Purifying stem cell‐derived red blood cells: a high‐throughput label‐free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration

Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and econom...

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Published in:	Biotechnology and bioengineering Vol. 117; no. 7; pp. 2032 - 2045
Main Authors:	Guzniczak, Ewa, Otto, Oliver, Whyte, Graeme, Chandra, Tamir, Robertson, Neil A., Willoughby, Nik, Jimenez, Melanie, Bridle, Helen
Format:	Journal Article
Language:	English
Published:	United States Wiley Subscription Services, Inc 01.07.2020 John Wiley and Sons Inc
Subjects:	Biopharmaceuticals CD34 antigen Cell Line Cell Separation - instrumentation Cord blood Deformability Equipment Design Erythrocytes Erythrocytes - cytology Erythropoiesis Filtration Filtration - instrumentation Fluorescence Formability Gene expression Humans Manufacturing industry Mechanical properties Membrane filtration Membranes Microfluidic Analytical Techniques - instrumentation Microfluidics Nuclei (cytology) Purification Purity Separation sorting spiral microchannel Stem cells Stem Cells - cytology stem cell‐derived red blood cells Umbilical cord deformability stem cell-derived red blood cells purification sorting spiral microchannel
ISSN:	0006-3592, 1097-0290, 1097-0290
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Abstract	Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability‐based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high‐throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products. To address the challenge of stem‐cell‐derived red blood cells purification, we propose a label‐free approach to separate cells at high throughput based on their morphological (size) and mechanical (deformability) properties. The process consists of two main steps: (a) a microfluidic separation using inertial focusing in spiral microchannel and (b) membrane filtration.
AbstractList	Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability‐based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high‐throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products. To address the challenge of stem‐cell‐derived red blood cells purification, we propose a label‐free approach to separate cells at high throughput based on their morphological (size) and mechanical (deformability) properties. The process consists of two main steps: (a) a microfluidic separation using inertial focusing in spiral microchannel and (b) membrane filtration. Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability‐based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high‐throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products. Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability‐based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high‐throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products. To address the challenge of stem‐cell‐derived red blood cells purification, we propose a label‐free approach to separate cells at high throughput based on their morphological (size) and mechanical (deformability) properties. The process consists of two main steps: (a) a microfluidic separation using inertial focusing in spiral microchannel and (b) membrane filtration. Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability-based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high-throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products.Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability-based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high-throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products.
Author	Guzniczak, Ewa Chandra, Tamir Jimenez, Melanie Otto, Oliver Bridle, Helen Willoughby, Nik Robertson, Neil A. Whyte, Graeme
AuthorAffiliation	5 Biomedical Engineering Division, James Watt School of Engineering University of Glasgow Glasgow Scotland 2 Centre for Innovation Competence – Humoral Immune Reactions in Cardiovascular Diseases University of Greifswald Greifswald Germany 3 Deutsches Zentrum für Herz‐Kreislaufforschung Partner Site Greifswald Greifswald Germany 4 MRC Human Genetics Unit, MRC Institute of Genetics & Molecular Medicine, The University of Edinburgh Western General Hospital Edinburgh Scotland 1 Department of Biological Chemistry, Biophysics and Bioengineering Edinburgh Campus, School of Engineering and Physical Science Heriot‐Watt University Edinburgh Scotland
AuthorAffiliation_xml	– name: 1 Department of Biological Chemistry, Biophysics and Bioengineering Edinburgh Campus, School of Engineering and Physical Science Heriot‐Watt University Edinburgh Scotland – name: 2 Centre for Innovation Competence – Humoral Immune Reactions in Cardiovascular Diseases University of Greifswald Greifswald Germany – name: 3 Deutsches Zentrum für Herz‐Kreislaufforschung Partner Site Greifswald Greifswald Germany – name: 4 MRC Human Genetics Unit, MRC Institute of Genetics & Molecular Medicine, The University of Edinburgh Western General Hospital Edinburgh Scotland – name: 5 Biomedical Engineering Division, James Watt School of Engineering University of Glasgow Glasgow Scotland
Author_xml	– sequence: 1 givenname: Ewa orcidid: 0000-0002-7380-2077 surname: Guzniczak fullname: Guzniczak, Ewa email: eg100@hw.ac.uk organization: Heriot‐Watt University – sequence: 2 givenname: Oliver surname: Otto fullname: Otto, Oliver organization: Partner Site Greifswald – sequence: 3 givenname: Graeme surname: Whyte fullname: Whyte, Graeme organization: Heriot‐Watt University – sequence: 4 givenname: Tamir surname: Chandra fullname: Chandra, Tamir organization: Western General Hospital – sequence: 5 givenname: Neil A. surname: Robertson fullname: Robertson, Neil A. organization: Western General Hospital – sequence: 6 givenname: Nik surname: Willoughby fullname: Willoughby, Nik organization: Heriot‐Watt University – sequence: 7 givenname: Melanie surname: Jimenez fullname: Jimenez, Melanie organization: University of Glasgow – sequence: 8 givenname: Helen surname: Bridle fullname: Bridle, Helen organization: Heriot‐Watt University
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Keywords	deformability stem cell-derived red blood cells purification sorting spiral microchannel
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Snippet	Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product... Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product...
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StartPage	2032
SubjectTerms	Biopharmaceuticals CD34 antigen Cell Line Cell Separation - instrumentation Cord blood Deformability Equipment Design Erythrocytes Erythrocytes - cytology Erythropoiesis Filtration Filtration - instrumentation Fluorescence Formability Gene expression Humans Manufacturing industry Mechanical properties Membrane filtration Membranes Microfluidic Analytical Techniques - instrumentation Microfluidics Nuclei (cytology) Purification Purity Separation sorting spiral microchannel Stem cells Stem Cells - cytology stem cell‐derived red blood cells Umbilical cord
Title	Purifying stem cell‐derived red blood cells: a high‐throughput label‐free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration
URI	https://onlinelibrary.wiley.com/doi/abs/10.1002%2Fbit.27319 https://www.ncbi.nlm.nih.gov/pubmed/32100873 https://www.proquest.com/docview/2412768896 https://www.proquest.com/docview/2365220409 https://pubmed.ncbi.nlm.nih.gov/PMC7383897
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