A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

Abstract

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

Keywords: Hemolymph; Long-term cell culture; Preparation of growth medium for shrimp cells; Shrimp; Shrimp cell medium.

Graphical abstract

Fig. 1

Diagram of the preparation process of shrimp tissue extracts.

Fig. 2

Diagram of the preparation process of the optimal shrimp cell growth medium.

Fig. 3

The volume percentages of each of the stock solutions and other supplements in the optimal shrimp cell growth medium.

Fig. 4

Diagram of the drawing of shrimp hemolymph using a 2.5-mL syringe. Panel A shows the sampling site at the ventral sinus between the pereiopods and pleopods of shrimps as indicated in red circle. Panel B shows the process of drawing out of shrimp hemolymph.

Fig. 5

The light micrographs of shrimp hemocytes cultured in the optimal shrimp cell growth medium for 36 (A) and 90 days (B), respectively. Scale bar, 50 µm.