Oxidation-deficient silkworm hemolymph as a medium supplement for insect cell culture |
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Authors: | Eun Jeong Kim Ji-Young Choi Sam-Eun Kim Tai Hyun Park |
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Institution: | (1) School of Chemical Engineering, Seoul National University, 151-742 Seoul, Korea;(2) Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, 441-100 Suwon, Korea |
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Abstract: | Hemolymph is oxidized and darkens visibly during the collection from silkworms due to the activity of tyrosinase in it. Toxic
quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the
oxidation; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. It makes the hemolymph
collection difficult especially on a large-scale preparation. Hemolymphs collected from 257 different strains of silkworms
were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as Lemone, TBO, Cre,
Y4, and wEb showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer.
The hemolymph of wEb showed the slowest oxidation. The absorbance of this mutant hemolymph reached the saturation value at 20°C in 450 min, whereas
the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. We tested if this mutant
hemolymph is useful as a medium supplement for insect cell culture. Cell growth rate and final cell concentration in the medium
supplemented with the wEb hemolymph were almost same as those in the medium supplemented with the wild-type hemolymph. Hemolymph is collected on a
small scale by clipping the abdominal leg; however, this method is not appropriate for large scale preparation. Centrifugation
after chopping the silkworm hemolymph by a blending mixer is a more appropriate procedure for large scale collection. Slowly
oxidized wEb hemolymph resulted in higher cell concentration than the wild-type hemolymph when hemolymph was collected by the large scale
preparation method. |
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Keywords: | silkworm hemolymph insect cell oxidation medium supplement |
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