Development of an N-1 perfusion medium to intensify seed train operation

Seed train expansion of cells before the final production step is often time-consuming and a major source of process variability. For the intensification of seed train operations there are several opportunities discussed across the biopharma industry today. One of the possibilities is to operate N-1 bioreactors in perfusion mode to shorten timelines and improve bioreactor utilization. In this work, we investigated the influence of using an expansion medium especially designed for N-1 perfusion to gain optimal results in the whole manufacturing campaign. For screening and designing an N-1 perfusion expansion medium, a scale down model which represents a typical production campaign, including the seed train, was established. Expansion medium prototypes were combined with different production media in the final production step, e.g. EX-CELL® Advanced HD Perfusion as medium designed for high-density perfusion, and Cellvento® 4CHO Medium and 4FEED as exemplary fed-batch process. After determining a suitable expansion medium formulation, the prototype was evaluated for solubility and streamlined to ensure a simple hydration and robust supply chain. Afterwards, results were confirmed using a simulated manufacturing process using benchtop bioreactor systems, showing that the positive impact of the expansion medium on the final yield is present both when using perfusion or fed-batch as final production stage. Our results indicate that using the right companion medium in seed train expansion - specifically designed for the purpose - can prepare the cells optimally for the final N-stage and increase productivity while using low CSPRs. Combining these findings with the application of a perfused N-1 step in the manufacturing campaign leads to a great opportunity for the intensification of the whole upstream process

Upstream process intensification using frozen high cell density intermediates

Typical seed train operations start by thawing of a single vial followed by several expansion steps. Reaching sufficient absolute cell numbers for production bioreactor inoculation is time-consuming and reduces plant flexibility. Besides long ramp up times, open cell culture operations are a major source of process variability. High cell density cryopreservation (HCDC) is a method of freezing cells in bags instead of vials and at higher cell densities. This offers the advantage of decoupling expansion and production: both steps can be separated in space and time. Room classification could be decreased due to fully closed processing and reproducibility increased due to a reduction of manual handling steps. Furthermore, these frozen seed train intermediates allow global distribution from a central expansion facility to decentralized global production facilities. Besides from advantages in production, these HCDC bags can be used in process development to ensure equal starting points in experimental setups. In this study, we developed a single-use bag assembly that supports closed filling, freezing, thawing, and inoculation. Before using the bag application, relevant parameters for this process from filling to inoculation were evaluated in vials with different cell lines. We found that the DMSO concentration for optimal freezing must not be higher than 7,5%. Furthermore, direct freezing at -80 °C instead of using a controlled rate freezing method is possible. Maximum concentration of DMSO in cell cultures should not be higher than 0,5 % when cryopreserved cells in bags are used for inoculation. For the idea of seed train intensification, we tested increasing freezing cell densities from 10 to 100 million cells/mL showing comparable growth. Functionality test of this HCDC method in comparison to vials was demonstrated in 4,2 L bioreactors simulating a manufacturing process. Applicability of this cryopreservation technology has been demonstrated using different bioreactors, perfusion systems, and various CHO cell lines

Simplifying the analysis of software design variants with a colorful alloy

Formal modeling and automatic analysis are essential to achieve a trustworthy software design prior to its implementation. Alloy and its Analyzer are a popular language and tool for this task. Frequently, rather than a single software artifact, the goal is to develop a full software product line (SPL) with many variants supporting different features. Ideally, software design languages and tools should provide support for analyzing all such variants (e.g., by helping pinpoint combinations of features that could break a property), but that is not currently the case. Even when developing a single artifact, support for multi-variant analysis is desirable to explore design alternatives. Several techniques have been proposed to simplify the implementation of SPLs. One such technique is to use background colors to identify the fragments of code associated with each feature. In this paper we propose to use that same technique for formal design, showing how to add support for features and background colors to Alloy and its Analyzer, thus easing the analysis of software design variants. Some illustrative examples and evaluation results are presented, showing the benefits and efficiency of the implemented technique.This work is financed by the ERDF - European Regional Development Fund - through the Operational Programme for Competitiveness and Internationalisation - COMPETE 2020 - and by National Funds through the Portuguese funding agency, FCT - Fundação para a Ciência e a Tecnologia, within project POCI-01- 0145-FEDER-016826. The third author was also supported by the FCT sabbatical grant with reference SFRH/BSAB/143106/2018

