Full-automatic cell electroporation system based on digital microfluidics and gene editing cell preparation method

Abstract

The invention discloses a full-automatic cell electroporation system based on digital microfluidics and a gene editing cell preparation method, and belongs to the technical field of microfluidic chip detection. The system comprises a digital micro-fluidic chip, a system control and driving module, an electroporation signal generation module, a cell culture module, a magnetic control module and an in-situ detection module. An electroporation area and a driving electrode array are arranged on the chip, so that accurate control and efficient electroporation of liquid drops can be realized. The invention further provides a gene editing cell preparation method based on the system. The method comprises the whole-process integrated operation of T cell activation, magnetic bead removal, electroporation gene editing, cell amplification, co-culture and in-situ killing function detection. The system has the advantages of full-process automation, low cell and reagent dosage, high editing efficiency, good cell activity, high-throughput parallel processing and the like, and is particularly suitable for efficient gene editing and CAR-T cell preparation under low cell input.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidic chip detection, and particularly relates to a full-automatic cell electroporation system based on digital microfluidics and a method for preparing gene edited cells. BACKGROUND

[0002] In recent years, the field of gene editing has attracted widespread attention due to its great therapeutic potential. In particular, gene editing therapies, such as CRISPR / Cas9-mediated gene editing, provide a promising and safe treatment option for a variety of diseases. Gene editing in primary human target cells has been proven to be an effective way for cell therapy. However, the technical challenge of efficiently obtaining a large number of gene editing therapy cells in a short time hinders the widespread clinical application of CRISPR / Cas9-mediated gene editing.

[0003] The electroporation gene transfection method, as a method that has emerged in recent years, has its unique advantages and significance. Compared with other common gene transfection methods (such as chemical transfection and viral transduction), the advantages of electroporation are as follows: first, the applicability of cells is wide, and electroporation is almost independent of cell type, with high transfection rate in both suspended cells and adherent cells; second, the range of transfection molecules is wide, and electroporation is effective for transfection of macromolecules such as DNA, mRNA, and protein complexes (Cas9 / sgRNA); third, the operation is simple and fast; and fourth, the experimental results have strong repeatability.

[0004] Although electroporation has the above advantages, the electroporation method also faces the following challenges: first, the high-voltage pulse of the electroporation method causes greater damage to cells and easily causes a large number of cell deaths; second, compared with the chemical transfection method, the electroporation method requires a larger number of cells, and the parameters must be carefully optimized; third, the electroporation transfection method lacks fully automated equipment manufacturing, which increases the labor and time costs; and fourth, the electroporation throughput is low, and the number of cells processed at a time is limited.

[0005] Digital microfluidics (DMF) is a programmable droplet manipulation platform that can achieve high-precision, independent manipulation of discrete droplets with microliter to picoliter volumes, has the characteristics of micro-reagent use, high-precision liquid control, easy integration, on-chip cultivation and detection, and high throughput. The latest research combines DMF and electroporation to reduce the damage of high-voltage electroporation to cells and reduce the amount of T cells. For example, Little et al. combined CRISPR-based knockout and knock-in editing in primary human cells by implementing Tri-Drop electroporation. In addition, petal et al. improved the high cell input required for electroporation, and their proposed electroporation platform combined DMF to achieve efficient gene delivery in 10,000 T cells, but the platform is not fully automated, and only the electroporation step is implemented on the chip.

[0006] Therefore, based on the advantages of DMF electric conversion and the lack of full automation of current equipment, there is an urgent need in the art for a full-automatic manufacturing platform that can realize small voltage damage, small amount of reagent use, and low cell input under in-situ detection and analysis of CAR-T from T cell activation to high-throughput parallel electric conversion, so as to greatly reduce the labor and time cost. SUMMARY

[0007] The present application aims to solve the technical problems of large cell damage, large amount of cells and reagents, low automation degree, and insufficient throughput in existing electroporation technology, and in particular to solve the problem of difficult efficient gene editing and CAR-T cell preparation under low cell input (such as 10,000 cells).

