Introduction

T cell activation via the αβ-T cell receptor (TCR complex) is required for in vitro expansion. Treatment of T cells with monoclonal anti-CD3 antibodies and anti-CD28 antibodies provide a co-stimulatory signal that engages the TCR which can be used for antigen-induced activation. Using functional antibodies, as described below, with or without growth factors is a more economical method to expand T cell populations. Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation can also be used for activation and explanation of human T-Cells. Dynabeads T cell activation and expansion beads provide a strong stimulus with an even cell expansion and is useful for gene edited therapies. PMA or concanavalin A can be used for shorter incubations to increase levels of intracellular transcription factors and cytokine production for flow cytometry and immunoassays.


Materials

  1. Sterile cell-culture treated 6-well plates (e.g., Nunc Cell-Culture Treated 6-well plates, Cat. No. 140675)
  2. 15 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
  3. Anti-CD3 (e.g., Anti-Human CD3 (OKT3), Functional Grade, Cat. No. 16-0037-81 or Anti-Mouse CD3 Monoclonal Antibody (17A2), Functional Grade, Cat. No. 16-0032-82)
  4. Anti-CD28 antibody (e.g., Anti-Human CD28 Monoclonal antibody (CD28.2), Functional Grade, Cat. No. 16-0289-81 or CD28 Monoclonal Antibody (37.51), Functional Grade, Cat. No. 16-0281-82)
  5. RPMI 1640 medium (e.g., BenchStable RPMI 1640 Medium, Cat. No. A4192301 or OpTmizer T-Cell Expansion, Cat. No. A3705001)
  6. Fetal Bovine Serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140087, or Fetal Bovine Serum One Shot format, Cat No. A3160401)
  7. L-glutamine (e.g., 200 mM L-Glutamine, Cat. No. 25030149)
  8. Optional: Penicillin-Streptomycin (e.g., Gibco Penicillin-Streptomycin, Cat. No. 15140148)
  9. Phosphate buffered saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
  10. Cell scraper (e.g., Nunc Cell Scrapers, Cat. No. 179693)


Procedure

  1. Prepare 6-well culture plates by labeling the top 3-wells: ‘activated T cells’ and the bottom 3-wells: ‘non-activated T cells’.
  1. Coat the activated T cell wells with the anti-CD3 antibody by diluting the anti-CD3 antibody at 1 µg/mL in sterile PBS. Add diluted antibody to the 3 wells at 2 mL/well. Incubate plate at 5% CO2 at 37°C for 2 hours.
  1. Prepare complete RPMI 1640 medium by supplementing RPMI 1640 medium with fetal bovine serum to a final concentration of 10% and 2 mM L-glutamine (if using medium not currently supplemented with GlutaMAX). Bring medium to 37°C. Optional: Supplement media with 1% penicillin-streptomycin (5,000 units/mL).
  1. Using aseptic techniques under sterile conditions, isolate PBMC from whole blood or lymphoid tissue (See supplemental protocol A or B below, respectively).
  1. Resuspend cells to a concentration of 3 x 106 cells/mL in complete RPMI 1640 medium.
  1. Divide the cell solution evenly into to 2 conical tubes; label one tube ‘activated’ and the other ‘non-activated’.
  1. Into the activated tube, add 5 µg/mL of anti-CD28, and set aside in culture hood (room temperature).
  1. Retrieve 6-well plate from incubator after 2 hours. Aspirate out the anti-CD3 solution and discard. Do not wash the plate.
  1. Divide activated cell solution (from step 7) evenly into the anti-CD3 treated wells (activated T cells) of the 6-well plate (2–3 mL per well).
  1. Transfer non-activated cell solution from conical tube to the non-treated wells (non-activated T cells) of the 6-well plate (2–3 mL per well).
  1. Incubate culture plate for 1–3 days in 5% CO2 incubator at 37°C. By day 1, cells will produce transcription factors and cytokines. Most T cells will require up to 3 days to divide. These cells will clump but will not be attached to the plate.
  1. Harvest the cells with a cell scraper.

