IP Library › Granted Patent US 12,359,158
Granted Patent B2
US 12,359,158 · App. 18/456,340 · Granted Jul 15, 2025

Methods and systems for mechanoporation-based high-throughput payload delivery into biological cells

Inventors: Sewoon Han (Alameda, CA); Ian Sicher (Alameda, CA); Alexander Alexeev (Alameda, CA); Ockchul Kim (Alameda, CA)
Assignee: CellFE, Inc.
C12M35/04C12M29/04C12N15/87
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Quick Facts
Patent No.
US 12,359,158
App. No.
18/456,340
Granted
Jul 15, 2025
Kind
B2
Abstract

Described herein are methods and systems for mechanoporation-based high-throughput payload delivery into biological cells. For example, one system can process at least 1 billion cells per minute or at least 25 billion cells per minute, which is substantially greater than conventional methods. A cell processing apparatus comprises a processing assembly formed by stacking multiple processing components. Each processing component comprises channels, which may be used for filtration, mechanoporation, and/or separation of cells in the cell media. This functionality depends on the configuration of each channel. For example, each channel comprises one or more ridges such that each ridge forms a processing gap with an adjacent one of the processing components. The ridges may extend to the side walls or form a bypass gap with the wall. The processing gaps can be specially configured to compress cells as the cells pass through these gaps thereby initiating the mechanoporation process.

Claims (39)

1. A cell processing apparatus for processing cells using mechanoporation, the cell processing apparatus comprising:

an inlet plate comprising an inlet opening;

an outlet plate comprising an outlet opening; and

a processing component stacked between the inlet plate and the outlet plate, wherein:

the processing component comprises dividers, each comprising a first divider wall, a second divider wall, and a first surface, extending between the first divider wall and the second divider wall,

the processing component further comprises a channel wall, ridges protruding from the channel wall, and a second surface,

the first surface of the processing component directly contacts one of the inlet plate and the outlet plate,

the first divider wall, the second divider wall, the channel wall of the processing component and the one of the inlet plate and the outlet plate define multiple channels, each fluidically coupled and extending between the inlet opening and the outlet opening and configured for flowing a combination of a cell media and the cells such that one or more of the ridges of the processing component extend into each of the multiple channels, and

each of the ridges of the processing component forms a gap with the one of the inlet plate and the outlet plate such that the gap is smaller than a diameter of at least one of the cells thereby causing compression of the at least one of the cells while the cells pass through the gap.

2. The cell processing apparatus of claim 1 , wherein the first surface of the processing component directly contacts the inlet plate.

3. The cell processing apparatus of claim 2 , further comprising a second processing component, wherein:

the processing component comprises a second surface opposite the first surface,

the second processing component further comprises dividers, each comprising a first divider wall, a second divider wall, and a first surface, extending between the first divider wall and the second divider wall of the second processing component,

the second processing component further comprises a channel wall, ridges protruding from the channel wall, and a second surface,

the first surface of the second processing component directly contacts the second surface of the processing component,

the first divider wall, the second divider wall, the channel wall of the second processing component and the second surface of the processing component define multiple channels, each fluidically coupled and extending between the inlet opening and the outlet opening and configured for flowing the combination of the cell media and the cells such that one or more of the ridges of the processing component extend into each of the multiple channels, and

each of the ridges of the second processing component forms a gap with the second surface of the processing component such that the gap is smaller than the diameter of at least one of the cells thereby causing compression of the at least one of the cells while the cells pass through the gap.

4. The cell processing apparatus of claim 1 , wherein the first surface of the processing component directly contacts the outlet plate.

5. The cell processing apparatus of claim 1 , wherein the channels of the processing component extend parallel to each other.

6. The cell processing apparatus of claim 1 , wherein the channels of the processing component extend perpendicular to the inlet opening and the outlet opening.

7. The cell processing apparatus of claim 1 , wherein at least one of the inlet plate and the outlet plate is integrated with the processing component.

8. The cell processing apparatus of claim 1 , wherein each of the inlet plate and the outlet plate is sealed against the processing component.

9. The cell processing apparatus of claim 1 , wherein each of the inlet opening and the outlet opening has a cross-sectional area that changes along a direction perpendicular to the channels of the processing component.

10. The cell processing apparatus of claim 1 , wherein the ridges are oriented at an angle (a) between 30 degrees to 90 degrees relative to a central flow axis in each of the channels.

11. The cell processing apparatus of claim 1 , wherein:

the processing component is operable as a separator for separating the cells from the cell media;

the ridges are configured to direct the cells in the combination of the cell media and the cells to the outlet opening of the outlet plate; and

the outlet plate comprises a second outlet opening for removing the cell media from the processing component.