[0008] In order to overcome the shortcomings of existing technology, the present application proposes a full-automatic cell electroporation system based on digital microfluidics, and the specific technical solutions are as follows: A full-automatic cell electroporation system based on digital microfluidics, characterized in that it comprises: Digital microfluidic chip: as the core reaction platform, the chip integrates driving electrodes for droplet manipulation, special electrodes for cell electroporation, and areas for cell culture and detection. Specifically, it includes an upper plate and a lower plate, the distance between the upper plate and the lower plate is 10-1000 microns, and a gap for manipulating droplets is formed therebetween; the lower plate is provided with an array of driving electrodes for moving, merging, and splitting droplets through dielectric wetting effect; the chip is provided with at least one independent electroporation area, which contains one or more pairs of independent electroporation electrodes, and the electroporation electrodes are driving electrodes stripped of dielectric layers. The electroporation electrodes are different from standard driving electrodes, and the size, shape, and spacing of these electrodes are optimized to generate a local, high-intensity, and uniform electric field. Interdigital electrodes or concentric circle electrodes can be used to focus the electric field in a small volume, so as to achieve efficient electroporation at a lower voltage, ensure that the cell membrane can be effectively penetrated when the cell suspension passes through, and at the same time, minimize cell damage caused by joule heat or electrolysis.

[0009] System control and driving module: electrically connected to the array of driving electrodes, used to control the automatic operation process of droplets, and can accurately apply voltage to specific electrodes according to the preset program to realize the movement, mixing, and splitting of droplets.

[0010] Electroporation signal generation module: Electrically connected to the electroporation electrode, used to generate programmable electroporation pulse signals; the pulse signals include a high-voltage, short-pulse-width piercing pulse and one or more lower-voltage, longer-pulse-width drive / transfection pulses, the drive pulse employing a decaying waveform. The voltage range of the piercing pulse is 0.1-500V, the pulse length is 0.01-200ms, the pulse interval is 0.01-200ms, and the number of pulses is 1-10; the drive pulse includes two types: square wave and decaying wave. The decaying wave ranges from 0.1-500V; the square wave voltage range is 0.1-300V, the pulse length is 0.05-1000ms, the pulse interval is 0.05-1000ms, and the number of pulses is 1-1000.

[0011] Cell culture module: integrated into the system or coupled to the chip environment, used to provide and maintain the temperature, humidity and CO2 concentration environment required for cell culture, ensuring high cell survival rate throughout the preparation process. The cell culture module can maintain the chip environment at a temperature of 37±1℃, a CO2 concentration of 5±0.5%, and a humidity of not less than 60%.

[0012] Magnetically controlled module: includes a movable or switchable magnet for separating magnetic beads from cells on the chip or for magnetic bead-based cell sorting.

[0013] In-situ detection module: integrated within the system, used for real-time or endpoint detection of cells or their secretions on the chip; preferably, the in-situ detection module includes a fluorescence microscopy imaging module and / or a photomultiplier tube detection module.

[0014] Based on the above system, the present invention also provides a fully automated method for preparing electroporation and gene-edited cells (taking CAR-T cell preparation as an example), characterized by comprising the following steps: S1: On a digital microfluidic chip, droplets containing T cells are combined and mixed with droplets containing T cell activating magnetic beads, and then automatically activated and cultured.

[0015] S2: Use the magnetocontrol module to remove the activation magnetic beads to obtain activated T cell droplets.

[0016] S3: Combine and mix the droplet containing the activated T cells with the droplet containing the gene editing component in the electroporation buffer solution; the gene editing component includes the electroporation buffer solution, the expression vector of the target gene or other molecules (usually exogenous DNA (such as plasmid DNA) or RNA (such as mRNA or siRNA)), the CRISPR / Cas9 ribonucleoprotein complex, and the donor DNA template.

[0017] S4: Move the mixed droplet to the electroporation region and apply an optimized electroporation pulse for gene editing. The electroporation pulse includes one or more pore pulses and a series of drive pulses using attenuated waveforms or square waves. The input amount of the target cells is 1,000 to 100,000 cells per editing unit. The volume of the droplet is 0.1 to 50 microliters.

