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In this interview, originally featured at ISSCR 2023 in Boston, we talked to Liliana Vertel, a researcher out of the Kannu Lab at the University of Alberta. Here she discusses the impact of newer technologies including Captivate Bio’s CET Cocktail and in understanding biological pathways that affect the growth and maintenance of the skeletal system.
Liliana Vertel, a researcher from the lab of Dr. Peter Kannu in the Molecular Genetics Department at the University of Alberta, focused on understanding the biological pathways that affect the growth and maintenance of the skeletal system. Specifically, Liliana is developing a human model to study the pathophysiology of genetic Type II collagenopathies by engineering the disease mutation in iPSC lines using CRISPR gene editing techniques. Liliana has always been interested in understanding the genetic mechanisms of disease and human development and finds the ability to modify the human genome and generate human tissue for disease research fascinating.
Vertel: I’m working to engineer hiPSCs to harbor the genetic mutations that cause certain phenotypes of Type II collagenopathy, and then to differentiate these cells to cartilage tissue. Ultimately, I’m trying to create an in vitro human disease model that will allow researchers to study the disease and use the generated tissue as a platform for drug screening. This is critical for Type II collagenopathies since there is currently no human model to study the disease.
Generating the modified cell lines involves gene editing iPSCs using CRISPR/Cas9 to generate a heterozygous mutation via homology directed repair. I’m also incorporating a labelled tracer RNA to identify positive clones. After transfecting the iPSCs with the CRISPR/Cas9 complex and donor template, the cells are sorted by flow cytometry, and those that are positive for the tracer RNA are plated as single cells in a 96-well plate. The resulting colonies from those clones will be expanded and further screened for incorporation of the desired edit. Those that contain the specific mutation will be cultured as a disease-modified cell line.
We know that iPSCs spontaneously differentiate or die when they are in a single cell state, so I’m supplementing the early cultures with the CEPT strategy to minimize the possibility of this happening and improve the survival of new clones.
Before trying the CET Cocktail Kit, I was using the ROCK inhibitor Y27632. I was looking for strategies to improve iPSC single cell survival, and found the CEPT publication by the NIH, which reported genetically stable cell lines. The Captivate Bio CET Cocktail Kit was a convenient way for me to incorporate this strategy.
For my research, improved cell survival means I can generate more clones and stable cell lines. Therefore, It is very important to minimize cell stress and spontaneous differentiation, in order to improve the survival of the single cells going through the CRISPR gene editing process and to continue culturing the positive single cells after being sorted by flow cytometry.
Before starting the gene editing process, I primed my cells for 7 passages by supplementing them with CET Cocktail Kit plus polyamine solution (to make CEPT) during each passage. I wanted them to “get used to it” so I could start the gene editing experiment with very happy cells. I also added the small molecule cocktail to the media in the 96-well plate where they were seeded as single cells after FACs sorting to improve the attachment and viability of the positive clones. I’ve been very happy to see multiple small colonies attached and growing in my 96-well plate in only a week after seeding the single-cell clones.
Once the supplemented cells had been dissociated, I saw improvement in cell viability and attachment. Surprisingly, these cells that had been passaged with CEPT had very few unattached colonies and dead cells. In contrast, the cells that had not been supplemented contained a considerable amount of cell debris. The difference was striking for me, since I was so used to seeing cell death and poor attachment, even when using a ROCK inhibitor.
CEPT supplementation improves our ability to generate stable clonal cell lines for important applications. Additionally, overall improvement in cell survival will save on lab resources – money, time, and hands-on time.
Representative images of RVR-COL2A1-GFP iPSC line, captured 24 hours post-passage. RVR-COL2A1-GFP iPSC cells (courtesy of the Guliak Lab) were cultured in mTeSR1 media on Matrigel. Colonies were passaged with and without CEPT cocktail supplemented in the culture medium for the first 24 hours of each of 7 consecutive passages. Left: iPSC colony passaged with CEPT cocktail, showing excellent cell attachment and very minimal cellular debris 24 hours after passaging. Right: iPSC colony passaged with Y27632 ROCK inhibitor, showing cellular debris in culture 24 hours post-passage. These representative images show that CEPT promotes cell attachment and survival, preventing the dissociation-induced apoptosis during passaging.
As the first to launch the CET cocktail for hPSC research, Captivate Bio is committed to supporting our customers with new technology to speed discovery. We are actively working with and recruiting labs in validating various combinations of the CET Cocktail Kit and our new Captivate Bio Neuro Cocktail Kit through our Collaboration Program, contact us today to learn more.
Learn more about how we support researchers with cell culture products, contract manufacturing and storage services, custom reagent sourcing, as well as marketing, sales, and distribution programs for our industry partners.
The CEPT cocktail is a ground-breaking supplementation strategy for improving hPSC viability and survival under stressful conditions. Read more to learn how CEPT can keep your cells happy with a chemical hug.
In this interview, we talk to EverCell Bio’s Philip Manos about the challenges with hPSC cultures and how ROCK Inhibitors including Captivate Bio's CET Cocktail compares to traditional methods.
CEPT supplementation improves hPSC single-cell passaging, differentiation, cryopreservation, cloning, and other manipulations. Meet the new CET Cocktail from Captivate Bio.
