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Gene Editing Human Unactivated T Cells with the CellPore™ Transfection System

8 Parts 10 Materials Print

CRISPR-Cas9 gene editing of unactivated (resting) T cells is increasingly valuable for advancing T cell biology, disease modeling, and the development of next-generation gene-engineered cell therapies (e.g. CAR-T) with improved cell longevity and functionality. Unactivated T cells serve as an ideal research tool for long-term studies of immune function because they maintain an undifferentiated state with high proliferative potential. However, their quiescent nature presents unique challenges for genetic manipulation.

Unlike activated T cells, which are more permissive to gene modification, unactivated T cells possess a highly condensed chromatin structure that limits CRISPR-Cas9 access and reduces editing efficiency. Additionally, their resistance to transfection often necessitates more aggressive approaches, such as increased electroporation voltages. This can result in negative impacts to cell viability and function, as well as triggering unintended activation, differentiation, and/or apoptosis.

This protocol describes a novel mechanoporation-based approach to overcome some of these challenges by using the CellPore™ Transfection System. We describe how to perform CRISPR-Cas9 genome editing in human unactivated T cells isolated from peripheral blood mononuclear cells (PBMCs). It includes specific details for T cell isolation, preparation, and delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes, and recommendations for post-transfection handling, culture, and analysis. Instructions are also provided for the optional activation of T cells after RNP delivery. When used under optimized conditions, the CellPore™ Transfection System can achieve robust, efficient, and reliable gene editing performance, with minimal perturbations to cell phenotype and function.

Figure 1. Experimental Workflow for Gene Editing of Human Unactivated T Cells with the CellPore™ Transfection System

Human pan T cells can be isolated from fresh or cryopreserved primary peripheral blood mononuclear cells (PBMCs) using EasySep™ negative immunomagnetic isolation. CRISPR-Cas9 RNPs are prepared by combining glycerol-free Cas9 with ArciTect™ sgRNA designed to target a gene of interest at an optimal ratio. RNPs are delivered to unactivated T cells using the CellPore™ Transfection System. Post-transfection, cells are cultured in supplemented ImmunoCult™ XF T Cell Expansion Medium until ready for analysis. After an appropriate culture period, unactivated T cells can be assessed for editing efficiency via established methods as permitted by the experimental design, such as flow cytometry or ArciTect™ T7 Endonuclease Kit (Catalog #76022). Optionally, transfected T cells can be activated downstream with ImmunoCult™ Human CD3/CD28/CD2 or CD3/CD28 T cell Activator and expanded for several days in ImmunoCult™-XF T Cell Expansion Medium before analysis.

Materials


Protocol

Navigation: Part I: Sample Preparation Part II: EasySep™ Human T Cell Isolation and Resting Part III: Preparation of sgRNA Working Solution Part IV: Preparation of CRISPR-CAS9 RNP Complex Part V: Preparation of T Cell Suspension for the CellPore™ Transfection System Part VI: Delivery of RNP Complexes to Unactivated T Cells Using the CellPore™ Transfection System Part VII: Cell Culture and (Optional) Activation Part VIII: Assessing Viability and Delivery Efficiency

Part I: Sample Preparation

For available fresh and frozen human peripheral blood leukopak, refer to Catalog #70500; 200-0130.

Leukapheresis

  1. Wash the peripheral blood leukapheresis sample by adding an equivalent volume of EasySep™ Buffer or phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS).
  2. Centrifuge at 300 x g for 10 minutes at room temperature (15 - 25°C).
  3. Remove the supernatant and resuspend the cells at 5 x 107 cells/mL in EasySep™ Buffer.

Peripheral Blood

  1. Prepare a peripheral blood mononuclear cell (PBMC) suspension from whole blood by centrifugation over a density gradient medium e.g. Lymphoprep™ (Catalog #18060). For more rapid PBMC preparation, use SepMate™ cell isolation tubes (Catalog #85450, or #85415).
  2. If using previously frozen PBMCs, incubate the cells with DNase I Solution (Catalog #07900) at a concentration of 100 µg/mL, at room temperature (15 - 25°C), for at least 15 minutes before proceeding to Part II. Filter aggregated suspensions through a 37 µm cell strainer (e.g. Catalog #27250) for optimal results.
  3. After preparation, resuspend cells at 5 x 107 cells/mL in EasySep™ Buffer.

