Agarose

In order to analyse micromass formation, amounts of 5000 hADSC suspended in Leibovitz’s L-15 medium (Gibco/ Life Technologies, USA) were plated into agarose (Biozym Scientific GmbH, Germany) coated 8-well plates. Using the ibidi-Heating-System (ibidi GmbH, Germany), the microscope camera DS-Fi1 (Nikon, Japan) and the software “micro trac” (PD. Dr. D. Dirksen, University of Muenster), the movement of cells was displayed with 2.5 times magnification.

For the experiments regarding the Cirl mutant, the larvae were placed in the centre of Petri dishes of 9 cm inner diameter (Sarstedt, Nümbrecht, Germany), filled with 1% agarose (Biozym Scientific GmbH, Hessisch Oldendorf, Germany). The Petri dish was placed in a custom-built arena lit up with infrared light (PeakTech DC Power Supply 6080). Always five larvae were placed on a Petri dish, and their behaviour was recorded for 1 min with a camera (Logitech C920 HD Pro).

Multicellular trophoblast spheroids were grown in agarose micromolds that were prepared by pouring 3% (w/v) agarose (Biozym Scientific GmbH, Hessisch Olendorf, Germany) dissolved in distilled sterile H₂O (B. Braun, Melsungen, Germany) into silicone stencils (#12-256, MicroTissues Inc., Sigma-Aldrich) and sterilized under UV light for 30 min. Molds were transferred into a 12-well plate (CytoOne, StarLab International GmbH, Hamburg, Germany) and equilibrated in culture medium for 30 min. Trypsinized AC-1M-88 cells were suspended at 1,000,000 cells/mL, seeded into the agarose molds (200 µL/mold), and allowed to settle for 10 min at 37 °C. Additional culture medium was added to the wells. Growth and spheroid formation were monitored on a Zeiss Axiovert 135 Inverted Fluorescence Phase microscope (Carl Zeiss Microscopy, Jena, Germany) for 72 h.

Pulp tissue derived from human wisdom teeth was macerated enzymatically by the use of collagenase to isolate the cells from the surrounding tissue. The DPC were cultivated up to the fourth passage in D-MEM (low glucose), 20% FCS, 2% HEPES, 100 u/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 2.5 μg/ml amphotericin B (all PAA, Cölbe, Germany) at 37 °C, 5% CO₂ and 95% humid atmosphere with a medium change twice a week.
Chambers of 96-well plates were prepared by applying 50 μl of a mixture of 20 mg/ml agarose (Biozym Scientific GmbH) in D-MEM per well to ensure a non-attachment environment for the cells. A population of 100,000 cells per well was seeded into the specifically treated cell culture dishes and incubated in the same way as the cells described above.

DPC were harvested by trypsin incubation, counted using CASY cell counting technology (Schärfe System GmbH, Reutlingen, Germany) and transferred to a non-attachment environment. For this purpose, chambers of 96-well plates were prepared by applying 50 μl of a mixture of 20 mg/ml agarose (Biozym Scientific GmbH) in DMEM (Biochrom)/HGEM per well. A population of 100 000 cells per well was seeded into the treated cell culture dishes and incubated in the medium mentioned above at 37°C and 8% CO₂. The medium was changed twice per week. In preliminary tests it was possible to differentiate the cultivated human pulp cell spheres in different tissue specific ways to validate their multipotent stem cell character as described previously [23 (link)].

Chemicals were obtained from Sigma and Carl Roth & Co. Agarose was from Biozym Scientific GmbH. All fatty acids were from Sigma or Cayman Chemical. Acetonitrile was from Fisher Scientific. Restriction enzymes were purchased from MBI Fermentas.

Purified influenza A/PR/8/34 (PR/8) virus was obtained from Charles River, diluted 1:10 in sterile PBS, aliquoted and stored at -80°C. PR/8-OVA [22 (link)] was provided by Adolfo García-Sastre (Icahn School of Medicine at Mount Sinai, New York). Viral titers were determined by plaque forming assay using MDCK cells [57 (link)]. One day before the assay, 1x10⁶ cells/well were seeded in 6-well plates and grown overnight in Dulbecco’s Modified Eagle Medium (DMEM; Sigma), supplemented with 10% fetal calf serum (FCS; Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin, to obtain monolayers with >90% confluency. Cells were then washed with PBS and serial 1:10 dilutions of virus in DMEM (without FCS and antibiotics) were added to the cells for 1 hour at 37°C (each dilution in duplicates). Inoculates were aspirated and overlay medium, consisting of DMEM with antibiotics supplemented with 0.005% DEAE-Dextran (Sigma), 0.2% endotoxin-free bovine serum albumin (Fisher Scientific), 0.2% TPCK-treated trypsin (Sigma) and 1% agarose (Biozym), was added. 3 days later, plaques were visualized by staining with 0.03% neutral red (Sigma) for 3 hours and quantified.