可靠的CCCadvanced FN1無異源耗材支持人間充質干細胞擴增表達
Reliable and Robust Animal-Component-Free hMSC-BM Expansion on Ready-to-Use Eppendorf CCCadvanced? FN1 Motifs Surface
Aurélie Tacheny1, Wiame Ben El Mostapha1, Nadine Mellies2, Fran?oise De Longueville1
1 Eppendorf Application Technologies S.A., Namur, Belgium
2 Eppendorf AG, Hamburg, Germany
Abstract
In the last decade, human mesenchymal stem cells (hMSCs) have generated
increasing interest in the scientifc world. Many fetal and adult tissues
harbor potential multipotent MSCs, which hold a long-term in vitro
culturing capacity across several passages without losing their
essential characteristics. Prior to their use as a powerful tool for
research applications, hMSCs must be expanded in order to reach an
adequate number of cells without losing their homing ability,
multi-lineage potential, secretion of anti-in?ammatory molecules and
immunoregulatory e?ects. Experiments require stable and completely de
fned hMSC culture systems consisting of growth surface and culture
medium.
The novel Eppendorf CCCadvanced? FN1 motifs surface represents a
completely synthetic cell adhesion-pro moting growth surface for the
long-term cultivation of hMSCs in xeno-free and restrictive culture
conditions, providing a defned culture system without any animal and
human components. This ready-to-use surface is made up of
fbronectin-derived motifs to support cell attachment in various
serum-free media by mimicking native extra-cellular matrix proteins
without additional preparation of the surface.
With its unique properties, the FN1 motifs
surface combines convenience with reliable hMSC cultivation: the
ready-to-use consumable signifcantly reduces labor time and e?ort for
scientists while o?ering a fully synthetic growth surface with a high
level of consistency during long-term hMSC expansion.
Introduction
First described in the 1970s by Friedenstein as bone marrow-derived
fbroblast-like precursors, hMSCs, also known as human mesenchymal
stromal cells, consist of a heterogeneous population of multipotent
cells which can be easily isolated from various tissues, such as adult
bone marrow, adult adipose tissue, dental pulp, fetal and neonatal
tissues [1-4]. hMSC isolation and identifcation rely exclu sively on in
vitro expanded cell properties. According to the criteria defned by the
Mesenchymal and Tissue Stem Cell Committee of the International Society
for Cellular Therapy, hMSCs must present ex-vivo plastic-adherent growth
abili ties under standard culture conditions and must express a specifc
set of cell surface antigens such as CD73, CD90 and CD105, while
lacking expression of CD11b, CD19, CD34, CD45 and HLA-DR [5]. Moreover,
to conform to minimal hMSC defnition criteria, cells must also be able
to di?eren tiate in vitro into osteogenic, chondrogenic and adipogenic
lineages [6].
Besides their characteristic mesodermal di?er- entiation potential, it
has also been reported that these cells are able to transdi?erentiate
into additional non-mesodermal cell types including hepatocytes,
cardiomyocytes, neuron-like cells and pancreatic-like cells [7]. Very
interestingly, these cells present low immunogenicity and possess the
ability to secrete soluble bioactive factors that can modulate the im
mune system and in?ammation process and promote tissue repair [8, 9].
This unique combination of properties makes hMSCs a promising stem cell
population in various cell ap plications.
hMSC culture conditions
Present at relatively low abundance in their tissue of ori gin, hMSCs
require a robust in vitro cell culture expansion process in order to
reach sufcient numbers of high-quality cells. Traditionally, hMSC in
vitro expansion occurs in a serum-containing culture system. In the
presence of serum associated proteins, hMSC culture can be performed on
tis sue culture (TC) treated plastic vessels without any specifc
coating.
Nevertheless, the common use of animal-derived materi als, such as
serum, represents a non-defned composition, accompanied by variable
lot-to-lot quality and purity as well as potential contamination risk,
which could be problematic in a wide range of basic and applied research
applications [10, 11]. For the past several years, a growing section of
the academic and industrial scientifc community has been leaning
increasingly towards the use of well-defned, serum free, xeno-free (XF)
or animal-component-free (ACF) hMSC culture systems.
More consistent and defned culture conditions are also of great interest
to those working with hMSC cultures in con junction with
biopharmaceutical production, drug screen ing or disease modeling, as
these felds require robust cell performances with a high level of
consistency and repro ducibility. In the absence of serum proteins,
hMSCs expand ed in serum-free culture systems necessitate additional
cell adhesion-promoting coating on the culture surface. To ensure the
defned nature of the culture system, coatings of biologi cal origin
should be excluded.
Synthetic FN1 motifs surface
Based on a proprietary coating technology, the Eppendorf CCCadvanced FN1
motifs surface consists of synthetic fbronectin-derived motifs
(including RGD), specifcally designed to mimic the cell attachment site
of native extra cellular matrix (ECM) proteins. Used in combination with
synthetic culture medium and dissociation solution, this surface
represents an e?ective animal- and human-compo nent–free alternative to
other biological coating-dependent culture systems.
Being ready-to-use, it constitutes a real improvement for stem cell
researchers, signifcantly reducing labor time and e?ort while o?ering
lot-to-lot consistency and more reliable performance in comparison with
self-coating solutions. The FN1 motifs surface is suitable for the
long-term expansion of bone marrow-derived hMSCs (hMSC-BM), and it is
com patible with various commercial xeno-free media.
Cultured on this novel synthetic surface, hMSC-BM main tain their
characteristic cell morphology, as well as linear cumulative population
doubling, without the appearance of replicative senescence-associated
signs, across 10 succes sive passages. Following successive passages in a
completely defned animal-component-free culture system, cells which
were expanded on the FN1 motifs surface maintain a typical hMSC surface
antigen marker expression profle and the ability to di?erentiate in
vitro into osteoblasts, adipocytes and chondrocytes, representing the
three mesoderm lineages.