SUMMARY: Teehniques are deseribed for the dissociation, fractionation through Percoll, and in vitro maintenance of mucosal epithelia from the human or guinea pig large bowel. Tissue recovered after surgeu is predigested with trypsin-citrate and treated subsequently with a mixturc of trypsin, citrate, and collagenase. The resulting suspension of single cells, cell clusters, and partially digested crypts is suspended over a Percoll solution and enriched in multicellular elements by two sequential centrifugations. The recovered multicellular complexes are inoculated to specially treated culture vessels in a serum-free medium supplemented with epidermal growth factor, insulin, transferrin, selenium, and bovine pituitary extract. Epithelia, characterized as such by transmission electron microscopy, adhere to the substrate, form colonies, and can be maintained routinely for study for at least 10 wk.
Thirty‐nine of 106 algal strains tested were successfully lyophilized using at least one of the following as suspending agents: 20% (w/v) skim milk, 12% (w/v) sucrose, 100% lamb serum and 100% horse serum. The majority of the Chlorella and Scenedesmusstrains tested (27/40) were amenable to freeze‐drying. Much less success (3/18) occurred with the Chlamydomonas strains tested. At least one strain of Ankistrodesmus, Bracteacoccus, Characium, Trebouxia, Haematococcus, Coccomyxa, Interfilum, Hormidium and Nephrodiella were also recovered. More strains were recovered with 20% skim milk as the protective agent than with 12% sucrose; however, 12% sucrose generally provided higher yields. Freeze‐drying appears to be an effective method of preservation far some algal strains.
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