Optimizing the generation of mature bone marrow-derived dendritic cells in vitro: a factorial study design.

IF 3.6 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal, genetic engineering & biotechnology Pub Date : 2023-11-29 DOI:10.1186/s43141-023-00597-4
Najla Alotaibi, Alia Aldahlawi, Kawther Zaher, Fatemah Basingab, Jehan Alrahimi
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Abstract

Background: Factorial design is a simple, yet elegant method to investigate the effect of multiple factors and their interaction on a specific response simultaneously. Hence, this type of study design reaches the best optimization conditions of a process. Although the interaction between the variables is widely prevalent in cell culture procedures, factorial design per se is infrequently utilized in improving cell culture output. Therefore, we aim to optimize the experimental conditions for generating mature bone marrow-derived dendritic cells (BMDCs). Two different variables were investigated, including the concentrations of the inducing factors and the starting density of the bone marrow mononuclear cells. In the current study, we utilized the design of experiments (DoE), a statistical approach, to systematically assess the impact of factors with varying levels on cell culture outcomes. Herein, we apply a two-factor, two-level (22) factorial experiment resulting in four conditions that are run in triplicate. The two variables investigated here are cytokines combinations with two levels, granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or with interleukin-4 (IL4). The other parameter is cell density with two different concentrations, 2 × 106 and 4 × 106 cells/mL. Then, we measured cell viability using the trypan blue exclusion method, and a flow cytometer was used to detect the BMDCs expressing the markers FITC-CD80, CD86, CD83, and CD14. BMDC marker expression levels were calculated using arbitrary units (AU) of the mean fluorescence intensity (MFI).

Results: The current study showed that the highest total viable cells and cells yield obtained were in cell group seeded at 2 × 106 cells/mL and treated with GM-CSF and IL-4. Importantly, the expression of the co-stimulatory molecules CD83 and CD80/CD86 were statistically significant for cell density of 2 × 106 cells/mL (P < 0.01, two-way ANOVA). Bone marrow mononuclear cells seeded at 4 × 106 in the presence of the cytokine mix less efficiently differentiated and matured into BMDCs. Statistical analysis via two-way ANOVA revealed an interaction between cell density and cytokine combinations.

Conclusion: The analysis of this study indicates a substantial interaction between cytokines combinations and cell densities on BMDC maturation. However, higher cell density is not associated with optimizing DC maturation. Notably, applying DoE in bioprocess designs increases experimental efficacy and reliability while minimizing experiments, time, and process costs.

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优化体外成熟骨髓来源树突状细胞的生成:一项析因研究设计。
背景:析因设计是一种简单而优雅的方法,用于同时研究多种因素及其相互作用对特定反应的影响。因此,这种类型的研究设计达到了工艺的最佳优化条件。虽然变量之间的相互作用在细胞培养过程中广泛存在,但因子设计本身很少用于提高细胞培养产量。因此,我们的目标是优化成熟骨髓源性树突状细胞(bmdc)的实验条件。研究了诱导因子浓度和骨髓单核细胞起始密度两个不同的变量。在本研究中,我们利用实验设计(DoE),一种统计方法,系统地评估了不同水平的因素对细胞培养结果的影响。在这里,我们应用了一个两因素,两水平(22)析因实验,导致四种情况,在三次重复中运行。这里研究的两个变量是两个水平的细胞因子组合,粒细胞-巨噬细胞集落刺激因子(GM-CSF)单独或与白细胞介素-4 (IL4)。另一个参数是2 × 106和4 × 106细胞/mL两种不同浓度下的细胞密度。然后,我们使用台锥蓝排斥法检测细胞活力,并使用流式细胞仪检测表达FITC-CD80、CD86、CD83和CD14标记的BMDCs。采用平均荧光强度(MFI)的任意单位(AU)计算BMDC标志物表达水平。结果:本研究显示,2 × 106个/mL、GM-CSF和IL-4处理的细胞组总活细胞数和细胞产量最高。重要的是,当细胞密度为2 × 106个细胞/mL (p6)时,共刺激分子CD83和CD80/CD86的表达具有统计学意义,在细胞因子混合物存在的情况下,分化和成熟为BMDCs的效率较低。通过双向方差分析的统计分析显示细胞密度与细胞因子组合之间存在相互作用。结论:本研究的分析表明细胞因子组合和细胞密度对BMDC成熟有重要的相互作用。然而,较高的细胞密度与优化DC成熟无关。值得注意的是,在生物工艺设计中应用DoE可以提高实验效率和可靠性,同时最大限度地减少实验、时间和工艺成本。
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