Studená R, Horák D, Baloun J, Plichta Z, Pospíšilová Š
Klin Onkol. 2017 Spring;30(Supplementum1):184-186.
Primary human B cells chronic lymphocytic leukemia undergoes apoptosis, from which they can be rescued by contact with stromal cells or by the addition of specific soluble factor, when cultured in vitro. For research purposes of the behavior of CLL cells we created 3D in vitro model in which we simulated appropriate microenvironment for CLL cells to allow study the mechanism of survival of these cells in long-term cultivation.
Our aim was the scaffold structure to be geometrically similar to the 3D morphology of supporting bone marrow tissue in a trabecular bone; the 3D scaffold was also designed to conform to biocompatibility, sufficiently large surface area for cell attachment, high porosity for cell migration, proliferation and transport of nutrients. Another requirement was a partial transparency for inspection of cell model with optical techniques. We prepared 3D scaffolds from porous hydrogel poly (2-hydroxyethyl methacrylate) (pHEMA), poly (2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) p (HEMA-co-AEMA) and p (HEMA-co-AEMA) modified with frequently used cell adhesion peptide Arg-Gly-Asp (RGD). All hydrogel scaffolds were manufactured in four pore diameters (125, 200, 300 and 350-450 μm). Scaffolds were tested with human bone marrow stromal cell line HS-5 and human embryonic kidney cell line HEK293.
Hydrogel scaffold p (HEMA-co-AEMA) modified with adhesion peptide Arg-Gly-Asp (RGD) with pore diameter of 350-450 μm demonstrated that it is a convenient system for 3D cell cultivation, since it promotes interaction between the cells and also between the cells and the material. This scaffold was used for seeding of co-cultivation system of HS-5 cells with CLL-cells, which were stimulated through the CD40L signaling pathway as well as via the IL-4 pathway. Viability of B-CLL cells was higher in the presence of both stimulators than with each alone.
We have shown that 3D scaffold technology is very useful for modeling of microsystems where the cancer cells behave like in their natural microenvironment.Key words: hematooncology - leukemia - hydrogel - stromal cells This work was supported by grant COST CZ LD15144 "Cellular and acellular grounds for regeneration of bones and teeth" awarded by the Ministry of Education, Youth and Sport of the Czech Republic. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 3. 2017Accepted: 26. 3. 2017.
原代人B细胞慢性淋巴细胞白血病细胞在体外培养时会发生凋亡，而与基质细胞接触或添加特定可溶性因子可使其免于凋亡。为了研究慢性淋巴细胞白血病（CLL）细胞的行为，我们创建了一种三维体外模型，在该模型中模拟了适合CLL细胞的微环境，以便研究这些细胞在长期培养中的存活机制。
我们的目标是使支架结构在几何形状上与小梁骨中支持性骨髓组织的三维形态相似；三维支架还设计为符合生物相容性，具有足够大的细胞附着表面积、高孔隙率以利于细胞迁移、增殖和营养物质运输。另一个要求是具有部分透明度，以便用光学技术检查细胞模型。我们用多孔水凝胶聚（甲基丙烯酸2-羟乙酯）（pHEMA）、聚（甲基丙烯酸2-羟乙酯-co-甲基丙烯酸2-氨基乙酯）p（HEMA-co-AEMA）以及用常用的细胞黏附肽精氨酸-甘氨酸-天冬氨酸（RGD）修饰的p（HEMA-co-AEMA）制备了三维支架。所有水凝胶支架均制成四种孔径（125、200、300和350 - 450μm）。用人类骨髓基质细胞系HS-5和人类胚胎肾细胞系HEK293对支架进行测试。
用黏附肽精氨酸-甘氨酸-天冬氨酸（RGD）修饰的孔径为350 - 450μm的水凝胶支架p（HEMA-co-AEMA）表明，它是一种方便的三维细胞培养系统，因为它促进了细胞之间以及细胞与材料之间的相互作用。该支架用于接种HS-5细胞与CLL细胞的共培养系统，这些细胞通过CD40L信号通路以及IL-4通路受到刺激。在两种刺激物同时存在的情况下，B-CLL细胞的活力高于单独使用每种刺激物时。
我们已经表明，三维支架技术对于模拟癌细胞在其自然微环境中行为的微系统非常有用。关键词：血液肿瘤学 - 白血病 - 水凝胶 - 基质细胞 本研究得到捷克共和国教育、青年和体育部授予的COST CZ LD15144“骨骼和牙齿再生的细胞和无细胞基础”资助。作者声明他们在研究中使用的药物、产品或服务方面没有潜在的利益冲突。编辑委员会声明该手稿符合ICMJE对生物医学论文的建议。提交日期：2017年3月6日 接受日期：2017年3月26日
