New method unlocks insights from preserved tissues

A century of preserved human tissue may soon become a far richer source of medical information. Researchers at Vanderbilt University have developed a method that helps scientists recover intact cells from formalin-fixed, paraffin-embedded tissue, or FFPE, and then analyze the genetic activity inside those cells using single-cell RNA sequencing. The approach, described in a study led by Vanderbilt researchers James Evans and Joey Simmons, could give modern molecular tools access to hospital archives filled with tissue samples collected long before today’s sequencing technologies existed.

For decades, hospitals and pathology laboratories have preserved human tissue by immersing it in formalin, a solution based on formaldehyde, and embedding it in paraffin wax. The process stabilizes tissue so that it can be sliced into extremely thin sections, stained and examined under a microscope. This routine has created vast archives containing samples from patients with cancer, inflammatory disease and many other conditions. In many cases, the clinical histories and treatment outcomes of those patients are also known, making the archives potentially invaluable for researchers seeking biological explanations for disease progression or differences in treatment response.

The challenge is that formalin preservation can chemically alter biological molecules. Formaldehyde creates crosslinks between proteins and between proteins and nucleic acids, helping preserve the physical structure of cells but damaging or obscuring molecular information. Paraffin embedding adds another obstacle because the wax must be removed before tissue can be processed. RNA is particularly vulnerable. Unlike DNA, which is relatively stable, RNA molecules are fragile and can fragment or become chemically modified during fixation and storage. These changes have made it difficult to apply single-cell RNA sequencing, or scRNA-seq, to older FFPE samples.

ScRNA-seq has transformed cell biology by allowing researchers to examine gene activity one cell at a time. In a conventional tissue analysis, RNA from thousands or millions of cells may be mixed together, producing an average molecular profile that can conceal important differences. Single-cell sequencing preserves those distinctions. It can reveal which genes are active in individual cells, identify rare cell populations and show how neighboring cell types respond differently to disease, aging or treatment. Because RNA reflects which genetic instructions a cell is using, scRNA-seq provides a molecular snapshot of cell identity and function rather than merely showing the cell’s appearance under a microscope.

Earlier methods for analyzing FFPE tissue often focused on isolating cell nuclei. Nuclei are more resistant to the damage caused by preservation and contain genetic material that can still be sequenced. However, isolating only the nucleus removes the surrounding cytoplasm, where substantial amounts of RNA and other molecular information are found. This loss can reduce the number of detectable genes and make it harder to distinguish closely related cell states. It can also bias the results toward cell types whose nuclei survive the isolation procedure particularly well, leaving researchers with an incomplete representation of the original tissue.

The Vanderbilt team built on an intact-cell dissociation procedure developed by Simmons to recover more complete cells from FFPE samples. The method is designed to release whole cells from preserved tissue while minimizing the loss of cellular material outside the nucleus. Once recovered, the cells can be processed for scRNA-seq. In practical terms, this means that sequencing libraries can contain RNA from both the nucleus and the cytoplasm, increasing the amount of information available from each cell. The researchers reported that their procedure improved cell recovery and gene detection, two critical measures of performance in single-cell experiments.

The study tested the approach in samples from the colon and thymus, tissues with markedly different cellular structures. The colon contains relatively large epithelial cells lining its surface, along with other cell types embedded in a complex tissue architecture. These cells contain substantial cytoplasmic material beyond the nucleus, so recovering them intact can preserve information that nuclear sequencing would miss. In the colon samples, the new procedure produced a clear improvement in whole-cell recovery and gene detection. It also helped recover cell populations that are often underrepresented in single-cell experiments, offering a more complete view of the tissue’s cellular composition.

The results were less striking in thymus tissue, which contains many smaller immune cells. These cells have less cytoplasmic material outside the nucleus, meaning that the advantage of preserving the entire cell may be more limited. The contrast between the two tissue types suggests that intact-cell dissociation will not provide the same benefit for every archived sample. Instead, its value may depend on cell size, tissue structure, the extent of fixation damage and the physical interactions holding cells together. The findings also show why sample-specific optimization will remain important as researchers attempt to apply molecular technologies to historical specimens.

The potential scientific payoff extends beyond improving a laboratory protocol. Researchers could use archived tumors to compare the individual cells of patients who responded well to a therapy with those of patients whose disease progressed. They could investigate whether rare cell populations were associated with relapse, determine how immune and epithelial cells interacted within diseased tissue, or identify molecular signatures linked to survival. Because many FFPE samples are accompanied by detailed clinical records, such studies could connect gene activity at the cellular level with real-world outcomes. That combination could help reveal how diseases develop, explain why treatments work for some patients but not others, and identify biomarkers or therapeutic targets that would be difficult to discover from newly collected samples alone.

The work also highlights a broader shift in biomedical research: technologies once designed for fresh tissue are increasingly being adapted to the preserved materials already stored in hospitals worldwide. The Vanderbilt researchers caution that FFPE samples still present technical limitations, including RNA degradation and preservation-induced chemical changes, and that the thymus results demonstrate the method’s boundaries. Nevertheless, by recovering intact cells rather than only nuclei, the procedure opens a path toward turning decades of largely inaccessible pathology material into high-resolution molecular maps. The study, published in Cellular and Molecular Gastroenterology and Hepatology, suggests that the next generation of discoveries may come not only from collecting new samples, but also from looking again at the vast biological record already sitting in medical archives.

News Publication Date: 13-Aug-2026

Web References: https://doi.org/10.1016/j.jcmgh.2026.101863; https://ncbi.nlm.nih.gov/geo/ (dataset accession: GSE338505)

References: Cellular and Molecular Gastroenterology and Hepatology; DOI: 10.1016/j.jcmgh.2026.101863