Part II: EasySep™ Human T Cell Isolation and Resting

  1. Isolate human pan T cells from the sample (prepared in Part I) using the EasySep™ Human T Cell Isolation Kit. Refer to the Product Information Sheet () for complete instructions.
  2. Centrifuge isolated cells at 500 x g for 5 minutes at room temperature (15 - 25°C).
  3. Resuspend pan T cells in pre-warmed (37°C) ImmunoCult™-XF T Cell Expansion Medium at 2 x 106 to 4 x 106 cells/mL in a 50 mL conical tube.
  4. Rest pan T cells in the conical tube for 1 hour in a humidified incubator at 37°C and 5% CO2.
    Note: For T cells isolated from cryopreserved samples, increasing the resting period to 2 hours is recommended. Ensure isolated T cells are in a single-cell suspension before proceeding. If cell aggregation is observed at this stage, it is not recommended to proceed with the affected donor, as this may impact performance and recovery.

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 them immediately. Do not re-freeze.

    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 For 5 x 105 - 1 x 107 Cells For > 1 x 107 - 2.5 x 107 Cells
    Volume (μL) Amount (pmol) Volume (μL) Amount (pmol)
    10 mg/mL Cas9 Nuclease (Glycerol-Free Formulation) 0.97 60 1.94 120
    100 μM sgRNA 1.50 150 3.00 300
    CellPore™ Delivery Medium 7.53 – 5.06 –
    Total 10 – 10 –
    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. Avoid introducing excessive bubbles.
  3. Incubate the 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 T Cell Suspension for the CellPore™ Transfection System

Each CellPore™ Delivery Cartridge 300 can process from 5 x 105 to 2.5 x 107 unactivated pan T cells per reaction. The following example is for preparing one Reaction Mixture (unactivated pan T cells + RNP Complex Mix + [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 T cell culture medium (e.g. ImmunoCult™-XF T Cell Expansion Medium) for the required number of reactions and cell culture wells.
    Note: To improve cell viability, supplement with 5% human AB serum and/or 10 ng/mL human recombinant IL-2. If desired, add antibiotics immediately before use (e.g. 50 μg/mL gentamicin).
  2. After resting the isolated pan T cells (Part II, Step 4), perform a viable cell count to determine concentration. Transfer the desired number of cells to a sterile DNase- and RNase-free microcentrifuge tube.
  3. Centrifuge at 500 x g for 5 minutes at room temperature (15 - 25°C).
  4. Aspirate the supernatant and resuspend T cells in CellPore™ Delivery Medium as follows:
    1. For 5 x 105 - 1 x 107 cells, resuspend in 40 μL.
    2. For > 1 x 107 - 2.5 x 107 cells, resuspend in 90 μL.
    Note: Optionally, CellPore™ FITC-Dextran may be co-delivered as a positive control to measure successful cargo delivery. Adjust the resuspension volume to accommodate for the additional volume of CellPore™ FITC-Dextran if applicable (Table 3).
  5. Add 10 μL of RNP Complex Mixture (Prepared in Part IV) to the resuspended pan T cells (with or without CellPore™ FITC-Dextran) according to Table 3.
  6. Gently mix the Reaction Mixture by pipetting it up and down. Avoid introducing excessive bubbles. Immediately proceed to Part VI.

    Table 3. Volumes for Preparing 50 μL or 100 μL of Reaction Mixture

    Component For 5 x 105 - 1 x 107 Cells For > 1 x 107 - 2.5 x 107 Cells
    Volume (μL) Volume with CellPore™ FITC-Dextran (μL) Volume (μL) Volume with CellPore™ FITC-Dextran (μL)
    Pan T Cell Suspension 40 37.5 90 85
    RNP Complex Mixture 10 10 10 10
    CellPore™ FITC-Dextran - 2.5 - 5
    Total Volume 50 100

Part VI: Delivery of RNP Complexes to Unactivated T Cells Using the CellPore™ Transfection System

  1. Remove the Cartridge Insert from a new CellPore™ Delivery Cartridge 300 (Figure 2) and add the appropriate volume of T cell culture medium (prepared in Part V Step 1) to the Collection Tube. Re-insert the Cartridge Insert into the Collection Tube.
    1. For 5 x 105 - 1 x 107 cells, add 150 μL
    2. For > 1 x 107 - 2.5 x 107 cells, add 100 μL

    Figure 2. Preload the Collection Tube of the CellPore™ Delivery Cartridge with Complete Culture Medium

  2. Transfer the entire volume of the Reaction Mixture (50 µL or 100 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 a loss in delivery performance. Gently tap the Delivery Cartridge several times to collect the volume at the bottom, if necessary.

