LABORATORY VIDEO DOCUMENTATION OF THE CELL CULTURE ISOLATION CONTROLS AND METHOD

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Materials and Methods

Cell Culture

Human Embryonic Kidney (HEK293T ATCC CRL-3216) cells were seeded onto 6-well culture dishes at passage 5-8 at a concentration of 1x10⁶ per 5mL and grown for 3 to 4 days at 37⁰C, 5 % CO₂ in a humidified incubator. Conditions were as follows:

1. DMEM, 10% FBS, 1x P/S

2. DMEM, 2% FBS, 1x P/S

3. DMEM, 2% FBS, 2x P/S

4. DMEM, 2% FBS, 3x P/S

5. DMEM, 1% FBS, 1x P/S

6. DMEM, 1% FBS, 2x P/S

7. DMEM, 1% FBS, 3x P/S

At day 3 or day 4 of growth, cells were imaged at 20x with an Evos XL Core Imaging System.

After imaging cells, cultures were washed with PBS, then 1 mL of TryplE was added to dissociate cells from the dishes. After 3 minutes, 5mL of DMEM+10%FBS+1xP/S was added and cells were harvested then centrifuged at 1000 x rpm for 3 minutes. The cell pellets were re-suspended with 5mL of DMEM+10%FBS+1x P/S and viability was calculated by Countess 3 FC Automated Cell Counter (Invitrogen).

After viability was recorded, cells were centrifuged at 1000 rpm for 3 minutes, then the DMEM was poured out and the cell pellet was re-suspended in 300-500ul of TRIzol and stored at -80⁰C until further analysis.

*LOUD Turn sound down or off, Only Flow Hood sounds as proof the correct protocols for prohibiting contamination were taken*

LABORATORY VIDEO METHOD

Taking Stock

The CRO technician shows the laboratory setting and moves over to the flow hood where the reagents will all be handled. The flow hood is calibrated to settings of protocol to prohibit any contamination. Stock of all reagents is taken showing the Cell Line, DMEM, FBS, Pen/Step, Amphotericin, Trizol, Trypsin and a selection of pipettes and tips.

Confirming the cells are Trypsinized

Trypsinizing the Cell Line in culture to detach adherent cells from the surface of the culture vessel before counting and seeding.

Trypsin-EDTA solution (commonly 0.25%)

• Phosphate-Buffered Saline (PBS) or another balanced salt solution

• Cell culture medium (with serum to inactivate trypsin)

• Hemocytometer or automated cell counter

• Centrifuge

• Sterile pipettes

• Culture flasks or dishes

The Cells are then taken over to the scope to verify that they have detached from the vessel and are free moving.

Centrifugation into Pellet for Cell Counting

After trypsinizing or otherwise detaching cells from a culture vessel, the cell suspension is often dilute. Centrifugation forces cells to the bottom of a tube, concentrating them into a pellet, which makes subsequent steps like re-suspension or counting more manageable and accurate Cell Pellet Re-suspension

The supernatant (which includes trypsin, serum, and other culture medium components) can be removed after centrifugation. This step is crucial for:

• Neutralizing trypsin to prevent cell damage.

• Removing debris or dead cells which might interfere with counting or new culture initiation.

• Changing media or preparing cells for different solutions or buffers needed for downstream applications.

After Re-suspension the Cells are counted using the Countess 3 FC Automated Cell Counter (Invitrogen).

Seeding into 6 well plates, 1m per well.

• Seeding the Cells:

  • Dispense the calculated volume of cell suspension to match 1x10⁶ per 5mL concentration into each well. Use a pipette with disposable tips for precision and to prohibit cross contamination.

  • If the cell suspension is not at the concentration , adjust the volume accordingly or dilute your cell suspension to make calculation easier.

• Add More Medium:

  • After adding the cells, add additional culture medium to each well to reach the desired volume. For most adherent cells, add enough medium to get to 5ml per well.

Plating Test Variable Concentrations

Plating all reagents with varying concentrations of Fetal Bovine Serum and Pen/Strep into 1x10⁶ per 5mL concentration into each well.

1. DMEM, 10% FBS, 1x P/S

2. DMEM, 2% FBS, 1x P/S

3. DMEM, 2% FBS, 2x P/S

4. DMEM, 2% FBS, 3x P/S

5. DMEM, 1% FBS, 1x P/S

6. DMEM, 1% FBS, 2x P/S

7. DMEM, 1% FBS, 3x P/S

Incubation and Scope

Plates were grown for 3 to 4 days at 37⁰C, 5 % CO₂ in a humidified incubator, then examined under scope to verify negative Control 10% FBS Growth Medium and varying concentration Test plates.

CRO live scope of 10% FBS Stock Flask

The stock flask of 10% FBS for the Negative Control plate is put under scope. The CRO technician describes it as almost confluent meaning that the cells have grown out to touch each other but the technician points to the gaps in the cell line to show that there are still spaces for the line to grow. This shows that the cells were grown to a healthy confluence and not overgrown.

They then point out a few floating cells but that this is considered a normal and healthy culture. They describe that when the flask is less confluent you may see less of these floating cells but again this is considered a normal and healthy line.

They then show the order in which the plates are laid out with negative control top left and then varying concentrations in other wells.

CRO Live Scope of Negative Control and Test Plates

1. First they look at 10% FBS Negative Control where they describe it as confluent. They point out the spaces where it is not at 100% confluent showing that the cells can grow and are not overgrown. They point out a few floating cells similar to the stock flask imaging.

2. Next looks at 2% FBS Test Plate they describe it as nowhere near confluent, the morphology looks a lot different. They point out clear plaques in the line from lysed cells, clumping and Syncytia, lots more floating dead cells, ballooning cells. They say there is room to grow if they could, again suggesting that this plates morphology was not a result of overgrowing.

3. Next is 2%FBS with 2x Pen/Strep. Noted plaques, ballooning, dead cells Clumping and Syncytia and suggests there are the morphological features inline with other papers showing Cytopathic Effect.

The technician points out that in the two reduced serum test plates there is still plenty of nutrient medium left as the colour is still red.

0.5 Million Cells Concentration Test Plate Live Scope

To asses and compare the seeding density of the cells, the plates were run at half the concentration of cells as measured by the auto-counter , Countess. If the higher seeding density were causing any sort of nutrient starvation through over crowding one would expect drastic differences from the observed morphology in this plate:

The CRO technician describes these comparison plates as having the exact same morphological features ascribed to Cytopathic effect as above. They point out Rounding, Ballooning, Syncytia, Plaque Formations and Floating/Dead cells all features that are described as “Infected with Virus” in cross-referenced papers submitted as part of the working brief to the project.

They note that there is plenty of space if the cells wanted to grow, there is no contact inhibition and the media is of red colour showing there are plenty of nutrients left yet at 2% FBS 1x Pen/Strep all of the morphological features of Cytopathic effect are seen in the Test Cultures.