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  • 1.
    Greuel, Selina
    et al.
    Charite Univ Med Berlin, Germany.
    Freyer, Nora
    Charite Univ Med Berlin, Germany.
    Hanci, Gungor
    Charite Univ Med Berlin, Germany.
    Bohme, Mike
    Charite Univ Med Berlin, Germany.
    Miki, Toshio
    Univ Southern Calif, CA USA.
    Werner, Johannes
    Stem Cell Syst GmbH, Germany.
    Schubert, Frank
    Stem Cell Syst GmbH, Germany.
    Sittinger, Michael
    Charite Univ Med Berlin, Germany.
    Zeilinger, Katrin
    Charite Univ Med Berlin, Germany.
    Mandenius, Carl-Fredrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska fakulteten.
    Online measurement of oxygen enables continuous noninvasive evaluation of human-induced pluripotent stem cell (hiPSC) culture in a perfused 3D hollow-fiber bioreactor2019Inngår i: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 13, nr 7, s. 1203-1216Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    For clinical and/or pharmaceutical use of human-induced pluripotent stem cells (hiPSCs), large cell quantities of high quality are demanded. Therefore, we combined the expansion of hiPSCs in closed, perfusion-based 3D bioreactors with noninvasive online monitoring of oxygen as culture control mechanism. Bioreactors with a cell compartment volume of 3 or 17 ml were inoculated with either 10 x 10(6) or 50 x 10(6) cells, and cells were expanded over 15 days with online oxygen and offline glucose and lactate measurements being performed. The CellTiter-Blue (R) Assay was performed at the end of the bioreactor experiments for indirect cell quantification. Model simulations enabled an estimation of cell numbers based on kinetic equations and experimental data during the 15-day bioreactor cultures. Calculated oxygen uptake rates (OUR), glucose consumption rates (GCR), and lactate production rates (LPR) revealed a highly significant correlation (p amp;lt; 0.0001). Oxygen consumption, which was measured at the beginning and the end of the experiment, showed a strong culture growth in line with the OUR and GCR data. Furthermore, the yield coefficient of lactate from glucose and the OUR to GCR ratio revealed a shift from nonoxidative to oxidative metabolism. The presented results indicate that oxygen is equally as applicable as parameter for hiPSC expansion as glucose while providing an accurate real-time impression of hiPSC culture development. Additionally, oxygen measurements inform about the metabolic state of the cells. Thus, the use of oxygen online monitoring for culture control facilitates the translation of hiPSC use to the clinical setting.

  • 2.
    Moelzer, Christine
    et al.
    Univ Aberdeen, Scotland.
    Shankar, Sucharita P.
    Univ Aberdeen, Scotland; UCB Pharma, England.
    Griffith, May
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Univ Montreal, Canada.
    Mirazul Islam, Mohammad Mirazul
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Harvard Med Sch, MA 02115 USA; Harvard Med Sch, MA 02115 USA.
    Forrester, John V
    Univ Aberdeen, Scotland.
    Kuffova, Lucia
    Univ Aberdeen, Scotland.
    Activation of dendritic cells by crosslinked collagen hydrogels (artificial corneas) varies with their composition2019Inngår i: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Activated T cells are known to promote fibrosis, a major complication limiting the range of polymeric hydrogels as artificial corneal implants. As T cells are activated by dendritic cells (DC), minimally activating hydrogels would be optimal. In this study, we evaluated the ability of a series of engineered (manufactured/fabricated) and natural collagen matrices to either activate DC or conversely induce DC apoptosis in vitro. Bone marrow DC were cultured on a series of singly and doubly crosslinked hydrogels (made from recombinant human collagen III [RHCIII] or collagen mimetic peptide [CMP]) or on natural collagen-containing matrices, Matrigel(TM) and de-cellularised mouse corneal stroma. DC surface expression of major histocompatibility complex Class II and CD86 as well as apoptosis markers were examined. Natural matrices induced low levels of DC activation and maintained a "tolerogenic" phenotype. The same applied to singly crosslinked CMP-PEG gels. RHCIII gels singly crosslinked using either N-(3-dimethylaminopropyl)-N -ethylcarbodiimide with the coinitiator N-hydroxy succinimide (EDC-NHS) or N-cyclohexyl-N-(2-morpholinoethyl)carbodiimide metho-p-toulenesulfonate with NHS (CMC-NHS) induced varying levels of DC activation. In contrast, however, RHCIII hydrogels incorporating an additional polymeric network of 2-methacryloyloxyethyl phosphorylcholine did not activate DC but instead induced DC apoptosis, a phenomenon observed in natural matrices. This correlated with increased DC expression of leukocyte-associated immunoglobulin-like receptor-1. Despite low immunogenic potential, viable tolerogenic DC migrated into and through both natural and manufactured RHCIII gels. These data show that the immunogenic potential of RHCIII gels varies with the nature and composition of the gel. Preclinical evaluation of hydrogel immunogenic/fibrogenic potential is recommended.

  • 3.
    Molzer, Christine
    et al.
    Univ Aberdeen, Scotland.
    Shankar, Sucharita P.
    Univ Aberdeen, Scotland; UCB Pharma, England.
    Masalski, Vlad
    Univ Aberdeen, Scotland.
    Griffith, May
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Univ Montreal, Canada; Maisonneuve Rosemont Hosp Res Ctr, Canada.
    Kuffova, Lucia
    Univ Aberdeen, Scotland.
    Forrester, John V
    Univ Aberdeen, Scotland.
    TGF-beta 1-activated type 2 dendritic cells promote wound healing and induce fibroblasts to express tenascin c following corneal full-thickness hydrogel transplantation2019Inngår i: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We showed previously that 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide hydrochloride (EDC) cross-linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4 years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers-bulky, aromatic N-cyclohexyl-N0-(2-morpholinoethyl)-carbodiimide (CMC), and nonbulky EDC-in a mouse corneal graft model. Murine corneas undergoing full-thickness implantation with these gels became opaque due to dense retro-corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c(+) dendritic cells (DC) were found, suggesting a hydrogel-specific innate immune response. More DC infiltrated the stroma surrounding EDC-N-hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis-associated tenascin c expression. The opposite was true for CMC-NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor beta 1 (TGF-beta 1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF-beta 1 neutralisation. Furthermore, tenascin c staining was found in 40- to 50 mu m long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF-beta 1 alternatively activated (tolerising) DC regulate fibroblast-mediated tenascin c secretion, possibly via local production of TGF-beta 1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.

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