High-throughput downstream process development for cell-based products using aqueous two-phase systems

Gretzinger

et al. 2017

Biotechnology Journal

Self Cite

The availability of clinical-scale downstream processing strategies for cell-based products presents a critical juncture between basic research and clinical development. Aqueous two-phase systems (ATPS) facilitate the label-free, scalable, and cost-effective separation of cells, and are a versatile tool for downstream processing of cell-based therapeutics. Here, we report the application of a previously developed robotic screening platform, here extended to enable a multiplexed high-throughput cell partitioning analysis in ATPS. We investigated the influence of polymer molecular weight and tie-line length on the resolution of five model cell lines in "charge-sensitive" polyethylene-glycol (PEG)-dextran ATPS. We show, how these factors influence cell partitioning, and that the combination of low molecular weight PEGs and high molecular weight dextrans enable the highest resolution of the five cell lines. Furthermore, we demonstrate that the separability of each cell line from the mixture is highly dependent on the polymer molecular weight composition and tie-line length. Using a countercurrent distribution model we demonstrate that our screenings yielded conditions that theoretically enable the isolation of four of the five cell lines with high purity (>99.9%) and yield.

Section: Methodsmentioning

confidence: 99%

“…Liquid handling calibration was performed for all buffers and stock solutions as described previously . Calibration was performed in the range of 50–1000 μL.…”

Section: Methodsmentioning

confidence: 99%

“…A HTS‐method for binodal determination based on could‐point titration has previously been established in our group and was performed as described …”

Section: Methodsmentioning

confidence: 99%

“…The automated HTS‐method was used as described in a previous publication . The entire HTS was performed on a Tecan Freedom Evo® 200 liquid‐handling station (LHS).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Cell Separation in Aqueous Two‐Phase Systems − Influence of Polymer Molecular Weight and Tie‐Line Length on the Resolution of Five Model Cell Lines

Gretzinger

et al. 2017

Biotechnology Journal

Self Cite

Adv Healthcare Materials

Emerging Biotechnology Applications of Aqueous Two‐Phase Systems

Teixeira

Agarwal

et al. 2017

Liquid–liquid phase separation between aqueous solutions containing two incompatible polymers, a polymer and a salt, or a polymer and a surfactant, has been exploited for a wide variety of biotechnology applications throughout the years. While many applications for aqueous two‐phase systems fall within the realm of separation science, the ability to partition many different materials within these systems, coupled with recent advances in materials science and liquid handling, has allowed bioengineers to imagine new applications. This progress report provides an overview of the history and key properties of aqueous two‐phase systems to lend context to how these materials have progressed to modern applications such as cellular micropatterning and bioprinting, high‐throughput 3D tissue assembly, microscale biomolecular assay development, facilitation of cell separation and microcapsule production using microfluidic devices, and synthetic biology. Future directions and present limitations and design considerations of this adaptable and promising toolkit for biomolecule and cellular manipulation are further evaluated.

High‐throughput downstream process development for cell‐based products using aqueous two‐phase systems (ATPS) – A case study

Scheeder

et al. 2016

Biotechnology Journal

Self Cite

The availability of preparative-scale downstream processing strategies for cell-based products presents a critical juncture between fundamental research and clinical development. Aqueous two-phase systems (ATPS) present a gentle, scalable, label-free, and cost-effective method for cell purification, and are thus a promising tool for downstream processing of cell-based therapeutics. Here, the application of a previously developed robotic screening platform that enables high-throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high-throughput screening (HTS)-data and countercurrent distribution (CCD)-modeling, and validated the CCD-model experimentally is designed. The obtained data are shown an excellent congruence between CCD-model and experimental data, indicating that CCD-models in combination with HTS-data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge-dependent separation of HL-60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions.