We have developed a method to facilitate the isolation and expansion of tumor cells from body fluids and tissue biopsies. Antibody-conjugated magnetic beads (immunobeads) were used to isolate tumor cells from blood, bone marrow, ascitic/pleural fluids, and enzyme-digested tissue biopsies. Filtration of the resulting cell suspension through a 20-micron nylon monofilament filter secured to the base of polystyrene 96-well strips purged the bead-rosetting cell fraction of contaminating normal cells and unbound beads. Tumor cells that bound the magnetic beads were retained on the membrane due to their increased size and concentrated into a small area (0.332 cm2), thus maintaining a high cell density. The filters provided a stable and uniform three-dimensional matrix for cell growth, with a total surface area of 1.42 cm2 available for cell attachment. The filters could be easily removed from the base of the 96-well strips to facilitate handling and transfer between culture vessels. Tumor cells grown on the filters could subsequently be harvested using trypsin/EDTA or left in situ for immunostaining with conventional immunohistochemical procedures. Filter-grown cells have shown extended passage in conventional cell culture in six cases. In two of five cases, the orthotopic implantation of confluent filters that contained approximately 10(4) cells/8 x 8 mm filter successfully produced tumors in nude mice after only 4 weeks. Our new approach may be of value in improving the success rate of generating long-term cultures from previously unproductive sources of tumor cells and thus may yield a greater variety of cell lines/strains for the study of malignant disease.
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机译:我们已经开发出一种促进从体液和组织活检物中分离和扩增肿瘤细胞的方法。抗体缀合的磁珠(免疫球蛋白)用于从血液,骨髓,腹水/胸水和酶消化的组织活检物中分离肿瘤细胞。通过固定在聚苯乙烯96孔条底部的20微米尼龙单丝过滤器过滤所得的细胞悬液,清除了污染正常细胞和未结合的珠子的珠子凝固细胞部分。结合磁珠的肿瘤细胞由于其尺寸增大而保留在膜上,并浓缩到较小的区域(0.332 cm2),从而保持较高的细胞密度。过滤器为细胞生长提供了稳定且均匀的三维矩阵,总表面积为1.42 cm2可用于细胞附着。可以轻松地从96孔条的底部取下过滤器,以方便在培养容器之间进行处理和转移。随后可以使用胰蛋白酶/ EDTA收获在滤膜上生长的肿瘤细胞,或原位留待常规免疫组织化学程序进行免疫染色。滤器生长的细胞在常规细胞培养中显示出六种情况下的传代延长。在五分之二的病例中,仅约4周后,原位植入融合滤膜(约含10(4)个细胞/ 8 x 8 mm滤膜)就成功在裸鼠中产生了肿瘤。我们的新方法可能对提高从以前非生产性的肿瘤细胞来源产生长期培养物的成功率具有价值,因此可能会产生更多种类的细胞系/菌株用于研究恶性疾病。
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