This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
This study did not generate/analyze datasets/code.
Reproducible and efficient expansion of different immune effector cells is required for pre-clinical studies investigating adoptive cell therapies against cancer. Here, we provide a protocol for the rapid expansion of mouse T cells, natural killer (NK) cells, and bone-marrow-derived macrophages (BMDMs). We describe steps for αCD3/αCD8 plate coating, isolating splenocytes, and expanding T cells and NK cells. Further, we detail procedures for bone marrow isolation and BMDM differentiation.
Subject areas: Cell Biology, Cell culture, Cell isolation, Flow Cytometry, Cancer, Immunology
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Reproducible and efficient expansion of different immune effector cells is required for pre-clinical studies investigating adoptive cell therapies against cancer. Here, we provide a protocol for the rapid expansion of mouse T cells, natural killer (NK) cells, and bone-marrow-derived macrophages (BMDMs). We describe steps for αCD3/αCD8 plate coating, isolating splenocytes, and expanding T cells and NK cells. Further, we detail procedures for bone marrow isolation and BMDM differentiation.
The current protocol enables a rapid and efficient expansion of murine T cells, NK cells and macrophages. These expansion procedures necessitate access to C57BL/6 mice for the extraction of spleens and bone marrow.
For T cell expansion αCD3 and αCD28 antibodies (Bio X Cell, #BE0001-1 and #BE0015-1) as well as mouse IL-2 are required. 1 NK cell isolation and expansion requires the EasySep Mouse NK Cell Isolation Kit (STEMCELL Technologies, #19855) 2 and mouse interleukin (IL)-15. 3 BMDM are differentiated from bone marrow cells using L929 conditioned medium as supplement. 4 L929 conditioned medium from the L929 fibroblast cell line needs to be prepared in advance. Alternatively, mouse macrophage colony-stimulating factor (M-CSF) can be used.
Before starting organ extractions prepare cytokine stocks of mouse IL-2, IL-15, if required M-CSF and freeze them at −80°C. Primary cell cultures described in this protocol require a strictly controlled environment for cell growth. A temperature of 37°C and a CO2 level of 5% should be maintained in the incubator during cell expansion. To prevent contamination and to ensure comparable functional states of immune effector cells it is required to ensure aseptic working conditions throughout the protocol.
All needed instruments, material and reagents should be purchased from well-established sources to ensure the integrity and reliability of the protocol.
All experiments were done in accordance with the guidelines of the Swiss federal law on animal protection and were approved by the cantonal veterinary office. Anyone who is interested in conducting described experiments needs to acquire permission from the relevant institutions before.
Thaw L929 cells and seed 5 × 10 6 cells in DMEM/F-12 complete medium in a T-150 flask for adherent cells and grow until confluent.
Remove the supernatant, wash 1× with DPBS and detach with TrypLE or Trypsin.Use new T-150 flasks for adherent cells and re-seed 5 × 10 6 cells per flask with 35 mL of DMEM/F-12 complete medium.
After 7 days, transfer the medium from the flasks to 50 mL centrifuge tubes and centrifuge at 400 g for 5 min.
Collect the supernatant in 15 mL or 50 mL centrifuge tubes and store at −20°C or −80°C. Repeat steps 3–5 if more L929-conditioned medium is needed.| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| InVivoMAb anti-mouse CD3ε (clone 145-2C11) | Bio X Cell | Cat#BE0001-1 |
| InVivoMAb anti-mouse CD28 (clone 37.51) | Bio X Cell | Cat#BE0015-1 |
| Anti-CD3-PerCP-Cy5.5 (clone 17A2) (1:100) | BD Biosciences | Cat#560527 |
| Anti-NK-1.1-PE (clone PK136) (1:100) | BD Biosciences | Cat#557391 |
