Chevaleyre Jean, Rodriguez Laura, Duchez Pascale, Plainfossé Marie, Dazey Bernard, Lapostolle Véronique, Vlaski Marija, Brunet de la Grange Philippe, Delorme Bruno, Ivanovic Zoran
1 Etablissement Français du Sang Aquitaine-Limousin, Bordeaux, France .
Stem Cells Dev. 2014 Aug 1;23(15):1820-30. doi: 10.1089/scd.2014.0046. Epub 2014 May 20.
During storage and transportation of collected cord blood units (CBUs) to the bank prior to their processing and cryopreservation, it is imperative to preserve the functional capacities of a relatively small amount of cells of interest (stem and progenitor cells) which are critical for graft potency. To improve CBU storage efficiency, we conceived an approach based on the following two principles: (1) to provide a better nutritive and biochemical environment to stem and progenitor cells in CB and (2) to prevent the hyperoxygenation of these cells transferred from a low- (1.1%-4% O2 in the CB) to a high-oxygen (20%-21% O2 in atmosphere) concentration. Our hypothesis is confirmed by the functional assessment of stem cell (hematopoietic reconstitution capacity in immunodeficient mice-scid repopulating cell assay) and committed progenitor activities (capacity of in vitro colony formation and of ex vivo expansion) after the storage period with our medium (HP02) in gas-impermeable bags. This storage procedure maintains the full functional capacity of a CBU graft for 3 days with respect to day 0. Further, using this procedure, a graft stored 3 days at +4°C exhibits better functional capacities than one currently used in routine storage (CBUs stored at +4°C for 1 day in gas-permeable bags and without medium). We provided the proof of principle of our approach, developed a clinical-scale kit and performed a preclinical assay demonstrating the feasibility and efficiency of our CBU preservation protocol through all steps of preparation (volume reduction, freezing, and thawing).
在采集的脐带血单位（CBUs）进行处理和冷冻保存之前，将其运输至血库的过程中，必须保存相对少量的目标细胞（干细胞和祖细胞）的功能能力，这些细胞对于移植物效力至关重要。为了提高CBU的储存效率，我们构思了一种基于以下两个原则的方法：（1）为脐带血中的干细胞和祖细胞提供更好的营养和生化环境；（2）防止这些从低氧浓度（脐带血中1.1%-4% O₂）转移至高氧浓度（大气中20%-21% O₂）的细胞发生过度氧化。我们的假设通过在不透气袋中使用我们的培养基（HP02）储存一段时间后对干细胞（免疫缺陷小鼠造血重建能力——重度联合免疫缺陷鼠再植细胞测定）和定向祖细胞活性（体外集落形成能力和体外扩增能力）的功能评估得到了证实。相对于第0天，这种储存程序可使CBU移植物在3天内保持其全部功能能力。此外，使用该程序，在+4°C储存3天的移植物比目前常规储存方法（在透气袋中且无培养基在+4°C储存1天的CBUs）具有更好的功能能力。我们提供了该方法的原理证明，开发了临床规模的试剂盒，并进行了临床前试验，通过制备的所有步骤（体积减少、冷冻和解冻）证明了我们的CBU保存方案的可行性和效率。
