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Membrane-less microfiltration using inertial microfluidics.

Warkiani ME, Tay AK, Guan G, Han J - Sci Rep (2015)

Bottom Line: Herein, we report the development of a membrane-less microfiltration system by massively parallelizing inertial microfluidics to achieve a macroscopic volume processing rates (~ 500 mL/min).We demonstrated the systems engineered for CHO (10-20 μm) and yeast (3-5 μm) cells filtration, which are two main cell types used for large-scale bioreactors.Our proposed system can replace existing filtration membrane and provide passive (no external force fields), continuous filtration, thus eliminating the need for membrane replacement.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Mechanical and Manufacturing Engineering, Australian Centre for NanoMedicine, University of New South Wales, Sydney, NSW 2052, Australia [2] BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.

ABSTRACT
Microfiltration is a ubiquitous and often crucial part of many industrial processes, including biopharmaceutical manufacturing. Yet, all existing filtration systems suffer from the issue of membrane clogging, which fundamentally limits the efficiency and reliability of the filtration process. Herein, we report the development of a membrane-less microfiltration system by massively parallelizing inertial microfluidics to achieve a macroscopic volume processing rates (~ 500 mL/min). We demonstrated the systems engineered for CHO (10-20 μm) and yeast (3-5 μm) cells filtration, which are two main cell types used for large-scale bioreactors. Our proposed system can replace existing filtration membrane and provide passive (no external force fields), continuous filtration, thus eliminating the need for membrane replacement. This platform has the desirable combinations of high throughput, low-cost, and scalability, making it compatible for a myriad of microfiltration applications and industrial purposes.

No MeSH data available.


Related in: MedlinePlus

Results demonstrating the effectiveness of the inertial filtration system for cell synchronization.(a) Left to right: Optical image of original cell population before sorting. CHO cells present displayed a range of sizes i.e. 11–20 μm. Optical image showing CHO cells arrested in G2/M phase with Nocodazole. Cell sizes were 18 ± 2 μm. Optical image showing CHO cells arrested in G0/G1 phase via contact inhibition. Cell sizes were 11 ± 2 μm. (b) Top & bottom: optical image showing CHO cells in the inner and outer outlets respectively after separation with their sizes compared to 10 μm beads at 1.5 mL/min. The cells at the inner outlet have larger sizes while that in the outer outlet have smaller sizes. (c) The positive slope of the forward and side scatter graphs indicated the correlation between cell size and cell-cycle phase. (d) Left: histogram of the DNA content showed that before sorting, the G0/G1:G2/M ratio was 1.82:1. Distinct peaks could be observed for cells in the G0/G1 and G2/M phase by their DNA content stained with Hoechst. Right: after sorting at 1.5 mL/min (1 × 106 cells/mL), the G0/G1:G2/M ratio improved close to three folds to 5.02:1 in the outer outlet. The G0/G1 purity in the outer outlet was ~ 80%, an improvement from ~ 60% in the unsorted sample. The increase in cell count in the outer outlet also indicated selective enrichment of cell population with lower DNA content in the G1 phase. This can be further improved by processing output from the inner outlet again (2nd or 3rd cycles).
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f4: Results demonstrating the effectiveness of the inertial filtration system for cell synchronization.(a) Left to right: Optical image of original cell population before sorting. CHO cells present displayed a range of sizes i.e. 11–20 μm. Optical image showing CHO cells arrested in G2/M phase with Nocodazole. Cell sizes were 18 ± 2 μm. Optical image showing CHO cells arrested in G0/G1 phase via contact inhibition. Cell sizes were 11 ± 2 μm. (b) Top & bottom: optical image showing CHO cells in the inner and outer outlets respectively after separation with their sizes compared to 10 μm beads at 1.5 mL/min. The cells at the inner outlet have larger sizes while that in the outer outlet have smaller sizes. (c) The positive slope of the forward and side scatter graphs indicated the correlation between cell size and cell-cycle phase. (d) Left: histogram of the DNA content showed that before sorting, the G0/G1:G2/M ratio was 1.82:1. Distinct peaks could be observed for cells in the G0/G1 and G2/M phase by their DNA content stained with Hoechst. Right: after sorting at 1.5 mL/min (1 × 106 cells/mL), the G0/G1:G2/M ratio improved close to three folds to 5.02:1 in the outer outlet. The G0/G1 purity in the outer outlet was ~ 80%, an improvement from ~ 60% in the unsorted sample. The increase in cell count in the outer outlet also indicated selective enrichment of cell population with lower DNA content in the G1 phase. This can be further improved by processing output from the inner outlet again (2nd or 3rd cycles).

Mentions: Previously, our group made use of inertial forces in spiral microchannel (i.e., with rectangular cross-section) to isolate an array of cells such as CHO and marrow-derived human mesenchymal cells (hMSCs)34. In this study, we demonstrate another application of our novel high-throughput system for large-scale cell synchronization suitable for industrial applications. In the fractionation mode (see Fig. 1a), cells typically ≥14 μm are isolated via the inner outlet while smaller cells, which are trapped in the strong vortices near outer wall, exit the spiral device via the outer outlet. Capitalizing on the size differences between cells in G0/G1 and G2/M phase (with the latter being larger, Fig. 4a,c), a single spiral microfluidic device is able to perform cell synchronization with a throughput of 1–2 mL/min (up to 1 × 106 cells/mL) while the multiplexed system presented here can deliver throughput of 150–300 mL/min or even higher (see video S4). This throughput is high enough to fractionate multiple cell culture flasks or a perfusion reactor (i.e., 1–5 L capacity) within few minutes for downstream characterization of cells or re-inoculation of the system.


