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Optimizing Delivery Efficiency with Fluorescent Dextran Using the CellPore™ Transfection System

Optimizing Delivery Efficiency with Fluorescent Dextran Using the CellPore™ Transfection System

4 Parts 4 Materials Print

The following protocol describes the general guidelines for conducting a pressure sweep (titration) to optimize cargo delivery using the CellPore™ Transfection System. The primary objective is to determine the specific pressure that achieves the highest delivery efficiency while maintaining maximum cell viability for a given cell type.

To facilitate rapid, same-day optimization, this protocol utilizes a fluorescent reporter cargo (e.g. FITC-Dextran). The pressure identified in this titration serves as the baseline for all subsequent experiments with this cell type. While this provides a strong starting point, in some cases, further fine-tuning or validation using your specific cargo of interest is highly recommended to ensure optimal biological results.

For complete instructions on using the CellPore™ Transfection System, refer to the CellPore™ User Reference Manual (Document #10000018433).

Figure 1. Rapid Workflow for Optimizing CellPore™ Delivery Parameters Using FITC-Dextran As a Cargo Surrogate

Cells are prepared as a single-cell suspension and mixed with a fluorescent reporter cargo (e.g. CellPore™ FITC-dextran), which mimics the uptake behavior of biological cargoes. The mixture is processed through the CellPore™ Transfection System across a range of delivery pressures. Because the surrogate provides an immediate readout, both cell viability and delivery efficiency can be analyzed via flow cytometry on the same day. This workflow allows users to quickly identify the pressure that achieves saturation of delivery efficiency with minimal impact on viability, establishing a baseline for subsequent functional experiments.

Part I: Preparation of Single-Cell Suspension

High cell viability and a single-cell suspension are critical for successful mechanoporation. Use of standardized cell isolation and/or culture methods are important considerations to obtain the best results using the CellPore™ Transfection System.

Alternatively, click here for information on available fresh and frozen samples.

FROM LEUKAPHERESIS or WHOLE BLOOD

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

  1. Prepare a peripheral blood mononuclear cell (PBMC) suspension from either a leukapheresis sample or whole blood samples.
    NOTE: For best results, we recommend using fresh cells. For cryopreserved samples, incubate 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 with isolation.
  2. If applicable, isolate the target cell population using EasySep™ negative cell isolation kits.
    NOTE: Refer to the Product Information Sheet (PIS) that comes with the EasySep™ kit for instructions on how to perform cell isolation. Click here for available cell isolation kits.
  3. (Recommended for primary cells) After isolation, rest cells in culture medium in a humidified incubator with 5% CO2 at 37°C for 1 hour.
    NOTE: Optimal resting and/or pre-culture periods prior to handling on the CellPore™ Transfection System will vary depending on the source and cell type. Longer resting periods (up to overnight) may be required for cryopreserved samples.
  4. Centrifuge at 300 x g for 5 - 10 minutes at room temperature (15 - 25°C).
  5. Remove the supernatant and gently resuspend cells in a small (e.g. 0.5 - 1 mL) volume of culture medium.
  6. Count cells using a hemocytometer or an automated cell counting method.

FROM CULTURE

  1. Use cells from a healthy, sub-confluent culture (e.g. 70 - 85% confluent for adherent cells).
    NOTE: When starting from cryopreserved cells, thaw cells and passage them at least 1 - 2 times under standard culture conditions before use.
  2. If applicable, add a minimal volume of a gentle cell dissociation reagent (e.g. Accutase™) and incubate at 37°C until cells detach from the cultureware (typically 3 - 5 minutes). Add at least 1 volume of complete culture medium to neutralize the dissociation reagent.
  3. Gently pipette to create a single-cell suspension and transfer to a conical tube.
    NOTE: Avoid excessive or harsh trituration, as this may adversely impact cell viability. If the cells do not readily detach, a longer incubation with the dissociation reagent may be required.
  4. Centrifuge at 300 x g for 5 - 10 minutes at room temperature (15 - 25°C).
  5. Remove the supernatant and gently resuspend cells in a small volume of culture medium.
  6. Count cells using a hemocytometer or an automated cell counting method.

Part II: Preparation of Reaction Mixture for CellPore™ Transfection

The following protocol is for preparing a master mix for 7 x 50 µL reactions to perform a five-point pressure sweep optimization experiment, including two controls. One extra reaction was included in the preparation of these samples to account for pipetting error.

  1. Thaw CellPore™ FITC-Dextran at room temperature (15 - 25°C). Vortex to mix.
  2. Transfer the required number of cells for the entire experiment into a new tube.
    NOTE: The recommended number of cells per reaction varies depending on the cell type. Refer to the CellPore™ Mechanoporation Guide available here for specific details for your cell type.
  3. Centrifuge at 300 x g for 5 - 10 minutes at room temperature (15 - 25°C).
  4. Remove the supernatant and gently resuspend the cell pellet in 400 μL of CellPore™ Delivery Medium.
  5. Gently pipette the cells to obtain a single-cell suspension.
  6. Aliquot 50 µL of the cell suspension into a labeled tube. This is the ”Untreated” control.
  7. To the remaining 350 µL of cell suspension, add 17.5 µL of CellPore™ FITC-Dextran to reach the final target concentration (0.1 mg/mL). Mix gently by pipetting. This is the ”Reaction Mixture.”
  8. Aliquot 50 µL of the Reaction Mixture into a labeled tube. This is the ”Endocytosis” control.
  9. Proceed immediately to Part III with the remaining 300 µL Reaction Mixture.

