Pulmozyme

A sample of sputum previously collected from a non-induced patient (p18) was thawed at room temperature and placed on a Petri dish inside an exposure box (12 × 8.3 × 6.7 cm). Pulmozyme^® (rhDNAse, 2500 IU/2.5 mL, Roche) was aerosolized using the eFlow^® Rapid Nebulizer (PARI, France). Rheological characterization was performed immediately after this treatment. Furthermore, Istendo^® (N-acetyl-cysteine, 1 g/5 mL, Laboratoires Delbert) was deposited on another sample of p18 sputum and directly analyzed in rheology.

All reagents were purchased from commercial sources, unless stated otherwise – magnesium sulfate, calcium chloride, and DNA from herring testes (type XIV) were purchased from Sigma-Aldrich. All the described reagents were used without further purification. Bacterial strain W1050 of S. marcescens was kindly provided by Prof. Michael Benedik (University of Houston, United States). A commercial preparation Pulmozyme^® (F. Hoffman-La Roche) contained 1 mg Dornase alfa, 8.77 mg NaCl, and 0.152 mg CaCl₂⋅2H₂O per 1 ml.

In order to precisely analyze cell composition and avoid a bias between the two isolation processes, an additional extensive enzymatic digestion process was applied to the two groups: the objective was to obtain a fully digested cellular suspension, to be analyzed by flow cytometry after filtration. Thus, each sample from mechanically isolated SVF and enzymatically isolated SVF was digested for 45 min at 37°C on a shaker using a solution with 10 ml α-MEM (32,561, Life Technologies), 100 UI/mL, and 100 μg/ml penicillin/streptomycin (15140122, Life Technologies), 200 UI/mL, 100 UI/mL, and 100 μg/ml penicillin/streptomycin (15140122, Life Technologies), 200 UI/mL of type IV CLS-4 collagenase (rLS00189, Worthington), 1.6 UI/mL dispase (LS02104, Worthington), 10 UI/ml DNase (Pulmozyme, ref 10139247, Roche, Basel, Switzerland), and 5 mM MgCl, (magnesium chloride, ref AM95306, Ambion, Thermo Fisher Scientific, Massachusetts, United States). At the end of the digestion process, all samples were washed with PBS and filtered at 100 µm, as before, and the pellet was transferred in a PBS and 10 U/ml of DNase solution, with a volume of 1 ml per gram of freshly harvested tissue.

The TIL expansion procedure was performed as previously published (Nguyen et al., 2019 (link)). Briefly, melanoma tissue was processed by cutting into ~1 mm³ fragments. Tissue fragments were either plated directly into 24-well plates or enzymatically dissociated in IMDM containing collagenase (Sigma) and Pulmozyme (Roche) and then plated in 24-well plates. Cells were cultured in complete medium (as previously described) and 6,000 IU/mL IL-2 and expanded for approximately 4 weeks prior to cryopreservation.
For the REP, TILs were thawed, rested, and seeded in T175 flasks with 30 ng/mL OKT3 (Miltenyi Biotec), irradiated (50 Gy) allogeneic PBMC feeder cells (1:200 TIL:PBMC), and 600 IU/mL IL-2 in ‘50/50’ media containing 50% complete medium prepared using human serum AB⁺ (Gemini Bio Products) and 50% AIM V media (Gibco). TILs were harvested on day 14 of the REP and cryopreserved before analysis.

Aliquots of recombinant DNase1L2 and rhDNase (Pulmozyme; Roche, Basel, Switzerland) were buffer exchanged into 20 mM Tris-HCl pH7.5 with 5 mM EDTA, using PD SpinTrap G-25 columns (GE Healthcare, Chicago, IL, USA) to eliminate divalent cations.
Purified DNase1L2 (14 μM) and rhDNase (14 μM) in PBS with 1 mM CaCl₂ or in 20 mM Tris-HCl pH7.5 with 5 mM EDTA were incubated with 0 or 100 mM βME at room temperature for 30 min. After that, samples were incubated in the dark with 0 or 5 mM iodoacetamide (IAM) for 30 min. The reaction mixtures were then buffer exchanged into 20 mM Tris-HCl pH7.5, using PD SpinTrap G-25 columns (GE Healthcare, Chicago, IL, USA). Protein concentrations were determined by absorbance at 280 nm (ε₂₈₀ = 32,890 M⁻¹·cm⁻¹ for DNase1L2; ε₂₈₀ = 46,090 M⁻¹·cm⁻¹ for rhDNase), and DNase activity was assayed in a standard reaction mix, containing 0.5 units of enzyme, 20 ng/μL of plasmid DNA, 3 mM CaCl₂, and 3 mM MgCl₂.

This study was approved by the local authority LANUV (Landesamt für Natur, Umwelt und Verbraucherschutz NRW; AZ84-02.04.2014.A103) and carried out in accordance with German and European guidelines of laboratory animal care. 24 male Wistar rats weighing between 510 and 525 g (Janvier Breeding Center, France) were randomly assigned to three experimental groups: one control group undergoing CPB with DHCA (group 1), one group undergoing CPB with DHCA that received an i.v. bolus of DNase I (Pulmozyme®, Roche, 5 mg/kg body weight) before CPB (group 2), and one group undergoing CPB with DHCA that received DNase I before CPB and a second bolus after rewarming/before reperfusion, respectively (group 3). This DNase I dose was previously found to effectively degrade plasma cfDNA/NETs^{18 (link),36 (link)}. Two animals with low haemoglobin concentration during the procedure (Hb < 5.0 mg/dL) caused by a diffuse bleeding at the insertion site of the arterial cannula developed a catecholamine-refractory hypotension and were subsequently excluded from the study. The study was conducted with 7 animals in groups 1 and 2 respectively, and 8 animals in group 3.