IP Library › Granted Patent US 12,709,727
Granted Patent B2
US 12,709,727 · App. 17/965,367 · Granted Aug 18, 2026

Devices and methods for increasing throughput of flow-based electroporation systems

Inventors: Thomas N. Corso (Groton, NY); Harold G. Craighead (Ithaca, NY); Jacob Vanderburgh (Ithaca, NY)
Assignee: CyteQuest, Inc.
C12M35/02C12N13/00C12N15/87
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Quick Facts
Patent No.
US 12,709,727
App. No.
17/965,367
Filed
Oct 13, 2022
Granted
Aug 18, 2026
Kind
B2
Art Unit
1798
USPC
435/285.2
Abstract

Disclosed are flow devices and methods for electroporation, which allow controlling the throughput of electroporation, for example by operating the electroporation process at selected throughput or operating at an increased or decreased level of throughput compared to a reference level of throughput by scaling a subset of electroporation parameters, while allowing maintaining cell viability and transfection efficiency.

Claims (22)

1 . A method of electroporation using a microfluidic device, the method comprising:

flowing a liquid through a channel in the microfluidic device in a flow direction, the fluid including a plurality of cells and molecules, wherein the channel comprises a channel height (H) and a channel width (W), the ratio of the channel height to the channel width being less than about 0.1;

generating a time-dependent electric field across the channel and perpendicular to the flow direction using electrodes arranged transverse to the direction of flow, wherein a polarity of the time-dependent electric field is configured to alternate as a function of time, and wherein a waveform of the time-dependent electric field comprises a series of alternating positive and negative peaks; and

coordinating a flow speed of the liquid through the channel and a waveform of the time-dependent voltage applied to the electrodes to generate the time-dependent electric field such that the molecules are moved into the plurality of cells after being subject to the time-dependent electric field, wherein the voltage wave form comprises a periodic bipolar waveform with alternating segments of symmetric reversed polarity, each of the alternating segments having a first segment portion followed by a second segment portion, the first segment portion having a shorter duration and a higher amplitude relative to the second segment portion.

2 . The method of claim 1 , further comprising selecting parameters for the electroporation, the parameters including:

a volume (V) of the liquid to be electroporated; and

a time (T) to perform the electroporation in.

3 . The method of claim 2 , where in the flow speed is a fluid flow speed(S), and a relationship exists where H×W×S equals a throughput of the electroporation, and wherein the throughput is defined as the volume of the liquid (V) to be electroporated per the time (T).

4 . The method of claim 3 , wherein the channel width and the fluid flow speed are proportionally modified by a same factor to maintain a same average linear flow velocity of the plurality of cells.

5 . The method of claim 1 , wherein the microfluidic device comprises 2-10 electrodes.

6 . The method of claim 1 , wherein the channel comprises multiple pairs of electrodes to apply the time-dependent electric field.

7 . The method of claim 1 , wherein the microfluidic device comprises one inlet.

8 . The method of claim 1 , wherein the microfluidic device comprises at least two inlets.

9 . The method of claim 1 , wherein the channel comprises one or more electrodes disposed on each of opposite sides of the channel, wherein a space is defined between the one or more electrodes.

10 . The method of claim 9 , wherein the plurality of cells pass through the space in a single layer.

11 . The method of claim 1 , wherein the plurality of cells are selected from the group consisting of lymphocytes, T cells, primary T cells, CHO, Hela, CD8+, CD4+, CD3+, PBMC, Huh-7, Renca, NIH 3T3, Primary Fibroblasts, hMSC, K562, Vero, HEK 293, A549, B16, BHK-21, C2C12, C6, CaCo-2, CAP-T, COS-1, Cos-7, CV-1, DLD-1, H1299, Hep G2, HOS, Jurkat, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, Mesenchymal Stem Cells, Min-6, Neuro2a, NIH3T3L1, NSO, Panc-1, PC12, PC-3, RBL, RLE, SF21, SF9, SH-SY5Y, SK-MES-1, SK-N-SH, SL3, SW403, THP-1, U205, U937, and combinations thereof.

12 . The method of claim 1 , wherein the microfluidic device further comprises a flow sensor configured to measure the flow speed and communicate with a feedback control mechanism.

13 . The method of claim 1 , wherein the channel width is constant across a length of the channel.

14 . The method of claim 1 , wherein the channel width varies across a length of the channel.

15 . The method of claim 1 , wherein the molecules are selected from the group consisting of DNA, RNA, protein, peptide, peptidomimetic, beads, dyes, or combinations thereof.

16 . The method of claim 1 , wherein the molecules comprise mRNA, and the mRNA is electroporated into at least a portion of the plurality of cells with a transfection efficiency of greater than 95% and less than 2% in loss of cell viability.

17 . The method of claim 1 , wherein the molecules are charged.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 29, 2022
From: CORSO, THOMAS N.; CRAIGHEAD, HAROLD G.; VANDERBURGH, JACOB
To: CYTEQUEST, INC.
Reel/Frame 062234/0716 →
Continuity (3)
Provisional Application 63399024 · Aug 18, 2022
Provisional Application 63255294 · Oct 13, 2021
Related Publication 20230114435A1 · Apr 13, 2023
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