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

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

8 Parts 11 Materials Print

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are powerful tools for regenerative medicine and disease modelling. Genome editing in hPSCs, particularly using CRISPR-Cas9, enables precise manipulation of genetic loci to study gene function or create engineered lines. However, maintaining high viability and functionality after editing requires gentle delivery methods.

The CellPore™ Transfection System uses mechanoporation to transiently permeabilize cell membranes via microfluidic channels, enabling the delivery of biomolecules such as CRISPR-Cas9 ribonucleoprotein (RNP) complexes. This streamlined protocol provides instructions for preparing hPSCs, forming RNP complexes, delivering them using the CellPore™ system, and handling post-delivery culture and analysis.

Cardiomyocyte Differentiation in 3D Suspension Culture Protocol Diagram

Figure 1. Experimental Workflow for Genome Editing of hPSCs Using CellPore™ Transfection System.

Start with a culture of high quality, genetically characterised human PSCs (hPSCs), healthy control cell lines may be purchased (Catalog #200-0945, -0944, -0769, -0511) from STEMCELL Technologies or other reputable vendors. Newly passaged hPSCs are initially cultured for 4-7 days in complete hPSC maintenance medium (mTeSR™1, mTeSR™ Plus or eTeSR™) until they reach approximately 70% confluence. CRISPR-Cas9 RNPs are prepared by complexing glycerol-free Cas9 nuclease with ArciTect™ sgRNAs at an optimal ratio. A single cell suspension of hPSCs is prepared using enzymatic dissociation, such as ACCUTASE™ (Catalog #07920). RNPs are delivered to the single-cell suspension of hPSCs via the CellPore™ Transfection System. Post-transfection, cells are then plated in complete hPSC maintenance medium supplemented with CloneR™2 (Catalog #100-0691) or 10 µM Y-27632 ROCK inhibitor (Catalog #72304) on a Corning® Matrigel®-coated plate for 24-48 hours, to enhance survival of hPSCs plated as single cells. Editing efficiencies can be subsequently analyzed via established methods as permitted by the experimental design, such as flow cytometry or ArciTect™ T7 endonuclease I Kit (Catalog #76002).

Materials

Protocol Overview

This protocol outlines the steps for performing gene editing in human pluripotent stem cells (hPSCs) using ribonucleoprotein (RNP) delivery with the CellPore™ Transfection System.

For first-time users, or when optimizing with a new hPSC line, it is highly recommended to first perform the optional pressure titration (see below) using CellPore™ FITC-Dextran in order to determine the most optimal delivery pressure. Experienced users who have already established their optimal pressure can proceed directly to Part I.

(Optional) Pressure Titration with 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 CellPoreTM Transfection System Part VII: Post-Delivery hPSC Culture Part VIII: Assessment of hPSC Viability and Gene Knockout Efficiency

(Optional) Pressure Titration with 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) will be tested using CellPore™ FITC-Dextran as a proxy for the RNP complex.

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 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 as follows:
    • 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. Preparation of RNP complex mixture for CellPore™ Transfection System delivery

    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 Notes and Tips).
  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

  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 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™ (for 6-well plate). Incubate the plate at 37°C and 5% CO2 for approximately 5 minutes, or until colonies appear to be dissociated.
    NOTE: To further facilitate the cellular detachment, gently tap the culture plate during incubation.
  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. Pipette the cell suspension directly into the bottom of the cartridge, avoiding contact with the sidewalls.

    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 (Document #10000018433)
  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 CellPore™ 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 Single Cell Plating Medium with 2mL of complete mTeSR™1 or mTeSR™ Plus or eTeSR™. Perform a full medium change every 48 hours with mTeSR™ Plus or eTeSR™ , or every 24 hours with mTeSR™1, thereafter until 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 10^5 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 line. The endocytosis condition indicates a control where cells are incubated in the presence of FITC-dextran without mechanoporation. Untreated indicates an unmanipulated sample. 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 optima

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