Smooth muscle cell growth medium 2

Human umbilical vein endothelial cells (HUVECs) and human umbilical arterial smooth muscle cells (HUASMCs) were purchased from PromoCell, Germany. HUVECs were maintained in Endothelial Cell Growth Medium 2 (PromoCell, Germany). HUASMCs were maintained in Smooth Muscle Cell Growth Medium 2 (PromoCell, Germany). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C. Cell passages 3–6 (HUVECs) and 4–5 (HUASMCs) were used for experiments.

Primary Human Aortic Smooth Muscle Cells (HAoSMCs; male, Caucasian; PromoCell, Heidelberg, Germany) were cultivated in Smooth Muscle Cell Growth Medium 2 (PromoCell, Heidelberg, Germany) containing 5% FCS, Epidermal Growth Factor (0.5 ng/ml), Basic Fibroblast Growth Factor (2 ng/ml) and Insulin (5 µg/ml) at 37 °C in a humidified atmosphere with 5% CO₂. HAoSMCs at passages 2–11 were used in experiments after reaching 70–80% confluence. Cell number was determined with a Casy® cell sorter and analyzer (Innovatis, Reutlingen, Germany). 50.000 cells/cm² were seeded for experiments and ~ 20.000 cells/cm² for cultivating growth on 10 cm petri dishes.
Prior to the experiments, HAoSMCs were synchronized by incubation in supplement- and serum free Dulbecco's modified eagle's medium (DMEM) medium (5.5 mM glucose, 24 mM NaHCO₃, 25 mM HEPES) for 24 h. Subsequently, cells were treated in DMEM medium for 48 h under the following conditions representing an acidotic priming: (i) control (pH 7.4); (ii) hydrochloric acid (HCl, pH 6.8); (iii) lactic acid (LA, pH 6.8, 24 mmol/l) and the respective controls (iv) mannitol (pH 7.4, 24 mmol/l) (hyperosmolar control) and (v) Na⁺-Lactate (pH 7.4, anion of lactic acid, 24 mmol/l).

Autologous UCs and SMCs were isolated as described previously [37 ]. Briefly, the urothelium and detrusor muscle were dissected from the bladder biopsy and the cells mechanically dissociated. After gentle agitation (1 h at 37 °C) in MEM with 400 µg/ml collagenase (Liberase^®, Roche Applied Sciences, Penzberg, Germany) the cell suspension was filtered through a stainless steel mesh (1.0 mm² pore size), washed three times with MEM containing 10% (v/v) fetal calf serum (FCS, Thermo Scientific) and transferred in collagen-coated (Biochrom AG, Berlin, Germany) cell culture flasks (Nunclon™, Thermo Scientific). The UCs were cultured in Keratinocyte-SFM (Life Technologies) and SMCs in a selection medium described elsewhere in a humidified incubator with 5% CO₂ at 37 °C [32 (link)]. The selection medium was used for 3 days to inhibit the growth of fibroblasts in the SMC population. For the subsequent cultivation, SMCs were treated with smooth muscle cell growth medium 2 (Promo Cell GmbH, Heidelberg, Germany). The cultured cells were regularly monitored via a Leica DMI 4000B (Leica Microsystems GmbH, Wetzlar, Germany) and integrated software (Diskus 4.80.5909, Hilgers, Technisches Büro, Königswinter, Germany).

All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Primary human saphenous vein–derived endothelial cells (HSVECs) were isolated by a modified version of the protocol described by Jaffe and colleagues^{18 (link)} and maintained in large-vessel endothelial cell culture medium supplemented with 20% fetal calf serum (Life Technologies, Paisley, UK). Primary human saphenous vein–derived smooth muscle cells (HSVSMCs) were isolated from medial explants^{19 (link)} and maintained in Smooth Muscle Cell Growth Medium 2 (PromoCell, Heidelberg, Germany) with supplements. Human coronary artery VSMCs were purchased from Lonza (Basel, Switzerland) and maintained in VSMC media as above.

Bronchial smooth muscle cells (BSMCs) from healthy subjects (Lonza, Basel, Switzerland) were cultured in Smooth Muscle Cell Growth Medium 2 (PromoCell, Heidelberg, Germany). Medium was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). All cultures were maintained at 37°C in an atmosphere containing 5% CO₂.

Human myometrial tissue samples from the lower uterine segment were obtained from nonlaboring patients at >37 weeks’ gestation during Cesarean section under spinal anesthesia. Human subjects research was performed in accordance with the principles stated in the Declaration of Helsinki. Participants signed consent forms approved by the Washington University in St Louis Internal Review Board (protocol no. 201108143). Tissues were cut into small pieces and incubated with 1 mg/ml collagenase IA and collagenase XI (Millipore Sigma) for 45 to 60 m at 37 °C with rotation. The collagenase-treated mixture was then passed through a 70 μm cell strainer, and collagenase was neutralized with 10% FBS. Cells were centrifuged, resuspended, and plated in DMEM/Ham’s F12 media without phenol red, supplemented with 5% Smooth Muscle Cell Growth Medium 2 (PromoCell). Cells were used at passage 0, 1, or 2.