12. The cell processing apparatus of claim 1 , wherein:

the inlet plate is configured to connect to a sterile bag operable as a cell media source; and

the outlet plate is configured to connect to an additional sterile bag operable as a cell media receiver.

13. The cell processing apparatus of claim 1 , wherein the inlet plate comprises a collector port fluidically coupled to each of the multiple channels and configured to connect to a gas flow source.

14. The cell processing apparatus of claim 1 , wherein the inlet opening is positioned at a top of the cell processing apparatus thereby reducing air bubbles in the combination of the cell media and the cells through the inlet opening.

15. The cell processing apparatus of claim 1 , wherein the inlet plate or the processing component comprises a distribution pathway fluidically coupled to each of the channels in the processing component.

16. The cell processing apparatus of claim 1 , wherein at least a portion of the processing component is formed from silicon.

17. The cell processing apparatus of claim 1 , wherein at least a portion of the processing component is formed from glass.

18. The cell processing apparatus of claim 1 , wherein the inlet opening comprises a level sensor for measuring a level of the combination of the cell media and the cells within the inlet opening.

19. The cell processing apparatus of claim 1 , wherein the inlet opening comprises a pressure sensor for measuring pressure within the inlet opening.

20. The cell processing apparatus of claim 1 , wherein the processing component is operable as one or more of a primary filter, a mechanoporator, or a separator.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 25, 2023
From: HAN, SEWOON; SICHER, IAN; ALEXEEV, ALEXANDER; KIM, OCKCHUL
To: CELLFE, INC.
Reel/Frame 064711/0302 →
Continuity (4)
Continuation 18162372 · Jan 31, 2023
Continuation PCTUS2021059856 · Nov 18, 2021
Provisional Application 63115507 · Nov 18, 2020
Related Publication 20230416781A1 · Dec 28, 2023
References Cited (72)
US 6544423B1 · Baurmeister et al. · 2003 [cited by applicant]
US 8356714B2 · Sulchek et al. · 2013 [cited by applicant]
US 10039885B2 · Chow et al. · 2018 [cited by applicant]
US 10717084B2 · Sulchek et al. · 2020 [cited by applicant]
US 11198127B2 · Sulchek et al. · 2021 [cited by applicant]
US 11268892B2 · Sulchek et al. · 2022 [cited by applicant]
US 11788050B2 · Han · 2023 [cited by examiner]
US 20070072290A1 · Hvichia · 2007 [cited by applicant]
US 20110081674A1 · Han et al. · 2011 [cited by applicant]
US 20110253224A1 · Linder · 2011 [cited by examiner]
US 20110256551A1 · Linder et al. · 2011 [cited by applicant]
US 20140227777A1 · Choi et al. · 2014 [cited by applicant]
US 20140273229A1 · Meacham et al. · 2014 [cited by applicant]
US 20140287509A1 · Sharei et al. · 2014 [cited by applicant]
US 20160193605A1 · Sharei et al. · 2016 [cited by applicant]
US 20160228647A1 · Chow et al. · 2016 [cited by applicant]
US 20160272961A1 · Lee · 2016 [cited by applicant]
US 20170233692A1 · Pawell · 2017 [cited by applicant]
US 20180003696A1 · Sharei et al. · 2018 [cited by applicant]
US 20180016539A1 · Ding et al. · 2018 [cited by applicant]
US 20180142198A1 · Sharei et al. · 2018 [cited by applicant]
US 20180155669A1 · Pawell · 2018 [cited by applicant]
US 20180201889A1 · Sharei et al. · 2018 [cited by applicant]
US 20180245089A1 · Sharei et al. · 2018 [cited by applicant]
US 20180327706A1 · Qin et al. · 2018 [cited by applicant]
US 20190017072A1 · Ditommaso et al. · 2019 [cited by applicant]
US 20190111082A1 · Gilbert et al. · 2019 [cited by applicant]
US 20190177677A1 · Jonas et al. · 2019 [cited by applicant]
US 20190275520A1 · Stewart et al. · 2019 [cited by applicant]
US 20190322976A1 · Williams et al. · 2019 [cited by applicant]
US 20190382796A1 · Gilbert et al. · 2019 [cited by applicant]
US 20200172845A1 · Baker et al. · 2020 [cited by applicant]
US 20200316604A1 · Dadgar · 2020 [cited by examiner]
US 20200332243A1 · Dadgar · 2020 [cited by examiner]