Part III: Delivery of FITC-Dextran via the CellPore™ Transfection System (Pressure Sweep)

Best results are obtained when limiting prolonged cell exposure to ambient temperature conditions. Work as quickly as possible and consider keeping unused cells in a humidified incubator at 37°C and 5% CO2 when performing larger experiments.

Instrument Setup

  1. Program the CellPore™ Transfection System to the desired pressure sweep. The optimal range is highly cell type-dependent.
    NOTE: Although the instrument supports pressures from 5 to 120 psi, we recommend starting in the range of 15 to 100 psi for initial experiments.

    Table 1. Example Pressure Sweeps

    CELL TYPE / SCENARIO SUGGESTED RANGE EXAMPLE 5-POINT SWEEP (psi)
    Standard / Broad Optimization 20 - 100 psi 20, 40, 60, 80, 100
    Sensitive Cells
    (e.g. CD34+ HSPCs)    		 15 - 60 psi 15, 25, 35, 45, 55
    Robust Cells
    (e.g. unactivated T cells)    		 50 - 90 psi 50, 60, 70, 80, 90

    HSPCs = hematopoietic stem and progenitor cells

  2. Set the run time according to the cell type protocol recommendation. If a specific recommendation is unavailable, initially set a longer run time per sample (e.g. 5 seconds) to ensure the full sample volume is processed.
    NOTE: The run time defines the duration of applied pressure. In subsequent experiments, this duration can be shortened based on the actual time observed for the sample to pass through the cartridge. Be aware that for lower pressures (< 20 psi), higher cell concentrations, or larger reaction volumes, longer run times (e.g. 10 seconds) could be required.

Running Samples

To determine which cartridge model is best suited for your cell type or application, refer to the CellPore™ Mechanoporation Guide available here.

  1. Remove the Cartridge Insert of a new CellPore™ Delivery Cartridge (Figure 2) and add 150 μL of an appropriate culture medium to the Collection Tube. Re-insert the Cartridge Insert into the Collection Tube.

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

  2. Gently mix the ”Reaction Mixture” and transfer 50 µL into the Cartridge Insert. Always insert the pipette tip to the bottom of the Cartridge Insert when dispensing the sample and avoid creating excessive bubbles (Figure 3).

    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.
  5. Run the first pressure setting.
  6. 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.
  7. Remove and discard the Cartridge Insert.
  8. Cap the Collection Tube and set aside at room temperature.
  9. Repeat steps 1 - 8 for all remaining pressures.
  10. Once complete, add 150 µL of culture medium to both the “Untreated” and “Endocytosis” control aliquots set aside earlier.
  11. Proceed to flow cytometry analysis.
    NOTE: Viability and recovery for some cell types may benefit from incubation at 37°C and 5% CO2 for 30 minutes to 2 hours. For adherent cell types, it is recommended to immediately proceed to analysis.

Part IV: Assessing Viability and Delivery Efficiency

Cell viability and dextran delivery efficiency may be assessed by flow cytometry, preferably on the same day of the experiment. If compensation is required, consider using an aliquot of a delivered cell sample.

Flow Cytometry Staining

  1. Wash the cells once in an appropriate flow cytometry buffer (e.g. 1X PBS containing 2% FBS), and resuspend in 100 µL.
  2. If staining for surface markers is required, add fluorochome-conjugated antibodies at this stage and incubate according to the manufacturer's protocol.
  3. (Important) Wash the cells twice using flow cytometry buffer to thoroughly remove excess fluorescent dextran and unbound antibodies.
  4. Add a viability dye (e.g. 7-AAD) to all samples prior to running on a flow cytometer.

Flow Cytometry Gating Strategy

  1. Gate 1: Total Cells. Use FSC-A vs. SSC-A to identify the cell population of interest.
  2. Gate 2: Singlets. Use FSC-A vs. FSC-H to exclude doublets.
  3. Gate 3: Viable.
    1. Use the ”Untreated” sample to set the viability gate (FSC-A vs. Viability Dye).
    2. Viable cells are generally gated on the Viability Dye-negative population.
  4. Gate 4: Delivery Efficiency.
    1. Create a plot from “Viable” (Gate 3) of FSC-A vs. FITC-Dextran.
    2. Use the “Endocytosis” control sample to set the FITC-positive gate. This sample defines background (natural) uptake.
    3. Cells in the FITC-positive population define the delivery efficiency.