Membrane-less microfiltration using inertial microfluidics.

Warkiani ME, Tay AK, Guan G, Han J - Sci Rep (2015)

Results demonstrating the effectiveness of the inertial filtration system for cell synchronization.(a) Left to right: Optical image of original cell population before sorting. CHO cells present displayed a range of sizes i.e. 11–20 μm. Optical image showing CHO cells arrested in G2/M phase with Nocodazole. Cell sizes were 18 ± 2 μm. Optical image showing CHO cells arrested in G0/G1 phase via contact inhibition. Cell sizes were 11 ± 2 μm. (b) Top & bottom: optical image showing CHO cells in the inner and outer outlets respectively after separation with their sizes compared to 10 μm beads at 1.5 mL/min. The cells at the inner outlet have larger sizes while that in the outer outlet have smaller sizes. (c) The positive slope of the forward and side scatter graphs indicated the correlation between cell size and cell-cycle phase. (d) Left: histogram of the DNA content showed that before sorting, the G0/G1:G2/M ratio was 1.82:1. Distinct peaks could be observed for cells in the G0/G1 and G2/M phase by their DNA content stained with Hoechst. Right: after sorting at 1.5 mL/min (1 × 106 cells/mL), the G0/G1:G2/M ratio improved close to three folds to 5.02:1 in the outer outlet. The G0/G1 purity in the outer outlet was ~ 80%, an improvement from ~ 60% in the unsorted sample. The increase in cell count in the outer outlet also indicated selective enrichment of cell population with lower DNA content in the G1 phase. This can be further improved by processing output from the inner outlet again (2nd or 3rd cycles).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495597&req=5

f4: Results demonstrating the effectiveness of the inertial filtration system for cell synchronization.(a) Left to right: Optical image of original cell population before sorting. CHO cells present displayed a range of sizes i.e. 11–20 μm. Optical image showing CHO cells arrested in G2/M phase with Nocodazole. Cell sizes were 18 ± 2 μm. Optical image showing CHO cells arrested in G0/G1 phase via contact inhibition. Cell sizes were 11 ± 2 μm. (b) Top & bottom: optical image showing CHO cells in the inner and outer outlets respectively after separation with their sizes compared to 10 μm beads at 1.5 mL/min. The cells at the inner outlet have larger sizes while that in the outer outlet have smaller sizes. (c) The positive slope of the forward and side scatter graphs indicated the correlation between cell size and cell-cycle phase. (d) Left: histogram of the DNA content showed that before sorting, the G0/G1:G2/M ratio was 1.82:1. Distinct peaks could be observed for cells in the G0/G1 and G2/M phase by their DNA content stained with Hoechst. Right: after sorting at 1.5 mL/min (1 × 106 cells/mL), the G0/G1:G2/M ratio improved close to three folds to 5.02:1 in the outer outlet. The G0/G1 purity in the outer outlet was ~ 80%, an improvement from ~ 60% in the unsorted sample. The increase in cell count in the outer outlet also indicated selective enrichment of cell population with lower DNA content in the G1 phase. This can be further improved by processing output from the inner outlet again (2nd or 3rd cycles).
Mentions: Previously, our group made use of inertial forces in spiral microchannel (i.e., with rectangular cross-section) to isolate an array of cells such as CHO and marrow-derived human mesenchymal cells (hMSCs)34. In this study, we demonstrate another application of our novel high-throughput system for large-scale cell synchronization suitable for industrial applications. In the fractionation mode (see Fig. 1a), cells typically ≥14 μm are isolated via the inner outlet while smaller cells, which are trapped in the strong vortices near outer wall, exit the spiral device via the outer outlet. Capitalizing on the size differences between cells in G0/G1 and G2/M phase (with the latter being larger, Fig. 4a,c), a single spiral microfluidic device is able to perform cell synchronization with a throughput of 1–2 mL/min (up to 1 × 106 cells/mL) while the multiplexed system presented here can deliver throughput of 150–300 mL/min or even higher (see video S4). This throughput is high enough to fractionate multiple cell culture flasks or a perfusion reactor (i.e., 1–5 L capacity) within few minutes for downstream characterization of cells or re-inoculation of the system.

Bottom Line: Herein, we report the development of a membrane-less microfiltration system by massively parallelizing inertial microfluidics to achieve a macroscopic volume processing rates (~ 500 mL/min).We demonstrated the systems engineered for CHO (10-20 μm) and yeast (3-5 μm) cells filtration, which are two main cell types used for large-scale bioreactors.Our proposed system can replace existing filtration membrane and provide passive (no external force fields), continuous filtration, thus eliminating the need for membrane replacement.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Mechanical and Manufacturing Engineering, Australian Centre for NanoMedicine, University of New South Wales, Sydney, NSW 2052, Australia [2] BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.

ABSTRACT
Microfiltration is a ubiquitous and often crucial part of many industrial processes, including biopharmaceutical manufacturing. Yet, all existing filtration systems suffer from the issue of membrane clogging, which fundamentally limits the efficiency and reliability of the filtration process. Herein, we report the development of a membrane-less microfiltration system by massively parallelizing inertial microfluidics to achieve a macroscopic volume processing rates (~ 500 mL/min). We demonstrated the systems engineered for CHO (10-20 μm) and yeast (3-5 μm) cells filtration, which are two main cell types used for large-scale bioreactors. Our proposed system can replace existing filtration membrane and provide passive (no external force fields), continuous filtration, thus eliminating the need for membrane replacement. This platform has the desirable combinations of high throughput, low-cost, and scalability, making it compatible for a myriad of microfiltration applications and industrial purposes.

No MeSH data available.


Related in: MedlinePlus