[0018] S5: Remove the cell droplets after electroporation from the electroporation area, add culture medium droplets containing cytokines, and carry out automated amplification culture, with programmed medium changes during the process.

[0019] S6: The expanded CAR-T cell droplets are combined with target cell droplets expressing specific antigens and co-cultured.

[0020] S7: Add fluorescent dye to the co-cultured cell droplets, mix well, and then use the in-situ detection module to detect the target cell survival rate and evaluate the killing efficacy of CAR-T cells.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. Integrated and automated process: For the first time, the entire process of gene-edited cell preparation and characterization has been integrated on a single DMF platform. Taking CAR-T editing as an example, it demonstrates the complete process from T cell activation, magnetic bead removal, electroporation, amplification, co-culture to in-situ detection, realizing true "sample in, result out", which greatly reduces manual operation, cross-contamination risk, and time and labor costs.

[0022] 2. High efficiency and low cell usage: Through optimized chip design and electroporation parameters (especially the drive pulse of the attenuation waveform), gene transfection / editing efficiency of more than 50% can still be achieved at extremely low cell input (such as 10,000 cells), breaking through the technical bottleneck of traditional plate electroporation where efficiency is close to the background level at low cell numbers.

[0023] 3. High cell viability and killing efficacy: Low-volume, low-current electroporation in a microfluidic environment effectively reduces damage from Joule heating and electrolysis byproducts, allowing edited cells to maintain high viability (e.g., >79%), and the prepared CAR-T cells exhibit superior target cell killing efficiency compared to traditional methods (approximately 70% vs. 60%).

[0024] 4. High throughput and parallelizability: The on-chip drive electrode array and multiple independent electrotransfer regions allow for the simultaneous execution of multiple independent or parallel experiments under different conditions, providing a powerful platform for the rapid optimization of CAR-T fabrication conditions and high-throughput production.

[0025] 5. Extremely low reagent consumption: Micro- and even nano-level droplet manipulation reduces reagent consumption (such as expensive mRNA, siRNA, sgRNA, plasmid DNA) to 1 / 50 of that of traditional methods, significantly reducing experimental costs. Attached Figure Description

[0026] Figure 1 This is a side view of the chip used in this invention; Figure 2 This is a timeline of CAR-T preparation and in-situ analysis on the platform of this invention; Figure 3 This is a schematic diagram of the entire process of fabricating CAR-T on a chip and performing in-situ analysis; Figure 4 This is a schematic diagram of the electroporation driving voltage attenuation method of the present invention; Figure 5 These are microscopic images of cells after electroporation and amplification. Figure 6 This is a Day 5 cell viability analysis graph; Figure 7 This is a flow cytometry image from three parallel experiments, used to detect the positive rate of cell electroporation. Figure 8 This is a comparison chart of the killing efficiency of CAR-T cells prepared by the present invention and CAR-T cells prepared by the traditional well plate method. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1 The core of this invention lies in integrating multiple independent steps of traditional cell gene editing into an automated digital microfluidic (DMF) platform. Using CAR-T cell editing as an example, the following details the complete experimental process from initial T cell processing to final functional verification, along with the key technical parameters and implementation details involved.

[0029] Step 1: T cell activation (Day 0) Pre-operative preparation for off-screen manipulation: PanCD3+ T cells were derived from peripheral blood mononuclear cells (PBMCs). The isolated T cells were prepared at a concentration of 1×10⁻⁶. 7 A suspension of cells / mL.

[0030] On the chip, one unit (1 μL) of T cell suspension and one unit of T cell activator (containing CD3+ activating magnetic beads) are combined and mixed, with a bead-to-cell ratio of 2:5. The culture medium contains 200 IU / mL IL-2, 10 ng / mL IL-7, and 5 ng / mL IL-15. The mixture is homogenized three times using a pre-defined circular motion, and then cultured in a 37°C, 5% CO2 incubator. The homogenization process is repeated 2-3 times during this period to promote cell-bead contact. This process corresponds to... Figure 3 Step (a) in the process.

