Practice question

Conversion of solid animal tissue into single cell suspension requires enzymatic disaggregation targeting extracellular matrix and intercellular junctions. Trypsin, serine protease from porcine pancreas, hydrolyzes peptide bonds on carboxyl side of basic residues lysine and arginine abundant in adhesion proteins fibronectin, laminin, and cadherin extracellular domains. Collagenase, derived from Clostridium histolyticum, specifically cleaves triple-helical native collagen rich in proline-hydroxyproline-glycine repeats found in connective tissue, basement membrane, and cartilage, requiring calcium cofactor and causing less damage to cell surface receptors than crude trypsin. Combined use enhances yield, for example collagenase pretreatment loosens stroma followed by trypsin for epithelial release. DNase I often supplemented to digest genomic DNA released from damaged cells preventing clumping. Amylase degrades starch, lipase hydrolyzes fats, and proteinase K excessively digests membrane proteins leading to low viability, making trypsin-collagenase combination standard for primary culture establishment from embryonic, hepatic, and neoplastic tissues. Viability assessed by trypan blue exclusion ensures disaggregation protocol gentle enough for downstream culture. This knowledge strengthens laboratory safety, protocol reproducibility, and regulatory compliance critical for translational research and clinical applications, ensuring reliable data and workforce protection.