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.

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.

Polyethylene glycols interact with membrane glycerophospholipids: is this part of their mechanism for hypothermic graft protection?

Polyethylene glycol (PEG), a high-molecular-weight colloid present in new organ preservation solutions, protects against cold ischemia injuries leading to better graft function of transplanted organs. This protective effect cannot be totally explained by immuno-camouflaging property or signaling-pathway modifications. Therefore, we sought for an alternative mechanism dependent on membrane fluidity. Using the Langmuir-Pockles technique, we show here that PEGs interacted with lipid monolayers of defined composition or constituted by a renal cell lipid extract. High-molecular-weight PEGs stabilized the lipid monolayer at low surface pressure. Paradoxically, at high surface pressure, PEGs destabilized the monolayers. Hypothermia reduced the destabilization of saturated monolayer whereas unsaturated monolayer remained unaffected. Modification of ionic strength and pH induced a stronger stabilizing effect of PEG 35,000 Da which could explain its reported higher effectiveness on cold-induced injuries during organ transplantation. This study sheds a new light on PEG protective effects during organ preservation different from all classical hypotheses.

Influence of colloid, preservation medium and trimetazidine on renal medulla injury.

In organ transplantation, preservation injury is an important factor which could influence short-term and long-term graft outcome. The renal medulla is particularly sensitive to oxidant stress and ischemia-reperfusion injury (IRI). Using an autotransplant pig kidney model, we investigated renal function and medullary damage determined between day 1 and week 2 after 24- or 48-h cold storage in different preservation solutions: University of Wisconsin solution (UW), Hopital Edouard Herriot solution (a high Na+ version of UW), ECPEG (high Na+ preservation solution with PEG) and ICPEG (a high K+ version of ECPEG) with or without trimetazidine (TMZ). TMZ improved renal preservation and increased renal function when added in each preservation solution (particularly HEH and ECPEG). Medullary damage led to the early appearance of trimethylamine-N-oxide (TMAO) followed by 1H-NMR in urine and plasma. TMZ and ECPEG is the most efficient association to reduce medullary damage. This study clarifies the role of colloid and polarity solution and the role of mitochondrial protection by TMZ.

Evidence for protective roles of polyethylene glycol plus high sodium solution and trimetazidine against consequences of renal medulla ischaemia during cold preservation and reperfusion in a pig kidney model.

BACKGROUND: The renal medulla is particularly sensitive to oxidant stress and to ischaemia-reperfusion injury (IRI). In organ transplantation, delayed graft function is an important problem and cold ischaemia is thought to be the most important factor in short- and long-term complications. Our aim was to study cold-induced damage in proximal tubular segments and renal medulla osmolite excretion during use of various preservation solutions, and to clarify the role of trimetazidine (TMZ) in limiting renal dysfunction. METHODS: Using an autotransplanted pig kidney model, we assessed renal tubule function, medullary osmolite excretion and renal damage between day 1 and week 2 after 24 or 48 h cold storage in University of Wisconsin solution (UW), Celsior and ECPEG (two new high Na(+) preservation solutions) or the Hopital Edouard Herriot solution (HEH; a high Na(+) version of UW). In additional groups, TMZ was added to these preservation solutions for 24 and 48 h cold storage. RESULTS: Renal function was reduced under these preservation conditions. Tubular injury was associated with aminoaciduria and with a limited Na(+) reabsorbtion. Medullary damage led to the early appearance of trimethylamine-N-oxide and dimethylamine in urine. However, renal damage was modulated by preservation conditions. In addition, TMZ added to each of the solutions efficiently protected against IRI even after prolonged preservation. CONCLUSION: TMZ efficiently protected kidneys against damage when added to the HEH and particularly ECPEG solutions, even after 24 h cold storage. These findings point to a role for drugs that target mitochondria, and demonstrate that TMZ may provide a valuable therapeutic tool against IRI and could be included in therapeutic protocols.

Cyclooxygenase 1-dependent production of F2-isoprostane and changes in redox status during warm renal ischemia-reperfusion.

