Allieri M A, Douay L, Deloux J, Smadja N, Najman A, Gorin N C
Laboratoire d'Etude de l'Hématopoièse-Unité de recherche sur les greffes de cellules souches hématopoiétiques, Formation Associée Claude Bernard, CHU St. Antoine, Paris, France.
Exp Hematol. 1990 Sep;18(8):911-5.
The use of a semisolid support like methylcellulose (MC) in a clonogenic assay prevents cell migration and nonspecific aggregation. However, the inhibitory effect of MC on myeloid cell lines has been reported. To assess the effect of MC on human leukemic progenitor cell growth (acute myeloblastic leukemia colony-forming units, AML-CFU), increasing concentrations of MC (0.36%, 0.72%, and 1.44%) were added in a double-feeder culture system. T-lymphocyte-depleted leukemic cells from 12 patients with AML were cultured in the presence of 2.5% phytohemagglutinin (PHA) in a liquid and a semisolid (MC) medium over a leukocyte feeder layer. The leukemic nature of the colonies was confirmed by cytogenetic studies. The median cloning efficiency in the optimal MC assay system was significantly higher (217 leukemic colony-forming units [CFU-L]/5 x 10(4) cells) than the one obtained in the liquid assay system (72.5 CFU-L/5 x 10(4) cells). However, three patterns of growth were observed: 1) colony formation was significantly better in MC than in the liquid assay system (seven of ten cases), 2) there was no difference in growth response (three of ten cases), and 3) colony formation was significantly better in the liquid assay system (one of ten cases). In the semisolid assay system, colony growth was dependent on MC concentration and varied among individual patients. A striking feature was the partial reduction of AML-CFU growth at 1.44% MC, with complete inhibition in 4/11 cases. This phenomenon was not observed for normal progenitors cultured under the same conditions. Cytological evaluation of AML-CFU showed an incomplete maturation to the myelocyte state, accompanied occasionally by macrophagic differentiation. In contrast, maturation of the granulocyte-macrophage colony-forming unit (CFU-GM) clones was harmonious, resulting in greater than 40% polynuclear cells, even from a 7-day culture. Despite a variable clonal response of leukemic progenitors from individual patients, we conclude that 0.72% MC is the optimal concentration of MC in our system, allowing clonal growth of AML-CFU.
在克隆形成试验中使用甲基纤维素（MC）等半固体支持物可防止细胞迁移和非特异性聚集。然而，已有报道称MC对髓系细胞系有抑制作用。为评估MC对人白血病祖细胞生长（急性髓性白血病集落形成单位，AML-CFU）的影响，在双饲养层培养系统中添加了浓度递增的MC（0.36%、0.72%和1.44%）。来自12例急性髓性白血病患者的T淋巴细胞耗竭的白血病细胞在含有2.5%植物血凝素（PHA）的液体和半固体（MC）培养基中，在白细胞饲养层上进行培养。通过细胞遗传学研究证实了集落的白血病性质。最佳MC试验系统中的中位克隆效率（217个白血病集落形成单位[CFU-L]/5×10⁴个细胞）显著高于液体试验系统中的克隆效率（72.5个CFU-L/5×10⁴个细胞）。然而，观察到三种生长模式：1）MC中的集落形成明显优于液体试验系统（10例中有7例），2）生长反应无差异（10例中有3例），3）液体试验系统中的集落形成明显更好（10例中有1例）。在半固体试验系统中，集落生长取决于MC浓度，且在个体患者之间存在差异。一个显著特征是在MC浓度为1.44%时AML-CFU生长部分降低，在11例中有4例完全抑制。在相同条件下培养的正常祖细胞未观察到这种现象。AML-CFU的细胞学评估显示向髓细胞状态的成熟不完全，偶尔伴有巨噬细胞分化。相比之下，粒细胞-巨噬细胞集落形成单位（CFU-GM）克隆的成熟是协调的，即使经过7天培养，多核细胞也超过40%。尽管个体患者的白血病祖细胞克隆反应存在差异，但我们得出结论，0.72%的MC是我们系统中MC的最佳浓度，可使AML-CFU进行克隆生长。
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