Ready-to-use Eppendorf CCCadvanced? FN1 Motifs Surface for Xeno-Free Expansion
of Human Pluripotent Stem Cells
Aurélie Tacheny1, Silvia Tejerina1, Wiame Ben El Mostapha1, Nadine Mellies2, Fran?oise De Longueville1
1 Eppendorf Application Technologies S.A., Namur, Belgium
2 Eppendorf AG, Hamburg, Germany
Abstract
Since the groundbreaking discovery of human induced pluripotent stem
cells (hiPSCs) by Shinya Yamanaka in 2006, the use of pluripotent stem
cells (PSCs) has experienced a boom, thus representing an important tool
for biological research. The ultimate goal during the cultivation of
PSCs is the preservation of their pluripotency, routinely characterized
by their morphology, growth, pluripotency marker expression, as well as
by their differentiation potential. The selection of a defned culture
system consisting of a growth surface and culture medium is therefore
crucial. The commonly used biological coating materials for PSC
expansion, based on feeder layers or animal-derived protein mixtures,
present a non-defned growth surface, which poses an obstacle when aiming
at applications requiring high consistency.
The ready-to-use Eppendorf CCCadvanced? FN1 motifs surface o?ers a
comfortable alternative for the long-term cultivation of PSCs in a
feeder-free as well as animal- and human-component-free environment.
This novel surface is made up of fbronectin-derived motifs to support
cell attachment by mimicking native extracellular matrix proteins. This
fully synthetic surface allows expansion of PSCs in xeno-free and
restrictive culture conditions, aiming to provide a completely defned
culture system for PSCs without any animal or human components.
The FN1 motifs surface combines convenience with reliable PSC performance: the ready-to-use consumable signifcantly reduces labor time and e?ort for scientists while o?ering a fully synthetic culture system with a high level of consistency during long-term PSC expansion.
Introduction
Due to their extensive in vitro self-renewal properties and their
ability to di?erentiate into all three specialized germ layer cell
types, PSCs, and especially hiPSCs, o?er consid erable and exciting
promises in a wide range of cell appli cations [1, 2]. As a critical but
essential milestone to fully exploit the potential of these stem cells,
PSC expansion conditions have progressively moved from the traditional
mouse embryonic fbroblast (MEF) feeder layer-based cul ture system
towards more defned feeder-free cell culture systems [3, 4].
Nevertheless, most common feeder-free alternatives are still based on
the use of complex animal-derived protein mixtures presenting a
non-defned composition, variable lot-to-lot quality and purity as well
as a potential pathogen contamina-tion risk. Gold-standard biological
coating types, for example Corning? Matrigel?, are enriched with unde
fned growth factors and extracellular matrix (ECM) compo nents known to
sustain cell adhesion and pluripotency [5]. To ensure robust cell
performances in downstream applica tions, consistent and defned culture
conditions are crucial. In this context, fully synthetic,
animal-component-free culture systems are of great interest [6, 7, 8].
Based on a proprietary coating technology, the Eppendorf CCCadvanced FN1
motifs surface is made up of synthetic fbronectin-derived motifs
(including RGD), specifcally designed to mimic the cell attachment site
of native ECM proteins. Used in combination with a well-defned culture
medium and dissociation solution, this surface represents an e?ective
animal- and human-component free alternative, not only to the
conventional feeder layer-based culture system, but also to other
culture systems that depend on biological coating. Being ready-to-use,
it constitutes a real improvement for stem cell researchers,
signifcantly reduc ing labor time and e?ort while o?ering better
lot-to-lot consistency and more reliable performance in comparison with
self-coating solutions. The FN1 motifs surface is highly suitable for
the long-term expansion of undi?erentiated hiPSCs. Throughout at least
20 successive passages, hiPSCs maintain their typical cell morphology,
stable doubling time and karyotype as well as pluripotent marker
expression, when cultured on this surface. The in vitro trilineage
di?eren tiation capacity provides defnitive proof of the preservation of
the functional pluripotency of these cells.