- Cells
- iPSCs, T cells, Cancer Cell Lines
- Cargos
- Antibody, Oligo, Peptide, DELs, Protein, small molecules
Publication text
Research-Scale System Gateway™ Consumable Delivery Cartridge Compact bench-top unit that controls delivery parameters and easily fits in hood MicroBooster™ Defined pore size for deformation 18mm 35mm Cell type-specific consumable for optimal mechanoporationmediated cargo delivery Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● Low pressure requirements enable integration into existing robotics ● Ready for early adopters 2024 High-Throughput Capabilities “Powered by Portal” Consumables ● Portal is creating a transformative technology for cellular engineering compatible across cell types and cargos ○ Small molecules, oligos, peptides, antibodies, DELs, mRNA, siRNA, DNA, RNPs, proteins, and other cargos ○ Primary and patient-derived cell lines including immune and stem cells ● Cells are boosted through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering Enabling Novel Live Cell Assays with a Silicon Membrane-based Platform for Mechanical Delivery of Impermeable Small Molecules, Peptides, and Probes to Accelerate Early Drug Discovery Sophia Hirsch, Andrew Larocque, Eleni Rogers, Darby Kreienberg, Zhihui Song, Crystal Tsui, Jacquelyn Hanson, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA. www.portal.bio
- Sharei, et al., PNAS, 2013
- Szeto, et al., Scientific Reports, 2015
- Sharei, et al., PLOS One, 2015
- Lee and Sharei, et al. Nano Letters 2012
- Sharei et al., Integrative Biology 2014
- DiTommaso, et al., PNAS, 2018
Background on Mechanoporation ● Previous work has shown that mechanical deformation of cells through a microfluidic channel enables cytosolic intracellular delivery1 ○ Delivers macromolecules2,3,4 to both primary cells and cell lines ● Cells are squeezed through a narrow channel at high pressure. The cell membrane is temporarily disrupted, allowing the target cargo to enter the cytosol before the membrane seals5. ○ Cells can be mixed with cargo or added before membrane re-sealing ● Mechanoporation has minimal impact on cell function and gene expression, in contrast to electroporation and viral delivery methods6
Summary
● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Efficient delivery of diverse impermeable molecules for use in downstream screening including: ● Compatible across broad cell types including: NK cells HSCs B cellsPBMCs T cells iPSCs Connect with Portal! Contact us for beta program eligibility Efficient Co-delivery of Antibody, Oligos, and Dextran to HeLa Cells Mixed isotype control antibody, labeled oligo, and fluorescent dextran Antibody Untreated Oligo Dextran Fluorescently-labeled IgG isotype control antibody, Cy5-labeled oligo, and fluorescent dextran were mixed and mechanoporated into HeLa cells. Flow cytometry was conducted post-boost to assess viability and delivery of the cargo types to the cells. Robust DNA Encoded Library Delivery in iPSCs and HeLa Cells iPSCs Fluorescent-tagged OR iPSC Boost HeLa Boost Induced pluripotent stem cells (iPSCs) or HeLa cells were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was measured using flow cytometry to detect the fluorescent tag immediately after boosting. Consistent Delivery of Cargo Across Pre-loaded Plates by Automated Dispensing Boost with no cargo Dispense into 96 well plates pre-loaded with dextran 24h post-boost Dextran Delivery to HeLa Cells by Automated Dispensing Well # (Plate 2) Well # (Plate 7) 10 min post-boost A cartridge was designed for integration of mechanoporation with a robotic single-head dispenser. HeLa cells were automatically dispensed into 96-well plates pre-loaded with fluorescent dextran. Flow cytometry was used to assess dextran delivery. Data for delivery across the 2nd (wells 97-192) and 7th (wells 577-672) 96-well plates are shown here. Phase contrast images (right) show morphology of HeLa cells 10 minutes (top) and 24 hours (bottom) post-boost. Scale bar (white) = 300 μm. Macrocycle Delivery to HeLa cells Shows Robust Dose Response Macrocycle Boost with no cargo Efficient Delivery of Labeled Macrocycle to HEK293 Cells HEK293 cells FITC-labeled macrocycle Untreated FITC Transmitted light HEK293 cells were mechanoporated with a FITC-labeled macrocycle. Flow cytometry was used to assess viability and macrocycle delivery to the untreated and boosted samples. Delivery was also assessed by fluorescence microscopy (right). Scale bar = 300 μm. Pipette into plate pre-loaded with labeled macrocycle HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with FITC-labeled macrocycle at 0 - 96 μM. Flow cytometry was conducted post-boost to assess viability and efficiency of macrocycle delivery into boosted cells.
- Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999
www.portal.bio ● Simple workflow enabling high throughput capabilities ● Can be used to deliver single or multiplexed cargoes ● Little impact on normal cellular function or morphology 0 μM 12 μM 24 μM 48 μM 96 μM Enabling High Throughput Screening by Automated Dispensing into Pre-loaded Cargo Plates Automated dispensing into multiwell plates ● Portal cartridge integrated in-line with the fluidics of an existing robot. The highlighted component is the only addition/modification ● Cells are porated as they transit through the membrane ● Cargo is pre-plated in the wells and the porated cells take up material upon entering the well HEK293 cells + Other cell lines DELs Peptides Macrocycle Antibodies Small molecules Robust Multiplex Delivery in Unstimulated T Cells GFP expression B2M expression Mixed dextran, mRNA, & RNP Naive T cells Naive T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. After 40h, expression of mRNA and B2M protein were assessed by flow cytometry. Delivering Promega’s LgBiT Reagents for Intracellular Protein Detection ● MiaPaCa-2 cells endogenously tagged with HiBiT at the KRAS(G12C) locus were treated with the PROTAC LC-2 for 24 hours (0-10 μM) and mechanoporated with LgBiT protein (Promega) ● Nano-Glo® Endurazine™ was added and luminescence was detected on a GloMAX plate reader to detect degradation of
Kras(g12c)
● Potential utility in other intracellular labeling assays to determine PPIs, degradation and other live cell kinetics Concentration LC-2 Endogenous protein with HiBiT LgBiT delivery for luminescence Degradation removes signal
Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.
# Publication
This page is a text version of a Portal Biotechnologies publication. The bibliographic record is labeled in the text so it can be read without the surrounding website.
## Bibliographic record
- Title: Publication
- Document type: Poster
- Year: 2024
- Outlet: Drug Discovery Chemistry Europe
- Cells: iPSCs, T cells, Cancer Cell Lines
- Cargos: Antibody, Oligo, Peptide, DELs, Protein, small molecules
- Page URL: https://portal.bio/publications/2024-publication--ajkGOxEAACkAc92Y
- Markdown URL: https://portal.bio/publications/2024-publication--ajkGOxEAACkAc92Y.md
- Original file: https://portal-bio.cdn.prismic.io/portal-bio/aFos-3fc4bHWiplR_DDC-EuropePosterTsui-1-.pdf
## Publication text
Research-Scale System Gateway™ Consumable Delivery Cartridge Compact bench-top unit that controls delivery parameters and easily fits in hood MicroBooster™ Defined pore size for deformation 18mm 35mm Cell type-specific consumable for optimal mechanoporationmediated cargo delivery Introduction to Portal’s Approach: Implementing a Universal Cell Engineering Solution Mechanical Disruption via Boosting Intracellular DeliveryCells and Cargo Cells Cargo ● Low pressure requirements enable integration into existing robotics ● Ready for early adopters 2024 High-Throughput Capabilities “Powered by Portal” Consumables ● Portal is creating a transformative technology for cellular engineering compatible across cell types and cargos ○ Small molecules, oligos, peptides, antibodies, DELs, mRNA, siRNA, DNA, RNPs, proteins, and other cargos ○ Primary and patient-derived cell lines including immune and stem cells ● Cells are boosted through a membrane at low operating pressure (3-15 PSI) ○ Membrane format1 enables high-throughput cellular engineering Enabling Novel Live Cell Assays with a Silicon Membrane-based Platform for Mechanical Delivery of Impermeable Small Molecules, Peptides, and Probes to Accelerate Early Drug Discovery Sophia Hirsch, Andrew Larocque, Eleni Rogers, Darby Kreienberg, Zhihui Song, Crystal Tsui, Jacquelyn Hanson, Alec Barclay, Armon Sharei Portal Biotechnologies, 134 Coolidge Ave, Watertown, MA 02472, USA. www.portal.bio
1. Sharei, et al., PNAS, 2013
2. Szeto, et al., Scientific Reports, 2015
3. Sharei, et al., PLOS One, 2015
4. Lee and Sharei, et al. Nano Letters 2012
5. Sharei et al., Integrative Biology 2014
6. DiTommaso, et al., PNAS, 2018
Background on Mechanoporation ● Previous work has shown that mechanical deformation of cells through a microfluidic channel enables cytosolic intracellular delivery1 ○ Delivers macromolecules2,3,4 to both primary cells and cell lines ● Cells are squeezed through a narrow channel at high pressure. The cell membrane is temporarily disrupted, allowing the target cargo to enter the cytosol before the membrane seals5. ○ Cells can be mixed with cargo or added before membrane re-sealing ● Mechanoporation has minimal impact on cell function and gene expression, in contrast to electroporation and viral delivery methods6
