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首页> 外文期刊>Biotechnology Progress >Sustained Growth of Explants from Mediterranean Sponge Crambe crambe Cultured In Vitro with Enriched RPMI 1640
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Sustained Growth of Explants from Mediterranean Sponge Crambe crambe Cultured In Vitro with Enriched RPMI 1640

机译：丰富的RPMI 1640体外培养的地中海海绵Crambe crambe外植体的持续生长

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Marine sponges are potential sources of many unique metabolites,including cytotoxic and anticancer compounds.Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest.It is commonly accepted that tissue (fragments,explants,and primmorphs)and in vitro cell cultivation show great potential.However,there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow.In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC)(e.g.,DMEM,RPMI,Ml99,L-15,etc.).One of the reasons for this failure is the use of hardly identifiable growth promoters,based on terrestrial animal sera.An alternative is the use of extracts from marine animals,since they may contain nutrients necessary for growth.In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe.It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins).Initially a new approach was developed in order to show consumption of DOC by explants.Thus,different initial DOC concentrations (300,400,700 and 1200 mg DOC L~(-1))were assayed.Consumption was evident in all four assays and was more marked in the first 6 h.The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates.Second,a protocol was established to cultivate explants in vitro.Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed.The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v)resulted in an evident sustained long-term growth of C.crambe explants.This growth enhancement produced high metabolic activity in the explants,as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose.The lactate accumulation increased with the size and age of explants.Prior to these experiments,we successfully developed a robust new alternative method,based on digital image treatment,for accurate determination of the explant apparent volume as growth measure.
机译：海洋海绵是许多独特代谢物的潜在来源，包括细胞毒性和抗癌化合物。天然海绵种群不足或无法产生商业量的目标代谢产物。组织（碎片，外植体和原形）和体外细胞通常被公认养殖显示出巨大的潜力。然而，人们对海洋海绵进行有效，持续的体外培养所需的营养知识知之甚少，而且进展缓慢。在海洋无脊椎动物中，使用典型的商业化方法对体外培养进行了许多不成功的尝试。基于溶解有机碳（DOC）来源（例如DMEM，RPMI，Ml99，L-15等）的动物细胞培养基。造成这种失败的原因之一是使用了基于陆生动物的难以识别的生长促进剂血清。另一种方法是使用海洋动物提取物，因为它们可能含有生长所需的营养。它是地中海海岸线上最丰富的海绵之一，是地中海海岸线上最丰富的海绵之一，并且还具有一系列潜在的活性代谢产物（Crambines和Crambescidins）。最初开发了一种新方法来显示食用通过外植体测定DOC。因此，测定了不同的初始DOC浓度（300,400,700和1200 mg DOC L〜（-1））。在所有四个测定中的消耗量均明显，并且在开始的6小时内更显着.DOC吸收数据调整为第二，建立了一种体外培养外植体的实验方案。分析了基于富含氨基酸和无机盐的RPMI 1640商业培养基模拟海水盐度的不同培养基配方。用章鱼水提取物以10％和20％（v / v）的比例富集该培养基会导致长期持续的明显生长由于C.crambe外植体的生长，这种生长的增强在外植体中产生了高代谢活性，这可以通过更新后几天培养基中的铵和乳酸含量高以及葡萄糖的消耗得到证实。在这些实验之前，我们成功地开发了一种可靠的新替代方法，该方法基于数​​字图像处理，可以准确确定外植体的表观体积作为生长量。

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