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Gene Editing Human Pluripotent Stem Cells (hPSCs) Using the CellPore™ Transfection System

8 Parts 11 Materials Print

Achieving precise genetic modification in human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), is fundamental for advancing regenerative medicine and disease modeling. Gene editing in hPSCs, particularly using CRISPR-Cas9, enables precise manipulation of genetic loci to study gene function or generate engineered lines. However, maintaining high viability and functionality after editing requires gentle delivery methods that minimize cellular stress.

The CellPore™ Transfection System addresses this challenge by using mechanoporation to transiently permeabilize cell membranes via microfluidic channels, enabling the gentle and efficient intracellular delivery of biomolecules such as CRISPR-Cas9 ribonucleoprotein (RNP) complexes. This streamlined protocol provides detailed instructions for preparing hPSCs, forming RNP complexes, delivering them using the CellPore™ Transfection System, and recommendations for post-delivery culture and analysis.

Experimental Workflow for Gene Editing hPSCs Using the CellPore™ Transfection System

Figure 1. Experimental Workflow for Gene Editing hPSCs Using the CellPore™ Transfection System

High-quality, genetically characterized hPSCs (Catalog #200-0945, #200-0944, #200-0769, #200-0511) are cultured for 4 - 7 days in complete hPSC maintenance medium e.g. mTeSR™1, mTeSR™ Plus, or eTeSR™ (Catalog #85850 or #100-0276 or #100-1215) until they reach approximately 70% confluence. CRISPR-Cas9 RNPs are prepared by complexing glycerol-free Cas9 nuclease with ArciTect™ sgRNAs. A single-cell suspension of hPSCs is created using enzymatic dissociation, such as ACCUTASE™ (Catalog #07920). RNPs are delivered to the single-cell hPSC suspension via the CellPore™ Transfection System. Post-transfection, cells are plated on a Corning® Matrigel®-coated plate in complete hPSC maintenance medium supplemented with CloneR™2 (Catalog #100-0691) or 10 µM Y-27632 ROCK inhibitor (Catalog #72304) for 24 - 48 hours to enhance single-cell survival. Editing efficiencies can then be analyzed using methods such as flow cytometry or the ArciTect™ T7 endonuclease I Kit (Catalog #76002).

Materials

Protocol

(Optional) Pressure Titration Using Fluorescent Dextran Part I: Preparation of Tissue Culture Plate and Single Cell Plating Medium Part II: Preparation of sgRNA Working Solution Part III: Preparation of Ribonucleoprotein (RNP) Complex Part IV: Preparation of Single-Cell Suspension Part V: Preparation of hPSCs for CellPore™ Transfection Part VI: Delivery of RNP Complexes Using CellPore™ Transfection System Part VII: Post-Delivery hPSC Culture Part VIII: Assessment of hPSC Viability and Gene Knockout Efficiency

(Optional) Pressure Titration Using Fluorescent Dextran

This procedure is designed to identify the optimal pressure for delivering cargo into your specific hPSCs by balancing delivery efficiency with cell viability. A range of pressures (10, 15, 20, and 25 psi) is tested using CellPore™ FITC-Dextran as a proxy for the RNP complex.

If you're using this system for the first time or working with a new hPSC line, it's advised to first conduct a pressure titration with CellPore™ FITC-Dextran to find the best delivery pressure. However, if you're an experienced user and have already determined your optimal pressure, you can skip this step and go directly to Part I.

A: Preparation of Single-Cell Suspension

  1. Follow the steps in Part IV

B: Preparation of hPSCs for CellPore™ Transfection

The following steps are for preparing a master mix for 6 x 50 µL reactions (4 conditions + 2 controls) to perform a four-point pressure sweep optimization experiment.

