In vitro culture of blood cells from the colonial protochordate Botryllus schlosseri

Primary cultures of circulatory blood cells from the colonial tunicate Botryllus schlosseri were cultivated in 96-well plates for up to 3 mo. in a medium based on Dulbecco's modified Eagle's medium, supplemented with salts to the botryllid ascidian hemolymph osmolarity, HEPES buffer, l-glutamin, fetal bovine serum, and antibiotics. Intercellular bridges between granular pigment cells were established within 24 h. The viability of these cells decreased slowly, and most died within 1 mo. without any sign of cell proliferation. Other cell types remained in an arrested state and were subjected to a weekly medium exchange. Spontaneous cell proliferation was randomly recorded in 6 to 10% of the wells from 2 wk to 1 mo. This proliferation was followed by the formation of masses of cell clumps, from which uniform hemocytes (5 μm, lymphocytelike cells) migrated peripherally. Stress conditions, which included longer intervals between medium exchanges and partial medium replacement, increased the probability of cell proliferation. From each proliferating primary culture, we successfully performed up to 10 plating cycles over a period of 15 wk, during which the cells differentiate in size but are uniformly structured. This produced the first Botryllus lymphocytelike cell line. From this stage, cell numbers remained constant for up to 6 mo. without increase in cell number. Several mitogenic factors were employed on primary cultures. Botryllus and sea cucumber hemolymphs and mixed interleukins were found to augment significantly proliferation of at least one specific cell size, whereas cells were not markedly responsive to lectins (Concavalin A, wheat germ agglutinin, Ulex europaeus agglutinin), insulin, and retinoic acid. The results are discussed with respect to future efforts in the development of tunicate blood cell cultures.