What can I do to improve the efficiency of isolation of urine-derived stem cell?
What are the factors that might impact the success rate of urine-derived stem cell isolation and culture?
Here is my protocol:
1. Approximately 100–200 mL urines were collected from the donors into a sterile container supplemented with 5ml Antibiotics-Antimycotics (sometimes urine samples were saved at 4 ℃ for up to 3 hours before they were transported to the lab)
2. Fresh urine samples were then centrifuged at 400 g for 10 minutes at room temperature. The supernatant was carefully discarded, leaving pellets and approximately 1 ml or less of urine in the tube.
3. Pellets were resuspended with phosphate-buffered saline (PBS). The samples were centrifuged at 400 g for 10 minutes. The supernatant was discarded, leaving only around 0.2 ml of sample.
4. Around 12 ml of primary medium was added to resuspend the cell pellet. The recipe for the primary medium contained DMEM and Renal epithelial cell growth medium at a mixture of 1:1, with SingleQuot kit supplement, 10 % of fetal bovine serum, ampothericinB and penicillin/streptomycin, Recombinant human fbroblast growth factor-basic, Recombinant human platelet-derived growth factor-AB, Recombinant human epidermal growth factor, Non-essential amino acid solution (NEAA) and GlutaMAX . The cells were then transferred onto 12 well plates coated with L-gelatine in 1 ml of primary medium.
5. The first full media change was made after the first cells/colonies were seen (within 7 days). Cells were then split onto a bigger surface aided by 0.25% trypsin when the culture grew confluent.
Sometimes i could get colonies, but the success rate was rather low.
Hi June,
My thoughts (may not be correct) are:
- Paradoxically, under certain conditions it may be better for cell viability to transport them at room temp rather than 4C, please see
Article The analysis of viability for mammalian cells treated at dif...
- When we did isolation of epithelial cells from cows milk we used 1 liter to isolate cells for a 6 well plate - perhaps you could get more urine?
- I would perform an isolation and try different culture media to see if any perform better. e.g
Article Isolation, Culture and Comprehensive Characterization of Bio...
Article Improved Isolation and Culture of Urine-Derived Stem Cells (...
(seems similar to yours), https://www.stemcell.com/reprogramming-human-urine-derived-cells-to-induced-pluripotent-stem-cells-using-an-episomal-vector-system-in-tesr-e7-or-reprotesr.html, https://www.nature.com/articles/s41598-020-57723-2
Also, what concentration of antibiotic/antimycotic are you using in step 1?
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