Protocols

1. Ficoll–Hypaque Solution

• Density: 1.077 g/L
• Storage: 4°C in the dark

2. Cell Culture Medium

• Common Types: RPMI 1640, Iscove’s Modified Dulbecco’s Medium, a-MEM, McCoy’s 5A
• Storage: 4°C in the dark
• Preparation: Prepare aliquots of complete medium (maximally 100–200 mL) in separate sterile glass bottles for each cell line. Store these at room temperature in the culture laboratory to quickly detect any microbiological contamination.

3. Fetal Bovine Serum (FBS)

• Pre-treatment: Inactivate toxic components by heating in a 56°C water bath for 45 minutes.
• Storage: Store aliquots of maximally 50 mL in plastic tubes at −20 to −30°C.

4. Neubauer Hemocytometer and Trypan Blue Solution

5. Recombinant Growth Factors

• Examples: Erythropoietin (EPO), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Granulocyte-CSF (G-CSF), Interleukin-2 (IL-2), IL-3, IL-6, Stem Cell Factor (SCF), Thrombopoietin (TPO).

6. Conditioned Medium (CM) from Tumor Cell Line Cultures

• Example: Human bladder carcinoma cell line 5637 is known to produce and secrete various growth factors.
• Preparation: Aliquot the CM in 30 or 50 mL tubes and store at −20°C.
• Testing: Test the 5637 CM in proliferation assays using an indicator cell line or determine the exact growth factor concentration with ELISAs.

Day 1 - Cell Culture and Establishment

1. Prepare the following reagents and materials:

   • Ficoll–Hypaque solution (density 1.077 g/L).
   • Cell culture medium (e.g., RPMI 1640, Iscove’s Modified Dulbecco’s Medium).
   • Fetal bovine serum (FBS) - inactivated.
   • Recumbent growth factors (e.g., EPO, GM-CSF).
   • Conditioned medium (CM) from 5637 cell line.
   • Sterile glass bottles for medium aliquots.
   • Cell culture flasks or plates.
   • Incubator (37°C, 5% CO2).

2. In a sterile environment:

   1. Prepare aliquots of complete medium (100-200 mL) in separate sterile glass bottles.
   2. Thaw FBS aliquots if frozen.
   3. Mix cell culture medium with 20% FBS and 10% 5637 CM (or appropriate growth factors).
   4. Adjust cell suspension of patient cells to 2-5×10^6/mL in culture medium.

3. Seed cells into culture flasks/plates:

   1. Place 5-10 mL of the cell suspension into culture flasks (80 cm2) or plates.
   2. Incubate at 37°C with 5% CO2.

Day 2 - Initial Culture Maintenance

1. Check cell culture:

   • Inspect cell growth and adherence.
   • Exchange half of the spent culture volume with fresh medium (20% FBS, 10% 5637 CM) to maintain cells.

Day 3 - Ongoing Culture Maintenance

1. Continue culture maintenance:

   • Exchange half of the medium with fresh complete medium once a week.
   • Monitor cell growth.

Day 10-14 - Cell Proliferation and Monitoring

1. Continue to maintain cell culture:

   • Exchange medium as needed.
   • Observe cell proliferation.

Day 1: Cell Plating and Initial Culture

1. Materials:

   • Cell culture plates (2 x 75 cm²).
   • Cell suspension (2 million cells/plate) in complete medium (DMEM + FBS + antibiotics, 18 ml).
2. Incubate at 37°C, 5% CO₂ for 48 hours (2 days).

Day 3: Washing and Differential Medium

1. Procedure:

   • Both plates were washed 3 times with 10 ml of PBS.
   • In one plate, 18 ml of complete medium was added, and in the other plate only DMEM.

Day 4: Cell Harvest and Freezing

1. Procedure:

   • Using cell scrapers, detach the cells from both plates.
   • Add 5 ml of PBS to each plate to collect the cells.
   • Transfer the cell suspensions from both plates to separate 10 ml tubes.
   • Centrifuge the tubes at 1200 rpm for 5 minutes to pellet the cells.
   • Carefully discard the supernatant.
   • Freeze the cell pellets at -20°C for future use.

