Mem neaa 100x

MOPC315.BM cells [20 (link)] was kindly provided by Prof. Bjarne Bogen (University of Oslo, Norway). They were cultured at 37 °C in 5% CO₂ in RPMI 1640 (Sigma-Aldrich, Rehovot, Israel) supplemented with 10% FBS, 1% MEM NEAA 100x (Gibco), 0.005% 1 M I-thioglycerol, 0.03% Gensumycin 40 mg/ml (Sigma-Aldrich) and 2 mM L-glutamine (Biological Industries, Beit Haemek, Israel). I.v. injection of MOPC315.BM cells results in tumor development in the bone marrow (BM) and spleen and is associated with osteolytic lesions, validating the model as resembling human MM disease [21 (link)]. In advanced disease stages (within 3–4 weeks), the mice develop paraplegia through spinal cord compression. They were sacrificed when presenting signs of paraplegia, deterioration of general condition or apathy.

HeLa cells (Homo sapiens cervix adenocarcinoma, CCL-2 ATCC) were cultured at 37°C with 5% CO₂ in growth culture medium for cell propagation. HeLa growth medium consisted of Minimal Essential Medium with Earle’s salts (MEM; GIBCO, Life Technologies, Carlsbad, CA, United States) supplemented with 4 mM GlutaMAX-I (GIBCO), 1% MEM Non-Essential Amino Acids (MEM NEAA; 100x, GIBCO) and 10% fetal calf serum (FCS; BioConcept, Allschwil, Switzerland). Medium used for infections contained the same components as growth medium, but was not supplemented with fetal calf serum. Cells were seeded directly into wells or on round glass coverslips (13 mm diameter, Thermo Fisher Scientific, Waltham, MA, United States) in 24-well plates [Techno Plastic Products AG (TPP), Trasadingen, Switzerland]. Infection experiments were performed 24 h post-seeding or 59 h post-seeding (Figure 1).

Vero 76 cells (African green monkey kidney cells, CRL 1587 American Type Culture Collection (ATCC), Manassas, VA, USA) and HeLa cells (Homo sapiens cervix adenocarcinoma, CCL-2 ATCC) were cultured at 37°C with 5% CO₂ in growth culture medium for cell propagation. Vero growth medium consisted of Minimal Essential Medium (MEM) with Earle's salts, 25 mM HEPES, without L-Glutamine (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, BioConcept, Allschwil, Switzerland), 4 mM GlutaMAX-I (200 mM, GIBCO) and 0.2 mg/ml gentamycin (50 mg/ml, GIBCO). HeLa cell culture media were further supplemented with 1% MEM Non-Essential Amino Acids (MEM NEAA, 100x, GIBCO). Medium used for cell propagation intended for infection experiments was without gentamycin [13] (link). Cells were seeded on round glass coverslips (13 mm diameter, Sterilin Limited (Thermo Fisher Scientific), Cambridge, UK) in 24-well plates (Techno Plastic Products AG (TPP), Trasadingen, Switzerland) at a density of 3×10⁵/well in 1 mL medium for infection experiments. Infection experiments were performed when cells reached at least 90% confluency.

Macroscopically tumor-free liver tissue samples were obtained from patients undergoing liver surgery at Leipzig University Hospital. All patients gave their informed consent according to the ethical guidelines of the Medical Faculty of Leipzig University (Ethical vote: registration number 322/17-ek, date 2020/06/10 ratified on 2021/11/30 and registration number 006/17-ek, date 2017/03/21 ratified on 2019/02/12). PHHs were isolated from the liver tissue samples by a two-step EGTA/collagenase perfusion technique as described previously (Pfeiffer et al., 2015[31 (link)]). The resulting cell pellet was washed with phosphate buffered saline (PBS; Gibco, Paisley, UK) and the cells were resuspended in PHH culture medium (William's Medium E with GlutaMAX™ (WME; Gibco), supplemented with 10 % fetal calf serum (FCS; Merck Biochrom, Berlin, Germany), 15 mM HEPES, 1 % nonessential amino acids (MEM NEAA 100x), 1 mM sodium pyruvate, 100 U/100 μM penicillin/streptomycin (all provided by Gibco), 40 U/ml insulin (Sanofi Aventis, Frankfurt am Main, Germany) and 1 μg/ml dexamethasone (JENAPHARM, Jena, Germany)). Afterwards, the cell number and viability were determined in a Neubauer counting chamber by Trypan blue staining (Sigma-Aldrich, St. Louis, MO, USA).

The hepatocyte attachment and culture medium was based on William's Medium E with GlutaMAX™ (WME, Gibco, Paisley, UK), supplemented with 10 % fetal bovine serum (FBS, Merck Biochrom, Berlin, Germany), 15 mM HEPES, 0.1 mM non-essential amino acids (MEM NEAA 100x), 1 mM sodium pyruvate, 100 U/100 µM penicillin/streptomycin (all provided by Gibco, Paisley, UK), 80 IU/l human insulin (Lilly Deutschland GmbH, Bad Homburg, Germany) and 1 µg/ml dexamethasone (Fortecortin^®, Merck, Darmstadt, Germany). Phosphate-buffered saline solution supplemented with calcium and magnesium (PBS) was purchased from Gibco and Trypan Blue was provided by Biochrom (Berlin, Germany). All other chemicals were purchased from Sigma-Aldrich (Munich, Germany), if not stated otherwise. Rat tail collagen was prepared in our laboratory according to the protocol established by Rajan et al. (2006[31 (link)]).

hAECs were isolated according to the method of Miki et al.^{46 (link)}. Briefly, the amnion was mechanically peeled from the underlying chorion and washed four times with cold HBSS supplemented with 100 U/ml penicillin, 100 mg/ml Streptomycin, and 0.25 mg/ml amphotericin B. To isolate hAECs, the amnion was incubated with 0.05% trypsin/EDTA for 40 min at 37 °C with gentle shaking. Dispersed cells were collected by centrifugation at 500 g and after four washes were plated at a density 20 × 10⁴ cells/cm² in DMEM/F-12 medium (ThermoFisher Scientific) supplemented 2 mmol/l L-Glutamin, 100 U/ml Penicillin, and 0.1 mg/ml Streptomycin (1% (v/v) of a L-Glutamin-Penicillin-Streptomycin stock solution from Sigma-Aldrich), 1 mmol/l sodium pyruvate (Sigma-Aldrich), 1% (v/v) MEM NEAA 100X (ThermoFisher Scientific), 0.1% fungin (InvivoGen, San Diego, CA), 10% FBS, 0.05 mmol/l 2-mercaptoethanol (ThermoFisher Scientific), 10 ng/ml human recombinant epidermal growth factor (EGF; Sigma-Aldrich) and cultured at 37 °C, 5% CO₂ in a humidified atmosphere for 48–72 h to form a confluent monolayer. The culture medium was changed three times a week. hAECs were harvested at 80% confluence by mild trypsinization and were either used fresh or cryopreserved in 90% FBS and 10% DMSO for later use. To generate spheroids hAECs were thawed and cultured for 5 days.