Hyaluronidase

Using sterile technique, freshly harvested human VS tissue was rinsed in phosphate buffered saline (PBS) and dissected in culture medium consisting of Dulbecco’s Modified Eagle’s medium with Ham’s F12 mixture (DMEM/F12), 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% GlutaMAX. VS tissue was then dissociated in hyaluronidase and collagenase (all Life Technologies, Carlsbad, CA, USA) overnight, and cultured for 2–4 weeks, as described previously32 (link). Informed consent was obtained from all patients. The study protocols were approved by the Human Studies Committee of Massachusetts General Hospital and Massachusetts Eye and Ear Infirmary, and conducted in accordance with the Helsinki Declaration.
In addition to a primary human VS cell culture, two cell lines were used: 1) HEI-193 cells, an immortalized cell line derived from the vestibular schwannoma of a human NF2 patient, and 2) Nf2^−/− SC4 cells, a mouse Schwann cell line (both gifts from the House Ear Institute, Los Angeles, CA, USA). The cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

Dulbecco’s minimum essential medium Eagle (DMEM), penicillin, streptomycin and fungizone were purchased from Highveld Biological (Pty) Ltd. (Sandringham, SA). Sterile cell culture flasks and plates were obtained through Lasec SA (Pty) Ltd. (Honeydew, Johannesburg, South Africa). Gibco® collagenase and hyaluronidase were purchased from Life Technologies (Johannesburg, Gauteng, South Africa). A lactate dehydrogenase cytotoxicity Assay Kit II kit from BioVision Inc. (Mountain View, California, USA) was supplied by BIOCOM biotech (Pty) Ltd. (Clubview, South Africa). All other chemicals of analytical grade, as well as heat-inactivated fetal calf serum (FCS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Ported PCL particles were manufactured according to a previously published protocol (18). Non-ported PCL particles (i.e. PCL particles with no ports) were manufactured as per the published protocol with the omission of the port forming reagents. In a previously unpublished study in rats, we observed that the biotoxicity of PCL particles in vivo is low to non-existent, and where present is reflective of a foreign body response (Figs A-E in S1 File and Table A in S2 File, all raw data is in S3 File). PS particles (mean particle diameter of 150 μm) were obtained from Corpuscular Inc. (Cold Spring, New York, USA).

PDA tumor growth in KPC mice was monitored every month using small-animal ultrasound (Vevo770, VisualSonics, Toronto, Ontario, Canada). The tumor was harvested when reaching 10 mm in size, minced, and dissociated in pre-warmed digest medium [5% FBS RPMI 1640 with collagenase (1500 U/ml), and hyaluronidase (1000 U/ml); Life Technology, Carlsbad, CA, USA] and incubated at 37 °C for 1 h. Tumor cells after digestion were filtered through a cell strainer, centrifuged, and washed with cold PBS.
TAMs were sorted by the Flow Cytometer. The cell suspension was stained with a mixture of PE-conjugated anti-mouse F4/80 antibody (Biolegend), APC-conjugated anti-mouse CD3 antibody (Biolegend), PE-Cy™7 rat anti-mouse CD8a (BD Biosciences, San Jose, CA, USA), FITC-conjugated anti-CD4 antibody (Biolegend), and PI (BD Biosciences) for 30 min. CD3-F4/80⁺ live cells were selected for analysis.

The tissue obtained during the BC biopsy was cut into small pieces of 1 mm³ in volume and then placed in 1 mL of digestion medium for overnight incubation [21 ]. The digestion medium consisted of DMEM, antibiotic agents penicillin/streptomycin at a final concentration of 1%, 0.14 mg/mL of hyaluronidase (Thermo Fisher Scientific, France), and 1.6 mg/mL collagenase IV. After incubation, the suspension was transferred to the tube containing 2 mL PBS. The tissue slurry was centrifuged at room temperature at 700 g for 5 min. Afterward, the supernatant was removed, the pellet was resuspended in the fresh culture medium, and seeded on 3 wells of a 12-well plate.

All enzymes were purchased from Sigma-Aldrich, unless stated otherwise. Tissues were isolated in 5% FBS (Gibco) in RPMI-1640 (Gibco). Tissues were cut into small pieces and digested in the appropriate enzyme mixture at 37 °C in a water-bath shaker: Aorta:^{59 (link)} 450 U/ml collagenase I, 125 U/ml collagenase XI 60 U/ml hyaluronidase and 60 U/ml DNase I (ThermoFisher Scientific) for 50 min; Lung: 0.4 mg/ml collagenase IV and 0.15 mg/ml DNase I for 40 min; Heart: 1 mg/ml collagenase II and 0.15 mg/ml DNase I for 30 min. The cells were retrieved by passing tissue pieces through a 70 μm cell strainer (Greiner Bio-One). Spleen and lymph nodes were mechanically dissociated by passing cells through a 70 μm cell strainer. For separating aortic layers into the intima/media and the adventitia, the whole aorta was pre-incubated with 125 U/ml collagenase II and 3.75 U/ml elastase at 37 °C for 10 min. The adventitia layer was separated from the inner tube (the intima and media) by pulling with two pairs of fine forceps. The tissues were then digested as performed in the aorta sample. For lungs, hearts, and spleens, erythrocytes were lysed using Red Cell Lysis Buffer (Sigma-Aldrich) at room temperature for 2 min.