Connexin30.2:<i>In vitro</i> interaction with connexin36 in hela cells and expression in AII amacrine cells and intrinsically photosensitive ganglion cells in the mouse retina

Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36 – both expressed in AII amacrine cells – are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners

AII amacrine cells discriminate between heterocellular and homocellular locations when assembling connexin36-containing gap junctions

Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (C×36) is the most abundant isoform; however, the mechanisms underlying formation of C×36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native C×36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-C×36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of C×36-EGFP clusters on an AII cell in the KO-C×36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced - as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling C×36. We suggest that employing different gapjunction- forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin

Differential Distribution of Retinal Ca2+/Calmodulin-Dependent Kinase II (CaMKII) Isoforms Indicates CaMKII-β and -δ as Specific Elements of Electrical Synapses Made of Connexin36 (Cx36)

AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to ON cone bipolar cells to enable scotopic vision. Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions underlie a remarkable degree of plasticity and are modulated by different signaling cascades. In particular, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling in AII cells. However, it is unclear which CaMKII isoform mediates this effect. To obtain a more detailed understanding of the isoform composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution of CaMKII isoforms: CaMKII-α was strongly expressed in starburst amacrine cells, which are known to lack electrical coupling. CaMKII-β was abundant in OFF bipolar cells, which form electrical synapses in the outer and the inner retina. CaMKII-γ was diffusely distributed across the entire retina and could not be assigned to a specific cell type. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and the four CaMKII isoforms and revealed that the composition of the CaMKII enzyme differs between gap junctions in the outer and the inner retina: in the outer retina, only CaMKII-β colocalized with Cx36-containing gap junctions, whereas in the inner retina, CaMKII-β and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and the outer retina may be regulated differently although they both contain the same connexin. Taken together, our study identifies CaMKII-β and -δ as Cx36-specific regulators in the mouse retina with CaMKII-δ regulating the primary rod pathway

Expression Pattern of Kv11 (Ether à-go-go-Related Gene; erg) K+ Channels in the Mouse Retina

In response to light, most retinal neurons exhibit gradual changes in membrane potential. Therefore K+ channels that mediate threshold currents are well-suited for the fine-tuning of signal transduction. In the present study we demonstrate the expression of the different Kv11 (ether-à-go-go related gene; erg) channel subunits in the human and mouse retina by RT PCR and quantitative PCR, respectively. Immunofluorescence analysis with cryosections of mouse retinae revealed the following local distribution of the three Kv11 subunits: Kv11.1 (m-erg1) displayed the most abundant expression with the strongest immunoreactivity in rod bipolar cells. In addition, immunoreactivity was found in the inner part of the outer plexiform layer (OPL), in the inner plexiform layer (IPL) and in the inner segments of photoreceptors. Immunoreactivity for Kv11.2 (m-erg2) was observed in the outer part of the OPL and throughout the IPL. Double-labeling for vGluT1 or synaptophysin indicated a mainly presynaptic localization of Kv11.2. While no significant staining for Kv11.3 (m-erg3) was detected in the neuronal retina, strong Kv11.3 immunoreactivity was present in the apical membrane of the retinal pigment epithelium. The different expression levels were confirmed by real-time PCR showing almost equal levels of Kv11.1 and Kv11.2, while Kv11.3 mRNA expression was significantly lower. The two main splice variants of Kv11.1, isoforms a and b were detected in comparable levels suggesting a possible formation of cGMP/cGK-sensitive Kv11.1 channels in photoreceptors and rod bipolar cells. Taken together, the immunohistological results revealed different expression patterns of the three Kv11 channels in the mouse retina supposing distinct physiological roles