Hbmsc

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Human Bone Marrow-Derived Mesenchymal Stem Cells (HBMSCs) are primary adherent cells isolated from human bone marrow. They exhibit a fibroblast-like morphology and have the capacity to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes.

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21 protocols using hbmsc

Human Bone Marrow Stem Cell Culture

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The hBMSCs (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in a 37 °C incubator with 5% CO₂ with full relative humidity. For in vitro experiments, hBMSC incubation was performed with proliferation medium (PM) encompassing minimum essential medium α (α-MEM, Gibco, Carlsbad, CA, USA), 10% (v/v) fetal bovine serum (FBS, ScienCell Research Laboratories), and 100 IU/mL antibiotics (Gibco).
As for osteogenic induction, hBMSC culture was conducted with osteogenic medium (OM) encompassing standard PM, 10 mmol/L β-glycerophosphate, 0.2 mmol/L ascorbic acid, and 100 nmol/L dexamethasone. All other materials were bought from Sigma-Aldrich (St Louis, MO, USA) unless stated otherwise.

Li Y., Wang J., Ma Y., Du W., Feng H., Feng K., Li G, & Wang S. (2020). MicroRNA-15b shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles binds to WWP1 and promotes osteogenic differentiation. Arthritis Research & Therapy, 22, 269.

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Expansion of Human Bone Marrow Stromal Cells

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hBMSCs were purchased from Lonza (donor: 33 Y, male) at Passage 2. These cells were positive for CD44, CD105, and CD90, negative for CD45, CD19, and HLA-DR, and express multipotent capabilities (COA, Lot#0000684888). hBMSCs were plated at 3000 cells/cm² in growth media containing High-Glucose (4.5 g/L) Dulbecco’s Modified Eagle’s Medium (HG-DMEM) (Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA), 1% penicillin streptomycin (PS) (Gibco, NY, USA), and 5 ng/mL basic fibroblast growth factor (bFGF) (PeproTech, NJ, USA) and incubated in a humidified environment (37 °C, 5% CO₂). Media was changed at day 1 and day 5, and cells were cultured until 90% confluent, approximately 7 days. Cells were passaged using 0.05% Trypsin–EDTA (Gibco) and the cell suspensions were counted using a hemocytometer. Cells were expanded and banked at Passage 3 and 4 and used for experimentation at Passage 5.

Thorp H., Kim K., Kondo M., Grainger D.W, & Okano T. (2020). Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets. Scientific Reports, 10, 20869.

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Osteogenic Differentiation of hBMSCs on Peptide-Grafted Surfaces

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Commercially available hBMSCs (Lonza, France) were grown on gelatin coated culture flasks in MSCs growth medium (MSCGM) (Lonza, France), subcultures using trypsin/EDTA 1x (Sigmaaldrich, France) and maintained in a humidified atmosphere containing 5 % CO2 at 37 °C. Modified glass substrate of 1x1 cm² were sterilized with 70 % ethanol and rinsed by PBS, then placed in cell culture plates 24 well. To induce osteogenic differentiation, hBMSCs at passage 4 were seeded on different peptides grafted surfaces at a density of 104 cells/cm2 for 6 h in serum free α-MEM medium (Life technology, France). This allows the interactions between grafted peptides and their cell surface receptors without hassle of serum proteins. Serum-free medium was then removed and replaced with α-MEM medium containing 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin. The culture media was changed twice per week during four weeks of cell culture.

Bilem I., Chevallier P., Plawinski L., Sone E.D., Durrieu M.C, & Laroche G. (2016). RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells. Acta biomaterialia, 36.

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Culturing Human Bone Marrow Stem Cells

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Approximately 5 × 10⁵ HBMSCs/mL (ThermoFisher Scientific, Pittsburgh, PA) were cultured in T75 vented cell culture flasks using AdvanceStem Mesenchymal Stem Cell Medium (GE Healthcare Hyclone, Logan, UT) with 10% mesenchymal stem cell growth supplement (GE Healthcare, Malborough, MA) and 1% penicillin and streptomycin (ThermoFisher Scientific) for growth and expansion. Cells were grown in a standard cell culture incubator operating with 5% CO₂ at 37°C with 95% humidity. HBMSCs culture expanded to passages 4–6 were utilized for subsequent studies.

