Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

1. Preparation of Test Materials

NOTE: In order to prepare the biochips for testing, several confluent flasks of RTgill-W1 cells need to be ready. A good estimate of the number of flasks needed is one confluent T175 flask for 16 biochips to be seeded.

1. Perform the following steps in a class II biological safety cabinet (biohood) using aseptic technique. Use 70% ethanol for disinfecting the biohood and any materials placed in the hood.
   1. Prepare fibronectin substrate for the fluidic biochips by thawing a 1 mg vial of fibronectin and diluting in 100 ml of sterile L-15 media for a concentration of 10 µg/ml. Freeze in 40 ml aliquots in sterile 50 ml conical polypropylene tubes at -20 °C. Thaw at room temperature several hours prior to seeding the biochips. Once thawed, do not refreeze.
   2. Prepare cell culture media by adding 50 ml of fetal bovine serum, 5 ml of 200 mM an L-alanyl-L-glutamine supplement, and 5 ml of a penicillin/streptomycin solution (10,000 units penicillin/ml and 10,000 µg streptomycin/ml) stock to 500 ml of L-15 media. This will yield 560 ml of cell culture media containing 9% serum. Refrigerate.
      Note: This will be the complete cell culture media used for the culture flasks and the biochips.
2. Powdered Media Vials
   1. Prepare ahead of time 0.1 dram snap-cap vials containing 60 mg ± 0.5% of L-15ex powder using an automated powder dispenser. Label the vials with the date the powder was dispensed and refrigerate vials (in quantities of 50) in recloseable metalized poly bags; each containing three 1 g silica gel desiccation packs.
      Note: L-15ex powdered media vials can be made and stored for up to 9 months in advance of testing.
3. Make a solution of 100 ml 20% bleach by diluting household bleach with deionized (DI) water. Estimate that 5 ml of bleach solution will be needed for each biochip.
4. Make biochip tubing assemblies by cutting 27 mm sections of autoclavable biocompatible tubing (2 sections for each biochip to be seeded) and fit both ends of the tube with polycarbonate slip luer fittings. Place tubing assemblies in an autoclavable bag and autoclave for 8 min at 134 °C. Also autoclave biochip plugs (2 per biochip) in a separate bag at the same settings.
5. Autoclave DI water for 30 min at121 °C.
   Note: This water will be used for rinsing biochips after seeding. Estimate that 10 ml will be needed for each biochip.
   Note: The actual volume of each biochip channel is 2 ml, but 5 ml of sterile water is rinsed through each channel after removal of bleach solution.
6. Make syringe injection tubing assemblies by cutting 27 mm sections of biocompatible tubing and attaching male slip luer fittings to both ends of the tubing. Place tubing assemblies in a paper heat-seal sterilization pouch and autoclave for 8 min at 134 °C.

2. Fluidic Biochip Seeding Procedure

Note: Perform all procedures where the biochips or the media is handled in a class II biological safety cabinet using aseptic technique.

1. Twenty-four hr prior to scheduled seeding, remove the biochips from manufacturer packaging in the biohood and place in sterilized plastic instrument cases.
2. Sterilize the biochips using a 20% bleach solution as follows
   Note: Historically, this bleaching procedure has prevented fungal growth in the biochips during long-term storage in the event that the plasma sterilization of the biochips done by the manufacturer was not effective.
   1. Using a 20 ml sterile syringe with a syringe injection tubing assembly attached and working in the biohood, inject 2 ml of the 20% bleach solution into each channel of the biochip. Allow the biochips to sit for 1 hr with the bleach solution.
   2. After one hour, vacuum aspirate the bleach solution from both channels using a sterile male slip luer assembly attached to the vacuum suction tubing. Use one of the biochip tubing assemblies as a drain when rinsing the biochips.
   3. Using a sterile 20 ml syringe attached to a sterile syringe injection assembly, flush each channel of the chip with 5 ml of sterile water, allowing the excess water to drain into a container in the biohood. Then vacuum-aspirate off the water as just described for the bleach and place the biochips back into the plastic instrument cases and leave in the biohood until seeded with cells the following day.
3. Sixty min prior to seeding the biochips, inject 2 ml of the 10 µg/ml fibronectin solution into each channel of the biochip. Leave the biochips in the biohood for 60 min, and then vacuum aspirate off the fibronectin (as described in step 2.2.3) before seeding the biochip with cells. Place two sections of the sterile biochip tubing assemblies (refer to section 1.4) on the ports of the biochips.
4. Trypsinize one confluent RTgill-W1 T175 flask for every 16 biochips using procedures outlined in the American Type Culture Collection (ATCC) product description sheet¹⁶.
   1. Aspirate off the media from the confluent flask(s) of cells.
   2. Rinse the cell layer with 15 ml of PBS and then aspirate off.
   3. Add 6 ml of trypsin/EDTA to the cell layer in each T175 flask and allow cells to trypsinize for ~5 min.
   4. Add 15 ml of complete L-15 cell culture media to each flask to stop trypsinization.
   5. Combine the cell suspensions in a sterile disposable container.
      Note: Container size can vary from 150-500 ml, dependent upon the number of biochips being seeded. Estimate that 5 ml of cell suspension will be needed per biochip.
5. Remove ~1 ml of the cell suspension and place into a microcentrifuge tube for counting. Using a brightfield microscope with a 10X objective and a hemocytometer, count a 10 µl aliquot of the cells and calculate the volume of complete L-15 cell culture media needed to attain a cell suspension of 2.5 x 10⁵ cells/ml.
   Note: If using the cell suspension to seed flasks to continue the culture, this would be the point to do so. Adjust the cell suspension concentration using complete L-15 cell culture media.
6. Using a sterile 20 ml syringeattached to a sterile syringe injection tubing assembly (see section 1.6), inject 2.5 ml of the cell suspension into the outer port of each channel of the biochip (i.e., the ports that do not have the tubing attached), allowing some of the extra cell suspension to flow out of the tubing into a waste container in the biohood.
   Note: This will ensure that the entire channel and attached tubing will be full of the cell suspension.
   1. Create an enclosed loop for each biochip channel by inserting the free end of the hose with the luer fitting into the outer ports for each channel.
   2. Wipe any excess media off of the closed loops with a paper towel moistened with 70% ethanol and place the biochips back in the plastic box in a 20 °C incubator. Give each biochip a unique identifying number.
7. On days 4 and 7, remove the biochips from the 20 °C incubator and replenish the media in all of the biochips with temperature-equilibrated complete L-15 cell culture media. Follow the same procedure as in step 2.6 except use just L-15 cell culture media instead (no cell suspension). Place the biochips back in the 20 °C incubator after the day 4 feeding.
   1. After the day 7 feeding, remove and discard the hoses from the chips and insert the autoclaved drain plugs in the biochips.
   2. Place the biochips in a box in a 6 °C incubator until used for testing.
      Note: The chips can be stored at refrigerated temperatures for up to 9 months and are still viable for testing in the ECIS readers.

