3.3.1 Cell Culture Conditions and Passaging
If not noted otherwise, cells where cultured in RPMI 1640 supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM) , antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin), 2 ml vitamins, non-essential amino acids and 50 µM β-mercaptoethanol. Media supplements were purchased from Life Technologies, Karlsruhe, Germany.
Cells were cultured at 37°C, 5% CO2 and 95% relative humidity in an incubator. FCS
was heat-inactivated for 30 min at 56°C before use.
3.3.2 Assessing Cell Vitality by Trypan Blue Exclusion
Required solutions and materials:
Trypan blue solution 0.2% (w/v) Trypan blue
in 0.9% NaCl solution Neubauer haemocytometer slide with coverslip
The number of viable and dead cells was determined by Trypan blue exclusion. The cell suspension was diluted with trypan blue solution and the cells counted in a Neubauer haemocytometer. The concentration of viable cells was then calculated using the equation:
Number of viable cells/ml C = N x D x 104
with N: average of unstained cells per corner square(1 mm2 containing 16 sub-squares) D: dilution factor
3.3.3 Freezing and Thawing Cells
Freeze medium 50% RPMI 1640
40% FCS
10% DMSO
Cells were harvested and suspended in ice-cold RPMI 1640 at 1-10 Mio cells/ml (following ATCC recommendations for cell lines), T lymphocytes at 25-50 Mio/ml, and 1 ml of cell suspension was added to 1 ml ice-cold freeze medium in cryo-vials which where closed and inverted twice to mix. To allow gradual freezing at a rate of - 1°C/min, the vials were placed in Styrofoam containers or isopropanol-filled cryo- containers (Nalgene) and frozen at –80°C for 24 h. For long-term storage, the samples were then transferred to liquid nitrogen (-196°C).
To recover frozen cells, the cell suspension was thawed in a water bath at 37°C. To dilute the toxic DMSO, the suspension was transferred to 10-25 ml serum-containing medium as soon as thawed and the cells were spun down (8 min, 300xg, 4°C) and resuspended in fresh medium.
3.3.4 Primary Cells
3.3.4.1 Dendritic Cells
Immature monocyte-derived dendritic cells (DCs) were generated by culturing elutriated monocytes in 10% FCS/RPMI1640 supplemented with 500 U/ml each of recombinant human IL-4 (Schering-Plough, New Jersey, USA or Promocell, Heidelberg, Germany) and recombinant human GM-CSF (Essex, Munich, Germany) as described earlier (Meierhoff et al., 1998). To generate mature DCs, immature monocyte-derived DCs were activated after five days of culture with 100 ng/ml LPS or 10 ng/ml recombinant human TNF (Knoll AG, Ludwigshafen, Germany) for two additional days.
3.3.4.2 Macrophages
In order to generate macrophages in vitro, monocytes were cultured in RPMI1640 in the presence of 2% human pooled AB-group serum. If the macrophages had to be detached for further experiments, the cells were cultured in Teflon bags on teflon foils (Biofolie 25; Heraeus, Hanau, Germany), from which they could be harvested after cooling to 4°C for 45 min (Andreesen et al., 1983). Otherwise, cells were seeded into petri dishes at a density of 1-2x105 cells/cm2.
3.3.5 Cell Lines
The cell lines THP-1, HL-60, HepG2 and HeLa were maintained in 10% FCS/RPMI1640 with the supplements detailed under 3.3.1.
NHDFC were cultured in Dulbecco's modified MEM (DMEM) supplemented with L- glutamine, antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin and 1 µg/ml amphotericin B), 10 mM HEPES and 10% FCS.
The colon carcinoma cell lines CaCo-2 and HT-29 were cultured in 10% FCS/DMEM supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM) , antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin), 2 ml vitamins and 5 ml non-essential amino acids.
Cells were split 1:3 to 1:8 into fresh medium every 2-4 days. Adherent cells were passaged by washing once with PBS and incubation with 3 ml 0.05% Trypsin/0.02% EDTA/PBS per 75 cm2 culture vessel area at 37°C for 5-10 min. The detached cells were washed once with 5 ml medium containing FCS and resuspended and split 1:3 to 1:10 into 13 ml complete medium/75 cm2.
