Materials

• CellPore™ Transfection System (Catalog #100-0946)
• CellPore™ Delivery Cartridge 500 (Catalog #100-2175)
• CellPore™ Delivery Medium B (Catalog #100-2224)
• CellPore™ FITC-Dextran (Catalog #100-1024)
• Cas9 Nuclease (glycerol-free recommended)
• Target gRNA (e.g. ArciTect™ sgRNA, Catalog #200-0013)
• mTeSR™1 or mTeSR™ Plus or eTeSR™ Complete Medium (Catalog #85850 or #100-0276 or
  #100-1215)
• CloneR™2 Supplement (Catalog #100-0691)
• ROCK Inhibitor Y-27632 (Catalog #72304)
• ACCUTASE™ (Catalog #07920)
• Corning® Matrigel® Matrix (Corning Catalog #354277)

Protocol

(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.

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).
   

   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 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:

• Anti-Human TRA-1-60, clone TRA-1-60R (Catalog #60064)
• Anti-Human OCT4, clone 3A2A20 (Catalog #60093)
• Viability Dye, including 7-AAD (Catalog #75001), or Propidium Iodide (Catalog #75002)