  1. Add 2.1 x106 cells to a sterile 1.5 mL microtube. Centrifuge at 300 x g for 5 minutes.
  2. Aspirate the supernatant and resuspend the cell pellet in 300 µL of CellPore™ Delivery Medium B.
  3. Set aside a 50 µL aliquot of the cell suspension. This represents the ‘Untreated’ control.
  4. Add 12.5 µL of CellPore™ FITC-Dextran (2 mg/mL) to the remaining 250 µL suspension. Gently mix by pipetting.

C: Delivery of FITC-Dextran via CellPore™ Transfection System (Pressure Titration)

  1. Transfer 50 µL of the prepared cell suspension into 4 new CellPore™ Delivery Cartridge 500. Always insert the pipette tip at the bottom when dispensing the sample (Figure 2).
  2. Set aside the last 50 µL aliquot. This represents the ‘Endocytosis’ control.
  3. Perform CellPore™ transfection as indicated in Part VI, by applying the recommended pressure to each sample as indicated in Table 1.

    Table 1. Recommended conditions for optimizing cargo delivery pressure

    Sample (50 µL each) Pressure (psi) Run Time (s)
    Sample 1 10 3
    Sample 2 15 3
    Sample 3 20 3
    Sample 4 25 3
  4. Once completed, immediately proceed with flow cytometry for data acquisition and analysis.

Part I: Preparation of Tissue Culture Plate and Single Cell Plating Medium

The following protocol outlines the CellPore™ delivery method for hESCs or hiPSCs in a 6-well tissue culture plate. Adjust all volumes accordingly if using other cultureware.

  1. Coat a 6-well plate with Matrigel® and bring to room temperature (15 - 25°C) for at least 30 minutes prior to use.
  2. Warm (15 - 25°C) sufficient volumes of mTeSR™1 or mTeSR™ Plus or eTeSR™. Thaw CloneR™2.
  3. Prepare 2 mL of Single-Cell Plating Medium per transfection by adding the desired plating supplement to an appropriate volume of complete mTeSR™1 or mTeSR™ Plus or eTeSR™ medium using one of the following options:
    • CloneR™2: Add at a 1 in 10 dilution (e.g. add 1 mL of CloneR™2 to 9 mL of complete mTeSR™1 or mTeSR™ Plus or eTeSR™ medium).
    • Y-27632 (Dihydrochloride): Add to a final concentration of 10 µM
  4. Add 2 mL of Single-Cell Plating Medium per well. Pre-warm by incubating at 37°C and 5% CO2, and immediately proceed with the next steps.

Part II: Preparation of sgRNA Working Solution

  1. Briefly centrifuge the vial of lyophilized sgRNA before opening.
  2. Add nuclease-free water to the vial to achieve a final concentration of 100 µM (see Table 2 for examples). Mix thoroughly.

    Table 2. Resuspension of sgRNA to 100 µM

    sgRNA (nmol) Volume of Nuclease-free water (µL)
    1.5 15
    10 100
    50 500

    *100 µM is equal to 100 pmol/µL

Part III: Preparation Of CRISPR-Cas9 RNP Complex

The following example illustrates the preparation of RNP complexes for a single reaction. Adjust accordingly based on the number of reactions required.

  1. To prepare the RNP Complex Mix, combine the components listed in Table 3 in a sterile microcentrifuge tube. Adjust volumes according to the number of reactions, including controls required.
    NOTE: A glycerol-free formulation of Cas9 is recommended for optimal performance.

    Table 3. Preparing the RNP Complex Mixture for Delivery Using the CellPore™ Transfection System

    Reagent Volume (µL) Amount (pmol)
    10 mg/mL Cas9 Nuclease (Glycerol-free formulation) 1.0 60
    100 µM sgRNA 1.5 150
    Total 2.5 N/A
    NOTE: The above example provides the required volumes for a 1:2.5 Cas9:sgRNA ratio. It is highly recommended to optimize the Cas9:sgRNA ratio and Cas9 amount for each gene target (see Tips for Further Optimization).
  2. Mix thoroughly by gently pipetting up and down.
  3. Incubate the RNP complex mixture at room temperature (15 - 25°C) for 15 minutes.
    NOTE: If not used immediately, keep on ice until use. Allow the RNP complex mix to warm to room temperature for 5 minutes before transfection.

