Microcarrier expansion of c-MycERTAM -modified human olfactory mucosa cells for neural regeneration.

Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work, we derived PA5 cells, a polyclonal population that retains functional attributes of primary human OMCs. Microcarrier suspension culture is an alternative to planar two-dimensinal culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on plastic compared to plastic plus microcarriers with upregulation of neural markers ß-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4 × 105 cells/ml and 4.9 × 104 cells/ml for plastic and plastic plus, respectively, after 7 days. p75NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, coculture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study shows successful expansion of PA5 cells using suspension culture on microcarriers, and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields.

Scale-up of an intensified bioprocess for the expansion of bovine adipose-derived stem cells (bASCs) in stirred tank bioreactors.

Cultivated meat is an emerging field, aiming to establish the production of animal tissue for human consumption in an in vitro environment, eliminating the need to raise and slaughter animals for their meat. To realise this, the expansion of primary cells in a bioreactor is needed to achieve the high cell numbers required. The aim of this study was to develop a scalable, microcarrier based, intensified bioprocess for the expansion of bovine adipose-derived stem cells as precursors of fat and muscle tissue. The intensified bioprocess development was carried out initially in spinner flasks of different sizes and then translated to fully controlled litre scale benchtop bioreactors. Bioprocess intensification was achieved by utilising the previously demonstrated bead-to-bead transfer phenomenon and through the combined addition of microcarrier and medium to double the existing surface area and working volume in the bioreactor. Choosing the optimal time point for the additions was critical in enhancing the cell expansion. A significant fold increase of 114.19 ± 1.07 was obtained at the litre scale in the intensified bioprocess compared to the baseline (**p < .005). The quality of the cells was evaluated pre- and post-expansion and the cells were found to maintain their phenotype and differentiation capacity.

Bioprocess development for scalable production of cultivated meat.

Traditional farm-based products based on livestock are one of the main contributors to greenhouse gas emissions. Cultivated meat is an alternative that mimics animal meat, being produced in a bioreactor under controlled conditions rather than through the slaughtering of animals. The first step in the production of cultivated meat is the generation of sufficient reserves of starting cells. In this study, bovine adipose-derived stem cells (bASCs) were used as starting cells due to their ability to differentiate towards both fat and muscle, two cell types found in meat. A bioprocess for the expansion of these cells on microcarriers in spinner flasks was developed. Different cell seeding densities (1,500, 3,000, and 6,000 cells/cm2 ) and feeding strategies (80%, 65%, 50%, and combined 80%/50% medium exchanges) were investigated. Cell characterization was assessed pre- and postbioprocessing to ensure that bioprocessing did not negatively affect bASC quality. The best growth was obtained with the lowest cell seeding density (1,500 cells/cm2 ) with an 80% medium exchange performed (p < .0001) which yielded a 28-fold expansion. The ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages was retained postbioprocessing and no significant difference (p > .5) was found in clonogenicity pre- or postbioprocessing in any of the feeding regimes tested.

Regulation of embryonic neurogenesis by germinal zone vasculature.

In the adult rodent brain, new neurons are born in two germinal regions that are associated with blood vessels, and blood vessels and vessel-derived factors are thought to regulate the activity of adult neural stem cells. Recently, it has been proposed that a vascular niche also regulates prenatal neurogenesis. Here we identify the mouse embryo hindbrain as a powerful model to study embryonic neurogenesis and define the relationship between neural progenitor cell (NPC) behavior and vessel growth. Using this model, we show that a subventricular vascular plexus (SVP) extends through a hindbrain germinal zone populated by NPCs whose peak mitotic activity follows a surge in SVP growth. Hindbrains genetically defective in SVP formation owing to constitutive NRP1 loss showed a premature decline in both NPC activity and hindbrain growth downstream of precocious cell cycle exit, premature neuronal differentiation, and abnormal mitosis patterns. Defective regulation of NPC activity was not observed in mice lacking NRP1 expression by NPCs, but instead in mice lacking NRP1 selectively in endothelial cells, yet was independent of vascular roles in hindbrain oxygenation. Therefore, germinal zone vascularization sustains NPC proliferation in the prenatal brain.

Development and Characterisation of a Human Chronic Skin Wound Cell Line-Towards an Alternative for Animal Experimentation.