    Figure 3. Proper Pipetting Technique for CellPore™ Delivery Cartridge

  3. Close the cap of the Cartridge Insert and ensure it 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 70 psi and run time to 3 seconds. Press Run.
    Note: The recommended pressure range for unactivated pan T cells is 50 - 90 psi. When delivering to freshly isolated pan T cells, a starting delivery pressure of 70 psi is recommended. 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.
  8. Cap the Collection Tube and incubate in a humidified incubator at 37°C and 5% CO2 for 2 hours undisturbed.
    Note: It is NOT recommended to transfer cells out of the collection tube at this stage. A rest period of less than 2 hours may result in decreased viability.

Part VII: T Cell Culture and (Optional) Activation

  1. Following the 2-hour incubation period, perform a viable cell count.
  2. Culture unactivated pan T cells at a density of 2 x 106 to 4 x 106 cells/mL in T cell culture medium (prepared in Part V, Step 1)
    Note (Optional): If proceeding with T cell activation with ImmunoCult™ Human T cell activators (e.g. Catalog #10970, #10971), follow these steps instead:
    1. Prepare a fresh complete T cell culture medium (e.g. ImmunoCult™-XF T Cell Expansion Medium) by adding cytokines (e.g. Human Recombinant IL-2), as desired.
    2. Seed viable cells in the above prepared T cell culture medium at 1 x 106 cells/mL
    3. Activate T cells by adding ImmunoCult™ Human T Cell Activator to the suspension. Refer to the ImmunoCult™ Human T cell Activator Product Information Sheet (, ) for details.
  3. Incubate cells in a humidified incubator at 37°C and 5% CO2 until ready for analysis.

Part VIII: Assessing Viability and Delivery Efficiency

The following fluorochrome-conjugated antibodies and dyes are recommended in order to facilitate the analysis of gene-edited, unactivated T cells:

Figure 4. Optimizing CellPore™ Transfection Pressure for Efficient Gene Knockout

To determine the optimal delivery pressure, (A) CellPore™ FITC-Dextran was delivered to 2 x 106 unactivated T cells at pressures ranging from 30 to 110 psi. Flow cytometry analysis measured optimal delivery between 70 and 90 psi. Subsequently, (B) Cas9 RNPs targeting the beta-2-microglobulin (B2M) gene for knockout of the MHC-I surface receptor were delivered to unactivated T cells at 70 or 90 psi using doses ranging from 25 to 60 pmol. MHC-I knockout was measured after 2 days of culture and determined to be most optimal when using 60 pmol Cas9 RNPs at either delivery pressure. The effect of delivery pressure on T cell phenotype was also assessed by analyzing the proportions of (C) CD4+ and CD8+ T cells, as well as the proportions of (D) naïve (CD45RO- CCR7+), central memory (CM; CD45RO+ CCR7+), effector memory (EM; CD45RO+ CCR7-), and terminal effector (TE; CD45RO- CCR7-) subsets. Higher pressures were associated with a greater loss of naïve T cells. Data are shown as mean ± SD, n = 3 - 13.

Figure 5. Optimizing CRISPR-Cas9 RNP Composition and Dosage for Efficient Gene Knockout Using the CellPore™ Transfection System

To determine the most optimal conditions for gene knockout in unactivated T cells, the impact of MHC-I knockout performance was assessed by evaluating the composition and dosage of Cas9 RNPs. Specifically, the Cas9:sgRNA ratio, Cas9 RNP dosage, and Cas9 nuclease vendor formulation were evaluated. (A) Cas9 nuclease was combined with a previously optimized sgRNA targeting B2M at varying Cas9:sgRNA ratios. The RNP complexes were delivered to 2 x 106 unactivated T cells at 90 psi, and MHC-I knockout was assessed via flow cytometry. Robust knockout was measured at a Cas9:sgRNA ratio of 1:2.5. (B) Cas9 nuclease from 3 vendors were complexed with B2M sgRNA at the optimal 1:2.5 ratio and delivered at doses ranging from 40 to 100 pmol RNPs. Higher doses of Cas9 RNPs were required for some vendors to achieve optimal gene knockout. Furthermore, (C) a comparative analysis from a single Cas9 vendor was conducted to evaluate the sensitivity to glycerol. Cas9 RNPs comprising Cas9 nuclease from either a glycerol-free or glycerol-containing formulation were prepared by complexing with B2M sgRNA at the 1:2.5 ratio. The glycerol-free Cas9 formulation enabled overall higher editing efficiency in unactivated T cells across the range of doses tested. Data are presented as mean ± SD, n = 1 - 13.

