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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 ()
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 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.

Figure 6. The CellPore™ Transfection System Enables Earlier Gene Editing of CD34+ HSPCs

B2M RNP complexes were delivered to 5 x 10⁴ cord blood-derived CD34+ HSPCs using the CellPore™ Transfection System at 30 psi or via an optimized electroporation protocol following pre-transfection culture durations of 1, 4, 24, or 48 hours. Four days post-transfection, viability, MHC-I marker expression (i.e. B2M knock-out efficiency), and frequencies of CD45+CD34+ HSPC and CD34+CD45RA-CD90+ HSC were assessed by flow cytometry. CellPore™ achieved ≥80% knock-out efficiency as early as 4 hours, compared to electroporation that required at least 48 hours of pre-transfection culture to consistently reach high editing efficiency. The shorter CellPore™ workflow presents a significant advantage in maintaining high CD34+ HSPC and primitive CD90+ HSC frequencies for further downstream assays. Data are shown as mean ± SD (n = 1 - 6).

Figure 7. The CellPore™ Transfection System Maintains the Proliferation and Differentiation Potential of CD34+ HSPCs

CD34+ HSPCs were pre-cultured for either 4 or 24 hours before delivery of B2M RNP complexes. Four days post-transfection, flow cytometry was used to assess CD45, CD34, CD45RA and CD90 expression. (A) Analysis of phenotype markers indicated no change in cord blood (CB) or mobilized peripheral blood (mPB) HSPC or HSC subset frequencies after transfection with CellPore™. 24 hours after transfection, in vitro colony-forming unit (CFU) assays were performed by plating triplicate wells in MethoCult™ H4435 Enriched Medium (Catalog #04435) followed by a culture period of 14 days before counting on the STEMvision™ Instrument (Catalog #22005). (B) Cloning efficiency in B2M-edited samples was slightly reduced, whereas (C) distribution of colony sub-types, and (D) colony sizes were comparable between B2M-edited and untreated controls. Data are presented as mean ± SD (n = 3 - 4).

Tips for Further Optimization

1. In case cell clumping is observed after thawing cryopreserved cells, it is recommended to filter aggregated suspensions through a 37 µm cell strainer (e.g. Catalog #27250) for optimal results. In this case, the run time on the CellPore™ Transfection System may need to be increased to 10 seconds.
2. The addition of UM729 to the complete StemSpan™ SFEM II culture medium is recommended but can be omitted. UM729 enhances expansion of CD34+ cells and more primitive CD34+CD45RA-CD90+ cells.
3. For better retention of the primitive HSC population (CD34+CD45RA-CD90+), the pre-transfection culture period can be reduced from 24 hours to 4 hours. This may result in lower editing efficiencies.
4. To ensure availability of sufficient nutrients for CD34+ HSPC growth, it is recommended to keep the culture concentration lower than 1 x 10⁶ cells/mL.
5. 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.
6. When designing guide RNAs, it is recommended to choose sequences that align with the transcription start site of the gene of interest where possible.
7. 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.
8. For best results, the total volume of cargo added should not exceed 10% of the reaction volume.
9. Reducing the reaction volume to less than 80 µL may result in lower editing efficiencies and cell recoveries. A minimum reaction volume of 50 µL is required for consistent performance with the CellPore™ Transfection System.
10. Lowering the instrument pressure to 20 or 25 psi can improve functionality of cells in certain applications. This may result in lower editing efficiencies. When using pressures <30 psi, it is recommended to increase the run time to 10 seconds.