Background: Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives: To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results: Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions: These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening.

An ultra scale-down methodology to characterize aspects of the response of human cells to processing by membrane separation operations.

Tools that allow cost-effective screening of the susceptibility of cell lines to operating conditions which may apply during full scale processing are central to the rapid development of robust processes for cell-based therapies. In this paper, an ultra scale-down (USD) device has been developed for the characterization of the response of a human cell line to membrane-based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. The cell line used to develop the measurements was a clinically relevant human fibroblast cell line. The impact was evaluated by cell damage on completion of membrane processing as assessed by trypan blue exclusion and release of intracellular lactate dehydrogenase (LDH). Similar insight was gained from both methods and this allowed the extension of the use of the LDH measurements to examine cell damage as it occurs during processing by a combination of LDH appearance in the permeate and mass balancing of the overall operation. Transmission of LDH was investigated with time of operation and for the two disc speeds investigated (6,000 and 10,000 rpm or ϵmax ≈ 1.9 and 13.5 W mL-1 , respectively). As expected, increased energy dissipation rate led to increased transmission as well as significant increases in rate and extent of cell damage. The method developed can be used to test the impact of varying operating conditions and cell lines on cell damage and morphological changes. Biotechnol. Bioeng. 2017;114: 1241-1251. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Identification of a transcriptional signature for the wound healing continuum.

There is a spectrum/continuum of adult human wound healing outcomes ranging from the enhanced (nearly scarless) healing observed in oral mucosa to scarring within skin and the nonhealing of chronic skin wounds. Central to these outcomes is the role of the fibroblast. Global gene expression profiling utilizing microarrays is starting to give insight into the role of such cells during the healing process, but no studies to date have produced a gene signature for this wound healing continuum. Microarray analysis of adult oral mucosal fibroblast (OMF), normal skin fibroblast (NF), and chronic wound fibroblast (CWF) at 0 and 6 hours post-serum stimulation was performed. Genes whose expression increases following serum exposure in the order OMF < NF < CWF are candidates for a negative/impaired healing phenotype (the dysfunctional healing group), whereas genes with the converse pattern are potentially associated with a positive/preferential healing phenotype (the enhanced healing group). Sixty-six genes in the enhanced healing group and 38 genes in the dysfunctional healing group were identified. Overrepresentation analysis revealed pathways directly and indirectly associated with wound healing and aging and additional categories associated with differentiation, development, and morphogenesis. Knowledge of this wound healing continuum gene signature may in turn assist in the therapeutic assessment/treatment of a patient's wounds.

Cooperation between osteoblastic cells and endothelial cells enhances their phenotypic responses and improves osteoblast function.

Osteogenesis requires close co-operation with angiogenesis to create vascularized bone tissue. In this study, an indirect co-culture model using osteoblasts (OBs), primary endothelial cells (ECs) and Matrigel interlayer was established to understand the impact of each cell type on the other. ECs synergistically enhanced osteoblastic gene expression by OBs, while OBs were capable of supporting tubule-like structures formed by ECs on Matrigel, enhancing mean tubule length from 146.5 ± 23.5 µm in ECs alone to 192 ± 28.6 µm in co-culture (p < 0.05). Similar improvements were noted in terms of tubule number. An applicability study of the co-culture model to bone tissue engineering, performed on a biopolymer fibrous membrane, showed substantially enhanced deposition of calcified nodules. These results demonstrate the efficacy of co-culture with ECs to improve osteogenesis for bone tissue engineering.

Assessment of thermally stabilized electrospun poly(vinyl alcohol) materials as cell permeable membranes for a novel blood salvage device.