Castellanos G., Nasim S., Almora D.M., Rath S, & Ramaswamy S. (2018). Stem Cell Cytoskeletal Responses to Pulsatile Flow in Heart Valve Tissue Engineering Studies. Frontiers in Cardiovascular Medicine, 5, 58.

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Culturing Human Bone Marrow Stromal Cells

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hBMSCs (cat. no. BNCC338194) were obtained from BeNa Culture Collection. hBMSCs were cultured in DMEM (Thermo Fisher Scientific, Inc.) with 100 mg/ml streptomycin and 100 U/ml penicillin (Wuhan Boster Biological Technology, Ltd.) and 10% FBS (Sigma-Aldrich; Merck KGaA). The cells were grown in an incubator at 37˚C and 5% CO₂. The medium was replaced every 3 days. The cells were digested using 0.05% EDTA and 0.25% trypsin (Sigma Aldrich; Merck KGaA) and passaged when they filled ~80% of the culture flask. hBMSCs at passage three were used in the present study.

Huang Y., Liao L., Su H., Chen X., Jiang T., Liu J, & Hou Q. (2021). Psoralen accelerates osteogenic differentiation of human bone marrow mesenchymal stem cells by activating the TGF-β/Smad3 pathway. Experimental and Therapeutic Medicine, 22(3), 940.

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Dopamine-Mediated Cell Adhesion on Biomaterials

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Before cell culture, the samples (scaffolds or Si thin films) are pretreated with dopamine solution [dopamine hydrochloride of 2 mg/ml in 10 mM tris-HCl buffer solution (pH 8.5)] for 30 min at room temperature, creating hydrophilic surfaces (fig. S4) for improved cell adhesion. The scaffolds or Si thin-film samples are placed into well plates, with suspensions of hBMSCs (#7500, ScienCell, Carlsbad, CA, USA) or MC3T3-E1 cells dropped onto sample surfaces. Cells are incubated in cell culture media [high-glucose Dulbecco’s modified Eagle’s medium (DMEM) for hBMSCs and minimum essential medium α (MEMα) for MC3T3-E1 cells], supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO₂. Cell morphologies are characterized by an SEM (Zeiss Merlin) and a fluorescence confocal microscope (LSM710, Zeiss) [green: phalloidin staining actin cytoskeleton; blue: nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI)]. Fluorescence images are analyzed with the ImageJ software.

Wang H., Tian J., Jiang Y., Liu S., Zheng J., Li N., Wang G., Dong F., Chen J., Xie Y., Huang Y., Cai X., Wang X., Xiong W., Qi H., Yin L., Wang Y, & Sheng X. (2023). A 3D biomimetic optoelectronic scaffold repairs cranial defects. Science Advances, 9(7), eabq7750.

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Multipotent hBMSC Culture and Differentiation

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hBMSCs were purchased from Lonza (catalog no.: PT-2501) and grown in MesenPro RS media (Thermo Fisher Scientific; catalog no.: 12746012). Accutase (Thermo Fisher Scientific; catalog no.: A110501) was used as a cell dissociation reagent, and hBMSCs were frozen in supplement-free MesenPro RS media containing 10% dimethyl sulfoxide and 4% human albumin for storage. To induce adipogenic differentiation, cells were grown in adipogenic differentiation medium (Thermo Fisher Scientific; catalog no.: A10410-01). To induce osteoblastic differentiation, cells were grown in complete MesenPro RS media supplemented with 10 mM β-glycerol phosphate (Sigma; catalog no.: 50020), 50 μg/ml ascorbic acid (Sigma; catalog no.: A4403), and 100 nM dexamethasone (Sigma; catalog no.: D4902) or in osteogenic media purchased from Lonza (catalog no.: PT-3924).

Pham T., Najy A.J, & Kim H.R. (2022). E3 ligase HUWE1 promotes PDGF D-mediated osteoblastic differentiation of mesenchymal stem cells by effecting polyubiquitination of β-PDGFR. The Journal of Biological Chemistry, 298(6), 101981.