Protocol tip:

Optimal concentration range of antibodies anti-CD3 and anti-CD28 should be determined empirically. For anti-CD3 (mouse and human), optimal concentration range is 1–3 µg/mL and for anti-CD28 (mouse and human), optimal concentration range is 3–5 µg/mL.

Protocol tip:

IL-2 stimulates the growth and differentiation of cytotoxic T cells. Adding 100 ng IL-2 recombinant protein (Cat. No. PHC0026) instead of anti-CD28 can help expand this population.

Protocol tip:

Anti-CD3 can be directly added to the media in step 3 at a final concentration of 1 µg/mL to save time.

Protocol tip:

This protocol can be scaled for larger cell quantities by using T75 flasks.


Supplemental protocol A: isolation of PBMC from whole blood

Materials

  1. Phosphate buffered saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
  2. 15 mL or 50 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
  3. Ficoll-Paque® density separation medium
  4. Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

  1. Dilute blood sample at least 1:1 with PBS in a conical tube.
  1. Underlay the diluted sample with a volume of Ficoll-Paque® medium that is equal to the original sample volume.
  1. Centrifuge at 400 x g for 20 minutes at room temperature with the brake OFF.
  1. Harvest PBMC located at the interface of the PBS and Ficoll-Paque® medium layers into a fresh tube.
  1. Fill the tube with PBS to wash the cells.
  1. Centrifuge the cells at 300–400 x g for 4–5 minutes at 2–8°C. Discard supernatant.
  1. Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or buffer of choice, and perform a cell count and viability analysis.
  1. Centrifuge cells as in step 6, and resuspend in appropriate volume of complete RPMI 1640 so that the final cell concentration is 3 x 106 cells/mL.

Note:

As cells will be cultured, perform all steps using aseptic technique, and use buffers that do not contain azide.

Protocol tip:

Significantly improve the accuracy of assessing cell health and concentration from freshly harvested peripheral blood mononuclear cells with automated counters. See detailed protocol on how to count PBMC using the Countess II FL Automated Cell Counter.


Supplemental protocol B: isolation of immune cells from lymphoid tissue

Materials

  1. 60 x 15 mm cell culture dish (e.g., Nunc Cell Culture Petri Dishes, Cat. No. 150340)
  2. Plastic 3-mL syringe or two frosted glass microscope slides
  3. Cell strainer (nylon mesh)
  4. 15 mL or 50 mL conical tube (e.g., Nunc 15 mL conical sterile centrifuge tubes, Cat. No. 339650)
  5. Flow cytometry staining buffer (e.g., eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26)

Procedure

  1. Harvest tissue (spleen; thymus; lymph nodes) into a cell culture dish containing 10 mL of Flow Cytometry Staining Buffer or buffer of choice. Tease apart into a single-cell suspension by pressing with the plunger of a 3 mL syringe. Alternatively, mash tissue between the frosted ends of two microscope slides using 10 mL of Flow Cytometry Staining Buffer.
  1. Place a cell strainer on top of a 15-mL conical tube. Pass cells from the cell culture dish through the cell strainer to eliminate clumps and debris.
  1. Centrifuge cell suspension at 300–400 x g for 4–5 minutes at 2–8°C. Discard the supernatant.
  1. Resuspend the cell pellet in an appropriate volume of Flow Cytometry Staining Buffer or buffer of choice and perform a cell count and viability analysis.
  1. Centrifuge cells as in step 3, and resuspend in appropriate volume of complete RPMI 1640 medium to the final cell concentration is 3 x 106 cells/mL.

Note:

Mechanical disruption of lymphoid tissue is generally sufficient to release cells into a single-cell suspension.

Note:

As cells are to be cultured, perform all steps using aseptic technique, and use buffers that do not contain azide.