The use of Intraoperative Cell Salvage (ICS) is currently limited in oncological surgeries, due to safety concerns associated with the ability of existing devices to successfully remove circulating tumour cells. In this work, we present the first stages towards the creation of an alternative platform to current cell savers, based on the extremely selective immunoaffinity membrane chromatography principle. Non-woven membranes were produced via electrospinning using poly(vinyl alcohol) (PVA), and further heat treated at 180 °C to prevent their dissolution in aqueous environments and preserve their fibrous morphology. The effects of the PVA degree of hydrolysis (DH) (98 % vs 99 %), method of electrospinning (needleless DC vs AC), and heat treatment duration (1-8 h) were investigated. All heat treated supports maintained their cytocompatibility, whilst tensile tests indicated that the 99 % hydrolysed DC electrospun mats were stronger compared to their 98 % DH counterparts. Although, and at the described conditions, AC electrospinning produced fibres with more than double the diameter compared to those from DC electrospinning, it was not chosen for subsequent experiments because it is still under development. Evidence of unimpeded passage of SY5Y neuroblastoma cells and undiluted defibrinated sheep's blood in flow-through filtration experiments confirmed the successful creation of 3D networks with minimum resistance to mass transfer and lack of non-specific cell binding to the base material, paving the way for the development of novel, highly selective ICS devices for tumour surgeries.

Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect.

Tissue engineering of stem cells in concert with 3-dimensional (3D) scaffolds is a promising approach for regeneration of bone tissues. Bioactive ceramic microspheres are considered effective 3D stem cell carriers for bone tissue engineering. Here we used evacuated calcium phosphate (CaP) microspheres as the carrier of mesenchymal stem cells (MSCs) derived from rat bone marrow. The performance of the CaP-MSCs construct in bone formation within a rat calvarium defect was evaluated. MSCs were first cultured in combination with the evacuated microcarriers for 7 days in an osteogenic medium, which was then implanted in the 6 mm-diameter calvarium defect for 12 weeks. For comparison purposes, a control defect and cell-free CaP microspheres were also evaluated. The osteogenic differentiation of MSCs cultivated in the evacuated CaP microcarriers was confirmed by alkaline phosphatase staining and real time polymerase chain reaction. The in vivo results confirmed the highest bone formation was attained in the CaP microcarriers combined with MSCs, based on microcomputed tomography and histological assays. The results suggest that evacuated CaP microspheres have the potential to be useful as stem cell carriers for bone tissue engineering.

Muscle-derived precursor cells isolated on the basis of differential adhesion properties respond differently to capillary flow.

Capillary shear stress can improve osteogenic differentiation in muscle-derived precursor cells (MDPCs). This has implications for large-scale bioprocessing of cell therapies where capillary transfer is needed. The recovery, viability, and osteogenic differentiation potential of two subsets of MDPCs, early-adherent pre-plate 1 (PP1) and late-adherent PP3 populations, have been examined: PP1 MDPCs produced a greater degree of osteogenic differentiation than PP3 MDPCs, quantified by Alizarin Red S staining intensity (P < 0.05). For both cell populations, capillary flow-induced significant increases in Alizarin Red S staining (P < 0.05). However, PP1 cells were more susceptible to capillary flow-induced damage than PP3 cells and this was dependent on duration of exposure. Overall, results indicate that different cell subsets, even from within a single tissue, can respond variably to capillary shear stress, necessitating its precise monitoring and control.

Impact of Dual Cell Co-culture and Cell-conditioned Media on Yield and Function of a Human Olfactory Cell Line for Regenerative Medicine.

Olfactory ensheathing cells (OECs) are a promising candidate therapy for neuronal tissue repair. However, appropriate priming conditions to drive a regenerative phenotype are yet to be determined. We first assessed the effect of using a human fibroblast feeder layer and fibroblast conditioned media on primary rat olfactory mucosal cells (OMCs). We found that OMCs cultured on fibroblast feeders had greater expression of the key OEC marker p75NTR (25.1 ± 10.7 cells/mm2) compared with OMCs cultured on laminin (4.0 ± 0.8 cells/mm2, p = 0.001). However, the addition of fibroblast-conditioned media (CM) resulted in a significant increase in Thy1.1 (45.9 ± 9.0 cells/mm2 versus 12.5 ± 2.5 cells/mm2 on laminin, p = 0.006), an undesirable cell marker as it is regarded to be a marker of contaminating fibroblasts. A direct comparison between human feeders and GMP cell line Ms3T3 was then undertaken. Ms3T3 cells supported similar p75NTR levels (10.7 ± 5.3 cells/mm2) with significantly reduced Thy1.1 expression (4.8 ± 2.1 cells/mm2). Ms3T3 cells were used as feeder layers for human OECs to determine whether observations made in the rat model were conserved. Examination of the OEC phenotype (S100ß expression and neurite outgrowth from NG108-15 cells) revealed that co-culture with fibroblast feeders had a negative effect on human OECs, contrary to observations of rat OECs. CM negatively affected rat and human OECs equally. When the best and worst conditions in terms of supporting S100ß expression were used in NG108-15 neuron co-cultures, those with the highest S100ß expression resulted in longer and more numerous neurites (22.8 ± 2.4 µm neurite length/neuron for laminin) compared with the lowest S100ß expression (17.9 ± 1.1 µm for Ms3T3 feeders with CM). In conclusion, this work revealed that neither dual co-culture nor fibroblast-conditioned media support the regenerative OEC phenotype. In our case, a preliminary rat model was not predictive of human cell responses.

