Garcia Camacho F, Chileh T, Cerón García M C, Sanchez Mirón A, Belarbi E H, Contreras Gómez A, Molina Grima E
Department of Chemical Engineering, University of Almería, 04120 Almería, Spain.
Biotechnol Prog. 2006 May-Jun;22(3):781-90. doi: 10.1021/bp050341m.
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, M199, 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.
海洋海绵是许多独特代谢产物的潜在来源，包括细胞毒性和抗癌化合物。天然海绵种群数量不足或难以获取，无法生产出商业数量的目标代谢产物。人们普遍认为组织（碎片、外植体和原肠胚）以及体外细胞培养具有巨大潜力。然而，对于海洋海绵进行高效且持续的体外培养所需的营养需求了解甚少，进展也很缓慢。在海洋无脊椎动物中，使用基于溶解有机碳（DOC）来源的典型商业动物细胞培养基（如DMEM、RPMI、M199、L - 15等）进行体外培养的尝试大多未成功。失败的原因之一是使用了基于陆生动物血清的难以确定的生长促进剂。另一种选择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物质。在这项工作中，我们对包被型海洋海绵Crambe crambe的外植体进行了体外培养。它是地中海海岸线上最丰富的海绵之一，还拥有一系列潜在的活性代谢产物（克拉明和克拉姆贝西丁）。最初，我们开发了一种新方法来显示外植体对DOC的消耗。因此，测定了不同的初始DOC浓度（300、400、700和1200 mg DOC L(-1)）。在所有四个试验中都明显观察到了消耗，且在前6小时更为显著。DOC同化数据被调整为一个广泛用于海洋无脊椎动物中有机溶解化合物吸收动力学的经验模型。其次，建立了一个体外培养外植体的方案。测定了基于RPMI 1640商业培养基并添加氨基酸和无机盐以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，导致C. crambe外植体明显长期持续生长。这种生长增强使外植体具有高代谢活性，这在培养基更新几天后培养基中高铵和乳酸含量以及葡萄糖消耗中得到证实。乳酸积累随着外植体的大小和年龄增加。在这些实验之前，我们成功开发了一种基于数字图像处理的强大新方法，用于准确测定外植体的表观体积作为生长指标。
