Fetal bovine serum (fbs)

Human NSCLC A549 cells were maintained in DMEM/F12 medium (Meilun, China) and mouse Lewis lung carcinoma cells (LLCs) were cultured in RPMI-1640 medium, which was obtained from Gibco (Shanghai, China). The culture medium was supplemented with 10% fetal bovine serum (FBS), which was purchased from Wisent Co., Ltd. (Nanjing, China), 100 U/mL penicillin and 100 µg/mL streptomycin, which were obtained from Macgene (Beijing, China). Cells were cultivated in a humidified incubator at 37 °C in 5% CO₂. C57BL/6 mice, weighing 16–20 g, were supplied by Vitonglihua Co., Ltd. (Beijing, China).

HEK293T cells were cultured in DMEM medium (Wisent Inc., Saint-Jean-Baptiste, QC, Canada) supplemented with 10% FBS (Wisent Inc., Saint-Jean-Baptiste, QC, Canada), and 1% penicillin–streptomycin (Wisent Inc., Saint-Jean-Baptiste, QC, Canada). Cells were kept at 37 °C with 5% CO₂ in a humidified incubator.
The human myoblasts were cultured in a homemade medium [43 (link),44 (link)] made of the following components: DMEM medium, 20% FBS (Wisent Inc.), 16% medium 199 (Invitrogen™ Inc., Carlsbad, CA, USA), 1% penicillin–streptomycin (Wisent Inc.), Fetuin 25 μg/mL (Life Technologies, Carlsbad, CA, USA), hEGF 5 ng/mL (Life Technologies), bFGF 0.5 ng/mL (Life Technologies), insulin 5 μg/mL (Sigma-Aldrich Canada Inc., Oakville, ON, Canada, 91077C-1G), and Dex 0.2 μg/mL (Sigma-Aldrich Canada Inc.).

HepG2, Hep3B, Huh7, LM3, Hep1-6, and MHCC-97H cell lines (Chinese Academy of Sciences, Shanghai, China) were tested for mycoplasma negativity. These cells were cultured in high-glucose DMEM (Wisent, Canada) containing 1% streptomycin (Beyotime, Shanghai, China), 1% penicillin (Beyotime), and 10% fetal bovine serum (Wisent). Cells were cultured at 37 °C with 5% CO₂. The medium was replaced as needed according to cell growth.

HeLa cells (human epithelial cervix, adenocarcinoma, American Type Culture Collection [ATCC] CCL-2) and bEnd.3 cells (mouse brain endothelial cells, ATCC CRL-2299) were grown in high-glucose Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal bovine serum (Wisent, Inc). SH-SY5Y cells (ATCC CRL-2266) were grown in Eagle’s minimum essential media (ATTC 30-2003) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. All cells were maintained in a 5% CO₂ incubator at 37 °C.

Myometrial biopsies (N = 4) were collected in 50 mL falcon tubes containing 25 mL of ice-cold HBSS with Ca²⁺ and Mg²⁺ (HBSS+/+) supplemented with 2.5% HEPES (Wisent, Montreal, QC, Canada) and 1% Penicillin/Streptomycin (Pen/Strep, Lonza, Basel, Switzerland). Protocol for isolation of myometrial smooth muscle cells is described in Srikhajon et al. [9 (link)]. Briefly, myometrial tissues were washed in HBSS+/+ to remove blood, cut into small pieces (approximately 1 mm³) using small scissors, and blood vessels were carefully removed. Next, tissues were washed twice in HBSS without Ca²⁺ and Mg²⁺ (HBSS−/−), supplemented with 2.5% HEPES (Wisent, Montreal, QC, Canada) and 1% Pen/Strep (Lonza, Basel, BS, Switzerland), transferred to enzymatic digestion solution containing HBSS−/−, with 10% fetal bovine serum (FBS) (Wisent, Montreal, QC, Canada), 1 mg/mL collagenase 1A (Sigma-Aldrich, Oakville, ON, Canada), 1 mg/mL bovine serum albumin (BSA) (Wisent, Montreal, QC, Canada), 0.15 mg/mL DNase 1 (Sigma-Aldrich, Oakville, ON, Canada), and 0.1 mg/mL Trypsin inhibitor (Sigma-Aldrich, Oakville, ON, Canada), and placed in the rocking water bath at 37 °C. Following 1 h incubation, the digested myometrial tissue was pipetted 30 times and passed through a 70-micron filter to collect a single cell suspension in ice-cold dissociation solution (HBSS−/−, with 10% FBS (Wisent, Montreal, QC, Canada) and 1 mg/mL BSA (Wisent, Montreal, QC, Canada) to stop the enzymatic reaction. Undigested tissue was transferred to fresh enzymatic digestion solution for 1 h in the rocking water bath at 37 °C. Two cell suspensions were combined after the second round of digestion, centrifuged at 200× g for 10 min, washed with Dulbecco Modified Media (DMEM, Invitrogen, Carlsbad, CA, USA) containing 20% FBS (Wisent, Montreal, QC, Canada) + 1% Pen/Strep (Lonza, Basel, Switzerland), and passed through a 23 G ¾ gauge needle. The cells were seeded in a 10 cm tissue culture plate (Eppendorf, Mississauga, ON, Canada) or Flexcell plates (Flexcell International Corp., Burlington, NC, USA). The cells were grown in an incubator at 37 °C in 20% oxygen.