25 cm2 culture flask

The SIBC cell suspension was placed in 25 cm² culture flasks (Corning) for Cytogenetic examination. 5 μg/mL colcemid (Gibco‐Life Technology) was added into the SIBCs suspension for 2 hours to arrest cell mitosis. Cells were treated with 0.075 mol/L KCl and then fixated with 3:1 methanol:glacial acetic acid (Sinopharm Chemical Reagent). Cell solution was dropped onto a slide glass and air‐dried. Then, cells were treated with 0.005% trypsin for 7 minutes and stained with 6% Giemsa stain solution for 3.5 minutes. The results were processed with Applied Spectral Imaging. The number of chromosomes was analyzed in 30~50 cells, and a detailed karyotype was analyzed in 10~11 cells. The definitions were followed the International System for Human Cytogenetic Nomenclature (ISCN) 2013.

Madin‐Darby canine kidney 2 (MDCK‐2, ATCC^® CRL‐2936^™) grew in minimum essential medium (MEM) with 5% foetal calf serum (FCS) in 25‐cm² culture flasks (Corning, Nuembrecht, Germany). For isolation of the positive field samples, medium was replaced with infectious material and incubated for 1 hour at 37°C. After incubation, MEM with 6‐ (1‐tosylamido‐2‐phenyl) ethyl chloromethyl ketone (TPCK)‐treated trypsin was added. After 72 hours, supernatants of the cultures were passaged once after a freeze‐thaw‐step, regardless of a recognizable cytopathic effect (CPE).

Bone marrow aspirate (10–20 mL) was harvested from the proximal end of femur while inserting the reamer and tapering awl into the femoral canal during THA. The 10 ml bone marrow samples were diluted with 10ml phosphate-buffered saline (PBS; HyClone, United States) and added over 10 ml Percoll solution (Sigma, United States) with a density of 1.073 g/ml in a 50 mL conical tube. Mononuclear cells from the bone marrow were isolated by density gradient centrifugation (472 g for 30 min). Then, cells were harvested and resuspended in complete culture medium containing low-glucose complete Dulbecco’s modified Eagle’s medium (L-DMEM; HyClone, United States), 10% fetal bovine serum (FBS; Gibco, United States), and 1% penicillin-streptomycin (Invitrogen). These cells were seeded at a density of 5000 cells/cm² in 25-cm² culture flasks (Corning, United States) and incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide. After 1 day interval, the non-adherent cells were removed and fresh cultures were added every 3 days. When cells reached ≥ 80% confluence, they were digested using 0.25% trypsin/EDTA (HyClone, United States) and replated at a 1:2 dilution for initial subculture. The cells were expanded by successive subculture in vitro, and cells from the second or third passage were ready for the following experiments.

NP cells were isolated from 2 M rats as previously described [54 (link)]. Isolated NP cells were cultured at 37 °C and 5% O₂ and incubated with DMEM/F12 medium (Thermo Fisher, CA, USA) mixed with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Solarbio, Beijing, CN). After 14 days of culture, the NP cells were digested and reseeded in 25-cm² culture flasks (Corning, NY, USA) for further experiments.

BMSCs were isolated from the New Zealand white rabbits as described previously [12 (link)]. The isolated BMSCs were seeded in 25 cm² culture flasks (Corning, USA) and cultured in complete basal culture medium, which was composed of low glucose Dulbecco's modified Eagle medium (LG-DMEM) supplemented with 10% fetal bovine serum (FBS), 10 U/mL of penicillin, and 100 mg/mL of streptomycin (all from Gibco) at 37°C under 5% CO₂ atmosphere. Cells at passage two were used in this study.

The experiments were performed using human epidermoid carcinoma cells A431 (All-Russian Collection of Cell Cultures, Institute of Cytology of the Russian Academy of Sciences, Saint-Petersburg, Russia), human keratinocyte cells HaCaT and human adenocarcinoma ovarian cells SKOV-3.ip (provided from the cell collection of the Institute of Bioorganic Chemistry at the Russian Academy of Sciences, Moscow, Russia). The cells were cultured in 25 cm² culture flasks (Corning, Corning, NY, USA) at 37 °C under 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM, HyClone, Logan, UT, USA) containing 2 mM L-glutamine (PanEco, Moscow, Russia) and 10% fetal bovine serum (FBS, Thermo Fisher, Waltham, MA, USA). For passaging, the cells were carefully detached using a trypsin-versene solution (1:1) (PanEco, Moscow, Russia).

Because chain colonies break up upon pipetting and because some of the mutants formed chains with very large clusters of cells, we attempted to visualize the chain phenotypes while manipulating the cells as little as possible. Cells were diluted at a 1:10 ratio into 10 ml of HN media in 25 cm² culture flasks (Corning, NY). 24 hr later, we imaged the chain colonies at the bottom of the flask at 10× using a Leica DMIL LED inverted compound microscope with a Leica DFC 300FX camera. Images were manually false colored to highlight the chain colonies that were in focus.