Part IV: Preparation of Single-Cell Suspension

When working with sensitive cells, refer to Tips for Further Optimization for instructions on improving post-delivery attachment if required.

  1. Collect cells during their exponential growth phase.
  2. Use a microscope to visually identify regions of differentiation (if any) in the wells. Mark these using a felt tip pen or lens marker on the bottom of the plate. Remove regions of differentiation by scraping with a pipette tip or by aspiration.
  3. Aspirate the remaining medium from the well and add 1 mL of ACCUTASE™ (if using a 6-well plate). Incubate the plate at 37°C and 5% CO2 for approximately 5 minutes, or until colonies appear to be dissociated.
    NOTE: Gently tap the culture plate after incubation to detach the cells. It is not recommended to pipette the cells for detachment at this stage.
  4. Add 1 mL of complete mTeSR™1 or mTeSR™ Plus or eTeSR™ medium to the well. Using a pipettor fitted with a 1000 μL tip, gently wash the cells from the surface of the plate by pipetting the solution directly onto the colonies. Gently, pipette the suspension 2 - 3 times to break up small aggregates into single cells.
    NOTE: Avoid excessive or harsh trituration, as this may adversely impact cell viability. If the cells do not readily detach, a longer incubation may be required.
  5. Transfer the cell suspension to a 15 mL conical tube. Centrifuge at 300 x g for 5 minutes.
  6. Aspirate the supernatant and gently flick the tube 3 - 5 times to resuspend the cell pellet.
  7. Resuspend cells in at least 2 mL of complete mTeSR™1 or mTeSR™ Plus or eTeSR™ medium. Mix gently by flicking the tube 2 - 3 times.
  8. Count cells using a hemocytometer or an automated cell counting method.
  9. Cells are ready to proceed to Part V.

Part V: Preparation of hPSCs for CellPore™ Transfection

Each CellPore™ Delivery Cartridge 500 can process from 3.5 x105 to 2 x106 hPSCs per reaction. The following example is for preparing one reaction mixture (hPSCs + cargo). Adjust volumes accordingly based on the number of reactions required. Include a small excess to account for pipetting error.

  1. Add 3.5 x105 cells from Part IV to a new 1.5 mL microtube for each CellPore™ delivery condition. Centrifuge at 300 x g for 5 minutes.
  2. Aspirate the supernatant and resuspend the cell pellet in 47.5 µL of CellPore™ Delivery Medium B.
  3. Add the prepared RNP mixture from part III (2.5 µL per reaction) to the resuspended cells, according to Table 4.
  4. Gently mix by pipetting. Proceed immediately to Part VI.

    Table 4 - Volumes for preparing 50 μL of Reaction Mixture

    Component For Cas9 RNP delivery
    hPSC suspension in CellPore™ Delivery Medium B 47.5 µL
    RNP Complex Mixture 2.5 µL
    Total Volume 50 uL

Part VI: Delivery of RNP Complexes to hPSCs Using CellPore™ Transfection System

  1. Transfer the entire reaction mixture to a new CellPore™ Delivery Cartridge 500. Always insert the pipette tip at the bottom when dispensing the sample (Figure 2).
    Proper Pipetting Technique for CellPore™ Delivery Cartridge.

    Figure 2. Proper Pipetting Technique for CellPore™ Delivery Cartridge.

    Pipette the cell suspension directly into the bottom of the cartridge, avoiding contact with the sidewalls.

