Introduction

This protocol is intended for separation and In vitro expansion of human T cells using Dynabeads® Human T-Expander CD3/CD28. Dynabeads® Human T-Expander CD3/CD28 are uniform 4.5 μm, superparamagnetic polystyrene beads coated with a mixture of monoclonal antibodies against the CD3 and CD28 cell surface molecules of human T cells.

Principle of Isolation and Expansion


Human T-Expander CD3/CD28 offer a simple method for isolation and expansion of human T cells. Firstly, CD3+ T cells are separated and concentrated from the apheresis product by magnetic cell separation using Dynabeads® Human T-Expander CD3/CD28. Following separation, the CD3+ T cells are cultured in the presence of the beads. By combining anti-CD3 and anti-CD28 antibodies on Dynabeads®, the beads will provide both the primary and co-stimulatory signals that are required for activation and expansion of T cells. Activated T cells produce IL2, GM-CSF, IFN-g and TNF-α. T cells activated with Dynabeads® Human T-Expander CD3/CD28 can be expanded 100-1000 fold over a 9-14 day culture period.

Materials Supplied

  • 10 ml of Dynabeads® Human T-Expander CD3/CD28 - Supplied as a suspension containing 1 × 108 Dynabeads®/ml in phosphate buffered saline (PBS), pH 7.4, w/0.1% human serum albumin (HSA).

    Additional Materials Required

    Materials that are not included, but are recommended to perform the entire protocol:

    • Dynabeads® M-450 Epoxy –(Cat. No.140.11)
    • PBS: Cat. No. 14190-094 (Ca2+ and Mg2+ free)
    • Pooled Human Serum (HS) heat inactivated at 56°C for 1 hour
    • Human Serum Albumin (HSA)
    • OpTmizerTM T-Cell Expansion SFM, Cat. No.0080022SA (or equivalent)
    • RPMI-1640, Cat. No. 12633-012 L-glutamine, Cat. No. 25030-0832
    • HEPES buffer, Cat. No. 15630-080
    • REC. HUMAN IL-2, Cat.No. PHC0024
    • Appropriate sterile culture vessels

    Media Preparation

    Buffer 1:  
    PBS, pH 7.4, with 5% heat-inactivated HS or 1% HSA.

    Incomplete Medium:
    OpTmizerTM T-Cell Expansion SFM serum free 1× formulation designed to support the culture and expansion of human T cells (equivalent).

    Complete Medium: Add 200 IU/ml IL-2 to the incomplete medium. Store at 2-8°C for a maximum of 7 days.
    [Note: Autologous serum may be used, but expect donor to donor variation, IL-2 concentrations can be increased to enhance cell growth, although in static culture there is no significant increase in expansion for normal T cells when IL-2 levels are increased beyond 200-500 IU. Regulatory T cells may benefit from use of IL-2 at ≥500IU/ml. For IL-2 products where activity is not available in IU, typically 6-10ng/ml bioactive IL-2 is sufficient for optimal T cell expansion.]


Important Information

The protocol below is specifically written for the activation and expansion of human T cells from cryopreserved human PBMC obtained from leukopheresis products or Ficoll® separated whole blood, cord blood or bone marrow. Cultures may also be initiated from non-cryopreserved fresh samples. As sample sources and T cell or blood collection methods may vary, procedures may require specific modifications to maximize cell recovery, viability, activation and expansion.

Ordering Information

Sku Name Size Price Qty
11141D Dynabeads® Human T-Expander CD3/CD28 10 mL USD 1,900.00

Protocols

Points to consider are listed in brackets ([…]) throughout the protocol. These identify areas where modifications should be considered for specific circumstances.

Dynabeads® Washing Protocol

Dynabeads® should be washed before use with the aid of a magnet.

  1.    Resuspend the Dynabeads® in the vial.

  2.    Transfer the desired volume of Dynabeads® to a tube.

  3.    Add the same volume of Buffer 1 as the initial volume of Dynabeads®, or at least 1 ml, and mix.

  4.    Place the tube in a magnet for 1 minute until the Dynabeads® are separated; discard the supernatant. Remove the tube from the magnet.

