While developing our glioma drug screening platform, we developed new protocols and methodologies that can be used by iGEMers and synthetic biologists for:
- Optimized transfecting of patient-derived glioma cells
- Establishing a glioma and minibrain co-culture system for drug screening
- Engaging in meaningful integrated human practices interactions through the DUKE cycle
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.
Figure 1. Fluorescent Microscopy images of transfected patient-derived glioma cells, using three different electroporation protocols to identify optimal conditions.
Our protocol can be used by other synthetic biologists and iGEMers interested in integrating their novel plasmids into primary glioma cells:
- 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.
- 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.
- 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).
- Pellet down the cells in the centrifuge for 5 minutes at 1200 rpm and aspirate off the excess media.
- 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).
- Pipette 300 µL of cells and the required amount of DNA into each of the cuvettes.
- 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).
- 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.
- 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 for drug screening
We have developed a protocol for co-culturing mature minibrains and primary cancer cell lines. This protocol can be used by researchers in disease modeling to observe how primary cancer cell lines behave in an in vitro tumor microenvironment.
The general outline of our protocol is documented below:
- Briefly, a 96-well ultra-low attachment U-bottom plate is used to set up the experiment.
- The plate should include positive and negative control wells, i.e. minibrain only and glioma cell only conditions, with at least 3 replicates for each to avoid within group variations.
- Use cerebral organoid media in the co-culture system to ensure best performance.
- A low density of glioma cells should be seeded into each well. We recommend the amount of 500 cells/well in consideration of the small volume of the well. If the cancer cell line is fast-growing or drug-resistant, decrease seeding density.
- Add an equal amount of minibrains in each well. As a U-bottom plate is used, the minibrains will automatically collect to the bottom of the well and should be ideal for imaging purposes.
- Observe the plate over the course of 3-5 days. Take images regularly to document invasion behavior.
Downstream: The plate can be dosed with different drugs for screening purposes:
- We recommend having at least 3 titrations of concentration for each drug tested to observe both the killing effect and the potential toxicity of the drug on normal brain cells.
- The drug and the glioma cells should be dosed at the same time on day 0 into the co-culture system.
- Set up control wells where minibrains are dosed with glioma cells but no drug. They monitor the invasiveness of the glioma cells, and if no invasion is observed, the other wells stand no significant meaning.
- Observe over the course of 3-5 days and take images regularly to document the number and status of cells, migrations, and invasion.
Methodology for Integrated Human Practices
Throughout the human practices process, we wanted to ensure that we actively engaged with individuals and groups outside of the research bubble to provide context to the work that we are doing. To ensure stakeholder concerns were implemented in a productive and considerate manner, the team developed a novel four step human practice engagement cycle, called the DUKE Cycle:
Figure 2. The human practices DUKE cycle
- Discovering the problem: In this phase, the team begins to define topics of concern to address and gain a deeper understanding of.
- Understanding the problem: In this phase, the team conducts stakeholder and expert interviews in order to learn more about current attitudes on the issues. The team also reflects upon information from the interviews, generates summaries, and creates connections between their project and the world.
- Kickstarting positive action to address stakeholder concerns: In this phase, the team collected the concerns and issues raised by stakeholders. The team then incorporated these concerns into their project, undertaking various efforts to explore and address these issues.
- Evaluating and Refining Project Plans: In this phase, the team evaluates the efforts that were undertaken and analyzes next steps that would allow for further engagement.
The DUKE cycle provides a simple four-step framework for considering how our work impacts the world and how the world impacts our research, allowing our investigations to remain focused and contextualized while considering the perspectives of all key stakeholders. This cycle facilitates the incorporation of human practices work into actual lab research in an integrated manner by encouraging users to actively apply what they learn.