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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

(a) Sample processing workflow showing process of cell enrichment using the high throughput filtration system from spinner flasks imitating condition of a perfusion bioreactor. Cell cultures are subject to inertial filtration system and used media are assessed for product e.g., IgG concentration. Fresh media and enriched cell samples are added back to the flask following each filtration process. (b) Separation efficiency of CHO cells as a function of concentration at 6 mL/min flow rate for a single spiral device. (c) Viable cell density and viability of hybridoma cells for 10 days continuous culture. The results indicated that both parameters are not significantly affected by inertial filtration. (d) Rate of IgG production by hybridoma cells over 10 days. A steady increase in IgG was observed, suggesting that cellular activities involved in IgG production were minimally affected by inertial filtration (e) Evaluation of stress levels of processed cells compared to cells incubated at 37 °C in cell culture media, or at 24 °C in the PBS buffer. Up-regulation of c-FOS gene was evident in response to cycloheximide (CHX) treatment38 which was considered a positive control. The gene expression was normalized to the GAPDH housekeeping gene for CHO cells for different conditions.
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f2: (a) Sample processing workflow showing process of cell enrichment using the high throughput filtration system from spinner flasks imitating condition of a perfusion bioreactor. Cell cultures are subject to inertial filtration system and used media are assessed for product e.g., IgG concentration. Fresh media and enriched cell samples are added back to the flask following each filtration process. (b) Separation efficiency of CHO cells as a function of concentration at 6 mL/min flow rate for a single spiral device. (c) Viable cell density and viability of hybridoma cells for 10 days continuous culture. The results indicated that both parameters are not significantly affected by inertial filtration. (d) Rate of IgG production by hybridoma cells over 10 days. A steady increase in IgG was observed, suggesting that cellular activities involved in IgG production were minimally affected by inertial filtration (e) Evaluation of stress levels of processed cells compared to cells incubated at 37 °C in cell culture media, or at 24 °C in the PBS buffer. Up-regulation of c-FOS gene was evident in response to cycloheximide (CHX) treatment38 which was considered a positive control. The gene expression was normalized to the GAPDH housekeeping gene for CHO cells for different conditions.

Mentions: To demonstrate the feasibility of our system as a clog-free cell microfiltration system, cell cultures were performed using 250 mL disposable spinner flasks inside a humidified incubator. Microfiltration tests were conducted daily by isolating the used media from cell cultures using our inertial filtration system in a sterile environment while fresh media was replenished to each flask along with enriched cells following each experiment (Fig. 2a). Cell densities, viability, glucose, antibody titers and pH were monitored in each sample separately. Microfiltration tests revealed utility of our system for continuous cell separation from bioreactors over a range of concentrations, with over 95% efficiency at flow rate of 6 mL/min for a single spiral. 6 mL/min flow rate was optimized after testing a flow rate from 1–10 mL/min for a single chip. To demonstrate the ease of multiplexing to achieve desired flow rate in our technology, 84 spiral chips are multiplexed to provide a combined flow rate of 500 mL/min instead. Due to the relatively large channel designs (~mm range) in our inertial microfiltration system, cell concentrations up to 107 cells/mL can be accommodated in our system with negligible effect on separation efficiency; however, for concentrations higher than this value, the separation efficiency decreased (Fig. 2b). These results have been confirmed for three different cells lines (Fig. S1). The viability of the sorted cells was similar to that of the unsorted (control), with more than 90% of the cells excluding the propidium iodide dye, suggesting minimum cellular damage during separation (Fig. 2c). These results were further confirmed by seeding a fraction of sorted cells for proliferation and growth observations. The morphologies and proliferation rate of the isolated CHO cells was similar to the control cells with no noticeable differences (Fig. S2). Our group also showed that inertial separation of mesenchymal stromal cells (MSCs) did not induce any detectable changes in their viability and differentiation potential into different lineages23. These results support that our developed inertial filtration technique has minimal effect on the cells during isolation while maintaining high post-sorting cell viability (also see video S1 &S2). Cell productivity was also assessed by measuring activity of the secreted IgG protein using an enzymatic assay (Fig. 2d). Our results demonstrated sustainable growth of the cells and antibody production over a period of 10 days, suggesting the value of this new technology for separation of animal cells from the culture medium. The expression profile of c-FOS gene was investigated as an indicator of shear in our system (Fig. 2e). The c-FOS is involved in the regulation of early signal transduction pathways by modulating expression of multiple genes as a part of the AP-1 transcriptional activator complex and the shear stress inducibility of c-FOS protein has been shown in human and animal cells lines of different origins2425. Expression of this gene was evaluated on CHO cells and obtained results showed that fluidic shear did not induce up-regulation of c-FOS gene as compared with the two negative controls: cells incubated in culture media at 37 °C and cells in the PBS buffer at 22 °C. This can be attributed to the low residence times of cells in the microfluidic channels (<0.1 sec).