| Anti-CD11b-APC-Cy7 (clone M1/70) (1:100) | BD Biosciences | Cat#557657 |
| Anti-CD4-FITC (clone RM4-5) (1:100) | Thermo Fisher Scientific | Cat#11-0042-86 |
| Anti-CD8a-APC (clone 53-6.7) (1:100) | Thermo Fisher Scientific | Cat#17-0081-83 |
| Anti-NKp46-APC (clone 29A1.4) (1:100) | BioLegend | Cat#137608 |
| Anti-CD49b-PE (clone DX5) (1:100) | BioLegend | Cat#108907 |
| Anti-F4/80-FITC (clone Cl:A3-1) (1:100) | CiteAb | Cat#MCA497F |
| Chemicals, peptides, and recombinant proteins | ||
| DPBS (Ca 2+ and Mg 2+ -free) | Thermo Fisher Scientific | Cat#14190094 |
| RPMI-1640 medium, HEPES, no glutamine | Thermo Fisher Scientific | Cat#42401042 |
| MEMα, nucleosides, no phenol red | Thermo Fisher Scientific | Cat#41061029 |
| DMEM/F-12, no glutamine | Thermo Fisher Scientific | Cat#21331020 |
| Fetal bovine serum, qualified, heat inactivated, Brazil | Thermo Fisher Scientific | Cat#10500064 |
| L-glutamine (200 mM) | Thermo Fisher Scientific | Cat#25030081 |
| Penicillin-Streptomycin | Sigma-Aldrich | Cat#P4333-100ML |
| ACK lysing buffer | Thermo Fisher Scientific | Cat#A1049201 |
| UltraPure 0.5 M EDTA, pH 8.0 | Thermo Fisher Scientific | Cat#15575020 |
| 2-Mercaptoethanol (55 mM) | Thermo Fisher Scientific | Cat#21985-023 |
| EasySep buffer | STEMCELL Technologies | Cat#20144 |
| Recombinant murine IL-2 | PeproTech | Cat#212-12 |
| Recombinant murine IL-15 | PeproTech | Cat#210-15 |
| Recombinant murine M-CSF | PeproTech | Cat#315-02 |
| Critical commercial assays | ||
| EasySep Mouse NK Cell Isolation Kit | STEMCELL Technologies | Cat#19855 |
| Experimental models: Organisms/strains | ||
| C57BL/6J, male and female | Janvier Labs | Cat#SC-C57N-F |
| Other | ||
| 6-well suspension plate, non-adherent | Sarstedt | Cat#83.3920.500 |
| Cell culture flask, T-75, surface: suspension, filter cap | Sarstedt | Cat#83.3911.502 |
| Cell culture flask, T-175, surface: suspension, filter cap | Sarstedt | Cat#83.3912.502 |
| Tissue culture flask with re-closable lid 115/150 cm 2 | TPP Techno Plastic Products AG | Cat#90552 |
| Centrifuge tubes, conical 50 mL | TPP Techno Plastic Products AG | Cat#91050 |
| Petri dish 100 × 15 mm standard Falcon | Milian SA | Cat#351029 |
| Falcon cell strainers for 50 mL tubes, 40 μm | Milian SA | Cat#352340 |
| Falcon cell strainers for 50 mL tubes, 70 μm | Milian SA | Cat#352350 |
| Falcon cell strainers for 50 mL tubes, 100 μm | Milian SA | Cat#352360 |
Note: Use aseptic techniques to ensure the sterility of all material used under a cell culture hood.
| Reagent | Final concentration | Amount |
|---|---|---|
| DPBS (Ca ++ and Mg ++ -free) | N/A | 48.9 mL |
| Fetal Bovine Serum | 2% | 1 mL |
| UltraPure 0.5 M EDTA, pH 8.0 | 1 mM | 100 μL |
| Total | N/A | 50 mL |
The solution can be stored for up to 6 months at 4°C.
DMEM/F12 complete medium
| Reagent | Final concentration | Amount |
|---|---|---|
| DMEM/F-12, no glutamine | N/A | 500 mL |
| Fetal Bovine Serum | 10% | 50 mL |
| L-Glutamine (200 mM) | 2 mM | 5 mL |
| Penicillin-Streptomycin (100×) | 1× | 5 mL |
| Total | N/A | 560 mL |
The medium can be stored for up to 6 months at 4°C.
RPMI complete medium
| Reagent | Final concentration | Amount |
|---|---|---|
| RPMI-1640 Medium, HEPES, no glutamine | N/A | 500 mL |
| Fetal Bovine Serum | 10% | 50 mL |
| L-Glutamine (200 mM) | 2 mM | 5 mL |
| Penicillin-Streptomycin (100×) | 1× | 5 mL |
| 2-mercaptoethanol (55 mM) | 0.05 mM | 500 μL |
| Total | N/A | 560.5 mL |
The medium can be stored for up to 6 months at 4°C.
MEMα complete medium
| Reagent | Final concentration | Amount |
|---|---|---|
| MEMα, nucleosides, no phenol red | 80% | 500 mL |
| Fetal Bovine Serum | 20% | 100 mL |
| Penicillin-Streptomycin (100×) | 1× | 5 mL |
| 2-mercaptoethanol (55 mM) | 0.05 mM | 550 μL |
| Total | N/A | 605.5 mL |
The medium can be stored for up to 6 months at 4°C.
