Mammalian Cell Culture Process Development, FUJIFILM Diosynth Biotechnologies, Billingham, UK.
School of Mathematics, Statistics and Physics, Newcastle University, Newcastle upon Tyne, UK.
Biotechnol J. 2022 Jan;17(1):e2100357. doi: 10.1002/biot.202100357. Epub 2021 Oct 25.
There is an expectation from regulatory agencies that cell lines used in the commercial production of biopharmaceuticals are derived from a single cell progenitor. Traditional methods of single cell cloning include the use of the limiting dilution cloning method which often requires multiple rounds of low cell density cell plating and either microscopic evaluation that wells contain single cells and/or the calculation of a statistically derived probability of monoclonality.
We have combined the single cell screening, deposition and picodroplet imaging ability of Sphere Fluidics' Cyto-Mine technology with the plate imaging capability of the Solentim Cell Metric to create a novel workflow for the generation of high producing clonal cell lines with both high probability and assurance of monoclonality. The efficiency of three key stages of the process (single cell picodroplet encapsulation, single picodroplet dispensation and single cell settling in the focal plane of the plate imager) was determined and a probability calculation was derived using the Wilson Score Interval method. The combined probability that a single cell is encapsulated into a picodroplet, is deposited into the correct well of a 96-well plate and that a cell settles into the focal plane of the plate imager yields a combined > 99% probability of monoclonality. Furthermore, visual verification of a single cell progenitor is obtained at multiple steps throughout the cloning workflow.
This novel methodology for the rapid creation of high quality clonal cell lines for biomanufacturing purposes has many advantages over more traditional approaches including improved assurance of single cell derivation, integrated imaging capability, assay flexibility, equipment utilization time and in-process cell line segregation.
监管机构期望用于商业生产生物制药的细胞系源自单个细胞祖代。传统的单细胞克隆方法包括使用有限稀释克隆方法，该方法通常需要多轮低细胞密度细胞接种，并且需要显微镜评估孔是否含有单细胞和/或计算统计学衍生的单克隆概率。
我们将 Sphere Fluidics 的 Cyto-Mine 技术的单细胞筛选、沉积和皮升级液滴成像能力与 Solentim Cell Metric 的平板成像能力相结合，创建了一种新的工作流程，用于生成高产克隆细胞系，具有高单克隆性概率和保证。该过程的三个关键阶段（单细胞皮升级液滴包封、单细胞皮升级液滴分配和单细胞在平板成像仪焦平面上沉降）的效率进行了确定，并使用威尔逊得分区间法进行了概率计算。单个细胞被封装到皮升级液滴中、被分配到 96 孔板的正确孔中和细胞沉降到平板成像仪的焦平面中的组合概率产生了>99%的单克隆概率。此外，在克隆工作流程的多个步骤中都可以获得单细胞祖代的视觉验证。
这种用于快速创建高质量克隆细胞系用于生物制造的新方法与更传统的方法相比具有许多优势，包括提高单细胞衍生的保证、集成成像能力、分析灵活性、设备利用时间和过程中的细胞系分离。
