MSCs were collected and cultured as previously described (14 (link)). hUC tissues were obtained immediately after full-term births following cesarean section with informed consent. The hUCs (CTI-195; Cell2in, Seoul, Korea) were washed with PBS to remove blood vessels and amnion. Wharton’s jelly tissues within the hUC were isolated and minced. These explants were incubated with an enzyme mixture at 37°C for 3 h (Miltenyi Biotec, Bergisch Gladbach, Germany), filtered through a 100 μM cell strainer (BD Biosciences, Franklin Lakes, NJ, USA), and pelleted by low-speed centrifugation at 200 ×g for 10 min. The isolated WJ-MSCs were cultured in CellCor™ CD medium (Xcell Therapeutics, Seoul, Korea) supplemented with 2% human platelet lysate (StemCell Technologies, Vancouver, BC, Canada) in a 37°C incubator. High-density MSCs were isolated from cultured MSCs using a fluorescent real-time thiol tracer (FreSHtracer, Cell2in) (15 (link)).
Enzyme mixture
Manufactured by Miltenyi Biotec
Sourced in Germany
The Enzyme mixture is a proprietary blend of enzymes designed for the digestion and disaggregation of various biological tissues. The core function of this product is to facilitate the release of cells from solid tissue samples, enabling their subsequent isolation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Human platelet lysate, by STEMCELL (1 mentions)
100 μm cell strainer, by BD (1 mentions)
Cd11c macs beads, by Miltenyi Biotec (1 mentions)
Gentlemacs dissociator and tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Gm csf, by Fujifilm (1 mentions)
Interleukin 4 (il 4), by Fujifilm (1 mentions)
Percoll, by Merck Group (1 mentions)
3 protocols using enzyme mixture
1
Isolation and Culture of hUC-MSCs
2
Isolation of Single-Cell Suspensions from CM Tissue
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Fresh CM tissue was dissected into small fragments, and subjected to a single‐cold phosphate‐buffered saline (PBS) wash. The tissue fragments were then transferred to a 15 mL centrifuge tube with a 10 mL enzyme mixture (130‐095‐929, Miltenyi Biotec) prepared according to the manufacturer's instructions (9.35 mL DMEM, 50 μL Enzyme A, 200 μL Enzyme R, and 400 μL Enzyme H). Then, the tube was placed on a table concentrator for 30 min (37°C, 100×g). Single‐cell suspensions obtained underwent filtration through strainers of 100 and 40 μm to eliminate cellular debris and tissue. Afterward, the cell suspensions underwent centrifugation, with subsequent double washes at 500×g for 3 min at 4°C. The resulting pellets were then resuspended in PBS. Measurement of cell concentration and viability was conducted using a Countstar Rigel S3 cell counter (Alit Biotech, Shanghai).
3
Isolation and Preparation of Tumor-Infiltrating Regulatory T Cells
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For tumor-infiltrating Treg cell preparation, tumor tissues were harvested 3 weeks after tumor inoculation. Tumor tissues were cut into small pieces, incubated in 5% FBS RPMI-1640 in the presence of an enzyme mixture (Miltenyi Biotec) at 37℃ for 45 min, and digested by using a gentleMACS Dissociator and tumor dissociation kit (Miltenyi Biotec), according to the manufacturer’s instructions. Cells were filtered through 70 μm nylon mesh and subsequently centrifuged using different concentrations of Percoll (Sigma-Aldrich) to exclude tissue debris and were washed with staining medium.
BMDCs were generated as described previously (Nakahashi-Oda et al., 2016 (link)). Briefly, bone marrow cells were cultured in a 10 cm culture dish in complete RPMI-1640 containing 10 % FBS in the presence of 10 ng/ml GM-CSF (WAKO) and 10 ng/ml IL-4 (WAKO) for 7 days. BMDCs were enriched by using CD11c MACS Beads (Miltenyi Biotec) to remove dead cells generated during BMDC development.
BMDCs were generated as described previously (Nakahashi-Oda et al., 2016 (link)). Briefly, bone marrow cells were cultured in a 10 cm culture dish in complete RPMI-1640 containing 10 % FBS in the presence of 10 ng/ml GM-CSF (WAKO) and 10 ng/ml IL-4 (WAKO) for 7 days. BMDCs were enriched by using CD11c MACS Beads (Miltenyi Biotec) to remove dead cells generated during BMDC development.
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