Status update (December 2024)

The following is a status update of the project as of December 2024. The project officially started in spring 2024 after we received enough funding and is still ongoing. This lab note is a rough summary of the progress and also what we plan to do next.

Highlights

• Received a $250,000 grant from GFI to fund the remaining cost of the experiment, as well as follow-up experiments. This will allow us to generate more multiomic data for other fish species, as well as improve desired phenotypes to create more robust fish cell lines. The grant also allows us to expand the scope of the project to focus more broadly on areas related to cell line development and optimization for cultivated fish. We're the first academic team in Canada to be working on cultivated fish.

• Antibody characterization: One of the goals of the GFI grant was to test and validate antibodies for fish. We tested a few desmin, Pax7, and myosin heavy chain primary antibodies in order to better understand the cell types in KFE-5 as well as to use them to characterize myogenesis. The hope was that this would give us a quick way of learning more about the cells prior to RNA-seq. However, most of these antibodies didn't perform too well and didn't give us signal. The lack of validated antibodies, especially in fish, is a common barrier in the cell-ag field. We think there's a lot of opportunity for future work in this area but as of now we think it's too time consuming and expensive for us to continue to test antibodies. Going forward, future work in 2025 will be more focused on the omics rather than antibodies.

  • One suggestion we would give to researchers in this field is to just start with existing antibodies found in literature, and use them one at a time. For example, a common myosin heavy chain antibody used in fish cell-ag projects is MF 20. We spent a lot of time trying to find ideal antibodies so we can combine multiple antibodies for multiple markers in a single experiment (such as measuring changes in pax7, desmin, and myosinHC over the course of differentiation). This complicates things and adds a lot of other considerations like secondary antibody signal overlap, steric hindrance, host species, conjugation, and additional controls. Try to keep things simple and logistically easy. Immunocytochemistry is known to be a good, reliable, and cheap method for antibody validation. Western blot is logistically harder and more time consuming.

• Thawed multiple versions of KFE-5: We found two different versions of the KFE-5 cell lines that behave differently. Both versions are immortalized. Qualitatively, it seems like version 1 has elements of spontaneous differentiation, while it seems like version 2 has lost a lot of differentiation potential. We want to confirm whether this is the case at the omics level. Going forward, in early 2025 we plan to set up a bulk RNA-seq comparison experiment to better understand the differences at a transcriptomic level. We believe this work will be valuable in understanding mechanisms of differentiation and also provide datapoints as to how fish cell lines behave post-immortalization.

  • There are a lot of additional simple experiments that we can do such as using conditioned media (for example: if we use the media for version 1, does it suddenly allow version 2 to differentiate), testing different growth factors, and trying different animal serums (we have access to trout serum).

  • We've received comments from researchers in the field that understanding abiotic factors such as temperature, oxygen, and pressure on cell line performance is also crucial. Fish exist in different environments and matching the environmental conditions to what their cells are used to may unlock a lot of performance.

  • There is also a lot of unmapped opportunity to incorporate bioelectricity and electrical stimulation into cultivated meat development.

• Modified research goal: While we originally had planned to do scRNA-seq, our new goal is to execute a bulk RNA-sequencing experiment on the two versions of KFE-5 first to help generate follow-up hypotheses. We believe that given the state of the cellular agriculture field, any new omics data would be useful and bulk RNA-seq data allows us to get results faster and at a lower cost. This is what we plan to do in 2025.

• Outreach: We were constantly in contact with genomic and Core facilities to understand logistics, pricing, and experimental considerations for omic experiments. We are also grateful to have many advisors help advise us on optimal experimental setup such as methodology, replicates, and bioinformatic considerations. We're also grateful to have started many collaborations with organizations such as the Good Food Institute and New Harvest to explore the connections of alternative protein development with public health and AI/ML.

  • There are a lot of exciting developments in the Canadian cultivated meat and alternative protein ecosystem that's worth tracking:

    • A new Institute of Cellular Agriculture at the University of Alberta, led by the first tenure-track academic for cellular agriculture in Canada

    •  $10 million grant co-led by 5 universities titled, “Omics guided technologies for scalable production of cell-cultivated meat”

    • A UofT project focused on recombinant growth factor production (with follow up work emerging at UCalgary)

    •  $2.5 million grant in developing sustainable bioprocesses that can convert waste into valuable commodities, such as for cultivated meat media recycling

    • Setting the blueprint for how machine learning can/will be used in cultivated meat 

    • Establishing robust safety standards for cell-based foods, used to inform global policy organizations 

    • Defining the field’s conversation around scale up