Part I: Preparation of Culture Medium

1. Prepare a sufficient volume of complete StemSpan™ SFEM II culture medium supplemented with 1X StemSpan™ CD34+ Expansion supplement and 1 µM UM729 for the experiment. Pre-warm to 37°C.
2. Pre-warm an additional volume of 15 - 20 mL basal StemSpan™ SFEM II medium (or IMDM + 2% FBS) to 37°C. This will serve as a wash medium.

Part II. Thawing and Expansion of CD34+ HSPCs

• For available fresh and frozen samples, refer to Catalog #70008, 70060
• When working with cells that have been freshly isolated using EasySep™ from cord blood (Catalog #17986) or mobilized peripheral blood (Catalog #17856), proceed from step 5 below.

1. Quickly thaw cells in a 37°C water bath by gently shaking the cryovial. Remove the vial when a small frozen cell pellet remains.
   Note: It is important to work quickly in the following steps to ensure high cell viability and recovery.
2. Wipe the outside of the cryovial with 70% ethanol or isopropanol.
3. Add pre-warmed wash medium (prepared in Part I, step 2) slowly to the cells in the cryovial at a 1:1 (v/v) ratio.
4. Mix gently before transferring cells to a conical tube containing 10 mL of pre-warmed wash medium.
   Note: Wash the pipette tip and cryovial with the wash medium at least 3 times to help improve recovery.
5. Centrifuge cells at 250 x g for 10 minutes, low brake at room temperature (15 - 25°C).
6. Resuspend cells in 1 mL of pre-warmed complete StemSpan™ SFEM II culture medium (prepared in Part I, step 1). Perform a viable cell count.
7. Adjust viable cell density to 1 x 10⁵ cells/mL with complete StemSpan™ SFEM II culture medium.
8. Transfer cells to an appropriate culture plate and incubate in a humidified incubator at 37°C and 5% CO₂ for 24 hours.
   Note: For better retention of the primitive hematopoietic stem cell (HSC) population (CD34+CD45RA-CD90+) the pre-transfection culture period can be reduced from 24 hours to 4 hours. See Tips for Further Optimization for more details.

Part III: 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 1 for examples). Mix thoroughly.
   Note: If not used immediately, aliquot the sgRNA working solution into DNase- and RNase-free microtubes and store at -20°C for up to 6 months. Alternatively, store at -80°C for long-term storage. After thawing the aliquots, use immediately. Do not refreeze.

   Table 1. 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 IV: Preparation of CRISPR-Cas9 RNP Complex

The following example is for preparing RNP complexes for 1 reaction. Adjust accordingly based on the number of reactions required.

1. To prepare the RNP Complex Mixture, combine the components listed in Table 2 in a sterile DNase- and RNase-free 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 2. Preparation of RNP Complex Mixture

   Reagent

   Volume (μL)

   Amount (pmol)

   10 mg/mL Cas9 Nuclease (Glycerol-free formulation)

   0.64

   40

   100 μM sgRNA

   1.00

   100

   CellPore™ Delivery Medium

   3.36

   –

   Total

   5

   –

   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 RNP Complex Mixture at room temperature (15 - 25°C) for 15 minutes.
   Note: If not used immediately after incubation, keep on ice until use. Allow the RNP Complex Mixture to warm to room temperature for 5 minutes prior to transfection.

Part V: Preparation of Reaction Mixture for Transfection

Each CellPore™ Delivery Cartridge 300 can process 1 x 10⁴ to 5 x 10⁵ cord blood-derived CD34+ HSPCs or 2 x 10⁴ to 1 x 10⁶ mobilized peripheral blood-derived CD34+ HSPCs per reaction. The following example is for preparing one Reaction Mixture (CD34+ HSPCs + RNP Complex Mixture + [optional] CellPore™ FITC-Dextran). Adjust volumes accordingly based on the number of reactions required. Include a small excess to account for pipetting error.

1. Pre-warm, to 37°C, a sufficient volume of complete StemSpan™ SFEM II culture medium (prepared in Part I, step 1) for the required number of reactions and cell culture wells.
2. Harvest cells from the culture plate (see Part II: Thawing and Expansion of CD34+ HSPCs) by gently pipetting up and down to resuspend cells that have settled down. Transfer cells to a 15 mL conical tube.
   Note: Rinse the culture wells with pre-warmed wash medium (Part I, step 2) 1 - 3 times to ensure full recovery of cells from the culture plate.
3. Perform a viable cell count to determine concentration. Transfer the desired number of CD34+ HSPCs for each reaction to a sterile DNase- and RNase-free microcentrifuge tube.
4. Centrifuge at 500 x g for 5 minutes at room temperature (15 - 25°C).
5. Aspirate the supernatant and resuspend CD34+ HSPCs in 75 µL of CellPore™ Delivery Medium.
   Note: Optionally, CellPore™ FITC-Dextran may be co-delivered as a positive control to measure successful cargo delivery. Adjust the resuspension volume to 71 µL to accommodate for the additional volume of CellPore™ FITC-Dextran if applicable (Table 3).
6. Add 5 µL of RNP Complex Mixture (prepared in Part IV) to the resuspended cells (with or without CellPore™ FITC-Dextran) according to Table 3.
7. Gently mix the Reaction Mixture by pipetting up and down.
   
