan sulfate	1 mg/mL in H2O	1:1,000	-20°C
Oleic acid/Gondoic acid	Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol	1:1,000	-20°C
Heparin	50 mg/mL in PBS	1:100	-20°C
Solutions	Recipe		Comments
Perfusion Buffer	50 μg/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +)		Use when ice-cold
Douncing Buffer	200 μL of 0.4% DNaseI in 50 mL of DPBS++		Use when ice-cold
Panning Buffer	2 mg/mL of peptone from milk solids in DPBS++
Microglia Growth Medium (MGM)	DMEM/F12 containing 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 5 μg/mL N-acetyl cysteine, 5 μg/mL insulin, 100 μg/mL apo-transferrin, and 100 ng/mL sodium selenite		Use ice-cold MGM to pan microglia off of immnopanning dish.
Collagen IV Coating	MGM containing 2 μg/mL collagen IV
Myelin Seperation Buffer	9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 μL 1 M CaCl2, 5 μL 1 M MgCl2		Mix well after the addition of  CaCl2 and MgCl2
TGF-b2/IL-34/Cholesterol containing growth media (TIC)	MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 μg/mL ovine wool cholesterol, 10 μg/mL heparan sulfate, 0.1 μg/ml oleic acid, and 0.001 μg/ml gondoic acid		Make sure to add cholesterol to media warmed to 37 °C and do not add more than 1.5 μg/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.