Larsberg Filip, Sprechert Maximilian, Hesse Deike, Brockmann Gudrun A, Kreuzer-Redmer Susanne
Albrecht Daniel Thaer-Institute, Breeding Biology and Molecular Genetics, Humboldt University of Berlin, Unter den Linden 6, 10099 Berlin, Germany.
Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria.
Animals (Basel). 2021 Dec 19;11(12):3600. doi: 10.3390/ani11123600.
Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na-citrate tubes revealed the highest count of vital cells compared to commercial Li-heparin ( < 0.01) and K3EDTA ( < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced ( < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) ( < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.
通过使用原代培养的外周血单个核细胞（PBMC）的可控但与动物相关的体外系统获得的关于免疫调节化合物（如病原体、药物或饲料添加剂，例如益生菌）作用模式的知识，将有助于制定有针对性的营养策略。此外，它有助于预防传染病和抗菌药物的使用，并进一步促进动物健康。然而，据我们所知，目前尚无从鸡血中分离血小板计数降低的PBMC并随后进行数天细胞培养以评估免疫调节化合物作用效果的方案。因此，我们建立了一种优化的方案，用于鸡PBMC的采血、免疫细胞分离、培养和表型分析。对于采血，与商用锂肝素管（<0.01）和K3乙二胺四乙酸管（<0.05）相比，商用柠檬酸钠管显示活细胞计数最高。与随后使用菲可分离的低速离心相比，使用葡聚糖和菲可联合密度梯度分离可显著降低血小板计数（<0.01）。对于细胞培养，与胎牛血清（FCS）相比，在RPMI-1640培养基中添加10%鸡血清导致血小板的相对细胞计数最低（<0.05）。为了验证细胞培养系统对刺激的反应能力，使用刀豆球蛋白A（conA）作为阳性对照。该优化方案允许从鸡血中分离和培养血小板计数降低的活PBMC，用于随后对免疫调节化合物作用模式的研究。