Membrane-less microfiltration using inertial microfluidics.

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

(a) Sample processing workflow showing process of cell enrichment using the high throughput filtration system from spinner flasks imitating condition of a perfusion bioreactor. Cell cultures are subject to inertial filtration system and used media are assessed for product e.g., IgG concentration. Fresh media and enriched cell samples are added back to the flask following each filtration process. (b) Separation efficiency of CHO cells as a function of concentration at 6 mL/min flow rate for a single spiral device. (c) Viable cell density and viability of hybridoma cells for 10 days continuous culture. The results indicated that both parameters are not significantly affected by inertial filtration. (d) Rate of IgG production by hybridoma cells over 10 days. A steady increase in IgG was observed, suggesting that cellular activities involved in IgG production were minimally affected by inertial filtration (e) Evaluation of stress levels of processed cells compared to cells incubated at 37 °C in cell culture media, or at 24 °C in the PBS buffer. Up-regulation of c-FOS gene was evident in response to cycloheximide (CHX) treatment38 which was considered a positive control. The gene expression was normalized to the GAPDH housekeeping gene for CHO cells for different conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: (a) Sample processing workflow showing process of cell enrichment using the high throughput filtration system from spinner flasks imitating condition of a perfusion bioreactor. Cell cultures are subject to inertial filtration system and used media are assessed for product e.g., IgG concentration. Fresh media and enriched cell samples are added back to the flask following each filtration process. (b) Separation efficiency of CHO cells as a function of concentration at 6 mL/min flow rate for a single spiral device. (c) Viable cell density and viability of hybridoma cells for 10 days continuous culture. The results indicated that both parameters are not significantly affected by inertial filtration. (d) Rate of IgG production by hybridoma cells over 10 days. A steady increase in IgG was observed, suggesting that cellular activities involved in IgG production were minimally affected by inertial filtration (e) Evaluation of stress levels of processed cells compared to cells incubated at 37 °C in cell culture media, or at 24 °C in the PBS buffer. Up-regulation of c-FOS gene was evident in response to cycloheximide (CHX) treatment38 which was considered a positive control. The gene expression was normalized to the GAPDH housekeeping gene for CHO cells for different conditions.
Mentions: To demonstrate the feasibility of our system as a clog-free cell microfiltration system, cell cultures were performed using 250 mL disposable spinner flasks inside a humidified incubator. Microfiltration tests were conducted daily by isolating the used media from cell cultures using our inertial filtration system in a sterile environment while fresh media was replenished to each flask along with enriched cells following each experiment (Fig. 2a). Cell densities, viability, glucose, antibody titers and pH were monitored in each sample separately. Microfiltration tests revealed utility of our system for continuous cell separation from bioreactors over a range of concentrations, with over 95% efficiency at flow rate of 6 mL/min for a single spiral. 6 mL/min flow rate was optimized after testing a flow rate from 1–10 mL/min for a single chip. To demonstrate the ease of multiplexing to achieve desired flow rate in our technology, 84 spiral chips are multiplexed to provide a combined flow rate of 500 mL/min instead. Due to the relatively large channel designs (~mm range) in our inertial microfiltration system, cell concentrations up to 107 cells/mL can be accommodated in our system with negligible effect on separation efficiency; however, for concentrations higher than this value, the separation efficiency decreased (Fig. 2b). These results have been confirmed for three different cells lines (Fig. S1). The viability of the sorted cells was similar to that of the unsorted (control), with more than 90% of the cells excluding the propidium iodide dye, suggesting minimum cellular damage during separation (Fig. 2c). These results were further confirmed by seeding a fraction of sorted cells for proliferation and growth observations. The morphologies and proliferation rate of the isolated CHO cells was similar to the control cells with no noticeable differences (Fig. S2). Our group also showed that inertial separation of mesenchymal stromal cells (MSCs) did not induce any detectable changes in their viability and differentiation potential into different lineages23. These results support that our developed inertial filtration technique has minimal effect on the cells during isolation while maintaining high post-sorting cell viability (also see video S1 &S2). Cell productivity was also assessed by measuring activity of the secreted IgG protein using an enzymatic assay (Fig. 2d). Our results demonstrated sustainable growth of the cells and antibody production over a period of 10 days, suggesting the value of this new technology for separation of animal cells from the culture medium. The expression profile of c-FOS gene was investigated as an indicator of shear in our system (Fig. 2e). The c-FOS is involved in the regulation of early signal transduction pathways by modulating expression of multiple genes as a part of the AP-1 transcriptional activator complex and the shear stress inducibility of c-FOS protein has been shown in human and animal cells lines of different origins2425. Expression of this gene was evaluated on CHO cells and obtained results showed that fluidic shear did not induce up-regulation of c-FOS gene as compared with the two negative controls: cells incubated in culture media at 37 °C and cells in the PBS buffer at 22 °C. This can be attributed to the low residence times of cells in the microfluidic channels (<0.1 sec).

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