Adhesion, proliferation and osteogenic differentiation of human MSCs cultured under perfusion with a marine oxygen carrier on an allogenic bone substitute.

Tissue engineering strategies have been developed to optimize osseointegration in dental implant surgery. One of the major problems is the non-homogeneous spatial cell distribution in the scaffold, as well as subsequent matrix production. Insufficient nutrient and oxygen supplies inside the scaffold are factors in this phenomenon. To mediate this gradient formation, we have implemented a perfusion culture method to seed human bone marrow mesenchymal stem cells (MSCs) into three-dimensional (3-D)-allogenic bone scaffolds in combination with a marine haemoglobin, HEMOXCell®, for oxygen delivery. Cell culture was performed under static and perfusion conditions, with standard and osteogenic media, with and without HEMOXCell®. The cell seeding efficiency, as well as MSC/scaffold cytocompatibly were assessed using viability and proliferation assays. Scaffolds' cellularization and extracellular matrix (ECM) formation were analyzed using scanning electron microscopy and histological staining. Cell differentiation was investigated with osteogenic biomarkers gene expression analysis. The perfusion culture was observed to significantly promote MSC proliferation and differentiation throughout the scaffolds, especially when using the induction medium w/HEMOXCell®. Our data suggest that perfusion culture of MSC into allogenic bone substitute with HEMOXCell® as a natural oxygen carrier is promising for tissue engineering applications to oxygenate hypoxic areas and to promote cellular proliferation.

HEMO2life as a protective additive to Celsior solution for static storage of donor hearts prior to transplantation.

BACKGROUND: Prior to heart transplantation, static storage of donor hearts is currently limited to 4-5 h, despite profound hypothermia (4-8 °C). Because heart transplantation is an emergency procedure, improved protection to extend safe storage duration would be advantageous. We investigated whether the naturally respiratory pigment HEMO2life®, which is effective at hypothermia for the passive release of oxygen via oxygen gradient, could improve long-term preservation. METHODS: Isolated Langendorff-perfused rat hearts (n = 12/group) were equilibrated (20 min) and function (left ventricular developed pressure: LVDP) measured by intraventricular balloon before arrest with cold (7.5 °C) Celsior® solution, either alone (control) or with the addition of HEMO2life® (Hemarina SA, Morlaix, France) at 1 g/L. Cold storage lasted 8 h prior to reperfusion (60 min) and recovery (as % of pre-ischemic function) was assessed. Hearts (minced and homogenized) were also assessed by TTC staining as a measure of viability and two hearts from each group were sliced and assessed by TTC staining for infarct size. Values are expressed as mean ± standard error of mean and analyzed by Student's t-test. RESULTS: Hearts recovered rapidly in both groups to a plateau by 20 min of reperfusion; control and HEMO2life® final recovery (60 min) was 45 ± 2% and 57 ± 1% (P < 0.05) respectively. Left ventricular end-diastolic pressure recovered to a similar extent in both groups (between 31 to 35 mmHg), as did heart rate (final recovery between 84 to 89% pre-ischemic value); however, coronary flow was significantly (P < 0.05) higher in HEMO2life® group (7.5 ± 0.7 ml/min) compared to control (5.4 ± 0.4 ml/min). Viability and infarct size measurements were similar between groups. CONCLUSION: The addition of the natural oxygen releasing pigment HEMO2life® to Celsior® preservation solution significantly improved post-ischemic recovery of heart function. This additive may have major therapeutic potential for clinical heart transplantation.

Comparison of Perfluorodecalin and HEMOXCell as Oxygen Carriers for Islet Oxygenation in an In Vitro Model of Encapsulation.

Transplantation of encapsulated islets in a bioartificial pancreas is a promising alternative to free islet cell therapy to avoid immunosuppressive regimens. However, hypoxia, which can induce a rapid loss of islets, is a major limiting factor. The efficiency of oxygen delivery in an in vitro model of bioartificial pancreas involving hypoxia and confined conditions has never been investigated. Oxygen carriers such as perfluorocarbons and hemoglobin might improve oxygenation. To verify this hypothesis, this study aimed to identify the best candidate of perfluorodecalin (PFD) or HEMOXCell® to reduce cellular hypoxia in a bioartificial pancreas in an in vitro model of encapsulation ex vivo. The survival, hypoxia, and inflammation markers and function of rat islets seeded at 600 islet equivalents (IEQ)/cm2 and under 2% pO2 were assessed in the presence of 50 µg/mL of HEMOXCell or 10% PFD with or without adenosine. Both PFD and HEMOXCell increased the cell viability and decreased markers of hypoxia (hypoxia-inducible factor mRNA and protein). In these culture conditions, adenosine had deleterious effects, including an increase in cyclooxygenase-2 and interleukin-6, in correlation with unregulated proinsulin release. Despite the effectiveness of PFD in decreasing hypoxia, no restoration of function was observed and only HEMOXCell had the capacity to restore insulin secretion to a normal level. Thus, it appeared that the decrease in cell hypoxia as well as the intrinsic superoxide dismutase activity of HEMOXCell were both mandatory to maintain islet function under hypoxia and confinement. In the context of islet encapsulation in a bioartificial pancreas, HEMOXCell is the candidate of choice for application in vivo.