Day 5 - Cell Harvest for Protein Isolation

1. Step 1: Check cell confluence

   • Ensure cells are proliferating actively.

2. Step 2: Harvest cells for protein isolation

   • Aspirate the culture medium.
   • Wash cells gently with PBS.
   • Add appropriate cell lysis buffer for protein extraction.
   • Scrape cells from flasks or plates.
   • Collect cell lysate in tubes.

3. Step 3: Centrifuge cell lysates

   • Centrifuge at an appropriate speed and time to pellet cell debris.
   • Collect the supernatant (cell lysate containing proteins).

4. Step 4: Quantify protein concentration

   • Use a protein assay (e.g., Bradford, BCA) to determine protein concentration.

5. Step 5: Store protein lysates

   • Store protein lysates at -80°C for Western blotting.

Day 6 - Western Blotting: Block and Antibody Incubation

Block and Antibody Incubation (Day 1):

1. Prepare samples

   • Thaw protein lysates.
   • Mix with loading buffer.

2. Run SDS-PAGE gel

   • Load samples onto gel.
   • Electrophorese proteins.

3. Transfer proteins to a membrane

   • Use a transfer apparatus (e.g., Western blot tank).

4. Block the membrane (90 minutes)

   • Prepare blocking buffer (e.g., 5% non-fat milk or BSA in TBS or TBST).
   • Submerge the membrane in the blocking buffer for 90 minutes.

Washing and Antibody Incubation (Day 2):

5. Wash the membrane (3 x 10 minutes)

   • Use TBS or TBST (Tris-buffered saline with Tween 20) for washing.
   • Perform three 10-minute washes with gentle agitation.

6. Incubate with the primary antibody (overnight)

   • Prepare the primary antibody solution in a suitable dilution with blocking buffer.
   • Incubate the membrane with the primary antibody overnight at 4°C.

Secondary Antibody Incubation (Day 2):

7. Wash the membrane (3 x 10 minutes)

   • Use TBS or TBST for washing.
   • Perform three 10-minute washes with gentle agitation.

8. Incubate with the secondary antibody (90 minutes)

   • Prepare the secondary antibody solution in a suitable dilution with blocking buffer.
   • Incubate the membrane with the secondary antibody for 90 minutes at room temperature.

Development:

9. Develop the Western blot

   • Follow the manufacturer's instructions for chemiluminescent or colorimetric detection.
   • Visualize and capture the protein bands using an imaging system or X-ray film.

Protein Preparation

Total protein extracts were prepared using 200 μl of RIPA lysis buffer (140 mM NaCl; 25 mM Tris-HCl; 1 mM EDTA; 0.5 mM EGTA; 1 mM PMSF; 1 mM NaF; 1% Triton X-100; 0.1% SDS; 0.1% Protease inhibitors cocktail; pH=7.4).

200 μl of this buffer was added to the cell pellets stored at -20°C. The lysis buffer and cell pellet were left on ice for 10 minutes. Afterward, both tubes were centrifuged at 12500 rpm for 5 minutes, and the supernatant was transferred to fresh tubes and stored on ice.

Protein concentration was measured with Bradford reagent. 20 μl of protein extracts were mixed with 1 ml of Bradford reagent and mixed until a blue color appeared. The color intensity was measured at 590 nm using a spectrophotometer. The protein concentration for the sample DMEM+FBS was 1.13 mg/ml, whereas the protein concentration for the sample DMEM only was 0.43 mg/ml.

Western Blot

From each sample, 10 μg of total proteins were mixed with 5 μl of loading buffer and denatured at 95°C for 5 minutes.