   

   Table 3. Volumes for Preparing 80 μL of Reaction Mixture

   Component

   Volume (μL)

   Volume (μL) with co-delivery of CellPore™ FITC-Dextran

   CD34+ HSPC Suspension

   75

   71

   RNP Complex Mixture

   5

   5

   CellPore™ FITC-Dextran

   -

   4

   Total Volume

   80

   80

Part VI: Delivery of RNP Complexes to CD34+ HSPCs Using CellPore™ Transfection System

1. Remove the Cartridge Insert of a new CellPore™ Delivery Cartridge 300 (Figure 2) and add 120 μL of complete StemSpan™ SFEM II culture medium to the Collection Tube. Re-insert the Cartridge Insert into the Collection Tube.
   

   Figure 2. Each CellPore™ Delivery Cartridge Comprises the Cartridge Insert and a Collection Tube

2. Transfer the entire volume of the Reaction Mixture (80 µL) into the Cartridge Insert. Always insert the pipette tip to the bottom of the Cartridge Insert when dispensing the sample (Figure 3).
   Note: Do not centrifuge the Delivery Cartridge at this stage as this will lead to loss in delivery performance. Gently tap the Delivery Cartridge several times to collect volume at the bottom if necessary.
   

   Figure 3. Proper Pipetting Technique for CellPore™ Delivery Cartridge

3. Close the cap and ensure the Cartridge Insert is securely placed in the Collection Tube.
4. Place the Delivery Cartridge into the Cartridge Holder of the CellPore™ Transfection System instrument.
5. Set instrument pressure to 30 psi and run time to 5 seconds. Press Run.
   Note: For complete instructions on performing sample runs, refer to the CellPore™ Transfection System User Reference Manual (Document #10000018433)
6. Once the run is complete, retrieve the 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 Delivery Cartridge in a mini-centrifuge for a few seconds for full volume recovery.
7. Remove and discard the Cartridge Insert. Perform a viable cell count.
8. Transfer the cell suspension to an appropriate culture plate and add complete StemSpan™ SFEM II culture medium to the desired culturing density (refer to the StemSpan™ CD34+ Expansion Supplement Product Information Sheet for guidance). Incubate cells in a humidified incubator at 37°C with 5% CO₂ until ready for analysis or downstream applications.

Part VII: Assessing Viability and Delivery Efficiency

The following fluorochrome-conjugated antibodies and dyes are recommended to facilitate analysis of gene-edited CD34+ HSPCs:

• Anti-Human CD45 Antibody, Clone HI30 (Catalog #60018)
• Anti-Human CD34 Antibody, Clone 581 (Catalog #60013)
• Anti-Human CD45RA Antibody, Clone HI100 (Catalog #100-0318)
• Anti-Human CD90 Antibody, Clone 5E10 (Catalog #60045)
• Viability Dye, including 7-AAD (Catalog #75001) or Propidium Iodide (Catalog #75002)

Figure 4. The CellPore™ Transfection System Enables Efficient Gene Knock-Out in CD34+ HSPCs

Cryopreserved cord blood-derived CD34+ HSPCs were cultured in complete StemSpan™ SFEM II medium for 24 hours. RNP complexes targeting the B2M or PTPRC gene were delivered to 5 x 10⁴ HSPCs. After a culture period of 4 days, flow cytometry was used to determine viability and (A) B2M knock-out (assessed via MHC-I surface expression, representative histogram shown) or (B) PTPRC knock-out (assessed via CD45 surface expression, representative histogram shown). (C) A pressure sweep was performed to identify the optimal delivery pressure for achieving robust gene knock out in CD34+ HSPCs, which was determined to be 30 psi. (D) When utilizing the optimal 30 psi delivery pressure, the average editing efficiency for MHC-I knock-out was 91.8% ± 1.4, and 84.2% ± 2.7 for CD45 knock-out, while retaining high viability. Data are presented as mean ± SD, n = 2 - 7.

Figure 5. CellPore™ Transfection System Enables Robust Knock-Out in HSPCs Derived from Cord Blood or Mobilized Peripheral Blood Across a Wide Range of Cell Numbers

Cryopreserved CD34+ HSPCs isolated from (A, B) cord blood or (C, D) mobilized peripheral blood were cultured for 24 hours in complete StemSpan™ SFEM II culture medium. RNP complexes targeting the B2M gene were delivered to a range of cell numbers per reaction. Four days post-transfection, cell viability and surface MHC-I marker expression were measured via flow cytometry in bulk CD34+ HSPCs and primitive CD34+CD45RA-CD90+ HSCs. Comparable viability and editing efficiency were obtained across the cell number range tested. Data are shown as mean ± SD, n = 2 - 5.