Figure 4. Representative Gating Strategy for Assessing Viability and Delivery Efficiency

Comparison of (A) Endocytosis control and (B) mechanoporated sample (e.g. 90 psi). Sequential gating identifies cells based on their forward scatter (FSC-A) and side scatter (SSC-A) properties. Singlets are gated using FSC-A vs. FSC-H to exclude doublets. Viable cells are subsequently identified as the dye-negative population (e.g. 88.7%) within the singlet gate. Delivery efficiency is quantified as the FITC-positive population (e.g. 92.7%) within the viable cells gate.

Figure 5. Rapid Identification of Optimal Delivery Pressures for Distinct T Cell Subsets Using CellPore™ FITC-Dextran

Pan T cells, CD4+ T cells, CD8+ T cells, and Regulatory T cells (Tregs) were isolated and subjected to a pressure sweep using FITC-Dextran to determine optimal delivery conditions for each subtype. Delivery was performed using CellPore™ Delivery Cartridge 300 and Delivery Medium A for all subtypes. The optimal delivery pressure ranges were (A) 70 - 90 psi for pan T cells, (B) 50 - 70 psi for CD4+ T cells, (C) 60 - 70 psi for CD8+ T cells, and (D) 50 - 70 psi for Tregs. All conditions were assessed by flow cytometry on the same day of delivery. The endocytosis control represents the natural uptake of CellPore™ FITC-Dextran in undelivered samples. Untreated refers to unmanipulated cells. These profiles serve as the predictive foundation for delivering biological cargoes to these subtypes. Data are shown as mean ± SD (n = 3 - 5).

Figure 6. Optimization with CellPore™ FITC-Dextran Accurately Predicts mRNA Delivery and Expression Efficiency in Human NK Cells

(A) FITC-dextran was delivered to 2 x 106 human NK cells using CellPore™ Delivery Cartridge 300 and Delivery Medium A across increasing pressure segments (30 to 90 psi). Delivery efficiency and viability was assessed by flow cytometry on the same day of the experiment and optimal performance was obtained at 70 psi (delivery 93 ± 3%; viability 85 ± 4%). To validate the predictive power of FITC-Dextran, both (B) eGFP and (C) mCherry mRNA were delivered to 2 x 106 human NK cells from the same donors. The high expression (> 90%) and viability (> 80%) obtained with the mRNA cargoes after 24 hours correlated directly with the optimization parameters established by the FITC-dextran screen. Data are shown as mean ± SD; n = 2 - 3.

Figure 7. CellPore™ FITC-Dextran Serves As a Predictive Surrogate for Cas9 RNP-Mediated Gene Editing in CD34+ Hematopoietic Stem and Progenitor Cells

(A) FITC-dextran was delivered to 5 x 104 cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) using CellPore™ Delivery Cartridge 300 and Delivery Medium A across increasing pressure segments (10 to 50 psi) to rapidly identify the pressure balancing delivery efficiency and viability. Flow cytometry analysis on the day of the experiment identified 30 psi as the optimal condition. (B) Functional validation was subsequently performed using Cas9 ribonucleoprotein (RNP) targeting the B2M gene across a similar pressure range. The maximal MHC-I knockout efficiency measured at Day 4 correlated precisely with the 30 psi condition identified before, confirming the utility of CellPore™ FITC-Dextran for optimizing gene editing workflows. “Non-Targeting” represents delivery of non-targeting RNP complexes. Data are presented as mean ± SD; n = 3 - 6.

Figure 8. Rapid Screening of Delivery Parameters for Human Pluripotent Stem Cells Using CellPore™ 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 105 hPSCs using CellPore™ Delivery Cartridge 500 and Delivery Medium B across increasing low pressure segments (5 to 25 psi). Immediate analysis allowed for the quick identification of 15 psi as the optimal condition across three different lines: (A) SCTi003-A iPSC, (B) WLS-1C iPSC, and (C) H9 ESC line. This highlights the utility of the surrogate cargo for establishing safe, effective delivery parameters for sensitive cell types prior to introducing valuable biological cargoes. Data are presented as mean ± SD (n = 3).


Tips for Further Optimization

  1. This protocol enables quick identification of the optimal delivery pressure for your cell type of interest. This is an excellent starting point. It is highly recommended to confirm and fine-tune this pressure (e.g. with a narrower sweep) using your cargo of interest.
  2. Other parameters, such as cell number and cargo concentration, should also be optimized.
  3. The reaction volume range for CellPore™ Delivery Cartridges is 20 - 200 µL. The recommended starting reaction volume is 50 µL, as smaller volumes may lead to suboptimal results for some cell types.
  4. CellPore™ Delivery Cartridges are recommended to be used with cells isolated from EasySep™ negative isolation kits. Positive selection may also be possible, but must be validated for the cell type of interest.
  5. Cells isolated from older PBMCs or leukapheresis samples (> 48 hours post draw) may result in lower viability for some donors.
  6. When using cryopreserved cells, care must be taken to fully wash away cryoprotectants (e.g. dimethyl sulfoxide [DMSO]) from the cell suspension. Cell viability may be improved by resting cells in culture medium in a humidified incubator with 5% CO2 at 37°C for at least 1 hour prior to handling on the CellPore™ Transfection System. Optim
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