Samples were loaded on a 10% SDS-PAGE gel. A protein marker (5 μl) was also added. The order on the gel was as follows:

• Well 1: Protein marker
• Well 2: Protein extract from sample DMEM+FBS
• Well 3: Protein extract from sample DMEM only
• Well 4: Empty
• Well 6: Protein extract from sample DMEM+FBS
• Well 7: Protein extract from sample DMEM only

The gel ran for 90 minutes at 120 mA. After the marker was well separated, the electrophoresis was stopped, and proteins were transferred to a nitrocellulose membrane using a transblot apparatus. The transfer was performed for 1 hour at 180 mA.

The nitrocellulose membrane was washed three times for 5 minutes in agitation with TBST (20 mM Tris; 150 mM NaCl; 0.1% Tween 20; pH 7.4). It was then cut vertically at the 4th well to obtain two identical lines of separated proteins.

The two nitrocellulose portions were placed in blocking buffers (5% milk in PBS) for 2 hours and then washed with TBST three times for 5 minutes.

The nitrocellulose portion containing the first 3 wells was incubated overnight at 4°C with an ERK antibody diluted 1:100 in the blocking buffer.

The nitrocellulose portion containing the wells 6th and 7th was incubated overnight at 4°C with a pERK antibody diluted 1:500 in blocking buffer.

After the overnight incubation, the primary antibody solution was removed, and both membranes were washed three times for 5 minutes in agitation with TBST. Subsequently, the secondary antibodies were added.

For the membrane incubated with the ERK antibody, a secondary anti-rabbit antibody conjugated to HRP was used at a dilution of 1:2000 in blocking buffer.

For the membrane incubated with the pERK antibody, a secondary anti-mouse antibody conjugated to HRP was used at a dilution of 1:2000 in blocking buffer.

Both membranes were incubated with the secondary antibodies for 1 hour at room temperature.

After the incubation time, both membranes were washed three times for 5 minutes in agitation with TBST.

Finally, detection of antibodies signal was performed using standard ECL reaction and photosensitive films to detect the chemiluminescence generated by the secondary antibodies.

2. Freezing and Storage of Cells

• Dimethylsulfoxide (DMSO).
• Freezing ampules (plastic cryo vials).
• Controlled rate freezer.
• Cryo Freezing Container (Nalgene Cryo 1°C Freezing Container).

3. Establishment of Cells

The isolation protocol was intended for all the volunteer samples, which we couldn't study as thoroughly as initially intended.

3.1 Acquisition of cells

1. Collect heparinized or otherwise anticoagulated specimens of peripheral blood or bone marrow or other samples in sterile tubes (see Notes 3 and 4). Place lymph nodes and other solid tissues in sterile containers.
2. Specimens should ideally be processed as soon as possible after receipt, but may be stored overnight at room temperature. Peripheral blood and bone marrow should remain undiluted, solid tissues should be placed in culture medium.
3. Cryopreserved samples can also be used for attempts to establish cell lines. But it appears to be of advantage to isolate the mononuclear cells prior to cryopreservation by Ficoll–Hypaque density gradient centrifugation.

3.2 Isolation of cells

1. Cut solid skin tissue with scissors and force the particles through a fine metal mesh. Suspend the cells in 50–100 mL culture medium (possibly also more depending on the size of the tissue specimen).
2. Dilute the blood and bone marrow samples 1:2 with culture medium. Isolation of cells from a leukapheresis collection requires dilution of the sample with culture medium at 1:4.
3. Pipette the Ficoll–Hypaque density gradient solution (density 1.077 g/L) into a 15 or 30 mL conical centrifuge tube. Slowly layer the mixture of medium and sample over the Ficoll–Hypaque solution. Use an equal volume of the sample mixture and Ficoll–Hypaque solution.
4. Centrifuge for 20–30 min at 450×g at room temperature (with the centrifuge brakes turned off). A layer of mononuclear cells should be visible on top of the Ficoll–Hypaque phase as they have a lower density than the Ficoll–Hypaque solution.
5. The anucleated erythrocytes and the polynucleated granulocytes are concentrated as a pellet below the Ficoll–Hypaque layer.
6. Using a sterile Pasteur pipette, transfer the interface layer containing the mononuclear cells to a centrifuge tube.
7. Wash the cells by adding culture medium plus 10% FBS (add about five times the volume of the mononuclear cell solution) and centrifuge for 5–10 min at 200×g at room temperature.
8. Discard the supernatant, resuspend the cell pellet in culture medium plus 10% FBS and repeat the washing procedure.
9. Finally, resuspend the cells in 20 mL of culture medium with 20% FBS. Count the cells and determine their viability.

