Single-cell isolation from full-thickness human intestinal tissue resections for single-cell RNA sequencing

Understanding cellular heterogeneity in complex tissues requires methods that preserve individual cell identity. Traditional bulk sequencing obscures rare cell types and interactions. Single-cell techniques offer a solution by analyzing transcriptomes at the single-cell level. However, isolating viable cells from dense tissues remains a challenge. Intestinal tissues are especially complex due to layered structures and diverse cell types. Prior research has shown that mechanical and enzymatic dissociation can damage cells. No prior work had resolved how to isolate full-thickness intestinal cells without compromising quality. This gap motivated the development of a refined protocol for single-cell isolation from human intestinal resections.

The aim of this work is to provide a detailed method for isolating single cells from full-thickness intestinal tissue. The specific problem is the difficulty in obtaining high-quality cell suspensions from such tissues. The motivation stems from the need to study cellular diversity in gastrointestinal diseases. A reliable protocol is essential for downstream single-cell RNA sequencing. This approach allows researchers to examine lamina propria and muscular layers separately. It also ensures that red blood cells are effectively removed. The method supports the use of the 10× Chromium platform for sequencing. This protocol fills a critical need in tissue dissociation techniques.

The protocol begins with pre-processing steps to prepare intestinal tissue resections. It then isolates lamina propria and muscular layers using mechanical and enzymatic methods. Red blood cell lysis is performed to remove erythrocytes from the cell suspension. Fixation of isolated cells is carried out to preserve their RNA integrity. Cell quality is assessed using viability and morphological criteria. The resulting suspension is compatible with the 10× Chromium platform. Each step is optimized to minimize cell damage and contamination. The method is designed for use with human surgical specimens.

The protocol successfully isolates viable single cells from full-thickness intestinal tissue. Lamina propria and muscular layers are separated with high efficiency. Red blood cell lysis reduces contamination to less than 1%. Fixed cells maintain RNA integrity suitable for sequencing. The cell suspension yields over 80% viability as measured by staining. The method is compatible with the 10× Chromium platform for sequencing. Quality control metrics indicate minimal cell stress or damage. The protocol is reproducible across multiple tissue samples.

The authors state that this protocol enables high-quality single-cell isolation from human intestinal tissue. It allows for the separation of lamina propria and muscular layers without significant cell loss. Red blood cell lysis is effective and does not compromise cell viability. Fixation preserves RNA integrity for downstream sequencing. The method is suitable for use with the 10× Chromium platform. The results suggest that this approach is reliable for studying intestinal cell heterogeneity. The protocol provides a standardized method for researchers in the field. The authors propose that this technique supports investigations into gastrointestinal diseases.