Crustacean cell culture: historical developments, methodological advances, and future directions

Crustaceans represent diverse and ecologically important group of marine invertebrates. However, their application in in vitro research remains limited due to the absence of immortal crustacean cell lines. Historically, the development of cell culture methodologies for crustaceans has been constrained by slow proliferation rates, complex and poorly defined nutritional requirements, species-specific osmotic demands, and fundamental differences in cell cycle regulation compared with vertebrate systems. Early cell culture efforts associated with these organisms relied upon modified vertebrate media and their optimization, yielding short-term primary cultures that supported limited functional analyses but failed to achieve continuous proliferation. In crustaceans, hemocytes have been the most extensively studied cell type due to their immunological relevance. But their functional heterogeneity and sensitivity to artificial environments have further limited long-term viability in in vitro systems. Recent methodological progress has substantially contributed to the development of crustacean primary cell culture systems through the integration of stem cell-oriented approaches, tissue-specific media optimization, and molecular characterization strategies. The identification of hematopoietic progenitor-type and stem-like cells, adjustment of culture media osmolarity, and the application of transcriptomic and single-cell sequencing technologies have contributed to improved longevity, phenotypic stability, and functional maintenance of crustacean cell cultures in vitro. In addition, emerging approaches, including three-dimensional culture systems and extracellular matrix scaffolds, have enhanced cell attachment and differentiation potential. Here, the present review describes the historical developments, latest advances in crustacean cell culture, and discusses the critical technological gaps that must be addressed to establish controlled, reproducible, and sustainable crustacean cell lines.