### Summary
● Portal’s mechanoporation technology can be used for drug discovery and cell engineering ● Efficient delivery of diverse impermeable molecules for use in downstream screening including: ● Compatible across broad cell types including: NK cells HSCs B cellsPBMCs T cells iPSCs Connect with Portal! Contact us for beta program eligibility Efficient Co-delivery of Antibody, Oligos, and Dextran to HeLa Cells Mixed isotype control antibody, labeled oligo, and fluorescent dextran Antibody Untreated Oligo Dextran Fluorescently-labeled IgG isotype control antibody, Cy5-labeled oligo, and fluorescent dextran were mixed and mechanoporated into HeLa cells. Flow cytometry was conducted post-boost to assess viability and delivery of the cargo types to the cells. Robust DNA Encoded Library Delivery in iPSCs and HeLa Cells iPSCs Fluorescent-tagged OR iPSC Boost HeLa Boost Induced pluripotent stem cells (iPSCs) or HeLa cells were mechanoporated with a fluorescently-tagged DNA encoded library (DEL) probe. Delivery efficiency was measured using flow cytometry to detect the fluorescent tag immediately after boosting. Consistent Delivery of Cargo Across Pre-loaded Plates by Automated Dispensing Boost with no cargo Dispense into 96 well plates pre-loaded with dextran 24h post-boost Dextran Delivery to HeLa Cells by Automated Dispensing Well # (Plate 2) Well # (Plate 7) 10 min post-boost A cartridge was designed for integration of mechanoporation with a robotic single-head dispenser. HeLa cells were automatically dispensed into 96-well plates pre-loaded with fluorescent dextran. Flow cytometry was used to assess dextran delivery. Data for delivery across the 2nd (wells 97-192) and 7th (wells 577-672) 96-well plates are shown here. Phase contrast images (right) show morphology of HeLa cells 10 minutes (top) and 24 hours (bottom) post-boost. Scale bar (white) = 300 μm. Macrocycle Delivery to HeLa cells Shows Robust Dose Response Macrocycle Boost with no cargo Efficient Delivery of Labeled Macrocycle to HEK293 Cells HEK293 cells FITC-labeled macrocycle Untreated FITC Transmitted light HEK293 cells were mechanoporated with a FITC-labeled macrocycle. Flow cytometry was used to assess viability and macrocycle delivery to the untreated and boosted samples. Delivery was also assessed by fluorescence microscopy (right). Scale bar = 300 μm. Pipette into plate pre-loaded with labeled macrocycle HeLa cells were boosted then manually added into wells of a 96-well plate pre-loaded with FITC-labeled macrocycle at 0 - 96 μM. Flow cytometry was conducted post-boost to assess viability and efficiency of macrocycle delivery into boosted cells.
1. Williams AR, Bao S, Miller DL. Biotechnol Bioeng. 1999
www.portal.bio ● Simple workflow enabling high throughput capabilities ● Can be used to deliver single or multiplexed cargoes ● Little impact on normal cellular function or morphology 0 μM 12 μM 24 μM 48 μM 96 μM Enabling High Throughput Screening by Automated Dispensing into Pre-loaded Cargo Plates Automated dispensing into multiwell plates ● Portal cartridge integrated in-line with the fluidics of an existing robot. The highlighted component is the only addition/modification ● Cells are porated as they transit through the membrane ● Cargo is pre-plated in the wells and the porated cells take up material upon entering the well HEK293 cells + Other cell lines DELs Peptides Macrocycle Antibodies Small molecules Robust Multiplex Delivery in Unstimulated T Cells GFP expression B2M expression Mixed dextran, mRNA, & RNP Naive T cells Naive T cells were isolated from PBMCs and mechanoporated with a multiplexed cargo containing GFP mRNA, fluorescently-labeled dextran polymers, and β2 microglobulin (B2M) CRISPR RNPs. After 40h, expression of mRNA and B2M protein were assessed by flow cytometry. Delivering Promega’s LgBiT Reagents for Intracellular Protein Detection ● MiaPaCa-2 cells endogenously tagged with HiBiT at the KRAS(G12C) locus were treated with the PROTAC LC-2 for 24 hours (0-10 μM) and mechanoporated with LgBiT protein (Promega) ● Nano-Glo® Endurazine™ was added and luminescence was detected on a GloMAX plate reader to detect degradation of
### Kras(g12c)
● Potential utility in other intracellular labeling assays to determine PPIs, degradation and other live cell kinetics Concentration LC-2 Endogenous protein with HiBiT LgBiT delivery for luminescence Degradation removes signal
## Provenance
Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.
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