Characterisation of osteogenic and vascular responses of hMSCs to Ti-Co doped phosphate glass microspheres using a microfluidic perfusion platform.

Using microspherical scaffolds as building blocks to repair bone defects of specific size and shape has been proposed as a tissue engineering strategy. Here, phosphate glass (PG) microcarriers doped with 5 mol % TiO2 and either 0 mol % CoO (CoO 0%) or 2 mol % CoO (CoO 2%) were investigated for their ability to support osteogenic and vascular responses of human mesenchymal stem cells (hMSCs). Together with standard culture techniques, cell-material interactions were studied using a novel perfusion microfluidic bioreactor that enabled cell culture on microspheres, along with automated processing and screening of culture variables. While titanium doping was found to support hMSCs expansion and differentiation, as well as endothelial cell-derived vessel formation, additional doping with cobalt did not improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled screening of culture parameters for cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing.

Mathematical and computational models for bone tissue engineering in bioreactor systems.

Research into cellular engineered bone grafts offers a promising solution to problems associated with the currently used auto- and allografts. Bioreactor systems can facilitate the development of functional cellular bone grafts by augmenting mass transport through media convection and shear flow-induced mechanical stimulation. Developing successful and reproducible protocols for growing bone tissue in vitro is dependent on tuning the bioreactor operating conditions to the specific cell type and graft design. This process, largely reliant on a trial-and-error approach, is challenging, time-consuming and expensive. Modelling can streamline the process by providing further insight into the effect of the bioreactor environment on the cell culture, and by identifying a beneficial range of operational settings to stimulate tissue production. Models can explore the impact of changing flow speeds, scaffold properties, and nutrient and growth factor concentrations. Aiming to act as an introductory reference for bone tissue engineers looking to direct their experimental work, this article presents a comprehensive framework of mathematical models on various aspects of bioreactor bone cultures and overviews modelling case studies from literature.

A parameterised mathematical model to elucidate osteoblast cell growth in a phosphate-glass microcarrier culture.

Tissue engineering has the potential to augment bone grafting. Employing microcarriers as cell-expansion vehicles is a promising bottom-up bone tissue engineering strategy. Here we propose a collaborative approach between experimental work and mathematical modelling to develop protocols for growing microcarrier-based engineered constructs of clinically relevant size. Experiments in 96-well plates characterise cell growth with the model human cell line MG-63 using four phosphate glass microcarrier materials. Three of the materials are doped with 5 mol% TiO2 and contain 0%, 2% or 5% CoO, and the fourth material is doped only with 7% TiO2 (0% CoO). A mathematical model of cell growth is parameterised by finding material-specific growth coefficients through data-fitting against these experiments. The parameterised mathematical model offers more insight into the material performance by comparing culture outcome against clinically relevant criteria: maximising final cell number starting with the lowest cell number in the shortest time frame. Based on this analysis, material 7% TiO2 is identified as the most promising.

Assessing behaviour of osteoblastic cells in dynamic culture conditions using titanium-doped phosphate glass microcarriers.

