Dextran

PBMCs were isolated using combined dextran–ficoll isolation as described previously [34 (link),35 (link)]. In brief, blood was diluted with Dulbecco’s phosphate-buffered saline (DPBS, Gibco™, ThermoFisher Scientific, Waltham, MA, USA) containing 2 mM ethylenediaminetetraacetic acid (EDTA, Carl Roth, Karlsruhe, Germany) 1:1, mixed with 3% dextran (Carl Roth) in a ratio of 1:0.4, and centrifuged at 50× g for 20 min. Thereafter, the upper phase was collected, layered onto an equal volume of Histopaque^®-1077 (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged at 900× g for 30 min. The PBMCs at the interphase were collected, washed twice with DPBS/EDTA in a new 50 mL tube, and centrifuged at 400× g for 10 min. Isolated PBMCs were adjusted to 5×10⁶ cells/mL living cells using Tali™ image-based cytometer (ThermoFisher Scientific) with propidium iodide (PI, ThermoFisher Scientific) as a viability marker. PBMCs were cultured in RPMI 1640 medium (Gibco™, ThermoFisher Scientific) with 10% chicken serum (Gibco™, ThermoFisher Scientific) and 1% penicillin (10,000 U/mL)-streptomycin (10,000 µg/mL) (Pen/Strep, Gibco™, ThermoFisher Scientific) at 41 °C with 5% CO₂. The next day, further experiments were performed.

PMNs were isolated from whole blood of healthy malaria-naïve individuals.⁴⁸ To this end, whole-blood from three malaria-naive healthy donors (18 ml each) was pooled, mixed 1:1 with 3% dextran (Carl Roth) in 0.9% NaCl, and incubated for 18 min at room temperature to pellet red blood cell. After centrifugation (500 × g for 10 min at 4 °C), the thin white layer was collected and resuspended in 0.9% NaCl. This suspension was layered on top of Ficoll-Histopaque (Sigma-Aldrich) and centrifuged at 400×g for 35 min at room temperature. The thin PMN layer was resuspended in ice-cold distilled water and kept on ice for 30 s to lyse remaining erythrocytes before an equal volume of 1.8% NaCl was added. After centrifugation (500 × g for 5 min at 4 °C), the pellet was washed with Hanks’ balanced salt solution (Thermo Fisher Scientific) and resuspended in cold PBS. The quality of the preparation and the number of PMN were determined in a hemacytometer after trypan blue staining. The PMN concentration was adjusted to 2.5 × 10⁷ PMNs ml⁻¹ in sterile PBS.

Different CDFM were prepared for cryopreservation of the adapted HuH-7 cells. The composition of each formula is shown in Figure S3. PVP (PVP360, Sigma, Steinheim, Germany), Dextran (92191, Roth), Pluronic F68™ (A1288, Applichem, Darmstadt, Germany) work as cryoprotectants. As control formulations, 90% FBS with 10% DMSO (D2650, Sigma, Steinheim, Germany) and 90% CDM with 10% DMSO were used. HuH-7 cells were cultured until they reached a confluence of 70–80%. Subsequently, cells were harvested using TrypLE and quantified before being centrifuged at 300× g for 3 min. The resulting cell pellets were then resuspended in ice-cold freezing media at a density of 1.5 × 10⁶ cells/mL. Next, 1 mL of the cell suspension was transferred to a Simport cryo-vial (E309, Roth). The NALGENETM cryo freezing container controlled a gradual cooling process at a rate of −1 °C/min until reaching −80 °C. Following a 24-h period at −80 °C, the samples were relocated to a liquid nitrogen tank for an additional 6 days before thawing. For retrieving cells from liquid nitrogen, the cryovials were incubated at 37 °C for 1–2 min in a water bath. Subsequently, the cells were resuspended and centrifuged at 300× g for 3 min. The cell pellets were resuspended in CDM and cultured in collagen-precoated T25 cell culture flasks.

Ferrous sulfate heptahydrate (FeSO₄·7H₂O), ferric chloride hexahydrate (FeCl₃·6H₂O), ammonium hydroxide 25% (NH₄OH), ethanol, and curcumin were purchased from Sigma-Aldrich Merck (Darmstadt, Germany). Dextran (M~500,000 g/mol) and phosphate buffer saline (PBS) were purchased from Carl Roth (Karlsruhe, Baden-Württemberg, Germany). All chemicals were of analytical purity and used with no further purification.
The antimicrobial assays were performed using three microbial strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Candida albicans ATCC 10231) obtained from the collection of the microbiology laboratory within the Faculty of Biology, University of Bucharest.
HT-29 human epithelial cells (colon adenocarcinoma, well-differentiated) (ATCC, Merck, Romania) were selected as the model for cytotoxicity assessments.

Bundling
of DNA filaments
was induced by addition of molecular crowders like dextran (molecular
weight: 6, 35, and 500 kDa, Carl Roth), polyethylene glycol (PEG,
molecular weight 8 kDa, Carl Roth), or methylcellulose (molecular
weight: 500 kDa, Carl Roth). The bundling agent was mixed with DNA
filaments directly before GUV formation. For transmission electron
microscopy and confocal imaging of DNA bundles in bulk, DNA filaments
were incubated for 5 min with the respective bundling agent.

The compositions of the different perfusate solutions are displayed in Table 1. Steen Solution™ and Perfadex™ were purchased from XVIVO Perfusion (Gothenburg, Sweden). Custodiol-N was acquired from Dr. F. Köhler Chemie (Bensheim, Germany). Pyrogen-free dextran 40 (AppliChem, Darmstadt, Germany) was added to the modified Custodiol-N solution in a concentration of 50 g/L. The D-EVLP with Custodiol-N solution supplied with dextran + albumin (D-EVLP/CDA) solution was additionally supplemented with 7 g/L of bovine serum albumin (Carl Roth, Karlsruhe, Germany). The sterilization process of the solution was performed by filtration using a 0.22 µm filter (Filtropur BT25, Sarstedt, Nümbrecht, Germany). Directly before use, a 10 ml of 10% glucose solution (G-10, B. Braun, Melsungen, Germany) was added to 1 L of the CD and CDA solutions.