US 20210292700A1 · Han et al. · 2021 [cited by applicant]
US 20210388390A1 · Bernstein et al. · 2021 [cited by applicant]
US 20220105166A1 · Sharei et al. · 2022 [cited by applicant]
US 20220204908A1 · Han et al. · 2022 [cited by applicant]
US 20220213422A1 · Zamarayeva et al. · 2022 [cited by applicant]
US 20220298461A1 · Zamarayeva et al. · 2022 [cited by applicant]
US 20230174918A1 · Han · 2023 [cited by applicant]
CN 110198785A · 2019 [cited by applicant]
CN 117015596B · 2024 [cited by applicant]
JP 2018025759A · 2018 [cited by applicant]
JP 2020503847A · 2020 [cited by applicant]
SG 11202303746 · 2024 [cited by applicant]
WO 2018089497A1 · 2018 [cited by applicant]
WO 2020026047A1 · 2020 [cited by applicant]
WO 2020037117A1 · 2020 [cited by applicant]
WO 2020210162A1 · 2020 [cited by applicant]
WO 2022109113A1 · 2022 [cited by applicant]
Singapore Application Serial No. 11202303746T, Decision to Grant mailed May 20, 2024, 2 pgs. [cited by applicant]
Williams, A.R. et al., “Filtroporation: A Simple, Reliable Technique for Transfection and Macromolecular Loading of Cells in Suspension,” Biotechnology and Bioengineering, vol. 65, No. 3, Nov. 5, 1999. [cited by applicant]
U.S. Appl. No. 18/162,372, Non Final Office Action mailed Jul. 13, 2023, 30 pgs. [cited by applicant]
U.S. Appl. No. 18/162,372, Notice of Allowance mailed Aug. 24, 2023, 10 pgs. [cited by applicant]
U.S. Appl. No. 18/162,372, Restriction Requirement mailed May 25, 2023, 7 pgs. [cited by applicant]
Barber “A Technic for the Inoculation of Bacteria and Other Substances into Living Cells” The Journal of Infectious Diseases, Apr. 12, 1911, vol. 8, No. 3 (Apr. 12, 1911), pp. 348-360. [cited by applicant]
Deng et al. “Intracellular Delivery of Nanomaterials via an Inertial Microfluidic Cell Hydroporator” Nano Lett. 2018, 18, 4, 2705-2710. [cited by applicant]
Di Carlo, “Enhanced Velocity Gradients within Microfluidics for Cellular Manipulation” In: Baba, Y., Shoji, S., van den Berg, A. (eds) Micro Total Analysis Systems 2002. Springer, Dordrecht. https://doi.org/10.1007/978-… [cited by applicant]
DiCarlo et al. “Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation” Lab on a Chip 3(4):287-91. [cited by applicant]
Ding et al. “High-throughput Nuclear Delivery and Rapid Expression of DNA via Mechanical and Electrical Cell-Membrane Disruption” Nat Biomed Eng. 2017; 1: 0039. [cited by applicant]
Hallow et al., “Shear-induced intracellular loading of cells with molecules by controlled microfluidics” Biotechnol Bioeng. Mar. 1, 2008; 99(4): 846-854. doi:10.1002/bit.21651. [cited by applicant]
Han et al. “CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation” Science Advances Aug. 14, 2015. [cited by applicant]
International Application Serial No. PCT/US2021/059856, Search Report and Written Opinion mailed Feb. 24, 2022. [cited by applicant]
Int'l Application Serial No. PCT/US21/59856, Int'l Preliminary Report on Patentability mailed 2023-06-016 pgs. [cited by applicant]
Jarrell et al. “Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding” Sci Rep 9, 3214 (2019). [cited by applicant]
Kang et al. “Intracellular Nanomaterial Delivery via Spiral Hydroporation” (ACS Nano 2020, 14, 3048-3058). [cited by applicant]
Liu et al “Cell mechanical and physiological behavior in the regime of rapid mechanical compressions that lead to volume change.” Small 16, 1903857-11, 2020. [cited by applicant]
Liu et al “Microfluidic generation of transient cell volume exchange for convectively driven intracellular delivery of large macromolecules. Materials Today 21, 703-712, 2018”. [cited by applicant]
Schmiderer et al. “Efficient and nontoxic biomolecule delivery to primary human hematopoietic stem cells using nanostraws” PNAS Sep. 1, 2020 117 (35) 21267-21273. [cited by applicant]
Sharei et al. “A vector-free microfluidic platform for intracellular delivery” Proc Natl Acad Sci U S A. Feb. 5, 2013; 110(6): 2082-2087. [cited by applicant]
Japanese Application Serial No. 2023-529914, Office Action mailed Jan. 28, 2025, 2 pgs. [cited by applicant]