Claësson M H, Andersen V, Sønderstrup-Hansen G
Clin Exp Immunol. 1978 Dec;34(3):364-73.
A method is described for the growth in semi-solid agar medium of human lymphocyte colonies in response to stimulation by lymphocytosis-promoting factor (LPF) derived from . Colony formation was dependent on (a) a liquid pre-culture step with LPF prior to agar seeding, (b) presence of LPF in the agar medium, (c) a cell density in the agar culture of more than 5000 cells per ml. Optimal colony formation was obtained with 30 μl LPF preparation in the liquid pre-culture step and 20 μl LPF preparation in the agar medium. Moreover, colony development improved after addition of 5 × 10M 2-mercaptoethanol and 0·6% human AB serum. The frequency of LPF-induced T-lymphocyte colonies in ten normal adult donors was 5450±1800 per 10 mononuclear blood cells. In comparison, the frequency of phytohaemagglutinin (PHA)-induced colonies was 20,000±2500 per 10 mononuclear cells plated directly in the agar medium. Lymphocytes seeded in agar medium with LPF started to divide within 2–3 days of culture and formed colonies of 30 to 200 cells on day 7 of culture. Cultures became moribund at day 8. The colony cells were negative for surface immunoglobulin and approximately 75% formed rosettes with sheep red blood cells (SRBC). No synergistic effect between LPF and PHA on colony formation was observed when PHA and LPF were used in the first and second step of culture respectively. Similarly, addition of LPF did not influence the growth of PHA-induced colonies. When mononuclear cells were depleted of monocytes prior to agar culture addition of supernate factor(s) from cultures of adherent blood mononuclear cells (AC-CM) was necessary to ensure optimal development of colonies. Mononuclear cells were separated by various rosette-depletion techniques. Colony-forming cells were recovered in the sheep red blood cell-rosetting fraction (E-RFC) of human blood lymphocytes indicating that these precursor cells are themselves T lymphocytes. E-RFCs were separated into Fc(γ)-receptor-positive and negative subpopulations. The frequency of colony-forming cells in the Fc(γ)-receptor-negative T-lymphocyte population was two to ten times higher than that of the Fc(γ)-positive T-cell population. Co-culture of equal numbers of Fc-positive and Fc-negative T lymphocytes reduced the frequency of T colonies by 20 to 95%. Mononuclear cells were separated by passage through Ig-anti-Ig-coated columns. Attempts to grow LPF-stimulated colonies from passaged lymphocytes failed, suggesting that the colony-forming cells or cells necessary for colony formation were trapped in the column. In contrast, PHA-induced colony formation was enriched in cells which had passed through Ig-anti-Ig-coated columns.
本文描述了一种在半固体琼脂培养基中培养人淋巴细胞集落的方法，该集落是对源自……的淋巴细胞增多促进因子（LPF）刺激的反应。集落形成取决于：（a）在琼脂接种前用LPF进行液体预培养步骤；（b）琼脂培养基中存在LPF；（c）琼脂培养中的细胞密度超过每毫升5000个细胞。在液体预培养步骤中加入30 μl LPF制剂，在琼脂培养基中加入20 μl LPF制剂可获得最佳集落形成。此外，加入5×10⁻⁵M 2-巯基乙醇和0.6%人AB血清后集落发育得到改善。10名正常成年供体中LPF诱导的T淋巴细胞集落频率为每10个单核血细胞5450±1800个。相比之下，直接接种在琼脂培养基中的植物血凝素（PHA）诱导的集落频率为每10个单核细胞20000±2500个。接种在含LPF的琼脂培养基中的淋巴细胞在培养2 - 3天内开始分裂，并在培养第7天形成30至200个细胞的集落。培养物在第8天开始濒死。集落细胞表面免疫球蛋白呈阴性，约75%与绵羊红细胞（SRBC）形成玫瑰花结。当分别在培养的第一步和第二步使用PHA和LPF时，未观察到LPF和PHA对集落形成的协同作用。同样，加入LPF不影响PHA诱导的集落生长。当在琼脂培养前单核细胞被去除单核细胞后，添加来自贴壁血单核细胞培养物的上清因子（AC-CM）对于确保集落的最佳发育是必要的。单核细胞通过各种玫瑰花结去除技术进行分离。集落形成细胞在人血淋巴细胞的绵羊红细胞玫瑰花结形成部分（E-RFC）中回收，表明这些前体细胞本身就是T淋巴细胞。E-RFC被分离为Fc（γ）受体阳性和阴性亚群。Fc（γ）受体阴性T淋巴细胞群体中集落形成细胞的频率比Fc（γ）阳性T细胞群体高2至10倍。等量的Fc阳性和Fc阴性T淋巴细胞共培养使T集落频率降低20%至95%。单核细胞通过Ig-抗Ig包被柱进行分离。从通过柱的淋巴细胞中培养LPF刺激的集落的尝试失败，表明集落形成细胞或集落形成所需的细胞被困在柱中。相反，PHA诱导的集落形成在通过Ig-抗Ig包被柱的细胞中富集。