3.3.6 Mycoplasma Assay
Cells were frequently checked for mycoplasma contamination by ELISA with a Mycoplasma Detection Kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.
3.3.7 Mixed Leukocyte Culture
When cultured together, leukocytes from two unrelated individuals begin to proliferate. Bain and colleagues (Bain et al., 1964) coined the term "Mixed Leukocyte Reaction" to describe this observation and suggested that this reaction might be useful as an indicator of compatibility between siblings prior to organ transplantation to assess the risk of rejection of the transplant. Indeed, later on it could be shown that the observed proliferation of the T lymphocytes is a response to the "allogenicity" of the two leukocyte populations and that an MLR is absent if the MLC is carried out between syngeneic populations which express the same MHC alleles. In a syngeneic MLC, the proliferative response depends on the presence of both non-self antigens and antigen presenting cells in the leukocyte populations. In an allogeneic setting, the
MHC incompatibility alone is sufficient to trigger T cell proliferation, the magnitude of the reaction being solely dependent on the costimulatory capacity of the antigen presenting cells.
To ascertain the superior antigen presenting capabilities of DCs compared to the monocytes the were generated from, the magnitude of the MLR induced by co- culture with allogeneic T lymphocytes was assessed as the amount of tritium-labeled thymidine incorporated during DNA synthesis by the proliferating T lymphocytes. Ascending numbers of stimulators were used to induce proliferation of a fixed number of responder cells and the stimulatory capacity of the antigen presenting cells was assessed in terms of the amount of 3H-thymidine incorporated by the proliferating T lympocytes.
T lymphocyte-rich elutriation fractions (IIa1) frozen previously from a different donor were used as responder cells. The cells were thawed as described under 3.3.3 and resuspended in 10%FCS/RPMI at a concentration of 1x106 responder cells/ml. A
fixed number of 1x105 responders (100 µl) was seeded into sterile roundbottom 96-
well plates, leaving one row empty for the stimulator-only control. As stimulator cells, either DCs prepared as described above (see 3.3.4.1) or their monocyte precursors were used, which had been frozen as above (see 3.3.3). To prevent cells in the stimulator population from proliferating, stimulators were irradiated with a single dose of 30 Gy from a 137Cs source. Stimulators were washed with 10% FCS/RPMI and resuspended at 3x105 cells/ml in 10% human pooled AB serum/RPMI and further dilutions of 1x105, 3x104, 1x104, 3x103 and 1x103 cells/ml were prepared in the same medium. MLCs were set up as depicted in Figure 3.1:
One hundred milliliters quadruplicates of each concentration were added to the wells containing the responder cells, except one row for the responder-only control. The wells for the stimulator proliferation control were filled with 100 µl of the 3x105 cells/ml suspension. The volume in single cell population-containing wells was adjusted to 200 µl by adding 100 µl of 10% AB serum/RPMI or 10% FCS/RPMI to FCS or AB serum-containing well, respectively and the microwell plates were centrifuged for 8 min at 300xg and RT.
Stim. A Stim. B Stim. C Stimulators Responders A OOOO OOOO OOOO 3x104 none
B OOOO OOOO OOOO 3x104 1x105
C OOOO OOOO OOOO 1x104 1x105
D OOOO OOOO OOOO 3x103 1x105
E OOOO OOOO OOOO 1x103 1x105
F OOOO OOOO OOOO 3x102 1x105
G OOOO OOOO OOOO 1x102 1x105
H OOOO OOOO OOOO none 1x105
Following 5 days of incubation at 37°C, 5% CO2 and 95% relative humidity, 1 µCi/well
[methyl-3H]thymidine was added (10 µl of 1 mCi/ml diluted 1:10 with RPMI 1640) and the cells were incubated as before for an additional 18 h. MLCs were harvested with onto H2O-prewetted 96-well fiber glass filter plates using a FilterMate 196 harvester
(Packard BioScience), washed three times with H2O and air-dried briefly. The bottom
of the glass filter plate was sealed, 50 µl scintillation cocktail were added to each well, the top of the plate sealed as well with transparent adhesive foil and the activity measured in a TopCount scintillation counter (Packard Bioscience).