Collagenase dispase solution

Exfoliated deciduous incisors were collected from two normal healthy donors and three CMD patients carrying a F377del mutation in ANKH. SHED cells were isolated as described previously^{44 (link)}. In brief, pulp was separated from the crown and digested in a mixture of collagenase/dispase solution (Roche) for 1 hour at 37 °C. Cell suspension was filtered through a 70 μm filter (Falcon). Cells were maintained in α-MEM growing medium supplemented with 15% FBS, Pen/Strep and 2 mM glutamine. Once confluent, cells were cultured in growing medium with ascorbic acid (50 μg/ml) and 8 mM β-glycerophosphate (Sigma-Aldrich). Bone explant cultures from one healthy donor and one CMD patient with a F377del mutation in ANKH were performed as previously published^{22 (link)}.

All patients were first diagnosed with primary NSCLC without other tumour occurrences. They did not receive any therapy before surgery. Samples were chopped into small pieces, and incubated in 1 mg ml⁻¹ collagenase/dispase solution (Roche, Indianapolis, IN) with 0.001% DNAse (Sigma-Aldrich, St Louis, MO) and 2% antibiotics (Sigma) in a water bath at 37 °C for 3 h. After incubation, the suspensions were passed through 70- and 40-μm cell-strainers (BD Falcon, San Jose, CA) and centrifuged at 122g for 5 min at 4 °C. Cells were then resuspended in red blood cell lysis buffer (eBioscience, San Diego, CA) for 4 min at room temperature with intermittent shaking. The cell viability was evaluated by trypan blue dye exclusion. Live single cells accounted for 90% of the whole population.

Lung tissue was excised from an anesthetized mouse (1-week-old), minced, and then incubated in Dulbecco’s Modified Eagle’s Medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 1 mg/ml collagenase/dispase solution (Roche, Basel, Switzerland) and 5 U/ml DNase I (Roche) for 45 min at 37 °C. The digested pieces were further minced by passing them through a 20-gauge needle and then filtered with a 70-μm cell strainer (BD Biosciences, San Jose, CA, USA). The filtrate was centrifuged for 5 min at 400×g at 20 °C, and the resulting cell pellet was suspended in PBS containing 0.1% BSA. The cells were incubated with Dynabeads (Invitrogen, Carlsbad, CA, USA) precoated with rat anti-mouse CD31 antibody (BD Biosciences) for 30 min at room temperature. ECs bound to the Dynabeads were collected with a magnet, washed using PBS with 0.1% BSA, and then cultured in a 60-mm dish coated with 2% gelatin in Endothelial Cell Growth Medium 2 (Takara). The purity of isolated ECs was > 95%, which was confirmed by immunofluorescence microscopy using anti-CD31 and anti-CD102 antibodies (BD Biosciences).

Healthy third molars (n = 3, 16 to 25 years old) were extracted for reasons not related to this study at the Center of Dental Medicine, University of Zurich. All respective patients had impacted teeth and gave informed consent that their teeth would be used for scientific purposes. Anonymized tooth samples obtained with written informed consent are exempt from the necessity of obtaining an individual ethics approval according to local law (Swiss Federal Council, Federal Act on Research involving Human Beings). Extractions were done carefully in order to minimize the risk of infection. Freshly extracted teeth were kept in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Paisley, UK) supplemented with 10% Pen Strep, and 10% fetal bovine serum (FBS, Gibco, Paisley, UK).
The tooth crown was removed using a diamond bur under water-cooling. The pulp tissue contained in the tooth was then harvested and cut into smaller pieces, washed in phosphate-buffered saline (PBS, Gibco), and finally digested in a collagenase/dispase solution (Roche, Mannheim, Germany). Cells were cultured in DMEM supplemented with 1% Pen Strep and 10% fetal bovine serum (FBS, Gibco). Cells at passages 3–6 at 80% confluence were used in the following experiments.

After 5 days in culture, the gonads were removed from the membrane dissociated in collagenase/dispase solution (10,269,638,001, Roche) at 37 °C, for 20 min. The cell suspension was incubated for 2 h in 12-well plates pre-treated with gelatin (G1890, Sigma) for somatic cell adhesion. The medium containing non-adhered cells (including PGC) was centrifuged and resuspended in 1X PBS (pH 7.2) containing magnetic beads pre-incubated with biotinilated Dolichus biflorus agglutinin (DBA). The suspension was incubated for 1 h at 4 °C rocking and placed in a magnetic rack (Invitrogen). The positive selected cells (PGC) and the negative selected cells (somatic cells) were submitted to RNA isolation using TRIzol® (Invitrogen). The cells that adhered to the 12-well plates in the first step of the selection were treated with 1X trypsin (59418C, Sigma) and combined with the magnetic negative selection cells for RNA isolation. The cDNA was obtained using the Superscript III Reverse Transcriptase (Invitrogen). Conventional PCR was performed using specific primers for the developmental genes Pax6 (FP 5′-agtgaatgggcggagttatg-3′ and RP 5′-aacaaccacatgagccaaca-3′) and Dazl (FP 5′-gaaatggcccacaaaagaaa-3′ and 5′-ttaagcactgcccgacttct −3′).

Fragments of healthy lung specimens were washed several times with phosphate buffer solution (PBS) and finely minced with scissors and subjected to enzymatic digestion for 75 minutes at 37°C with 1 mg/mL collagenase/dispase solution (Roche, Basel, Switzerland). The resulting digestion product was squeezed over a 100 μm nylon mesh (BD Biosciences, San Josè, CA, USA) to remove aggregates. The harvested cells were washed, seeded on collagen coated wells of a 6-well plate (BD Biosciences) and cultured in complete growth medium consisting of endothelial growth medium-MV (EGM-MV; Lonza, Walkersville, MD, USA) plus 5% fetal bovine serum (FBS; Lonza) and 2 ng/mL recombinant human vascular endothelial growth factor-C (VEGF-C; ReliaTECH).
Twenty-four hours later, nonadherent cells and debris were removed, whereas the adherent cell population was washed with PBS and cultured in complete medium until 70–80% confluence (usually reached in 5–7 days).

Fin tissue was dissociated by vigorous shaking in 1 mg mL⁻¹ collagenase/dispase solution (Roche) for 20 min at 37 °C. Collagenase was inactivated with 20% FBS/DMEM, and the suspension was passed through a 40-μm mesh. The suspension was centrifuged for 10 min, and the cell pellet was resuspended and incubated in 2% paraformaldehyde/PBS for 10 min at room temperature. Afterwards, cells were precooled for 1 min on ice and then permeabilized with ice-cold methanol, which was added dropwise while shaking. After incubation for 30 min on ice, the cell suspension was washed with PBS and stained with 0.05% Hoechst/PBS. After a final wash step with PBS, cells were analysed with the flow cytometer FACSCanto II (BD Biosciences, Heidelberg, Germany) using forward and side scatter parameters to exclude cell debris. Five to six animals were used for analysis per time point and age group, while 10–14 animals were used for analysis of uninjured fin tissue. Data were analysed with FLOWING Software (version 2.5.0, www.flowingsoftware.com). Significant differences between age groups were analysed at each time point using ANOVA followed by pairwise comparison using Tukey’s HSD test.