Process analytical technology tools for perfusion cell culture

During cell cultivation processes for the production of biopharmaceuticals, good process performance and good product quality can be ensured by online monitoring of critical process parameters (e.g. temperature, pH, or dissolved oxygen). These data can be used in real‐time for process control, as suggested by the process analytical technology (PAT) initiative. Today, solutions for real‐time monitoring of parameters such as concentrations of cells, main nutrients, and metabolism by‐products are developing, but applications of these more complex tools in industrial settings are still limited. Here, we evaluated the use of dielectric spectroscopy (DS) and near‐infrared spectroscopy (NIRS) as PAT tools for a perfusion PER.C6^® cultivation process. We showed that DS enabled predictions of viable cell density from the cultivation vessel, with a root mean square error of prediction (RMSEP) of 4.4% of the calibration range. Additionally, predictions of glucose and lactate concentrations from the cultivation vessel (RMSEP of 10 and 14%, respectively) and from the perfusion stream (RMSEP of 12 and 10%, respectively) were achieved with NIRS. We also showed that the perfusion stream offers great opportunities for noninvasive, yet frequent process monitoring. Accurate online monitoring of critical process parameters with PAT tools is the essential first step toward increased control of process output.     

Expressing and purifying an anti-atherosclerosis polypeptide vaccine in Escherichia coli

A chimeric polypeptide of TTP-CETPC was successfully expressed as inclusion bodies in Escherichia coli by fusing it with the C-terminus of asparaginase and a basic amino acid-rich peptide (KR). After partially purified by washing with 0.5% (v/v) Triton X-100 in 10 mM PB, the pellet was solubilized in 8 M urea. The solution was precipitated with single volume and double volumes of cold ethanol for removing impurities. The fusion protein in solution was precipitated with triple volumes of ethanol to increase purity and then hydrolyzed with 50 mM hydrochloric acid at 55 degrees C for 72 h. The TTP-CETPC polypeptide was released after the unique acid-labile aspartylprolyl bond in the fusion protein was cleaved by acid. After impurities were removed by adjusting the hydrolysis solution pH to 9.45 and then to 8.37, the TTP-CETPC polypeptide was further purified by DEAE-cellulose column. The TTP-CETPC containing fractions were eluted at 60-80 mM NaCl. The purified TTP-CETPC cysteines were oxidized to form into intermolecular disulfide bonded dimers for immunizing mice. Specific anti-CETP antibodies in mice serum were assayed by ELISA and Western blot to verify that antibodies against CETP had been successfully induced and lasted for more than seventeen weeks in vivo.     

Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide

To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine.     

3D-model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart.     

Growth and multiplication of white skeletal muscle fibres in carp larvae in relation to somatic growth rate

A morphometric analysis of white axial muscle of common carp Cyprinus carpio was undertaken in order to quantify increase in fibre size, fibre nuclei and fibre number in relation to somatic growth rate during early life. In fast-growing carp larvae fed zooplankton, length and height of fibres from the central part of dorsolateral muscle increased at the same rate (0.75) relative to the total length of the larvae during the first 2 weeks of feeding. During this period, the number of nuclei per fibre increased threefold while the number of nuclei per unit fibre surface remained constant. In fast-growing larvae fed a formulated diet, the total cross-sectional area of one epaxial quadrant of white muscle and the total area of white fibres increased at almost the same rate (3.15; 3.23) relative to larval total length during the first 28 days of exogenous feeding. The total number of white fibres increased faster (2.07) relative to the total length of larvae than the mean area of white fibres (1.16). Hyperplasia accounted for 64% of muscle growth in these larvae. The proportion of fibres with a width < 10 μm decreased from 72% at first feeding to 14% 28 days later, while the proportion of fibres with a width >20 μm which was 0% at first feeding increased up to 34% in the same time. The recruitment of new white fibres seemed to be almost the same in the whole muscle quadrant at first feeding and 18 or 28 days later but, 8 days after first feeding, a transient significant recruitment of new fibres was shown at the apex of the myotome. Comparisons between fast- and slow-growing groups of larvae showed that for a given larval total length: (1) the mean width of central white fibres was higher and the proportion of central fibres with a width <10 μm was lower in slow-growing larvae than in fast-growing ones; (2) the total number of white fibres was lower for a higher total cross-sectional area of white muscle in slow-growing larvae than in fast-growing ones. These results suggest that, in Cyprinus carpio larvae, slow-growing conditions are related to a decreased contribution of hyperplasia to muscle growth.     

Early thermal history significantly affects the seasonal hyperplastic process occurring in the myotomal white muscle of <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> juveniles

The effect of early (embryonic and larval) thermal history on subsequent (juvenile) white muscle hyperplasia was studied in a teleost fish, the European sea bass (Dicentrarchus labrax L.). D. labrax, incubated and reared at constant temperatures of 13°C, 15°C or 20°C from the embryonic stage of half epiboly up to 18–19 mm in total length, were transferred to ambient seawater temperature and reared for the subsequent 14 months on commercial feed. The somatic growth of juveniles was linked to annual variations of ambient seawater temperature and inversely related to early rearing temperature, so that, after 14 months, the juveniles originally reared at low temperatures had compensated for the growth retardation experienced during early life. The white muscle growth process of juveniles was quantified after two periods of growth opportunity at ambient seawater temperature (100 and 400 days post-transfer) as well as, in order to follow total-length-dependent effects of early temperature and to discriminate total-length-independent effects of early temperature, on juveniles from the three batches sampled at six successive equivalent total lengths (31–33, 84–88, 141–145, 166–172, 196–206 and 211–220 mm). Our data demonstrate the existence of a seasonal recruitment of new white muscle fibres when seawater temperature increases and of a shrinkage of the largest white muscle fibres during the winter months. The seasonal recruitment of new white muscle fibres occurring in juveniles is linked to their early rearing temperature. Juveniles originating from low temperatures have a higher and longer capacity to recruit new white muscle fibres when seawater temperature increases, supporting their better somatic growth. This finding is discussed in relation to the early (embryonic and larval) myogenic processes of the three populations and is related to their sex ratio. This work was financed by the European Commission, FAIR Program no. PL96–1941 “Muscle Ontogeny in Sea bass and Trout”.     

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, ∼63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.     

Microglia-derived pronerve growth factor promotes photoreceptor cell death via p75 neurotrophin receptor

Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its possible role in degenerative retinal disease. Freshly isolated activated retinal microglia expressed high molecular weight forms of neurotrophins including that of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. Conditioned media from cultured retinal microglia (MGCM) consistently yielded a approximately 32-kDa NGF-reactive band when supplemented with bovine serum albumin (BSA) or protease inhibitors (PI); and promoted cell death that was suppressed by NGF immunodepletion in a mouse photoreceptor cell line (661w). The approximately 32 kDa protein was partially purified (MGCM/p32) and was highly immunoreactive with a polyclonal anti-pro-NGF antibody. Both MGCM/p32 and recombinant pro-NGF protein promoted cell death in 661w cultures. Increased levels of pro-NGF mRNA and protein were observed in the RCS rat model of retinal dystrophy. MGCM-mediated cell death was reversed by p75NTR antiserum in p75NTR(+)/trkA(-) 661w cells. Our study shows that a approximately 32 kDa pro-NGF protein released by activated retinal microglia promoted degeneration of cultured photoreceptor cells. Moreover, our study suggests that defective post-translational processing of NGF might be involved in photoreceptor cell loss in retinal dystrophy.