  2. Close the cap of the cartridge insert and ensure it is securely placed in the collection tube.
  3. Place the Delivery Cartridge into the Cartridge Holder of the CellPore™ Transfection System instrument.
  4. Set instrument pressure to 15 psi, and run time of 3 seconds. Press Run.
    NOTE: The recommended pressure range could vary between 10 - 25 psi. A starting delivery pressure of 15 psi is recommended. For complete instructions on performing sample runs, refer to the CellPore™ Transfection System User Reference Manual ()
  5. Once the run is complete, retrieve the CellPore™ Delivery Cartridge from the instrument. The cell sample should be at the bottom or side of the collection tube.
    NOTE: It is recommended to spin down the CellPore™ Delivery Cartridge in a mini-centrifuge for a few seconds for full volume recovery.
  6. Discard the Cartridge Insert and proceed immediately to Part VII.

Part VII: Post-Delivery hPSC Culture

  1. Immediately after delivery, transfer cells to the pre-warmed (37°C) plate prepared in Part I.
  2. Move the plate in several, quick, short, back-and-forth and side-to-side motions to distribute the hPSCs across the surface of the well.
  3. Incubate cells in a humidified incubator at 37°C with 5% CO2. Do not disturb the plate for 24 hours.
  4. Replace the Single-Cell Plating Medium with 2 mL of complete mTeSR™1, mTeSR™ Plus, or eTeSR™. Perform a full medium change every 48 hours with mTeSR™ Plus or eTeSR™, or every 24 hours with mTeSR™1, until the cells are ready for analysis.

Part VIII: Assessment of hPSC Viability and Gene Knockout Efficiency

The following fluorochrome-conjugated antibodies and dyes are recommended to facilitate analysis:

Optimizing CellPore™ Delivery Pressure for Efficient Transfection of hPSCs Using FITC-Dextran

Figure 3. Optimizing CellPore™ Delivery Pressure for Efficient Transfection of hPSCs Using FITC-Dextran.

Newly passaged hPSC lines were cultured in complete mTeSR™ Medium for 7 days or until reaching 70% confluency. FITC-dextran was delivered to 3.5 x 105 cells per reaction in increasing pressure (5 - 25 psi). Immediately after delivery, cells were analyzed by flow cytometry to determine cargo delivery efficiency and cell viability. Conditions that maximized delivery efficiency with limited impacts to hPSC viability were determined to be 15 psi across 3 lines tested: (A) SCTi003-A hiPSC, (B) WLS-1C hiPSC, and (C) H9 hESC lines. The ”Endocytosis” condition indicates a control where cells are incubated in the presence of FITC-dextran without mechanoporation. “Untreated” indicates an unmanipulated sample without FITC-dextran. Data are presented as mean ± SD (n = 3).

Optimization of CRISPR-Cas9 RNP ratio for Enabling Efficient hPSC Gene Knockout Using the CellPore™ Transfection System

Figure 4. Optimization of CRISPR-Cas9 RNP ratio for Enabling Efficient hPSC Gene Knockout Using the CellPore™ Transfection System.

To determine the optimal RNP composition, a previously optimized sgRNA targeting β2-microglobulin (B2M) was mixed at varying Cas9:sgRNA ratios. (A) The most optimal MHC-I knockout was measured at the 1:2.5 ratio. (B) Cas9:sgRNA ratios up to 1:2.5 demonstrated robust total fold expansion. Results were assessed at Day 2 post-transfection by flow cytometry. Data are presented as mean ± SD (n = 2).

Optimization of CRISPR-Cas9 RNP Concentration for Efficient Gene Knockout Using the CellPore™ Transfection System

Figure 5. Optimization of CRISPR-Cas9 RNP Concentration for Efficient Gene Knockout Using the CellPore™ Transfection System

RNP complexes were formed by combining glycerol-free Cas9 with B2M targeting sgRNA at an optimal 1:2.5 mol ratio and delivered to hPSCs at doses ranging from 22.5 to 80 pmol in 50 µL reactions at 15 psi. Cells were analyzed by flow cytometry after 2 days post-transfection to allow sufficient time for MHC-I surface marker knockout. Conditions that maximized MHC-I knockout with minimal impacts to hPSC viability and downstream expansion were determined to be (A) 60 pmol for the SCTi003-A hiPSC line, (B) 60 - 80 pmol for WLS-1C hiPSC line, and (C) 60 pmol for the H9 hESC line. Scramble control represents delivery of non-targeting RNP complexes. Data are presented as mean ± SD (n = 1 - 5).

