Ivanovic Zoran, Duchez Pascale, Dazey Bernard, Hermitte Francis, Lamrissi-Garcia Isabelle, Mazurier Frédéric, Praloran Vincent, Reiffers Josy, Vezon Gérard, Boiron Jean-Michel
French Blood Establishment Aquitaine-Limousin, Bordeaux; CNRS UMR 5164, University of Bordeaux 2, Bordeaux, France.
Transfusion. 2006 Jan;46(1):126-31. doi: 10.1111/j.1537-2995.2005.00675.x.
The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum-free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical-grade IS medium is no longer available, a new serum-free medium, Maco Biotech HP 01 (Macopharma), was evaluated.
Purified CD 34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD 34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP 01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO(2), at 37 degrees C, and at 100 percent humidity.
A higher amplification of total nucleated cells (NCs) and colony-forming cells (CFCs) was obtained with HP 01 medium than with IS medium (42+/-16.6-fold vs. 20.5+/-5.9-fold for NCs and 26.7+/-7.4-fold vs. 15.5+/-2.5-fold for CFCs, respectively), whereas an increase in CD 34+ cells (3.5+/- 1.2-fold for HP 01 vs. 2.7+/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP 01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD 41+ cells after expansion in HP 01 than in IS medium was also observed.
Maco Biotech HP 01 medium is suitable for clinical-scale expansion of CD 34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.
在无血清条件下体外扩增的CD 34+细胞自体移植可显著减轻骨髓瘤患者清髓性中性粒细胞减少症。在我们的细胞治疗单元，用于该临床检测的细胞已在良好生产规范（GMP）条件下，使用含干细胞因子（SCF）、粒细胞集落刺激因子（G-CSF）和巨核细胞生长发育因子（MGDF，均为100 ng/mL）的无血清欧文科学（IS）培养基进行扩增。由于这种临床级IS培养基已不再可用，因此对一种新的无血清培养基——Maco Biotech HP 01（Macopharma公司）进行了评估。
从骨髓瘤患者动员的外周血样本中纯化的CD 34+细胞（Isolex 300i，百特公司）经解冻、洗涤后进行培养，方法与之前的临床检测相同。每1 L添加SCF、G-CSF和MGDF的培养基（HP 01或IS）中重悬2000万CD 34+细胞，装入3 L培养袋（AFC），在5%二氧化碳、37℃和100%湿度条件下培养10天。
与IS培养基相比，HP 01培养基能使总核细胞（NCs）和集落形成细胞（CFCs）获得更高倍数的扩增（NCs分别为42±16.6倍和20.5±5.9倍，CFCs分别为26.7±7.4倍和15.5±2.5倍），而CD 34+细胞的增加倍数（HP 01为3.5±1.2倍，IS为2.7±1.5倍）无显著差异。IS培养基能部分维持重症联合免疫缺陷（SCID）重建细胞（SRC），而在HP 01培养基中培养10天可完全维持干细胞活性。还观察到在HP 01中扩增后的CD 41+细胞频率高于IS培养基。
Maco Biotech HP 01培养基适用于使用SCF、G-CSF和MGDF细胞因子组合进行CD 34+细胞的临床规模扩增，可使CFCs大量扩增并维持SRCs。