[0031] Step 2: Activate magnetic bead removal (Day 1) One day later, an external magnetic field (small magnet, incubated for 30 seconds to 1 minute) was applied through the magnetic enrichment module to separate the activation magnetic beads from the cells. The separated cells were used for the next step of electroporation, while the magnetic beads were resuspended in a resuspension solution and then moved to the drain tube connected to the chip for removal. This process corresponds to... Figure 3 Step (b) in the process.

[0032] Step 3: Preparations before electroporation a. Preparation of electroporation buffer: TRACRNPs were assembled on ice for 20 minutes using 110 pmol TRACsgRNA and 33 pmol SpCas9 outside the chip. The test concentrations of the anti-HER2CAR DNA donor template were 10 ng, 5 ng, 0.625 ng, 0.0375 ng, and 0 ng (stock solution and diluent were added on-chip; other concentrations were diluted on-chip). Each edit contained one unit of transfection buffer (1.1 pmol of guide, 0.33 pmol of Cas9, and the anti-HER2CAR concentration as described above).

[0033] b. Preparation of cell sap: Each edit contains one unit (1 μL) of a 1×10⁻⁶ solution. 7 T cells / mL.

[0034] Step 4: Electroporation (Day 1) Combine one unit of transfection buffer with one unit of activated T cell suspension and mix thoroughly three times following the pre-defined circular motion. Transfer the mixed cell suspension to the electroporation zone in preparation for electroporation. Once in place, perform electroporation according to the pre-defined electroporation parameters. The electroporation parameters are as follows: Perforation voltage: 75V, pulse length: 5ms, pulse interval: 1ms, number of pulses: 1, attenuation: none; Drive voltage: 15V, pulse length: 50ms, pulse interval: 50ms, number of pulses: 5, mode: decay (dynamic decay: alternating positive and negative decay of square wave + exponential wave).

[0035] The voltage attenuation method is referenced. Figure 4 Incubate for 5 minutes after electroporation. This process corresponds to... Figure 3 Step (d) in the process.

[0036] Step 5: Amplification (Day 1-5) After electroporation, T cells were removed from the electroporation area and expanded by applying T cell culture medium (a medium containing IL-2 and Alt-RHDR enhancer V2 added to the basal medium). Automated medium replenishment and replacement were performed every 12-24 hours, maintaining expansion for 5 days. This process corresponds to... Figure 3 Step (e) in the process. Cell morphology after electroporation and amplification. Figure 5 Day 5 cell viability analysis showed that the viability of un-electroplated T cells was 92%, while the viability of electropollinated CAR-T cells was 76%. Figure 6 .

[0037] Step 6: Co-cultivation (Day 5) One unit of target cell suspension (target cells expressing HER2 protein) and two units of expanded anti-HER2 CAR-T cell suspension were combined and mixed (target cell to CAR-T ratio 1:1). The mixture was then moved three times via a pre-defined circular motion to ensure homogeneity. The resulting cell suspension was then incubated statically in a co-culture zone for one day. This process corresponds to... Figure 3 Step (f) in the process.

[0038] Step 7: Kill Detection (Day 6) One unit of fluorescent dye was combined and mixed with three units of co-cultured cell suspension. The mixed fluorescent dye and cell suspension were circulated three times along a designated path to ensure thorough mixing. After mixing, the viability of the target cells was detected by scanning with a PMT detection module. This process corresponds to... Figure 3 Step (g) in the process.

[0039] Results analysis: Flow cytometry analysis in three parallel experiments showed positive rates of electroporation of 51.90%, 54.55%, and 56.52%, respectively. Figure 7 The killing effect test results showed that the killing efficiency of control T cells was below 5%; the killing efficiency of the CAR-T cells prepared on the substrate in this invention was approximately 70%, which is higher than the approximately 60% of that obtained by traditional plate electroporation. Figure 8 .

[0040] The positional relationships described in this embodiment are for illustrative purposes only and should not be construed as limiting this patent. Clearly, the above embodiments of the present invention are merely examples to clearly illustrate the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of this invention.

