Department of Parasitology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
PLoS One. 2013 Aug 20;8(8):e72084. doi: 10.1371/journal.pone.0072084. eCollection 2013.
The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.
本研究试图在含有最低必需培养基（MEM）、氨基酸（AA）、胺（AM）、脂肪酸（FA）、碳水化合物（CA）和 20%胎牛血清（FCS）的完全定义培养基中，从第三期幼虫（AcL3）体外培养到第四期幼虫（AcL4），从而成功地培养广州管圆线虫（Angiostrongylus cantonensis）。当 AcL3 在 37°C、5%CO2 气氛的完全定义培养基中培养时，幼虫在培养 30 天后开始发育成 AcL4，并被包裹在生命周期的第三次蜕皮鞘内。在这些条件下，幼虫均匀发育，36 天到达第四期。记录和分析 AcL3 发育为 AcL4 的形态。然后比较体外培养在完全定义培养基中和体内感染 BALB/c 小鼠培养的广州管圆线虫幼虫形态和发育。体外培养的幼虫比感染 BALB/c 小鼠的 AcL4 幼虫小。然而，在不完全定义培养基（MEM 加 20%FCS 加 AA+AM、FA、CA、AA+AM+FA、FA+CA、CA+AA+AM 或不添加任何物质）中培养的 AcL3 不能充分存活和发育。因此，可以推断只有完全定义的培养基才能使 AcL3 在体外发育为 AcL4。一些线虫已经成功培养成成熟的蠕虫，但只有少数研究成功地在体外培养广州管圆线虫。本研究首次成功地通过体外培养将 AcL3 发育为 AcL4。最后，本文的体外培养研究结果有助于改善广州管圆线虫的有效发育和生长的培养基。因此，解决了幼虫在体外从第三期幼虫到第四期幼虫培养过程中广州管圆线虫生命周期的空白。