Alternatives: STEMCELL Technologies provides the EasySep Buffer (#20144) as an alternative to Recommended Medium.
Plate coating αCD3/αCD28 needs to be performed one day before splenocyte isolation.
Dilute both antibodies in 12 mL DPBS to a final concentration of 1 μg/mL for αCD3 and 5 μg/mL for αCD28.
Transfer 2 mL of the antibody dilution per well on each well of a 6-well suspension plate and make sure that the wells are fully covered.
Wrap the plate in aluminum foil and incubate for 8–16 h at 4°C.The spleen of one C57BL/6 mouse will give ∼30–80 × 10 6 splenocytes with ∼20%–30% T cells and 1%–2% NK cells.
Prepare a 50 mL centrifuge tube with DPBS on ice. Sacrifice one mouse and disinfect the abdominal skin with 70% ethanol. Open the peritoneum using a forceps and scissors. Expose the spleen by pulling associated white connective tissue with forceps. Remove unwanted tissue with scissors and transfer the spleen into the 50 mL centrifuge tube.Pause point: The spleen can be kept in DPBS on ice for transport. Try to process within 2 h after isolation.
Place a 100 μm cell strainer in a new 50 mL centrifuge tube and mash the spleen with a plunger of a 2 mL syringe. Continuously moisten the cell strainer with DPBS while mashing. Fill the tube with DPBS.
Spin at 400 g for 5 min and aspirate the supernatant. Use 500 μL ACK lysis buffer/spleen. Incubate for 2–3 min and fill the tube with DPBS.CRITICAL: Ensure that the tissue stays moistened with DPBS throughout this step.
Pass cells through a 40 or 70 μm cell strainer to remove cell clumps and debris. Moisten the cell strainer with DPBS or medium before use.
Spin at 400 g for 5 min and aspirate the supernatant.Resuspend the pellet in RPMI complete medium (for T cell expansion) or recommended medium (for NK cell expansion) to get ∼8–12 × 10 6 cells/mL.
Count cells with a hemocytometer at a 1:20 dilution.Note: Proceed with T cell or NK cell expansion after this step.
Splenocytes will be activated with αCD3/αCD28 antibodies for 2 days and further expanded with IL-2. During activation, T cells will form clusters and will increase in size. After 3–5 days, an increased proliferation rate, an elongated shape and clonal clusters can be observed.
CRITICAL: Ensure you have splenocytes counted and ready for seeding before starting.
Aspirate the remaining supernatant of the αCD3/CD28-coated 6-well suspension plates. Wash one time with DPBS to remove unbound antibodies and repeat aspiration.
Seed 5 × 10 6 isolated splenocytes in 5 mL RPMI complete medium per coated well. On day 1, supplement the medium with 50 U/mL IL-2.Detach the cells by pipetting up and down with a serological pipette and transfer the cell suspension into a 50 mL centrifuge tube.
Add 1 mL DPBS to remaining cells and detach with a 1000 μL pipette. Combine cell fractions.Spin the cell suspension at 400 g for 5 min, resuspend in appropriate volume of RPMI complete medium and count. Reseed cells at a concentration of 1 × 10 6 cells/mL with 0.5 mL/cm 2 . Supplement the medium with 50 U/mL IL-2.
Note: Activated T cells can be easily detached by resuspension.
From now on, count cell numbers every 1–2 days and expand with additional RPMI complete medium to maintain a concentration of 1 × 10 6 cells/mL with 0.5 mL/cm 2 . Supplement additional medium with 50 U/mL IL-2.
T cells show >95% purity and high viability 5–7 days after isolation ( Figure 1 ) and can be used for in vitro and in vivo experiments.
T cell proliferation and phenotypic characterization during expansion (A) Expansion of T cells over time starting from 1 × 10 6 splenocytes. Error bars are based on n = 3 replicates. Mean cell numbers (in ×10 6 ) are indicated for each measurement time point. (B) Microscopic image of T cells on day 6 of expansion. As expected, T cells show elongated shape and clonal clusters indicative for a proliferative state. Scale bar is 100 μm. (C) Flow cytometry of CD3 + T cells on day 3, 5 and 7 of expansion including the relative proportion of CD4 + and CD8 + T cells. SSC, side scatter.