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Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

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Human bone marrow mesenchymal stem cells (hBMSCs) (HUXMA-01001) were purchased from Cyagen Biosciences (Soochow, China). In the standard culture medium (CM) group, the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 100 U/mL penicillin, 10% fetal bovine serum (FBS, Gibco), and 100 μg/mL streptomycin. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂. For osteogenic induction, hBMSCs were cultured in osteogenic medium (OM), which consisted of Alpha Modified Eagle's Medium (α-MEM) containing 10% FBS, 1% penicillin–streptomycin, 50 ng/mL ascorbic acid, 10 mmol/mL β-glycerophosphate, and 4 ng/mL dexamethasone (Thermo Fisher, USA) [22 (link)]. The medium was changed every 3 days, and the cells were cultured for 14 days for an observation of osteogenic differentiation by alkaline phosphatase (ALP) staining and Alizarin Red staining.

Yu Y.F., Yao P.Q., Wang Z.K, & Xie W.W. (2023). MiR-137 promotes TLR4/NF-κB pathway activity through targeting KDM4A, inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells and aggravates osteoporosis. Journal of Orthopaedic Surgery and Research, 18, 444.

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Osteoclast and Osteoblast Cell Culture

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The hBMSCs were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The hBMSCs were maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG; Gibco BRL, Rockville, MD, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (P/S, Gibco). The BMMs were isolated from C57BL/6 mice (8-week-old) by flushing the bone marrow of the tibiae and femurs. The BMMs were cultured in α-MEM (Gibco) containing 10% FBS, 1% P/S, and 30 ng/mL M-CSF (416-ML/CF, R&D Systems, Minneapolis, MN, USA) for 3 days. Subsequently, adherent cells were used as the osteoclast precursor cells^{40 (link)}. The RAW 264.7 cells were obtained from Korean Cell Line Bank (Seoul, Korea) and were cultured with α-MEM (Gibco) containing 10% FBS and 1% P/S. MC3T3-E1 (mouse pre-osteoblasts) cells were purchased from ATCC and maintained in alpha-modified minimum essential medium (Alpha-MEM, Gibco) supplemented with 10% FBS (Gibco) and 1% PS (Gibco). 3′-SL was provided by GeneChem Inc. (Daejeon, Korea).

Baek A., Baek D., Cho Y., Jo S., Kim J., Hong Y., Cho S., Kim S.H, & Cho S.R. (2024). 3′-Sialyllactose alleviates bone loss by regulating bone homeostasis. Communications Biology, 7, 110.

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Isolation and Culture of hASCs and hBMSCs

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Primary hASCs and human bone marrow mesenchymal stem cells (hBMSCs) were obtained from ScienCell Company. Cells were cultured at 37°C in an incubator with 5% CO₂ atmosphere and full relative humidity. To minimize the exogenous exosomes, hASCs were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) free of exosomes through ultracentrifugation at 100 000 g overnight with an Optima L‐90K Ultracentrifuge (Beckman Coulter, Inc). For the in vitro experiments, hBMSCs were cultured in proliferation medium (PM), which consisted of minimum essential medium α (α‐MEM, Gibco), 10% (v/v) foetal bovine serum (FBS, ScienCell) and 100 IU/mL antibiotics (Gibco). For osteogenic induction, hBMSCs were cultured in osteogenic medium (OM), which consisted of standard PM supplemented with 10 mmol/L β‐glycerophosphate, 0.2 mmol/L L‐ascorbic acid and 100 nmol/L dexamethasone. All other materials were purchased from Sigma‐Aldrich unless otherwise mentioned, and all experiments conducted with hBMSCs were extracted from three donors (Catalog#15901, #6881, #6890).

Chen S., Tang Y., Liu Y., Zhang P., Lv L., Zhang X., Jia L, & Zhou Y. (2019). Exosomes derived from miR‐375‐overexpressing human adipose mesenchymal stem cells promote bone regeneration. Cell Proliferation, 52(5), e12669.

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