Research Spotlight: Comparison of Single-Cell Profiling Methods Reveals Strengths and Limitations

David A. Drew, PhD, of the Clinical & Translational Epidemiology Unit and Division of Gastroenterology at Massachusetts General Hospital, is the corresponding author of a paper published in Cellular and Molecular Gastroenterology and Hepatology, “Droplet vs. Picowell: Considerations for single-cell transcriptomic profiling of human colon biopsies

How would you summarize your study for a lay audience?

Research on gastrointestinal diseases, especially cancer, has mainly looked at the epithelial cells, which line the surfaces of organs, are important for various functions, and are believed to be the cells that go awry to cause these diseases. However, studying these cells using single-cell RNA sequencing (scRNA) methods is tricky because the cells are delicate and can be damaged when taken out of their natural environment in the mucosal lining of the gut.

Droplet-based scRNA (D-scRNA) methods have provided many insights into these cell environments, but this approach can stress the epithelial cells and requires a lot of resources. Picowell-based (P-scRNA) platforms have emerged as a potentially gentler and more resource-efficient alternative to D-scRNA methods. However, no direct comparison between D-scRNA and P-scRNA methods for studying human mucosal biopsies, which are crucial for gastrointestinal research, has been done yet.

In our study, we directly compared these two approaches.

Our results highlight that there are unique advantages and disadvantages to each of the platforms. These variations pose significant challenges in maintaining rigor and reproducibility across studies that use different techniques. We anticipate that this study will help researchers more efficiently approach projects employing scRNA on human biopsy samples.

What approach did you use?

Human colon biopsies were collected and prepared using identical methods to allow for the analysis of the same set of cells with both D-scRNA and P-scRNA to directly compare the results. Factors such as the number of cells, gene coverage and mitochondrial DNA contamination were included in our analysis to better understand the strengths and limitations of each method.

What did you find?

We found clear differences between the D-scRNA and P-scRNA platforms and that each method has significant differences in the types of cells captured and the genes measured within them.

D-scRNA appears to provide more detailed insights into the environment of the mucosal lining and within individual cells, by capturing more cells and providing higher gene coverage. However, it requires more resources (i.e. time, money), adds stress to the cells and there was a higher rate of contamination from mitochondrial DNA, both of which may influence results if not carefully accounted for.

P-scRNA is gentler on cells, more resource efficient and better at preserving cell quality in terms of mitochondrial contamination. Although, it isn’t as effective in capturing large quantities of cells or the full, wide array of cell types present in the mucosal lining, especially in rare cell-types, which may have clear importance for the disease a researcher is studying.

What are the implications?

Our results can help scientists better understand the context for interpreting their scRNA findings when using either D-scRNA or P-scRNA methods. Additionally, by sharing these strengths and weaknesses we believe we can help researchers determine the appropriate platform to use for their own studies without them requiring additional testing on their own.

What are the next steps?

We seek to apply this knowledge to optimize our approach for analyzing biopsies collected from patients enrolled in our precision prevention clinical trials. These clinical trials aim to better understand the effects of potential anti-cancer and protective agents, including aspirin, omega-3-fatty acids, coffee and incretin mimetics (GLP1-receptor agonists), on the colon tissue microenvironment and prevent the occurrence of early-onset colorectal cancer and gastrointestinal cancer.

Authorship: In addition to Dr. Drew, Mass General Brigham authors include Jonathan M. Downie, Connor M. Geraghty, Alexander Caraballo, Shijie He, Kylor Lachut and Andrew T. Chan.

Paper cited: Downie, J. M. et al., “Droplet vs. Picowell: Considerations for single-cell transcriptomic profiling of human colon biopsies,” Cellular and Molecular Gastroenterology and Hepatology DOI: 10.1016/j.jcmgh.2025.101503

Funding: This work was supported by the National Institutes of Health-National Cancer Institute (NCI) (R01 CA257523; R35 CA253185), Harvard School of Public Health (T32 CA009001 (NCI)), the NIH Loan Repayment Program (NIDDK; L30DK137289), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (K01DK120742).

Disclosures: N/A