MC3T3-E1 cells, established as an osteoblastic cell line, were provided from Sigma (99072810). Osteoblasts were grown in alpha-modified minimal essential medium (α-MEM; Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies), 1% L-glutamin (Life Technologies) and 1% penicillin/streptomycin (Life Technologies) in an incubator at 5% CO2 heated at 37°C. The medium was changed twice weekly, and the cells were subcultured into 75 cm2 culture flasks by detaching them gently after a brief PBS rinsing step followed by Trypsin treatment once the cells were reaching subconfluency. For mechanical stimulation, MC3T3-E1 cells were plated into Ibidi μ-slide device (μ-Slide I 0.4 Luer, ibiTreat from Ibidi) at a concentration of 1.5 × 104 cells/mL. After overnight culture, the medium was replaced by α-MEM (powder exempt of Bicarbonate, Life Technologies) 10% FBS 1% L-glutamin 1% penicillin/streptomycin 25 mM HEPES (Life Technologies) adjusted to pH 7.4 for 4 hours in the incubator without CO2 at 37°C.
Mc3t3 e1 cells
Manufactured by Merck Group
Sourced in United States
MC3T3-E1 cells are a well-established mouse pre-osteoblastic cell line derived from newborn mouse calvaria. They are commonly used as an in vitro model to study osteoblast differentiation and function.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Ascorbic acid, by Merck Group (4 mentions)
Mc3t3 e1 cell, by American Type Culture Collection (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
β glycerophosphate, by Merck Group (3 mentions)
α mem, by Thermo Fisher Scientific (3 mentions)
μ slide i0.4 luer, by Ibidi (1 mentions)
L glutamin, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Thermo Fisher Scientific (1 mentions)
Rhbmp 2, by Merck Group (1 mentions)
Opg fc, by Amgen (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Glycerophosphate, by Merck Group (1 mentions)
α mem, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Clark Bioscience (1 mentions)
9 protocols using mc3t3 e1 cells
1
Mechanical Stimulation of MC3T3-E1 Osteoblasts
2
Murine Pre-Osteoblastic Cells: PTH Stimulation
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Murine pre-osteoblastic MC3T3-E1 cells were purchased from American Type Culture Collection (ATCC) agency (Shanghai, China), and cultured in α-MEM (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin within a humidified incubator at 37°C containing 5% CO2. Synthesized human PTH (1–34) (Sigma USA) was dissolved in PBS (phosphate buffered saline) to a concentration of 1.21 × 10− 4 M (stock solution) and then dissolved to a final concentration of 10− 8 M, 10− 9 M and 10− 10 M.
MC3T3-E1 cells were trypsinized and plated at a density of 2 × 10−4 cells/well in 6-well plates and grown to 70% confluency for PTH stimulation. The cells were divided into three groups: the continuous, intermittent and control groups. The continuous group was treated with PTH at 10− 8 M, 10− 9 M and 10− 10 M for 24 h. The intermittent group was exposed to PTH of different concentrations for the first 6 h and then replaced with culture medium free from PTH during the remainder of the cycle. The control group was cultured with common medium supplemented with 10% FBS only. Culture media of all groups were changed every 24 h (one cycle). This experiment consisted of three cycles.
MC3T3-E1 cells were trypsinized and plated at a density of 2 × 10−4 cells/well in 6-well plates and grown to 70% confluency for PTH stimulation. The cells were divided into three groups: the continuous, intermittent and control groups. The continuous group was treated with PTH at 10− 8 M, 10− 9 M and 10− 10 M for 24 h. The intermittent group was exposed to PTH of different concentrations for the first 6 h and then replaced with culture medium free from PTH during the remainder of the cycle. The control group was cultured with common medium supplemented with 10% FBS only. Culture media of all groups were changed every 24 h (one cycle). This experiment consisted of three cycles.
3
Osteoblast Differentiation of MC3T3-E1 Cells
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Preosteoblastic MC3T3-E1 cells used in this study were purchased from American Type Culture Collection (Manassas, VA, USA). Culture solutions were created by adding 10% fetal bovine serum (Gibco, Grand Island, NY, USA) to 1% penicillin/streptomycin α-minimum essential medium (without ascorbic acid; Gibco). With replacement in 2 to 3 day intervals, the solution was cultivated at 37°C, in a 5% CO2 incubator.
To induce differentiation from MC3T3-E1 cells to osteoblasts, 50 μg/mL of ascorbic acid, and 10 mM of β-glycerophosphate (both from Sigma-Aldrich, St. Louis, MO, USA) were added to the culture solution. Moreover, 50 ng/mL of RhBMP-2 (Sigma-Aldrich) and 0~200 ng/mL of OPG-Fc (Amgen, Thousand Oaks, CA, USA) were added to the culture solution.
To induce differentiation from MC3T3-E1 cells to osteoblasts, 50 μg/mL of ascorbic acid, and 10 mM of β-glycerophosphate (both from Sigma-Aldrich, St. Louis, MO, USA) were added to the culture solution. Moreover, 50 ng/mL of RhBMP-2 (Sigma-Aldrich) and 0~200 ng/mL of OPG-Fc (Amgen, Thousand Oaks, CA, USA) were added to the culture solution.
