Human BMMSCs were purchased from Lonza (Walkersville, MD, USA) and cultured in Mesenchymal Stem Cell Growth Medium (MSCGM) (Lonza). The cells were re-plated at optimal initial cell density, cultured, and harvested with 0.25% Trypsin/EDTA solution (Mediatech Manassas, Herndon, VA, USA) every 14 days. Bi-lineage (osteogenic and adipogenic) differentiation abilities were tested by hMSC Osteogenic BulletKit (Lonza), and hMSC Adipogenic BulletKit (Lonza), respectively. Human ACL cells were prepared from cadaveric knees provided by tissue banks. Minced ACL tissues were digested with 5 mg/ml collagenase V (SIGMA-ALDRICH, St. Louis, MO, USA), and filtered by 70 μm mesh. Cells were then collected, cultured in MSCGM, and passaged every 14 days. Passage 2 (P2) cells were used in the experiments.
Hmsc osteogenic bulletkit
Manufactured by Lonza
The HMSC Osteogenic BulletKit is a laboratory tool designed for the isolation and culture of human mesenchymal stem cells (hMSCs). The kit provides a standardized and optimized system for the differentiation of hMSCs into osteoblasts, the cells responsible for bone formation.
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Lab products found in correlation
Hmsc adipogenic bulletkit, by Lonza (4 mentions)
Collagenase 5, by Merck Group (1 mentions)
Mscgm, by Lonza (1 mentions)
Oil red solution, by Merck Group (1 mentions)
Alizarin red staining, by Merck Group (1 mentions)
Alcian blue staining, by Merck Group (1 mentions)
Tgf β3, by Lonza (1 mentions)
Hmsc chondro bulletkit, by Lonza (1 mentions)
Alizarin red s, by Nacalai Tesque (1 mentions)
Oil red o, by Muto Pure Chemicals (1 mentions)
Recombinant human basic fibroblast growth factor, by Thermo Fisher Scientific (1 mentions)
5 protocols using hmsc osteogenic bulletkit
1
MSC and ACL Cell Isolation Protocol
2
Multilineage Differentiation of Mesenchymal Stem Cells
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Twenty‐four hours after NP loading, hAMSC and hCV‐MSC were counted and differentiated toward chondrogenic, adipogenic and osteogenic lineages. For chondrogenic differentiation, 2.5 × 105 cells were grown for 2–3 weeks in tubes with hMSC Chondrogenic bulletkit (Lonza) supplemented with 10 ng/ml TGFβ3 (Lonza). The differentiation was detected with Alcian Blue staining (Sigma‐Aldrich, Italy). For adipogenic differentiation, cells were plated at 5 × 103/cm2 and when 80% confluency was reached, differentiation was induced using hMSC Adipogenic bulletkit (Lonza). Three weeks after the start of differentiation, the cytoplasmatic lipid vacuoles were stained with Oil red solution (Sigma‐Aldrich, Italy). For osteogenic differentiation, cells were plated at 5 × 103/cm2 and when 80% confluency was reached, differentiation was induced using hMSC Osteogenic bulletkit (Lonza). Calcium deposition was detected 3 weeks after the start of differentiation by Alizarin red staining (Sigma‐Aldrich, Italy). CHANG medium C by itself and cells without NPs were used as controls.
3
Multipotency Validation of iPSC-Derived Neural Crest Cells
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To validate whether iPSC-NCCs maintain multipotency, iPSC-NCCs were plated at a density of 5 × 105 cells in 35 mm dishes. Cells were fed every 2 days by completely replacing the medium with NCC induction medium (3–5 days). At 100% confluence, we performed adipogenic differentiation by treating the iPSC-NCCs with hMSC Adipogenic BulletKit (Lonza, Basel, Switzerland; catalog #PT-3004) for 3 weeks according to the manufacturer's protocol, followed by staining with oil red O (Muto Pure Chemicals, Tokyo, Japan; catalog #40491) to detect lipids. A pellet culture system was used to evaluate the capacity of iPSC-NCCs to differentiate into chondrocytes. iPSC-NCCs were applied to a pellet at a density of 5 × 105 cells and cultured in hMSC Chondro BulletKit (Lonza; catalog #PT-3003). The medium was changed every 3–4 days. After 4 weeks in culture, the induced cartilage tissue was fixed with 4% formaldehyde, sliced, and stained with 1% Alcian Blue (Amresco, Solon, OH; catalog #0298). To investigate the osteogenic differentiation capacity of iPSC-NCCs, confluent iPSC-NCCs in 35 mm dishes were treated with hMSC Osteogenic BulletKit (Lonza; catalog #PT-3002). The medium was changed every 2–3 days. After 2–3 weeks, the cells were stained with Alizarin Red S (Nacalai Tesque, Kyoto, Japan; catalog #01303-52), rinsed with water, and examined by microscopy.
4
Adipogenic and Osteogenic Differentiation of MSCs
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For the adipogenic differentiation 1 × 105 MSCs were seeded on two each of cBSA-fibers, BSA-fibers, 1 mg/mL cBSA-coated and 1 mg/mL BSA-coated wells of a 24-well cell culture plate as well as on untreated cell culture plate. All samples were incubated in DMEM supplemented with 5% (v/v) PL and 2 U/mL heparin for 24 h at 37 °C 5°% CO2. Afterwards, the medium of one of each sample was replaced by adipogenic induction medium (hMSC Adipogenic BulletKit, Lonza, Basel, Swizerland). The cells were incubated in the induction medium for 3 days followed by 2–3 days of incubation in maintenance medium. Three cycles of induction/maintenance stimulated the adipogenic differentiation of the cells. The medium of the other samples (control samples) was replaced by adipogenic maintenance medium (hMSC Adipogenic BulletKit, Lonza) and incubated for 3 days. Afterwards, the medium was changed with fresh maintenance medium and incubated for 2–3 days. This cycle was repeated three times.
For the osteogenic differentiation, 1 × 104 MSCs were seeded as described above. The cells were cultured in DMEM supplemented with 5% (v/v) PL and 2 U/mL heparin at 37 °C, 5°% CO2. After 24 h the medium of one sample each was replaced with osteogenic medium (hMSC Osteogenic BulletKit, Lonza) and the other samples with DMEM supplemented with 5% (v/v) PL and 2 U/mL heparin. The medium was changed every 2–3 days.
For the osteogenic differentiation, 1 × 104 MSCs were seeded as described above. The cells were cultured in DMEM supplemented with 5% (v/v) PL and 2 U/mL heparin at 37 °C, 5°% CO2. After 24 h the medium of one sample each was replaced with osteogenic medium (hMSC Osteogenic BulletKit, Lonza) and the other samples with DMEM supplemented with 5% (v/v) PL and 2 U/mL heparin. The medium was changed every 2–3 days.
5
Optimizing hMSC Culture and Differentiation
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