Abstract
Human mesenchymal stem cells (hMSCs) present an attractive target for cell therapy given their wide availability, immunomodulatory properties, and multipotent nature for differentiation into chondrocytes, osteocytes, and adipocytes. With the progression of hMSC clinical studies, there is an increasing demand for development of technologies that enable efficient cell scale-up into clinically relevant quantities. Commercial scale manufacturing of hMSCs will require a large surface area which is not cost effective with available two-dimensional culture vessels. Recent studies showed that microcarriers provide a three-dimensional culture environment suitable for hMSC expansion. Traditionally, biological coatings and/or serum-containing medium are required to facilitate hMSC attachment and expansion in dynamic conditions. These limitations may hinder the use of microcarriers as a scale-up technology for hMSC therapeutics, where cell products, and therefore patient safety, are more controlled with the use of xeno-free, defined culture conditions. Here we report the long term culture of hMSCs on novel synthetic Synthemax II microcarriers in two different xeno-free media. Cells were maintained over 40 days on sterile, ready-to-use microcarriers in spinner flasks with programmed agitation. hMSC expansion was obtained by addition of fresh beads without the need for enzymatic dissociation. We achieved a cumulative cell expansion of >10,000 fold, and cells retained normal hMSC phenotype, karyotype, and tri-lineage differentiation potential. To our knowledge, this report is the first example of long term culture of hMSCs on synthetic microcarriers in xeno-free, defined conditions.
Highlights
Human mesenchymal stem cells are multipotent adult stem cells able to differentiate to adipogenic, osteogenic or chondrogenic lineages [1]; the properties of hMSCs make them attractive cell therapy agents
We observed 80–90% of cells attached to the microcarriers after 24 hours in both Mesencult XF and stemgro hMSC media, and exponential growth was seen from days 2–5
In this study we report a method for large scale expansion of human mesenchymal stem cells on synthetic microcarriers in defined, xeno-free culture medium
Summary
Human mesenchymal stem cells (hMSCs) are multipotent adult stem cells able to differentiate to adipogenic, osteogenic or chondrogenic lineages [1]; the properties of hMSCs make them attractive cell therapy agents. Commercially-available microcarriers include two common classes: rigid particles made of glass or plastic (polystyrene) and soft, swellable particles (gelatin, alginate, or dextran) Both types can be functionalized chemically or coated with extracellular matrix (ECM) proteins to further promote cell adhesion. Healthcare) and gelatin-coated CultispherS (Percell) microcarriers are most commonly used with success for hMSC production in serum-containing media [11,12] [13] These studies define optimized culture conditions for large-scale hMSC production, they require biological coatings and serum-containing medium to facilitate cell attachment and expansion in stirred conditions. The microcarriers require time-consuming and labor intensive preparation (e.g. pre-swelling in water or buffer, autoclave sanitization) prior to cell seeding These limitations hinder the use of microcarriers for hMSC therapeutics, where cell products are more reproducible with the use of defined culture conditions [14] [15]. We demonstrate that cells present normal karyotype after long term culture on Synthemax II microcarriers
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