4
Osteogenic Differentiation of MC3T3-E1 Cells and BMSCs
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MC3T3-E1 cells were obtained from the American Type Culture Collection (Manassas, VA, United States) and cultured in modified essential medium (α-MEM, Gibco, Life Technologies, Carlsbad, CA, United States) with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA, United States) and 1% (v/v) penicillin/streptomycin (Gibco, Life Technologies, Carlsbad, CA, United States). All cells were cultured in an incubator at 37°C with 5% CO2. Mouse BMSCs were isolated from 4- to 6-week-old male mice as previously described (Han et al., 2015 (link)). BMSCs used to investigate the long-period HFD were isolated from 18-week-old HFD and ND mice. BMSCs were maintained in Dulbecco’s modified Eagle medium (Gibco, Life Technologies, Carlsbad, CA, United States) with 10% FBS and 1% (v/v) penicillin/streptomycin. Osteogenic differentiation of MC3T3-E1 cells and BMSCs was achieved by using an osteogenic medium containing ascorbic acid (50 mg/ml; Sigma-Aldrich, St. Louis, MO, United States), 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, United States), and 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, United States) for 4 days.
5
Modulating Osteogenic Differentiation via miR-125b and TRAF6
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For in vitro studies we selected MC3T3-E1 cells which were bought from Sigma Aldrich USA. The cells were stored and maintained in MEM which was added to glutamine (4 mM), fetal bovine serum (10%) and penicillin (1%) at room temperature under humid condition with 5%CO2. The cells were added to ascorbic acid (50 mg/L) and β‐glycerophosphate (10 mM) to induce osteogenic differentiation. To inhibit or promote the expression of miR-125b, the cells received transfection of miR-125b mimic or inhibitor for 48 hours with the help of Lipofectamine transfection reagent (Life Techno). To induce the overexpression of TRAF6, to build the overexpressing pcDNA3.1-TRAF6 vector, the cDNA of TRAF6 was cloned in pcDNA3.1 (Invitrogen, USA). The MC3T3-E1 cells were transfected with siRNA-TRAF6 for knock-down expression levels of TRAF6.
6
Osteoblastic Differentiation of MC3T3-E1 Cells
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Osteoblastic murine MC3T3-E1cells (Sigma-Aldrich) were cultured for 7 days in culture medium consisting of α minimum essential medium, supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (all from ThermoFisher Scientific). The medium was refreshed every 2–3 days. Before cell seeding, the implants were cut to 1 cm length and sterilized at 110 °C for 1 h in an oven (Nabertherm GmbH, Lilienthal, Germany). Cell seeding was performed by inserting an implant in a 0.2 ml tube with 1.5 × 105 MC3T3-E1 cells in 100 μl culture medium. Subsequently, the implants were incubated at 37 °C and 5% CO2 in a horizontal position and tilted every 20 min for 2 h in total. After seeding, the implants were placed in a 48-well plate with 200 μl fresh medium. After 2 days of culturing, osteogenic differentiation was induced by the addition of 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate (all from Sigma-Aldrich). Thereafter, the medium was refreshed every 2–3 days. Two independent experiments were performed (each time in quadruplicates).
7
Osteoblast Cell Culture on PLA Scaffolds
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For biological tests, pre‐osteoblastic MC3T3‐E1 cells (Sigma–Aldrich) were utilized. Cells were grown in Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% of fetal bovine serum, 1% of glutamine, and 1% of streptomycin/penicillin at 37°C with 5% CO2. Pure PLA, PLA/AS 1%, PLA/AS 5%, PLA/CLO 1%, and A PLA/CLO 5% scaffolds were cut to square‐shaped samples, with a surface of about 1 cm2. The scaffolds were then sterilized under UV for 2 hr and soaked for 12 hr in a complete culture medium. Twenty microliters of cellular suspension were inoculated onto each scaffold in order to reach a seeding concentration of 5 × 104 cells/cm2. After 90 min of incubation at 37°C and 5% CO2 (to promote cell adhesion), each scaffold was transferred into a medium‐filled well of a 24 multiwell plate.
8
Osteogenic Differentiation of MC3T3-E1 Cells on Ti Disks
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To evaluate the cellular response on Ti disks, MC3T3-E1 cells (CRL-2593, ATCC, Manassas, VA, USA) were cultured in Alpha Minimum Essential Medium; α-MEM (Sigma Aldrich) supplemented with 2 mM Glutamine, 10% Fetal Bovine Serum (FBS), 100 mg/mL penicillin/ streptomycin, and 0.25 mg/mL amphotericin B (Sigma Aldrich). To analyze the differentiation of osteoblast, MC3T3-E1 cells were cultured in osteogenic medium consisted of 50 µg/mL ascorbic acid and 10 mM ß-glycerophosphate (Sigma Aldrich).
9
Osteogenic Differentiation of MC3T3-E1 Cells
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In this study, we used MC3T3-E1 cells (Zhongqiaoxinzhou Biotechnology, Shanghai, China), a physiologically relevant system for studying OBs (29) (link). The MC3T3-E1 cells were cultured and passaged in ␣-minimum essential medium (␣-MEM; Hyclone, Logan, UT) containing 10% fetal bovine serum (Clark Bioscience, Richmond, VA) and 1% penicillinstreptomycin (Hyclone) at 37°C in a 5% CO 2 humidified incubator (Fisher Scientific, Waltham, MA). To investigate osteogenic differentiation, MC3T3-E1 cells (2 ϫ 10 5 cells/well) were seeded in six-well plates containing 2 mL of ␣-MEM supplemented with 50 g/mL ascorbic acid-2-phosphate (Sigma-Aldrich, St. Louis, MO), 5 mM -sodium glycerophosphate (Sigma-Aldrich), and dexamethasone (Xinhua Pharmaceutical, Shandong, China). For morphologic experiments, MC3T3-E1 cells(2 ϫ 10 5 cells/well) were seeded on glass slides.
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