Adipose tissues (AD) were sectioned from abdominal area by surgical doctor at Vinmec International Hospital and transferred to the laboratory. The tissues were minced and digested by 200 U/mL collagenase type I solution (Gibco, Massachusetts, USA) and 0.1% human albumin solution for 1 h at 37°C with shaking. Cells were pelleted by a centrifugation at 500 × g for 10 min and supernatant was discarded. Subsequently, pellets were resuspended in the MSC culture media (StemMACSTM MSC Expansion Media, Miltenyi Biotec, Bergisch Gladbach, Germany) and then centrifuged at 300 × g for 5 min to collect MSCs. The harvested MSCs then seeded into cell culture flask (Nunc, Thermo Scientific, Massachusetts, United States) coated with solution CTSTM CELLstartTM substrate (diluted in PBS at ratio 1: 300) (Gibco, Massachusetts, USA) with initial density of 3,000–5,000 cells/cm2 in the MSC culture media and cultured at 37°C and 5% CO2. The media were replaced by every 2 days of culture. When the cells reached 80% confluency, the cells were split by 0.05% trypsin for the next passage.
Ctstm cellstart substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CTSTM CELLstartTM substrate is a cell culture surface coating designed to promote cell attachment, proliferation, and differentiation. It provides a chemically defined, animal-component-free environment for culturing a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 1 solution, by Thermo Fisher Scientific (2 mentions)
Stemmacs msc expansion media, by Miltenyi Biotec (2 mentions)
T75 cell culture flask, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Mesencult acf plus medium, by STEMCELL (1 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ctstm tryple select enzyme, by Thermo Fisher Scientific (1 mentions)
5 protocols using ctstm cellstart substrate
1
Isolation and Expansion of Adipose-Derived MSCs
2
Isolation and Expansion of UCMSCs
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Primary umbilical cord-derived mesenchymal stem cells (UCMSCs) were supplied by the EV group (Vinmec Center for Applied Sciences and Regenerative Medicine). Cells were expanded to passage five in DMEM/F12 (Gibco, Waltham, MA, USA) supplemented with 10% FBS (Gibco, MA, USA) (v/v) in T75 cell culture flasks (Nunc, Thermo Scientific, Waltham, MA, USA) surface-coated with CTSTM CELLstartTM substrate (Gibco, MA, USA) under 5% CO2 and at 37 °C. Cells were split when they reached 80% confluency using CTSTM TrypLETM Select Enzyme (Life Technologies, New York, NY, USA).
At P5, UCMSCs were seeded in T75 flask (5000 cell/cm2) with EV-depleted culture media (DMEM/F12 supplemented with 10% EV-depleted FBS). EV-depleted FBS was prepared by centrifuging FBS at 120,000× g/18 h/4 °C to remove FBS EVs. The UCMSCs were designated as the control when UCMSCs were cultured in normal conditions, and as the cytokine treatment when UCMSCs were treated with 10 ng TGFβ/1 mL culture media for two days. After two days of incubation, UCMSCs from both groups reached 80% confluency, and the conditioned medium was collected for EV isolation.
At P5, UCMSCs were seeded in T75 flask (5000 cell/cm2) with EV-depleted culture media (DMEM/F12 supplemented with 10% EV-depleted FBS). EV-depleted FBS was prepared by centrifuging FBS at 120,000× g/18 h/4 °C to remove FBS EVs. The UCMSCs were designated as the control when UCMSCs were cultured in normal conditions, and as the cytokine treatment when UCMSCs were treated with 10 ng TGFβ/1 mL culture media for two days. After two days of incubation, UCMSCs from both groups reached 80% confluency, and the conditioned medium was collected for EV isolation.
3
Isolation and Expansion of Human Mesenchymal Stem Cells
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Bone marrow (BM) was aspirated from the bilateral posterior iliac crest under general anesthesia. A total of 30 mL of BM was collected using a 10 mL syringe with heparin sodium as anticoagulants. The mononuclear cells-enriched fraction was separated using the density gradient centrifugation on Ficoll-Paque Premium (GE Healthcare Life Sciences, Pennsylvania, USA). After Ficoll separation, the mononuclear cells were plated into T75 cell culture flasks (Nunc, Thermo Scientific, Massachusetts, United States) coated with solution CTSTM CELLstartTM substrate (diluted in PBS at ratio 1: 300) (Gibco, Massachusetts, USA) with the density 22.5 × 106 cells/flask. Cells were expanded in the MesenCultTM ACF Plus Medium (StemCell Technologies, Vancouver, Canada) at 5% CO2 and 37°C. After 48 h, media containing non-adherence cells was replaced with new media for cell growth. When the cells reached 80% confluency, the cells were split by 0.05% trypsin for the next passage.
4
Isolation and Culture of Umbilical Cord MSCs
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Umbilical cords (UC) were collected from pregnant women after baby delivery at the surgery of Vinmec International Hospital. The UCs were washed three times with ethanol 70% and then 3–5 times with PBS to sterilize and remove blood. The UC was cut into small pieces and transferred into a 50 mL conical tube following by an incubation with 500 U collagenase Type I solution (Gibco, Massachusetts, USA) for 1 h at 37°C with shaking. After digestion, solution was diluted in chilled PBS and centrifuge at 1,400 × g/10 min at 4°C to collect cell pellet. The cell pellet was resuspended in a MSC culture media (StemMACSTM MSC Expansion Media, Miltenyi Biotec, Bergisch Gladbach, Germany) and transferred into cell culture flasks (Nunc, Thermo Scientific, Massachusetts, United States) coated with solution CTSTM CELLstartTM substrate (diluted in PBS at ratio 1: 300) (Gibco, Massachusetts, USA) and incubated at 37°C and 5% CO2 for cell expansion. With each 5 mg of UC, cell pellets were seed into 1 T25 cell culture flask. Culture media were replaced by every 2 days of culture. When the cells reached 80% confluency, the cells would be split by 0.05% trypsin for the next passage.
5
Expansion and EV Isolation from UCMSCs
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UCMSCs were received from the EV groups at passage two (P2). The cells were thawed in the water bath (37 o C) and seeded into T75, or T225 cell culture flasks (Nunc, Thermo Scientific, Massachusetts, United States) containing DMEM/F12 (Dulbecco's Modified Eagle's Medium, Gibco, USA) supplemented with 10% FBS (Foetal Bovine Serum, Gibco, USA) with a density of 375 × 10 3 cells/cm 2 as passage 3. The culture flask was surface-coated with CTS TM CELLstart TM substrate (Gibco, Massachusetts, USA) diluted in PBS at the rate of 1: 300 and washed twice with PBS before cell seeding. UCMSCs were then incubated at the condition of 5% CO 2 /37 o C for the expansion. When cells reach 80% confluency, they were split using CTS TM TrypLE TM Select Enzyme (Thermo Fisher Scientific, USA) for the next passage. At the culture of P5, UCMSCs were incubated to reach 80% confluency and released EVs into conditioned media (supernatant) that were harvested for further EV isolation. The UCMSCs were split for marker analysis.
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