Quantitatively measuring the cytotoxicity of viscous hydrogels with direct cell sampling in a micro scale format “MicroDrop” and its comparison to CCK8

IF 4.2 3区 医学 Q2 ENGINEERING, BIOMEDICAL Journal of Materials Science: Materials in Medicine Pub Date : 2024-06-20 DOI:10.1007/s10856-024-06800-3
Anna Marie Margot, Andreas Engels, Michael Sittinger, Tilo Dehne, Shabnam Hemmati-Sadeghi
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Abstract

Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels.

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利用微尺度格式 "MicroDrop "直接进行细胞取样,定量测量粘性水凝胶的细胞毒性,并与 CCK8 进行比较。
组织工程有望利用水凝胶等粘性材料开发治疗应用。然而,由于非特异性相互作用,用传统方法评估此类材料的细胞毒性可能具有挑战性。为了解决这个问题,我们优化了一种用于定量评估的活体/死体染色方法,并将其与传统的 CCK8 检测法进行了比较。我们的 MicroDrop 方法是在 12 孔板上的 10 µl 培养基中加入含有 5000 个细胞的液滴。十二烷基硫酸钠(SDS)稀释系列比较了 MicroDrop 和 CCK8 检测法。研究结果显示,10 毫克/毫升透明质酸(HA)具有细胞类型特异性,其中 MC3T3-E1 细胞在 72 小时内保持 95% 的存活率,而 L929 细胞的存活率则逐渐下降到 17%。在所有时间点和细胞系中,2 毫克/毫升 HA 的存活率始终高于 90%。同样,纤维蛋白在不同稀释度和不同时间点的存活率均为 90%,只有未稀释样品的存活率从 85% 降至 20%。明胶-甲基丙烯醇在 5%和 10%浓度下,在所有时间点的存活率都高于 70%。活力(MicroDrop)和代谢活性(CCK8)测定之间的 SDS 稀释系列比较显示相关系数为 0.95。这项研究验证了所建立的检测方法的可行性,为研究人员提供了评估粘性材料细胞毒性的有效工具。此外,它还有可能为众所周知的水凝胶提供更精确的数据。
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来源期刊
Journal of Materials Science: Materials in Medicine
Journal of Materials Science: Materials in Medicine 工程技术-材料科学:生物材料
CiteScore
8.00
自引率
0.00%
发文量
73
审稿时长
3.5 months
期刊介绍: The Journal of Materials Science: Materials in Medicine publishes refereed papers providing significant progress in the application of biomaterials and tissue engineering constructs as medical or dental implants, prostheses and devices. Coverage spans a wide range of topics from basic science to clinical applications, around the theme of materials in medicine and dentistry. The central element is the development of synthetic and natural materials used in orthopaedic, maxillofacial, cardiovascular, neurological, ophthalmic and dental applications. Special biomedical topics include biomaterial synthesis and characterisation, biocompatibility studies, nanomedicine, tissue engineering constructs and cell substrates, regenerative medicine, computer modelling and other advanced experimental methodologies.
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