4. Culture Conditions

1. Adjust the cell suspension of the original patient cells to a concentration of 2–5×10^6/mL in culture medium with 20% FBS plus an additional 10% conditioned medium (CM) of cell line 5637 or with an appropriate concentration of purified or recombinant growth factors.
2. Place 5–10 mL of the cell suspension in the complete culture medium in an 80 cm^2 plastic culture flask. If 24-well plates are used, add 1–2 mL cell suspension into each well. Add 100–200 mL of cell suspension into wells of 96-well flat-bottomed microplates.
3. Place the cells in a humidified incubator at 37°C and 5% CO2 in air.
4. Expand the cells by exchanging half of the spent culture volume with culture medium plus 20% FBS plus 10% 5637 CM (or with appropriate concentrations of recombinant growth factors) once a week. After a few hours, some cells become adherent.
5. During the first weeks, the neoplastic cells may appear to proliferate actively. If the medium becomes acidic quickly (yellow in the case of RPMI 1640 medium), change half of the volume of medium at 2–3 days interval (seldom daily). If the number of the cells increases rapidly, readjust the cells weekly to a concentration of at least 1×10^6/mL in fresh complete medium by dilution or subdivision into new flasks or wells of the plate. The neoplastic cells from the majority of patients undergo as many as four doublings in 2 weeks, but after 2–3 weeks most malignant cells cease proliferating. Following a lag time of 2–4 weeks (crisis period), a small percentage of cells from the total population may still proliferate actively and may continue to grow forming a cell line.

5. Sensor

The sensor includes 2 separate components connected via electrical wires. The first component is the biological part which is able to physically interact with kinase enzymes; the second is the electronic part that detects the interaction between the antibodies and kinase enzymes via changes of capacitance and impedance.

For this interaction the following components have been used:

• Ab-Anti-ERK1 (purchased brand name: Santa Cruz) was dissolved in distilled water at a concentration of 0.2 mg/ml.
• Kinase enzyme-ERK1 Human Recombinant Protein (purchased brand name: OriGene) was dissolved in distilled water at a concentration of 0.02 mg/ml.
• L-cysteine (purchased brand name: Sigma-Aldrich) was dissolved in distilled water at a concentration of 25 mM.
• EDC (N-(3- Dimethylaminopropyl)-N′-ethylcarbodiimide) (purchased brand name: Sigma Aldrich) at a concentration of 0.877 g/ml was dissolved in water at a concentration of 0.1 M.
• MES (2-(N-morpholino)ethanesulfonic acid) (purchased brand name: Sigma Aldrich) was dissolved in water at a concentration of 0.1 M.
• PBS (Phosphate-buffered saline) was dissolved in a ratio 1:1 in water.
• Gold interdigitated electrodes were purchased from DropSens (Asturias, Spain), cat. N.: PW-IDEAU50. Each IDE has a finger width and spacing of 50 μm, with a total number of 70 fingers, a total electrode length of 7 mm, and electrode surface area of 8.45 mm^2.

References

[1] Cree, I. A. (2011). Cancer Biology. Methods in Molecular Biology, 1-11. https://doi.org/10.1007/978-1-61779-080-5_1.
[2] Georgas, A., Lampas, E., Houhoula, D. P., Skoufias, A., Patsilinakos, S., Tsafaridis, I., ... & Hristoforou, E. (2022). ACE2-Based Capacitance Sensor for Rapid Native SARS-CoV-2 Detection in Biological Fluids and Its Correlation with Real-Time PCR. Biosensors and Bioelectronics, 202, 114021. https://doi.org/10.1016/j.bios.2022.114021.