可靠的CCCadvanced FN1无异源耗材支持人间充质干细胞扩增表达

Reliable and Robust Animal-Component-Free hMSC-BM Expansion on Ready-to-Use Eppendorf CCCadvanced™ FN1 Motifs Surface

Aurélie Tacheny¹, Wiâme Ben El Mostapha¹, Nadine Mellies², Françoise 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-inflammatory molecules and immunoregulatory effects. 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 effort for scientists while offering 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 differen tiate in vitro into osteogenic, chondrogenic and adipogenic lineages [6].
Besides their characteristic mesodermal differ- entiation potential, it has also been reported that these cells are able to transdifferentiate 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 inflammation 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 effective 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 effort while offering 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 differentiate in vitro into osteoblasts, adipocytes and chondrocytes, representing the three mesoderm lineages.