Tissue engineering is a promising approach for bone regeneration; yet challenges remain that limit successful translation to patients. It is necessary to understand how real-world manufacturing processes will affect the constituent cells and biomaterials that are needed to create engineered bone. Bioactive phosphate glasses processed into microspheres are an attractive platform for expanding bone-forming cells and also for driving their osteogenic differentiation and maturation. The aim of this study was to assess whether Ti-doped phosphate glass microspheres could support osteoblastic cell responses in dynamic cell culture environments. Dynamic culture conditions were achieved using microwell studies under orbital agitation. Dimensionless parameters such as the Froude number were used to inform the choice of agitation speeds, and the impact on cell proliferation and microunit formation was quantified. We found that phosphate glass microspheres doped with titanium dioxide at both 5 and 7 mol% provided a suitable biomaterial platform for effective culture of MG63 osteoblastic cells and was not cytotoxic. Dynamic culture conditions supported expansion of MG63 cells and both 150 and 300 rpm orbital shake resulted in higher cell yield than static cultures at the end of the culture (day 13). The Froude number analysis provided insight into how the microunit size could be manipulated to enable an appropriate agitation speed to be used, while ensuring buoyancy of the microunits. These small-scale experiments and analyses provide understanding of the impact of fluid flow on cell expansion that will have increasing importance when scaling up to process technologies that can deliver clinical quantities of cell-microsphere units. Such knowledge will enable future engineering of living bone-like material using processing systems such as bioreactors that use mixing and agitation for nutrient transfer, therefore introducing cells to dynamic culture conditions.

Generation of c-MycERTAM-transduced human late-adherent olfactory mucosa cells for potential regenerative applications.

Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive "off-the-shelf" alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycERTAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycERTAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75NTR, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycERTAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.

Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation.

BACKGROUND INFORMATION: The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient-matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). RESULTS: Both the HOF and HDF cell types underwent TGF-beta1 (transforming growth factor-beta1)-induced myofibroblastic differentiation [upregulation of the expression of alpha-sma (alpha-smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of alpha-sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits alpha 5 (fibronectin) or alpha v (vitronectin) were used to determine whether the effects of TGF-beta1 were regulated via integrin signalling pathways. alpha-sma expression in both HOFs and HDFs was down-regulated by antibodies against both alpha 5 and alpha v. Functionally, TGF-beta1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF-beta1 (P<0.05). When TGF-beta1-stimulated cells were incubated with blocking antibodies against alpha 5 and alpha v, gel contraction was decreased to that of non-stimulated cells; however, blocking alpha v or alpha 5 could not restore cellular migration in both HOFs and HDFs. CONCLUSIONS: Despite intrinsic differences in their basal state, the cellular events associated with TGF-beta1-induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up-regulation of alpha-sma expression and increases in collagen gel contraction are vitronectin- and fibronectin-receptor-dependent processes, whereas wound re-population is not.

Manufacturing Exosomes: A Promising Therapeutic Platform.

Extracellular vesicles, in particular the subclass exosomes, are rapidly emerging as a novel therapeutic platform. However, currently very few clinical validation studies and no clearly defined manufacturing process exist. As exosomes progress towards the clinic for treatment of a vast array of diseases, it is important to define the engineering basis for their manufacture early in the development cycle to ensure they can be produced cost-effectively at the appropriate scale. We hypothesize that transitioning to defined manufacturing platforms will increase consistency of the exosome product and improve their clinical advancement as a new therapeutic tool. We present manufacturing technologies and strategies that are being implemented and consider their application for the transition from bench-scale to clinical production of exosomes.

Label-free detection of hypoxia-induced extracellular vesicle secretion from MCF-7 cells.

Nanoscale extracellular vesicles (EVs) including exosomes (50-150 nm membrane particles) have emerged as promising cancer biomarkers due to the carried genetic information about the parental cells. However the sensitive detection of these vesicles remains a challenge. Here we present a label-free electrochemical sensor to measure the EVs secretion levels of hypoxic and normoxic MCF-7 cells. The sensor design includes two consecutive steps; i) Au electrode surface functionalization for anti-CD81 Antibody and ii) EVs capture. The label-free detection of EVs was done via Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS). The working linear range for the sensor was 102-109 EVs/ml with an LOD 77 EVs/mL and 379 EVs/ml for EIS and DPV based detection. A blood-abundant protein, RhD was used for the selectivity test. In order to assess the performance of the biosensor, the level of EVs secretion by the human breast cancer MCF-7 cell line was compared with enzyme-linked immunosorbent assays (ELISA) and Nanoparticle Tracking Analysis (NTA). Designed label-free electrochemical sensors utilized for quantification of EVs secretion enhancement due to CoCl2-induced hypoxia and 1.23 fold increase with respect to normoxic conditions was found.