  5.    Resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume of Dynabeads®.

Starting Material

The preferred starting material is cryopreserved human PBMC obtained from leukapheresis product or Ficoll® separated whole blood. The starting material can also be enriched for specific T cell subsets, such as CD4+ T cells or CD25+ T cells.

Alternatively, PBMC from freshly obtained leukapheresis products or Ficoll separated whole blood may be activated and expanded without cryopreservation. If fresh samples are used, deplete monocytes with Dynabeads® M-450 Epoxy.

For maximum activation and expansion of T cells in PBMC magnetically capture CD3+ T cells prior to culture initiation.  Magnetic concentration is not required if T cells or T cell subsets have been enriched prior to activation and expansion.

Magnetic Seperation and Expansion of Cryopreserved CD3+ T Cells

[Note: Monocytes can rapidly phagocytose beads at 37°C, thereby reducing the absolute number of beads capable of contacting T cells, in turn reducing the level of T cell activation and expansion.]

  1. Determine the percentage of CD3+ T cells in the sample by flow cytometry or other suitable methods.

  2. For PBMC, resuspend cells in a tube at roughly 2-5 × 107 CD3+ cells/ml in Buffer 1, but not exceeding a maximum of 2 × 108 total nucleated cells/ml. For previously enriched/purified T cells (e.g. CD4+), resuspend the cells at  5-10 × 107 cells/ml.

  3. Add washed Dynabeads® Human T-Expander CD3/CD28 at a 3:1 bead: CD3+ T cell ratio. [Note: Bead:CD3+   cell ratio can be dropped as low as 1:1 when the starting T cell pool is more prone to activation induced cell death or where short term activation is desired.]

  4. Rotate the sample at 1 rpm for 30 min at between 4-25°C. Optimise the mixing temperature between 4-25°C for each application.

  5. Increase the volume in the tube to sufficient levels for magnetic selection by adding Incomplete Medium or Buffer 1 before placing the tube in a magnet for 1-2 min.

  6. Remove supernatant and stain with anti-CD3 antibody for flow cytometric analysis to calculate depletion efficiency.

  7. Gently resuspend bead: cell complexes to an estimated 0.5 x 106 -1 × 106 CD3+ T cells/ml in Complete Medium.

  8. Plate cells at 2.5-5 × 106 CD3+ cells in a total volume of 5 ml/well (0.5-1.0 × 106 cells/ml) of a 6 well culture plate in a humidified incubator at 37°C/ 5% CO2 for 3 days.

[Note: Smaller or larger culture vessels/wells can be used – adjust volumes accordingly. It is important that static cultures do not exceed ~1.2 cm in volume depth, otherwise expansion potential and viability may be compromised due to reduced gas exchange.]

Counting and Splitting of Cultures

Evaluate cell concentration daily, beginning on day 3 of culture. It may be useful to conduct regular phenotypic analyses by flow cytometry (e.g. days 3, 5, 8, and end of culture) to track T cell purity, phenotype and activation state. Similarly, supernatants may be collected to measure cytokine secretion patterns to further characterize T cell activity.

  1.    Gently mix contents of wells to dissociate beads and cells. Mixing should be sufficient to disrupt all visible bead:cell complexes.

  2.    Remove 50 μl sample and mix in 50 μl trypan blue (do not remove beads before counting).

  3.    Count cells manually using a hemocytometer.

  4.    When CD3+ T cell density is >1 × 106 cells/ml, dilute cells to approximately 0.5 × 106 CD3+ T cells/ml in culture medium.

  5.    At the end of culture (day 9-14) count cells and remove beads with a magnet.

Culture Initiation Without Magnetic Concentration of CD3+ T Cells

  1. Add 5 × 106 enriched or purified T cells to 1.5 × 107 washed Dynabeads® Human T-Expander CD3/CD28 in a final volume of 10 ml culture media, placing 5 ml/well in a 6 well tissue culture plate to give a final concentration of 0.5 x 106 CD3+T cells/ml. Mix gently.