CellPore™ Transfection System Enables Reliable Cas9-RNP Editing Across a Wide Range of Cell Numbers

Figure 6. The CellPore™ Transfection System Enables Reliable Cas9-RNP Editing Across a Wide Range of Cell Numbers

RNP complexes targeting the B2M gene (1:2.5 Cas9:sgRNA mol ratio) were delivered to a range of hPSC numbers per reaction (from 3.5 × 105 up to 2 × 106) in 50 µL reactions at 15 psi. Cells were cultured for 2 days post-transfection to allow sufficient time for MHC-I surface marker knockout. Comparable viability, editing efficiency, and expansion were obtained across the cell number range tested for (A) SCTi003-A hiPSCs, (B) WLS-1C hiPSCs, and (C) H9 hESCs. No cargo indicates mechanoporation of hPSCs without RNP complexes at 3.5 × 105 cells per reaction. Data are presented as mean ± SD (n = 3).

Gene-Edited hPSCs Manipulated by CellPore™ and Maintained in mTeSR™ Plus are of High Cell Quality

Figure 7. Gene-Edited hPSCs Manipulated by CellPore™ and Maintained in mTeSR™ Plus Are of High Cell Quality

(A) Gene-edited hESC (H9) and hiPSC (WLS-1C, SCTi003-A) maintained in mTeSR™ Plus on Corning® Matrigel® Matrix continue to exhibit classic hPSC morphology throughout the culture with densely packed, multilayered colonies. (B) hPSCs manipulated by CellPore™ (orange bars) harvested at Day 14 post-transfection were characterized for markers of the undifferentiated state, with > 90% of cells manipulated by CellPore™ (orange bars) staining positive for TRA-1-60 (left) and OCT4 (right), as determined by flow cytometry. Graphs show average expression (± standard deviation) results from analyses of 3 independent experiments. Genetic characterization using single nucleotide polymorphism array (SNPa) profiling confirmed no genetic perturbation across all 3 lines (data not shown).

Tips for Further Optimization

  1. To improve post-delivery attachment efficiency for more sensitive hPSCs, cells should be pre-incubated with 10 µM of ROCK inhibitor (Y-27632; Catalog #72308) or culture medium containing 1X CloneR™2 (Catalog #100-0691) for 30 minutes prior to single-cell dissociation prior to mechanoporation.
  2. In case cell clumping is observed prior to transfection, it is recommended to filter aggregated suspensions through a 37 µm cell strainer (e.g. Catalog #27250) for optimal results.
  3. Best results are obtained when limiting prolonged cell exposure to ambient temperature conditions. Consider keeping unused cells in a humidified incubator with 5% CO2 at 37°C when performing larger experiments.
  4. Titration of Cas9 RNP may be required to obtain optimal editing efficiencies. Similarly, titration of the Cas9:sgRNA ratio (from 1:1 - 1:8) may also be required.
  5. For best results, the total volume of cargo added should not exceed 10% of the reaction volume.
  6. Reducing the reaction volume to less than 50 µL may result in lower cell recoveries. A minimum reaction volume of 50 µL is required for consistent performance with the CellPore™ Transfection System.
  7. For reaction volumes > 50 µL, mechanoporation may take > 3 s. Increase run time accordingly for larger volumes.
  8. Lowering the instrument pressure to 10 psi can improve viability and early cell expansion. However, this may result in lower gene editing efficiencies.
  • Document #PR00111
  • Version 1.0.0
  • January 2026


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