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- ItemStatic and dynamic 3D culture of neural precursor cells on macroporous cryogel microcarriers(Amsterdam [u.a.] : Elsevier, 2020) Newland, Ben; Ehret, Fanny; Hoppe, Franziska; Eigel, Dimitri; Pette, Dagmar; Newland, Heike; Welzel, Petra B.; Kempermann, Gerd; Werner, CarstenNeural precursor cells have been much studied to further our understanding of the far-reaching and controversial question of adult neurogenesis. Currently, differentiation of primary neural precursor cells from the mouse dentate gyrus via 2-dimentional in vitro culture yields low numbers of neurons, a major hindrance to the field of study. 3-dimentional “neurosphere” culture allows better 3D cell-cell contact, but control over cell differentiation is poor because nutrition and oxygen restrictions at the core of the sphere causes spontaneous differentiation, predominantly to glial cells, not neurons. Our group has developed macroporous scaffolds, which overcome the above-mentioned problems, allowing long-term culture of neural stem cells, which can be differentiated into a much higher yield of neurons. Herein we describe a method for culturing neural precursor cells on RGD peptide functionalized-heparin containing cryogel scaffolds, either in standard non-adherent well-plates (static culture) or in spinner flasks (dynamic culture). This method includes: • The synthesis and characterization of heparin based microcarriers. • A “static” 3D culture method for that does not require spinner flask equipment. • “Dynamic” culture in which cell loaded microcarriers are transferred to a spinner flask. © 2020 The Authors
- ItemPoly(ethylene glycol) based nanotubes for tuneable drug delivery to glioblastoma multiforme(Cambridge : Royal Society of Chemistry, 2020) Alghamdi, Majed; Chierchini, Filippo; Eigel, Dimitri; Taplan, Christian; Miles, Thomas; Pette, Dagmar; Welzel, Petra B.; Werner, Carsten; Wang, Wenxin; Neto, Catia; Gumbleton, Mark; Newland, BenGlioblastoma multiforme (GBM) is the most aggressive type of malignant brain tumour, which is associated with a poor two-year survival rate and a high rate of fatal recurrence near the original tumour. Focal/local drug delivery devices hold promise for improving therapeutic outcomes for GBM by increasing drug concentrations locally at the tumour site, or by facilitating the use of potent anti-cancer drugs that are poorly permeable across the blood brain barrier (BBB). For inoperable tumours, stereotactic delivery to the tumour necessitates the development of nanoscale/microscale injectable drug delivery devices. Herein we assess the ability of a novel class of polymer nanotube (based on poly(ethylene glycol) (PEG)) to load doxorubicin (a mainstay breast cancer therapeutic with poor BBB permeability) and release it slowly. The drug loading properties of the PEG nanotubes could be tuned by varying the degree of carboxylic acid functionalisation and hence the capacity of the nanotubes to electrostatically bind and load doxorubicin. 70% of the drug was released over the first seven days followed by sustained drug release for the remaining two weeks tested. Unloaded PEG nanotubes showed no toxicity to any of the cell types analysed, whereas doxorubicin loaded nanotubes decreased GBM cell viability (C6, U-87 and U-251) in a dose dependent manner in 2Din vitroculture. Finally, doxorubicin loaded PEG nanotubes significantly reduced the viability ofin vitro3D GBM models whilst unloaded nanotubes showed no cytotoxicity. Taken together, these findings show that polymer nanotubes could be used to deliver alternative anti-cancer drugs for local therapeutic strategies against brain cancers. © The Royal Society of Chemistry 2020.
- ItemThermo-responsive cell culture carriers based on poly(vinyl methyl ether) - The effect of biomolecular ligands to balance cell adhesion and stimulated detachment(Abingdon : Taylor & Francis, 2015) Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V.; Noll, Thomas; Funk, Richard H.W.; Engelmann, Katrin; Werner, CarstenTwo established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.