Note: For long-term culture and higher number of cells increase IL-2 dose to 200 U/mL from day 6 onward. With increased IL-2 concentration T cells can be cultured for up to 11 days.
The following steps are written for the processing of splenocytes from two C57BL/6 mouse spleens of which ∼1.5 × 10 6 NK cells can be isolated. NK cells show an increased proliferation rate after 3–5 days.
Spin splenocytes in recommended medium at 400 g for 5 min and aspirate the supernatant.CRITICAL: Ensure you have splenocytes counted and ready for seeding before starting. Cell concentration and medium volume are important. Deviations will hamper proliferation and viability.
Resuspend the splenocyte pellet at 1 × 10 6 cells/mL in recommended medium and follow the instructions of the EasySep Mouse NK Cell Isolation Kit.
Spin the isolated NK cell suspension at 400 g for 5 min and aspirate the supernatant. Resuspend the pellet in 1 mL medium to get ∼1–2 × 10 6 cells/mL. Count cells with a hemocytometer at a 1:5 dilution.Seed NK cells at a concentration of 0.5 × 10 6 cells/mL in 800 μL MEMα complete medium/well on a 24-well suspension plate. Supplement medium with 150 ng/mL IL-15.
On day 3, count the cell suspension with a hemocytometer and expand with additional MEMα complete medium to reach 0.4 × 10 6 cells/mL with 0.25 mL/cm 2 . Supplement additional medium with 150 ng/mL IL-15.
From now on, count cell numbers every 2 days and expand with additional MEMα complete medium to maintain a concentration of 0.4 × 10 6 cells/mL with 0.25 mL/cm 2 . Supplement additional medium with 150 ng/mL IL-15.
NK cells show >95% purity 4 days after isolation ( Figure 2 ). High viability is maintained for 12 days after isolation. NK cells are recommended to be used 7–12 days after isolation for in vitro and in vivo experiments.
NK cell proliferation and phenotypic characterization during expansion (A) Expansion of NK cells over time starting from 1 × 10 6 splenocytes. Error bars are based on n = 3 replicates. Mean cell numbers (in ×10 6 ) are indicated for each measurement time point. (B) Microscopic image of NK cells on day 8 of expansion. As expected, NK cells show elongated shape indicative for a proliferative state. Scale bar is 100 μm. (C) Flow cytometry of CD3 - NKp46 + NK cells on day 4, 8 and 12 of expansion including the relative expression CD49b expression.
CRITICAL: Keep NK cells on uncoated suspension plates/flasks.
Note: The viability of NK cells will reduce after 1 day if not stimulated or deprived from IL-15.
Both femora and tibiae of one C57BL/6 mouse will give ∼50–90 × 10 6 bone marrow cells.
Prepare a 50 mL centrifuge tube with DPBS on ice. Sacrifice one mouse and disinfect the skin at the legs and abdomen with 70% ethanol.Make an incision at the top of each hind leg and pull the skin down towards the foot to expose the muscle. Cut the cartilage between femur and tibia.
Use scissors to free the hind legs from muscles and perform the amputation.Use a paper tissue sprayed with 70% ethanol to remove remaining muscle tissue from the bones.Note: Twisting will help to isolate the femur. But be careful not to break the bones.
Spray the bones with 70% ethanol to disinfect and wait 30 s. Transfer them into the 50 mL centrifuge tube filled with DPBS for transport.
Pause point: The bones can be kept in DPBS on ice for transport. Try to process within 2 h after isolation.
Place the bones on a petri dish under a sterile working bench. Remove remaining connective tissue with forceps and scalpel by scratching. Cut the cartilage at both sides of the bone to allow excess to the bone marrow.
CRITICAL: Process bones of max. 2 mice at the same time to not let the bone marrow dry out.
Attach a 25G needle to a 10 mL syringe. Flush the bone cavity with DMEM/F12 complete medium into a 50 mL centrifuge tube until the bone cavity appears white.
Spin at 400 g for 5 min and aspirate the supernatant. Use 500 μL ACK lysis buffer/spleen. Incubate for 2–3 min and fill with DPBS.Pass the cells through a 40 μm or 70 μm cell strainer to remove cell clumps and debris. Moisten the cell strainer with DPBS or medium before use.
Spin at 400 g for 5 min and aspirate the supernatant. Resuspend the pellet in DMEM/F12 complete medium to get ∼4–6 × 10 6 cells/mL. Count the bone marrow cells with an automated cell counter or a hemocytometer at a 1:10 dilution.Note: Proceed with BMDM differentiation and expansion after this step.