Figure 6. Robust CRISPR-Cas9 Gene Knockout in Unactivated T Cells Using the CellPore™ Transfection System

Cas9 RNPs (1:2.5 Cas9:sgRNA ratio) targeting (A, B) B2M or (C, D) TRAC genes were delivered to a range of unactivated T cells (0.5 to 25 x 106) at 90 psi. Flow cytometry was used to assess viability and knock-out efficiency of surface MHC-I (after 2 days in culture) or TCRαβ (after 6 days in culture). Results show minimal loss in viability and high knockout efficiency across the entire range tested. Untreated control refers to unmanipulated T cell samples. Scramble represents the delivery of non-targeting Cas9 RNPs. M = million. Data are presented as mean ± SD, n = 3.

Figure 7. T Cells Manipulated by the CellPore™ Transfection System Retain Their Unactivated Phenotype

Unactivated T cells were analyzed 24 - 48 hours after manipulation using either the CellPore™ Transfection System or electroporation. (A) The concentrations of 21 pro-inflammatory cytokines and chemokines were measured from the culture supernatants of 3 donors. CellPore™-treated cells exhibited a similar cytokine profile compared to the untreated control. Electroporated samples consistently measured increased levels of several pro-inflammatory cytokines (e.g. IFNγ, IL-2, TNF-α) and chemokines (e.g. MIP-1α, MIP-1β). (B) This result was further confirmed via RT-qPCR with measured increase of IFNγ and IL-2 mRNA transcripts (relative to untreated cells) in electroporated cells. Furthermore, (C) large increases in the early T cell activation marker CD69 were also measured after 2 days for electroporated samples. In contrast, unactivated T cells manipulated by CellPore™ resembled untreated controls. Data are presented as mean ± SD, n = 3.

Figure 8. CellPore™ Maintains the Activation and Expansion Capability of Unactivated T Cells

Gene-edited T cells were expanded with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator in ImmunoCult™-XF T Cell Expansion Medium, supplemented with Human Recombinant IL-2. Unactivated T cells were first transfected with 60 pmol of B2M Cas9 RNP using the CellPore™ Transfection System or electroporation. After 2 hours, 1 x 106 T cells were activated and expanded over 10 days. (A) Flow cytometry analysis of CD25 marker expression on subculture days showed normal levels of activation. (B) Total fold expansion at Day 10 demonstrated that T cells manipulated by CellPore™ expanded to levels similar to electroporated cells. Importantly, (C) Flow cytometry assessment of MHC-I surface marker expression confirmed sustained gene knockout efficiency throughout the expansion period, and resulted in (D) comparable numbers of viable edited cells recovered on Day 10. These results indicate that the CellPore™ Transfection System efficiently delivers gene editing complexes to unactivated T cells and preserves their ability to be activated and expanded on demand, while maintaining the desired gene knockout. Data are shown as mean ± SEM, n = 5.


Tips for Further Optimization

  1. This protocol is not recommended for transfecting activated human T cells.
  2. This protocol is not optimized for working from cryopreserved T cells. It is recommended to isolate T cells from cryopreserved PBMCs.
  3. Cells isolated from older PBMCs or leukapheresis samples (> 48 hours post-draw) may result in lower viability for some donors. Editing results may be donor-dependent.
  4. When using cryopreserved samples, it is important to promptly and thoroughly wash away cryoprotectants (e.g. DMSO) from the thawed cell suspension before proceeding.
  5. If cell clumping is observed before the T cell isolation step, it is recommended to filter the cell suspension through a 37 µm cell strainer (e.g. Catalog #27250) for optimal results. If clumping is observed after the T cell isolation step, it is not recommended to proceed, as this may impact performance.
  6. The best results are obtained when limiting prolonged cell exposure to ambient temperature conditions. Consider keeping unused cells in a humidified incubator at 37°C and 5% CO2 when performing larger experiments.
  7. Guide RNA (sgRNA) screening may be required in order to obtain optimal editing efficiency in unactivated T cells. When designing guide RNAs, it is recommended to choose sequences that align with the transcription start site of the gene of interest, where possible.
  8. A glycerol-free formulation of Cas9 nuclease is recommended for optimal performance.
  9. Titration of Cas9 RNP may be required in order to obtain optimal editing efficiencies. Similarly, titration of the Cas9:sgRNA ratio (from 1:1 - 1:8) may also be required.
  10. Lowering the delivery pressure can improve retention of T cell subsets and improve downstream functionality in certain applications. However, this may result in lower editing efficiencies.
  11. A minimum reaction volume of 50 µL is required for consistent performance with the CellPore™ Transfection System.
  12. For best results, the total volume of cargo added to a reaction should not exceed 10% of the volume of CellPore™ Delivery Medium.
  13. To ensure accurate results, consider including controls such as Cas9 only, sgRNA only, scrambled sgRNA, a mock transfection without cargo, and an untreated sample.
  • Document #PR00101
  • Version 1.0.0
  • May 2025
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