Claims

1. A fully automated cell electroporation system based on digital microfluidics, characterized in that, include: A digital microfluidic chip includes an upper electrode and a lower electrode, with a gap between them for manipulating droplets; the lower electrode is provided with a driving electrode array; the chip is provided with at least one independent electroporation region, which contains an independent electroporation electrode; The system control and drive module is electrically connected to the drive electrode array and is used to control the automated operation of the droplets; An electroporation signal generation module is electrically connected to the electroporation electrode and is used to generate a programmable electroporation pulse signal. A cell culture module is used to provide and maintain the temperature, humidity, and gas environment required for cell culture in the chip. The magnetron module is used to separate magnetic beads from cells on the chip or to perform cell sorting based on magnetic beads. An in-situ detection module, integrated within the system, is used to detect cells or cell secretions on the chip.

2. The system according to claim 1, characterized in that, The upper electrode of the digital microfluidic chip includes an upper substrate, a ground electrode, and an upper hydrophobic layer; the lower electrode includes a lower substrate, a driving electrode, a dielectric layer, and a lower hydrophobic layer; the distance between the upper electrode and the lower electrode is 10-1000 micrometers; the electroporation electrode is not covered by the dielectric layer.

3. The system according to claim 1, characterized in that, The pulse signal generated by the electroporation signal generation module includes a high-voltage, short-pulse-width piercing pulse and one or more lower-voltage, longer-pulse-width drive pulses, wherein the drive pulses employ a decaying waveform.

4. The system according to claim 3, characterized in that, The voltage range of the perforation pulse is 0.1-500V, the pulse length is 0.01-200ms, the pulse interval is 0.01-200ms, and the number of pulses is 1-10. The driving pulse includes two types: square wave and attenuated wave. The attenuated wave ranges from 0.1-500V; the square wave voltage ranges from 0.1-300V, the pulse length is 0.05-1000ms, the pulse interval is 0.05-1000ms, and the number of pulses is 1-1000.

5. The system according to claim 1, characterized in that, The cell culture module can maintain the chip environment at a temperature of 37±1℃, a CO2 concentration of 5±0.5%, and a humidity of not less than 60%.

6. The system according to claim 1, characterized in that, The magnetic control module includes a magnet that can be moved by the control system or whose magnetic field can be switched on or off.

7. The system according to claim 1, characterized in that, The in-situ detection module includes a photomultiplier tube and / or a fluorescence microscope imaging system.

8. A method for preparing gene-edited cells using a fully automated cell electroporation system according to any one of claims 1-7, characterized in that, Includes the following steps: S1: On the digital microfluidic chip, droplets containing target cells to be edited are combined with droplets containing target cell sorting magnetic beads, mixed, and then automatically sorted and cultured. S2: Use the magnetocontrol module to remove the sorting magnetic beads and obtain the sorted target cell droplets; S3: Combine and mix the sorted target cell droplets with the electroporation buffer droplets containing gene editing components; S4: Move the mixed droplets to the electroporation region and apply an electroporation pulse to perform gene editing; S5: Automated expansion and culture of cells after electroporation; S6: Combine the expanded cells with indicator cell droplets for co-culture; S7: Perform in situ detection on co-cultured cells to assess target cell function.

9. The method according to claim 8, characterized in that, The gene editing component in step S3 includes an electroporation buffer solution, an expression vector for the target gene or other molecules, and exogenous DNA or RNA; the electroporation pulse in step S4 includes one or more pore pulses and a series of drive pulses using attenuated waveforms or square waves; the input amount of the target cells is 1,000 to 100,000 cells per editing unit; and the volume of the droplet is 0.1 to 50 microliters.

10. The method according to claim 8 or 9, characterized in that, The in-situ detection described in step S7 involves adding chemical or fluorescent dyes to co-cultured cells and assessing the survival rate of target cells by detecting chemiluminescence or fluorescence signals, thereby reflecting the gene editing efficacy of the target cells.

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