3. ECIS Testing with Biochips

1. Prepare test chemicals if using. (See Brennan, et al., 2012⁷ for preparation of test chemicals).
2. Remove ECIS reader and test supplies from the carrying case. Remove a prepared biochip from the 6 °C incubator. Place the biochip on a paper towel. Turn on the ECIS reader.
3. Using 10 ml syringes, dispense 10 ml of control water into a labeled 0.5 ounce clear plastic control jar and 10 ml of the test sample into a labeled 0.5 ounce clear plastic test jar.
   Note: 1) Be sure to remove bubbles for an accurate measurement. 2) The syringes and jars are color-coded; blue for control and red for test.
4. Remove two powdered media vials from the foil bags. Open one of the powdered media vials (use multipurpose tool if needed) and pour entire contents of one vial into the jar with control water, dropping the entire vial into solution as well. Repeat this procedure with the test jar. Cap and shake the jars to ensure that the powder is dissolved.
5. Fill each of the colored 10 ml syringes with 9 ml of either control (blue syringe) or test (red syringe) solutions from respective vials. Remove air bubbles from syringes.
6. Remove the plugs from the biochip ports and attach the drain.
7. Place the biochip into the removable plastic tray in the ECIS reader and close the lid.
8. Attach filled syringes to the outer biochip ports and attach the syringe plunger.
9. From the main screen, select "NEXT" to initiate pre-test.
   Note: Reader software will check impedances. If impedances are within range set by the user (usually between 1,000 and 3,000 ohms) the screen will register "Cartridge Passed" as indicated in Figure 3, and instruct the user to select "Next" and the software will proceed to step 3.10). If the impedances are not within the set range because of a defective biochip or faulty connection of the biochip to the reader (usually due to a misalignment of the electrodes), then the screen will register "Cartridge Failed" and the user will have the option to either "Abort" or "Verify" the test.
   1. Select "Abort" to go back to step 3.9). Select "Verify" to proceed to step 3.10) after receiving a "Cartridge Passed" message and selecting "Next".
      Note: It is best to take the biochip out and visually inspect it for defects or leaking if a "Cartridge Failed" message is received before proceeding with a new biochip.

Figure 3: ECIS Reader Screenshot of a Biochip with Acceptable Impedance Readings. The screenshot shows initial impedance readings in ohms of each of the 4 control electrodes (CE) and 4 test sample electrodes (SE) within the fluidic biochip. Please click here to view a larger version of this figure.

10. When prompted by the reader, enter sample information using a soft keyboard and then select "Accept" when finished.
    Note: The ECIS reader software will automatically stamp each dataset with the date, time and other information entered by the user; such as the biochip number, and the type of chemical and concentration. Two min of impedance data will be collected from the inserted biochip and an on-screen timer will count-down time.
11. After two min when prompted by on-screen instructions that are flashing within a red box, hit "NEXT". When prompted by a flashing green box to "Inject samples now", inject the control and test media from the attached syringes simultaneously into the biochip channels. Leave the syringes in place on the biochips when finished.
    Note: Impedance values will be collected once per minute for 60 min. If the ECIS reader software determines that the treatment channel is statistically different from the control channel at any point between 10 and 60 min after the start of test, then the on-screen display will indicate that the sample is "Contaminated". If the treatment is not different from the control channel, then the screen will indicate "No Contamination Detected" at the end of the test run.
12. Record test results as contaminated or not contaminated for each sample.
13. At the end of the test run, remove and discard the biochip. Rinse and air-dry the syringes, test vials and removable plastic tray which housed the biochip during the tests.
    Note: The ECIS reader has 4 GB of onboard storage for the operational software, control models and the test files generated from running a test. This allows for several thousand test files to be stored on the reader. Files can be retrieved with a USB jump drive and transferred to a computer for further analysis for research purposes if desired.