Abstract
IntroductionFacing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags.MethodsWe cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs.ResultsWhen cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing β-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling.ConclusionsHere, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine.
Highlights
Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies
Afc-FEP bags 2PF-0002 (Afc)-FEP cell culture bags are suitable for three-dimensionalcultivation of human inferior turbinate stem cells (ITSCs) In order to investigate the potential suitability for ITSCexpansion, Afc-FEP bags were filled with stem cellcontaining medium supplemented with human blood plasma (BP) (Figure 1A)
To further elucidate the distribution of ITSCs growing within the cell culture bags, ITSCs were transiently transfected with green fluorescent protein (GFP)
Summary
Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. VueLife bags are gas permeable and certified to clinical-Good Manufacturing Practice (cGMP) grade, as recommended by the Food and Drug administration (FDA) for stem cell based products [5] Applying such Afc-FEP bags to cell expansion prior to transplantation, Lima and colleagues showed successful cultivation of cord blood cells followed by transplantation into ten patients with advanced hematological malignancies [6]. The suitability of Afc-FEP-bags to cell cultivation was further demonstrated by Krause et al using natural killer cells [7] as well as by Hendrikx and coworkers, who applied such bags for bone marrow cells before intramyocardial transplantation [8] Extending these promising findings, we applied Afc-FEP bags for the cultivation of neural crest-derived stem cells isolated from the adult human nose. The abandonment of animal sera is highly recommended by the FDA [5,12], clinically approvable production processes for ITSCs, including the use of cGMP-graded cell culture containers, is necessary for the safety of the stem cellbased products [4]
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