On day 1–3 of differentiation dead floating cells can be observed. Adherent macrophages will become visible on day 3–4 of differentiation. Each 10 cm Petri dish will give ∼8 × 10 6 BMDMs on day 5 and ∼18 × 10 6 BMDMs on day 7.
CRITICAL: Ensure you have bone marrow cells counted and ready for seeding before starting.
Dilute bone marrow cells to a concentration of 0.2–0.25 × 10 6 cells/mL in 12 mL DMEM/F-12 complete medium supplemented with 20% L929 conditioned medium and seed 12 mL cell suspension per non-adherent 10 cm petri dish.
Carefully tilt the plate and gently remove 7 mL medium with a serological pipette.Note: Alternative to 20% L929 conditioned medium also 20 ng/mL mouse M-CSF can be added to the medium.
Replace with fresh DMEM/F-12 complete medium supplemented with 20% L929 conditioned medium to again reach a total volume of 12 mL.
After 5–7 days, aspirate the medium from the plate. Gently wash the cells 1× with 5 mL DPBS. Add 5 mL of cold DPBS with 2.5 mM EDTA per plate. Incubate for 10–20 min on ice or at 4°C in the fridge.CRITICAL: Differentiating BMDMs are not firmly attached on day 3. Be careful to aspirate medium from the top.
Gently resuspend detaching cells with a serological pipette and collect them in a 50 mL centrifuge tube. Add additional DPBS + 2.5 mM EDTA to collect remaining cells.
Centrifuge at 350 g for 5 min and discard the supernatant. Flick the pellet and resuspend the cells in required buffer or medium.BMDMs show >95% purity 5 days after isolation ( Figure 3 ). They might be kept in culture for up to 2 weeks but will become more differentiated and adherent with time. With respect to the seeding concentration in this protocol, BMDMs are recommended to be used 5–7 days after isolation for in vitro and in vivo experiments.
BMDM proliferation and phenotypic characterization during expansion (A) Expansion of BMDMs over time starting from 1 × 10 6 bone marrow cells. Error bars are based on n = 3 replicates. Mean cell numbers (in ×10 6 ) are indicated for each measurement time point. (B) Microscopic image of BMDMs on day 5 of expansion. As expected, cells are attached to the plate indicative for a successful differentiation of bone marrow cells to BMDMs. Scale bar is 100 μm. (C) Flow cytometry of CD11b + F4/80 + BMDMs on day 3, 5 and 7 of expansion.
The present protocol has been validated in multiple differentiation experiments and cell expansion numbers and phenotypic characteristics remained stable ( Figures 1 , ,2, 2 , and and3). 3 ). However, we recommend confirming cell phenotypes when the protocol is established.
Expanded immune effector cells can be used for a variety of in vitro and in vivo studies. These can be immunological in vitro studies to investigate immune cell interactions in co-culture experiments as well as in vitro studies to analyze changes in phenotype and intracellular signaling after stimulation or inhibition. In a pre-clinical setting, the high number expansion allows to use the immune effector cells with or without modification for adoptive cell transfer in vivo. Studies using the same protocol for the generation of mRNA-based chimeric antigen receptor (CAR) expressing mouse T cells and the investigation of the effect of immunocytokines on T cell killing have already been published. 5 , 6 A study comparing all three immune effector cells transfected with mRNA coding for a CAR is ongoing.
Overall, this manuscript provides protocols for the rapid and high number expansion of mouse T cells, NK cells and BMDMs and serves as a valuable template to better standardize their phenotype on the day of use.
These protocols are referring to organ harvesting of C57BL/6 mice. The total numbers of splenocytes and bone marrow cells that can be isolated might be reduced if other mouse strains are used. Variations might also occur depending on the age of mice as splenocyte numbers decrease from 3- to 5-months old mice. 7 Therefore, for harvesting a maximal splenocyte number, 3-months old mice are recommended. Processing times should be held short to allow maximal expansion of cells.
One has to consider that the protocol aims to standardize the phenotypical state of the immune effector cells on the day of use. However, after long-term expansion it does not reflect every phenotypical state of T cells, NK cells and BMDMs in vivo as it is influenced by many factors such as development stage, tissue context and physiological condition of the animal. The usability of long-term expanded immune effector cells for own studies should be evaluated before conducting them. Otherwise, further adaptions need to be investigated such as complex cell culture, different culture media and the supplementation with additional cytokines.
Low number of isolated splenocytes, related to step 6–7.
