Ying Nie, Patrick Walsh, Diana L Clarke, Jon Rowley and Thomas Fellner look at a new development in stem cells cultivation.
Prevailing human pluripotent stem cells (hPSC) cultivation practices continuously enrich the undifferentiated cell population through visual selection and mechanical transfer of small undifferentiated multi-cellular aggregates from hPSC colonies. This labour intensive method results in substantial post-detachment cell injury and death, ultimately affecting the rate of cell expansion. Several alternative methods have been proposed and include the use of one or more enzymes to dissociate adherent hESCs and hiPSCs to single cells. However, continuous cultivation using single cell passaging methods often promotes chromosomal aneuploidy in conjunction with aberrant gene expression.
The subculture and continued maintenance of hPSCs as colony aggregates maintains lateral intercellular contacts important in cell to cell communication. This is thought to reduce cell stress, minimise spontaneous cell differentiation and promote rapid attachment and survival of cells to newly supplied substrates and matrices.
Preserving this important element of conventional hPSC cultivation requires the development of a reagent that produces an equivalent colony aggregate size and the high post-dissociation viability and replating efficiency required for efficient pluripotent stem cell expansion.
Our search to improve and simplify conventional hPSC dissociation methods resulted in the formulation of a scalable, hypertonic solution of sodium citrate, a mild chelating agent. Citrate promotes cell dissociation by binding the divalent cations present in the aqueous extracellular environment and intercellular space between cells. This disrupts molecules involved in maintaining cell adhesion such as calcium-dependent cadherins and calcium- and magnesium-dependent integrins. Citrate solutions, formulated to high osmolalities, favour the production of multi-cellular colony aggregates. Dissociating cultures with 1mM citrate solutions shows the preservation of colony aggregate integrity and the suppression of small colony aggregates as osmolality increases toward 800 mOsmol/kg. This suggests greater versatility in solution formulation and process development without reducing the colony aggregates to single cells.
Using a hypertonic citrate solution for open, small-scale hPSC dissociation, we were able to demonstrate multiple advantages over traditional enzymatic methods.
These advantages include:
- Higher post-detachment viability (97 per cent+/-1 per cent)
- Greater numbers of cells at each passage
- More uniform colony aggregate sizes
- Supporting the continuous generation of pluripotent cells for over 25 cell passages
In 2013 Lonza launched L13 hPSC Passaging Solution, a chemically-defined, non-enzymatic cell detachment formulation based on a hypertonic sodium citrate solution. L13 hPSC Passaging Solution gently dislodges multi-cellular colony fragments from a substrate without need for mechanical manipulation of the cultures. This user friendly method simplifies routine maintenance of your valuable hESC and iPSC lines.
L13 hPSC Passaging Solution is an essential component of Lonza's defined, Reprogramming and Culture System that will be launched in 2014.
For more information at www.scientistlive.com/eurolab
Ying Nie, Patrick Walsh, Diana L Clarke, Jon Rowley and Thomas Fellner are with Lonza Walkersville Inc, Walkersville, MD, USA. www.lonza.com/L13