The detrimental role of oxidative stress has been widely described in tissue damage caused by ischemia-reperfusion. A nonenzymatic, reactive oxygen species-related pathway has been suggested to produce 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), an epimer of prostaglandin F(2alpha) (PGF(2alpha)), which has been proposed as an indicator of oxidative stress. Using an in vivo ischemia-reperfusion model in rat kidneys, we investigated intrarenal accumulation of 8-iso-PGF(2alpha) and PGF(2alpha). Both prostanoids accumulated in the ischemic kidney and disappeared upon reperfusion. In addition, a nonselective (acetylsalicylic acid) or selective cyclooxygenase (COX) 1 inhibitor (SC-560) completely abrogated the 8-iso-PGF(2alpha) and PGF(2alpha) formation in kidneys subjected to ischemia. COX2 inhibition had no effect on the production of these prostanoids. Therefore the two metabolites of arachidonic acid seemed to be produced via an enzymatic COX1-dependent pathway. Neither COX overexpression nor COX activation was detected. We also investigated renal glutathione, which is considered to be the major thiol-disulfide redox buffer of the tissue. Total and oxidized glutathione was decreased during the ischemic period, whereas no further decrease was seen for up to 60 min of reperfusion. These data demonstrate that a dramatic decrease in antioxidant defense was initiated during warm renal ischemia, whereas the 8-iso-PGF(2alpha) was related only to arachidonate conversion by COX1.

Protective roles of polyethylene glycol and trimetazidine against cold ischemia and reperfusion injuries of pig kidney graft.

Ischemia-reperfusion injury (IRI) represents an allo-independent risk factor which favors chronic allograft nephropathy (CAN). Here we analyzed the influence of preservation solutions on the function of autotransplanted pig kidneys over 1-16 weeks after surgery. Kidneys were cold-flushed and cold-stored for 24 or 48 h either in University of Wisconsin (UW), modified-UW Hôpital Edouard Herriot, polyethylene glycol 20 kDa (PEG)-supplemented preservation solutions with low K+ (ECPEG) or high K+ (ICPEG) content. Animals autotransplanted with kidneys cold-stored for 24 h in ECPEG exhibited the greatest levels of creatinine clearance (Ccr: 161 +/- 12 mL/min, n=10) and the lowest levels of proteinuria (0.5 +/- 0.03 mg/mL) 16 weeks after surgery as compared with pigs autotransplanted with kidneys cold-stored in the other solutions tested (Ccr ranging from 80 and 140 mL/min). Similar differences, but with lower Ccr levels, were achieved after a prolonged period of cold-storage(48 h). ECPEG better preserved the kidneys from monocytes/macrophages and CD4+ T cells infiltrations, VCAM-1 and MHC class II overexpressions and occurrence of renal interstitial fibrosis (2%) as compared with the other preservation solutions (5%-20%). Adding the anti-ischemic drug trimetazidine (TMZ) to the preservation solutions, particularly ECPEG, further improved the quality of the week-16 post-transplanted kidneys (Ccr: 182 +/- 12 mL/min, n=10). These findings demonstrated that adding PEG to extracellular-like (with low K+ content) preservation solutions in combination with TMZ significantly improved the long-term outcome of kidney grafts in this model of autotransplanted pig kidney.

Evidence for a mitochondrial impact of trimetazidine during cold ischemia and reperfusion.

In organ transplantation, ischemia-reperfusion injury (IRI) has been implicated in delayed graft function (DGF) as well as in short- and long-term complications. Using an autotransplant pig kidney model, changes in renal function and morphology were determined after different periods of cold ischemia in kidneys preserved in the University of Wisconsin solution (UW), high-Na(+) version of UW (HEH) or Celsior (CEL) a newly developed high-Na(+) solution, with or without trimetazidine (TMZ). Kidney function was better preserved in CEL, UW and particularly HEH in combination with TMZ, particularly after 48 and 72 h. Mitochondria integrity was improved in TMZ-preserved groups. This study indicates that TMZ is efficiently protective against IRI even after prolonged preservation and in different preservation solutions.

Evidence for a protective role of trimetazidine during cold ischemia: targeting inflammation and nephron mass.

Ischemia-reperfusion injury (IRI) is associated with an increased risk of acute rejection, delayed graft function, or chronic graft dysfunction. Mitochondria plays a central role in this process. Using an autotransplant pig kidney model, changes in renal function and morphology were determined after different periods of cold ischemia in kidneys preserved in the University of Wisconsin solution (UW), high-Na(+) version of UW (HEH) or Celsior (CEL) a newly developed high-Na(+) solution, with or without trimetazidine (TMZ). Kidney function was better preserved in HEH after 24 hr and particularly 48- and 72-hr cold storage than in CEL and UW. TMZ improved the preservation quality when added to the different solutions tested, particularly after 48- and 72-hr cold storage. Interstitial fibrosis and tubular atrophy were reduced in HEH with TMZ. CD4(+) T-cell infiltration was also modulated by the preservation conditions. Peripheral-type benzodiazepine receptor (PBR) positive cells infiltration was also modulated by preservation conditions. TMZ was efficient to reduce IRI when added in the various preservation solutions. These results suggest that protection of the mitochondrial function should be a major target to limit IRI. In addition, this study outlines the role of CD4(+) T cells and PBR expression in inflammatory responses after IRI.