64 BIOTECHNOLOGY
hiPSCs to single cells. However, continuous cultivation using single cell passaging methods often promotes chromosomal aneuploidy in conjunction with aberrant gene expression.
Te subculture and continued maintenance of hPSCs as colony aggregates maintains lateral intercellular contacts important in cell to cell communication. Tis 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. Tis 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. Tis 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.
Tese advantages include:
n Higher post- detachment viability (97 per cent ±1 per cent)
n Greater numbers of cells at each passage
n More uniform colony aggregate sizes
n 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. Tis 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
Next generation sequencing bioinformatics heats up
The decreasing cost of genome sequencing lends momentum to the global next-generation sequencing (NGS) informatics market. As affordability drives adoption of genome sequencing in multiple end- user segments, an enormous output of NGS data to store and analyse is an overwhelming result. While primary and secondary data analysis tools are likely to become a commodity as pipelines standardize, the high-value tertiary data analysis segment used for biological interpretation and clinical reporting will drive revenue growth. New analysis from Frost & Sullivan’s Global Next Generation Sequencing
www.scientistlive.com
Informatics Market finds the market earned revenue of $170 million in 2012 and estimates this to reach $580 million in 2018 at a compound annual growth rate (CAGR )of 22.7 per cent. The research covers commercial
primary and secondary data analysis tools, storage, computing, commercial biological interpretation and reporting tools, NGS informatics services, and laboratory information management systems across North America, Europe, Asia, and rest of world. “With sequencing data production
forecast to grow at a CAGR of more than 75 percent between 2012 and 2018, researchers will need efficient NGS
informatics solutions to manage, analyse and interpret this escalating amount of data,” said Frost & Sullivan Life Sciences Senior Industry Analyst Christi Bird. “As the number of applications for NGS continues to grow, the implementation of NGS informatics will go up.” However, customers employ NGS for
a broad range of applications, and there is no ideal product for NGS informatics. Therefore, customers may struggle to identify the appropriate tools for their needs. The wide variety of free public
domain and commercial tools provided by the 100 plus competitors in this market has also led to low barriers to
entry, ever-intensifying competition, and commoditisation in certain product segments. Moreover, given the array of public
and open source software available along with readily accessible internal bioinformatics resources, NGS users are often hesitant to make additional investments in commercial informatics tools. Therefore, beyond the challenges of commoditisation and standardisation, suppliers must convince NGS users their commercial tools are better than free ones.
For more information, visit
www.lifesciences.frost.com
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