CONTRIBUTION

Overview

While developing our glioma drug screening platform, our team developed new protocols and methodologies that can be used by other iGEMers and synthetic biologists for:

1. Optimizing transfection of patient-derived glioma cells
2. Establishing a glioma and minibrain co-culture system for drug screening

Protocol for Transfecting Non-Standard Cell Lines

Although transfections are widely used to transfer plasmid DNA into common cell lines such as HEK 293T cells, plasmid transfections of glioma cells have not been well studied. Limited synthetic biology work has been performed on primary cells, including glioma cell lines and in organoid co-culture settings. In phase 1 of our project, we have successfully developed a protocol to introduce recombinant plasmids via non-viral delivery into patient-derived glioma cells. We found that performing electroporation using the 250V condition produced the best combination of transfection efficiency and fluorescence expression (Figure 1). See Proof of Concept to learn more.

Our protocol can be used by other synthetic biologists and iGEMers interested in integrating their novel plasmids into primary glioma cells:

1. Count the cells and get the total cell count by multiplying the volume of cells by the density of the culture. If the colonies had been expanded prior, they can go into a common flask for preparing the electroporation. Ideally, each well/condition will have 1,000,000 cells so that 100,000 will survive after electroporation; therefore, make sure that you have enough cells for all the conditions you want to test before starting.
2. Prepare the DNA that will be introduced into the cells at the right concentration. For the electroporation on 7/23/21, 500 ng/µL was the concentration that was used.
3. Prepare a 6 well plate with 2 mL of media in each well that will be needed (ex: for three conditions, fill three wells with media).
4. Pellet down the cells in the centrifuge for 5 minutes at 1200 rpm and aspirate off the excess media.
5. Resuspend the excess cells in enough media such that each sample will have 300 µL of media (for example, for three samples, resuspend in 900 µL of media).
6. Pipette 300 µL of cells and the required amount of DNA into each of the cuvettes.
7. Take the cuvettes to the electroporation machine and use the exponential mode; ensure that the settings are all correct before placing the cuvette in the holder and pressing the pulse button (look for blinking P icon). Be sure to note down the time constant (ideally around 30 to 40 ms).
8. Quickly bring the cells to the hood where the 6 well plate is. Using a transfer pipette, move some of the media from the correct well into the cuvette and pipette to mix. Draw up all of the content in the cuvette (including the white film on the top) and mix into the correct well.
9. Return the well with electroporated cells into the cell culture cabinet; the cells will require a media change in about 24 hours.

Protocol for establishing a glioma and minibrain co-culture system

Our project required a physiologically accurate system that models the human brain microenvironment to aid in the drug screening process for potential glioma therapeutics. To satisfy this need, we worked to develop a protocol for co-culturing mature minibrains and primary brain cancer cell lines.

The general outline of our protocol is documented below:

1. Set up

   Using a 96-well ultra-low attachment U-bottom plate and assign positive and negative control wells (i.e minibrain only and glioma cell only conditions). Plan to operate with 3 replicates for each control/dosage condition to avoid in-group variations.

2. Seed Glioma Cells

   Seed glioma cells into each well of the 96 well plate, aiming for approximately 500 cells/well. If the glioma cell line is drug resistant/fast-growing, seed fewer cells to avoid overgrowth. Use cerebral organoid media to ensure best performance

3. Integrate Minibrains

   Add an equal amount of minibrains in each well (we recommend 1 organoid/well) Note: U-bottom plate will allow the minibrains to collect at the bottom of the well (ideal for imaging purposes)

4. Observe

   Document changes in the plate over the course of 3-5 days imaging regularly and observing invasion behavior. We recommend using the IncuCyte or other plate scanners for regular whole-plate scanning.

Applications

The plate can be dosed with different drugs for screening purposes:

1. Create at least 3 titrations for concentration for each drug tested

   Observe both the killing effect and potential toxicity of the drug on normal brain cells

2. Set up control wells (Minibrains are dosed with with glioma cells but no drug)

   Control wells serve to monitor the invasiveness of glioma cells, If no invasion is observed, the other wells will not produce valid results

   Important Note: The drug and glioma cells should be dosed at the same time on day 0 into the co-culture system

3. Observe

   Imaging regularly over 3-5 days to document the number and status of cells, migrations and invasion

This protocol can be used by researchers in disease modeling to observe how primary cancer cell lines behave in an in vitro tumor microenvironment.