- No feeding required Saturdays or Sundays – Work-free weekends!
- No strict passaging schedule
- The cells maintain high pluripotency expression and proliferation rate
- Easy, efficient and reliable single cell passaging enables standardized experiments
- Defined and animal component-free long-term propagation of hPSC
- Protocol validated with a low bFGF concentration medium
Figure 2. Weekend-free cultured cells maintain characteristic, pluripotent cell morphology and protein expression
Expression of pluripotent stem cell marker OCT-4 (red staining) was high after 6 passages for both the human ES cells (HS181 and HS980) and iPS cells (C3) maintained in Nutristem using standard or weekend-free feeding protocols. DAPI staining (blue) was used as control. The cells also consitently maintained undifferentiated cell morphology (bright-field pictures).
LN-521 facilitates long-term self-renewal of human pluripotent stem cells without weekend feeding
Most established human pluripotent stem cells (hPSC) culture protocols require daily medium replacement, demanding researchers to routinely work weekends. Based on the biorelevant support from LN-521, we developed a protocol that eliminates weekend feeding. The protocol is flexible - just feed your cells daily during weekdays and perform a simple LN-521 single cell passage on Thursday or Friday. Splitting frequency will be cell line dependent, therefor simply adjust the seeding density, optimal for your cell line and work schedule. The only important guideline is that the cells must be passaged late in the week (e.g. Thursday or Friday). Your hPSC cultures will maintain characteristic cell morphology, high pluripotent cell marker expression and high expansion rates.
We validated the weekend-free feeding protocol (feeding only weekdays) for support of long-term maintenance of human ES and iPS cells by com- paring it to standard, every day feeding protocol (feeding 7 days/week). In both cases, cells were routinely passaged as single cells twice/week but always with a passage at the end of the week (Thursday or Friday). More information about the easy and reliable LN-521 single-cell passage protocol here. Experiments were performed using two hESC lines (HS181 and HS980) and one iPS cell line (C3), maintained in Nutristem XF medium on LN-521 stem cell matrix for 6 passages. Both culturing protocols were performed in parallel with 4 independent wells (n=4) for each cell line and protocol.
Compared to the standard protocol, all three cell lines cultured with the weekend-free culture protocol maintained the same high, long-term expansion rate for 6 passages (Figure 1). Independent of culture protocol, all three cell lines grew as a homogenous monolayer with characteristic hES and iPS cell morphology with prominent nucleoli and a high nuclear to cytoplasmic ratio (Figure 2, bright-field pictures). The cells also maintained the features of undifferentiated human ES and iPS cells as illustrated by high expression of the pluripotent stem cell marker OCT-4 (Figure 2, red staining).
Figure 1. Cell expansion rates are just as high under weekend-free feeding condition
Human ES cells (HS181 and HS980) or iPS cells (C3) were maintained in Nutristem under standard, every day-feeding (filled lines) or weekend-free feeding conditions (dashed lines) for 6 passages. Fold expansion at each passage was determined by comparing the number of cells generated to the amount seeded.