Advancement in recombinant protein production using a marine oxygen carrier to enhance oxygen transfer in a CHO-S cell line.

Recombinant proteins, particularly proteins used as therapeutics, are widely expressed for bioprocessing manufacturing processes. Mammalian cell lines represent the major host cells for bioproduction, according to their capacities of post-translational modifications and folding of secreted proteins. Many parameters can affect cell productivity, especially the rate of oxygen transfer. Dissolved oxygen, in high or low proportions, is a crucial parameter which can affect cell viability and thus productivity. HEMARINA has developed a new technology, commercially proposed as HEMOXCell(®), to improve cell culture at a large production scale. HEMOXCell(®) is a marine oxygen carrier having properties of high oxygen sensitivity, to be used as an oxygen additive during cell culture manufacturing. In this study, we investigated the effects of HEMOXCell(®) on the culture of the commonly used CHO-S cell line. Two main objectives were pursued: 1) cell growth rate and viability during a batch mode process, and 2) the determination of the effect of this oxygen carrier on recombinant protein production from a CHO-transfected cell line. Our results show an increase of CHO-S cellular growth at a rate of more than four-fold in culture with HEMOXCell(®). Moreover, an extension of the growth exponential phase and high cell viability were observed. All of these benefits seem to contribute to the improvement of recombinant protein production. This work underlines several applications using this marine-type oxygen carrier for large biomanufacturing. It is a promising cell culture additive according to the increasing demand for therapeutic products such as monoclonal antibodies.

In vivo biodistribution and oxygenation potential of a new generation of oxygen carrier.

Natural giant extracellular hemoglobins (Hbs) from polychaete annelids are currently actively investigated as promising oxygen carriers. Their powerful oxygenating ability and their safety have been demonstrated in preclinical studies, motivating their development for therapeutic and industrial applications. HEMARINA-M101 (M101) is derived from the marine invertebrate Arenicola marina. It is formulated as a manufactured product designated HEMOXYCarrier(®) (HEMARINA SA, France). The aim of the present study was to unveil the fate of M101 after a single intravenous (i.v.) injection in mice. For this purpose, M101 was tagged with a far-red fluorescent dye. Repeated non-invasive fluorescent imaging revealed a rapid diffusion of M101 in the whole body of animals, reaching all the examined organs such as brain, liver, lungs and ovaries. Functional M101 was circulating in bloodstream for several hours, without inducing any obvious side-effects. Last, a single i.v. injection of M101 in mice bearing human-derived subcutaneous tumors demonstrated the ability of this Hb to reduce hypoxia in poorly vascularized tissues, thus supporting the biological relevance of M101 oxygen release to vertebrate tissues. Altogether, these results further encourage the development of M101 as an oxygen carrying therapeutic.

Dose-ranging study of the performance of the natural oxygen transporter HEMO2 Life in organ preservation.

The intensity of ischemia-reperfusion injury of the donor organ during the preservation phase and after anastomosis is acknowledged as being a key factor for long-term graft outcome. We previously showed that the addition of 5 g/L of the natural oxygen carrier HEMO2 Life was beneficial for the cold static preservation of kidney grafts in both University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate solutions. Herein, we refined these findings by evaluating HEMO2 Life at various dose levels in UW, both in vitro with endothelial cells and in vivo in a pig kidney autotransplantation preclinical model. We showed in vitro that cells were significantly better preserved with HEMO2 Life in a dose-dependent manner, with benefits in terms of survival, metabolic activity, and cellular integrity. In vivo, serum creatinine measurements at reperfusion confirmed the important benefits of HEMO2 Life treatment on function recovery at the dose levels of 1, 2, and 5 g/L. Likewise, histological analysis of kidney parenchyma biopsies from day 7 confirmed the superiority of HEMO2 Life-supplemented UW over UW alone, and there was no difference between the doses. Three months' follow-up confirmed the trend of the first 2 weeks, with creatinine and fibrosis levels similar to those in pretransplant kidneys.

Assessment of cellular actin dynamics by measurement of fluorescence anisotropy.

To study cellular actin dynamics, a cell-free assay based on fluorescence anisotropy was developed. Using G-actin-Alexa as a probe, we found that anisotropy enhancement reflects F-actin elongation. Anisotropy enhancement varies with the concentration of magnesium and calcium cations and with ethylenediaminetetraacetate or well-known effectors of the polymerization. This assay gives the overall status of actin dynamics in cell extracts which are the closest conditions to in vivo, implying most of the regulating proteins that are missing in purified actin measurements. It can be used in a large-scale screening for chemical compounds which modulate actin polymerization.

In vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotides-nanospheres complexes.

The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS-Fli-1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB-coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS-Fli-1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.