  2. Culture cells in a humidified incubator at 37°C/5% CO2 for 3 days.

  3. Cell concentration should be evaluated daily beginning on day 3 of culture.

  4. Counting of cells and splitting of culture

Separation and Expansion of Fresh Samples

[Note: During the thawing process of cryopreserved PBMC, contaminating granulocytes tend to lyse, thereby diminishing any inhibitory effects upon subsequent T cell expansion. If working with fresh, non-cryopreserved samples, take into consideration the level of contaminating granulocytes when designing a culture initiation protocol. For initiation of cultures with fresh samples, use the Magnetic Concentration method described.

Overall T cell activation and expansion can be further improved by specifically depleting phagocytic cells, such as monocytes, using Dynabeads® M-450 Epoxy prior to magnetic concentration and culture initiation. This is particularly useful when monocyte levels are >15% of total PBMC.]

  1. Dilute PBMC to 5-10 × 106 cells/ml in Incomplete Medium (pre-warmed to 37°C).

  2. Wash Dynabeads® M-450 Epoxy according to the protocol.

  3. Add Dynabeads® M-450 Epoxy to the PBMC at 1-2 beads per total nucleated cells in a sterile culture vessel. 2 beads per nucleated cell can be used when monocyte levels are >15% of total PBMC.

  4. Place the culture vessel containing beads and cells into a humidified incubator at 37°C/5% CO2 for one hour. If using a culture flask, lay the flask on its side to maximize culture sur-face area.

  5. Remove Dynabeads® M-450 Epoxy and any cells that have ingested beads by applying a magnet for 1-2 min.

  6. Collect non-selected cells and initiate culture as described above, depending upon whether you want to incorporate the magnetic concentration step.

  7. Count cells and measure CD3+ T cell content of sample by flow cytometry.

  8. Adjust the concentration of cells to 1-5 × 107 CD3+ T cells/ml in Incomplete Medium.

  9. Capture CD3+ T cells as described above.

  10. Resuspend captured T cells and Dynabeads® Human T-Expander CD3/CD28 to approximately 0.5-1 × 106 CD3+2 until Day 3 of culture.

  11. Count and split cell culture as described above. T cells/ml in Complete Medium containing IL-2 and transfer to an appropriate culture vessel. Incubate at 37°C/5% CO


Procedures Incorporating Gene Transduction


Typically, for all culture conditions described earlier, T cells from normal donor tissues begin cycling and start to divide between day 2 and 3 of culture. Days 2, 3 and/or 4 of culture are recommended as optimal days for transduction using moloney based vectors, whereas day 1, 2 and/or 3 are recommended for lentivirus based vectors. Magnetic removal of beads prior to transduction will diminish overall cell expansion, but should not affect viability. Leaving beads in during the retroviral transduction process should be acceptable for most transduction applications.

[Note: T cells obtained from patients with various diseases and/or undergoing various treatments may be slower to enter cell cycle and cell division may not commence until 1, 2 or even 3 days later than typically observed for tissues from healthy donors. Thus it is important to monitor T cell activation markers, such as CD25, as well as cell division to determine optimal splitting schedules and timing for gene modification.]
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General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Warnings And Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.

Avoid pipetting by mouth!

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .
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References

  1. Kalamasz D et al (2004) Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and Anti-CD28 antibodies. J Immunother. 27(5):405-418.

  2. Bonyhadi M et al (2005) In vitro engagement of CD3 and CD28 corrects T cell defects in chronic lymphocytic leukemia. J Immunol. 174(4):2366-2375.

  3. Earle KE et al (2005) In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation. Clin Immunol. 115(1):3-9.

  4. Bondanza A et al (2006) Suicide gene therapy of graft-versus-host disease induced by central memory human T lymphocytes. Blood 107(5): 1828-36.

  5. Noonan K et al (2005) Activated marrowinfiltrating lymphocytes effectively target plasma cells and their clonogenic precursors. Cancer Res. 65(5):2026-2034.

  6. Huang et al (2008) DNA transposons for modification of human primary T lymphocytes. Methods in Molecular Biology, vol 506:115-126, DOI: 10.1007/978-1- 59745-409-4_9
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111.41D.indd    Rev 004    5-May-2009