Claudio Alberto Torres Suazo

Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development

In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered.

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier-based system.

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost‐effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier‐based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM‐MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM‐MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ∼4.82 ± 1.18 × 105 cell mL−1 at day 7, representing a 3.9‐fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP‐compliant large‐scale production system for hMSCs for cellular therapy.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained approximately 1.5-3 x 10(7)cells/mL after 3-4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 microg/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 microg/10(7) cells at days 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 microg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.

Characterization of growth and metabolism of Drosophila melanogaster cells transfected with the rabies‐virus glycoprotein gene

In the present study, the growth and key metabolic features of a gene‐transfected Drosophila melanogaster (fruitfly) S2 (Schneider 2) cell population (S2AcRVGP cells), cultured in Sf900‐II medium, have been evaluated to provide substantial support for the development of a bioprocess to produce RVGP (rabies‐virus glycoprotein). Experimental cultures were grown both in a 100 ml Schott flask incubated in a shaker at 28 °C and 100 rev./min and in a 3 litre stirred‐tank bioreactor at 28 °C, with increasing agitation. In small‐scale culture, S2AcRVGP cells reached a maximum cell concentration of 1.13×107 cell/ml, presented a μmax (maximum specific growth rate) of 0.037 h−1 and the growth was limited by oxygen deprivation. An early and remarkably long stationary phase was observed under hypoxia. Cell cultures grown in the bioreactor without oxygen limitation exhibited a maximum cell concentration of 2.2×107 cells/ml and μmax values as high as 0.048 h−1. The main substrate consumed in order to reach such a high growth rate was the amino acid proline, which seems to play an important role as a source of metabolic energy in the culture of S2AcRVGP cells. Under conditions of hypoxia, the cells were able to survive for 15 h without apparent damage, recovering their previous metabolic activity.

Spatio-temporal resolution enhancement of vocal tract MRI sequences based on image registration

Dynamic magnetic resonance imaging is an emerging technique for studying speech production. Vocal tract image sequences acquired during the speech of words or phonemes allow the identification of shapes taken by the vocal tract during speech production. However, there is no prior knowledge about the spatial and temporal resolution requirements, which are expected to vary depending on the speech task. Available approaches try to enhance resolution by empowering the acquisition devices. However, there are several cost and hardware limitations. In this paper, we propose an alternative approach to enhance spatio-temporal resolution using only digital image processing techniques. We use a previous non-rigid image registration method to identify displacements and deformations among the observed images. Based on a motion compensated interpolation approach, temporal resolution is increased by generating intermediate images which are coherent with the movement present in the observed sequence. Moreover, a MAP-MRF super-resolution image reconstruction approach is used to increase spatial resolution of the whole sequence. Results indicate the effectiveness of our approach.

Performance evaluation of CHO-K1 cell in culture medium supplemented with hemolymph

Desenvolvimento de meios de cultura isentos de soro fetal bovino (SFB) e uma das grandes prioridades de pesquisa em desenvolvimento de processos com celula animal. O objetivo do presente trabalho foi realizar uma analise do potencial de uso da hemolinfa como suplemento do meio utilizado no cultivo da celula animal ancorante CHO-K1. Para isso, foi adicionado 1% v/v de extrato de hemolinfa ao meio DMEM contendo 10% v/v de SFB e 1,0 ou 4,5 g/L de glicose. O cultivo foi realizado em frascos tipo spinner em um ambiente de 10% v/v de CO2, a 37oC, utilizando o microcarregador Cytodex 1. Comparando os resultados obtidos no ensaio com hemolinfa com um sem hemolinfa pode-se notar uma influencia positiva da hemolinfa no cultivo, ja que o ensaio com hemolinfa apresentou uma concentracao maxima de celulas 52% maior e uma produtividade maxima de ate 40% maior.

Three-dimensional noisy image restoration using filtered extrapolation and deconvolution

The problem of restoration in fluorescence microscopy has to deal at the same time with blurring and photon noise. Their combined effects corrupt the image by inserting elements that do not belong to the real object and distort the contrast. This hinders the possibility of using the images for visualization, recognition, and analysis using the three-dimensional data. The algorithms developed to restore the lost frequencies and perform band extrapolation, in general, assume absence of noise or an additive noise. This paper presents a restoration approach through band extrapolation and deconvolution that deals with the noise. An extrapolation algorithm using constraints on both spatial and frequency domains with a smoothing operator were combined with the Richardson-Lucy iterative algorithm. The results of the method for simulated data are compared with those obtained by the original Richardson-Lucy algorithm and also regularized by Total Variation. The extrapolation of frequencies is also analyzed both in synthetic and in real images. The method improved the results with higher signal-to-noise ratio and quality index values, performing band extrapolation, and achieving a better visualization of the 3D structures.

Nomenclature and guideline to express the amount of a membrane protein synthesized in animal cells in view of bioprocess optimization and production monitoring.

Studies of a bioprocess optimization and monitoring for protein synthesis in animal cells face a challenge on how to express in quantitative terms the system performance. It is possible to have a panel of calculated variables that fits more or less appropriately the intended goal. Each mathematical expression approach translates different quantitative aspects. We can basically separate them into two categories: those used for the evaluation of cell physiology in terms of product synthesis, which can be for bioprocess improvement or optimization, and those used for production unit sizing and for bioprocess operation. With these perspectives and based on our own data of kinetic S2 cells growth and metabolism, as well as on their synthesis of the transmembrane recombinant rabies virus glycoprotein, here indicated as P, we show and discuss the main characteristics of calculated variables and their recommended use. Mainly applied to a bioprocess improvement/optimization and that mainly used for operation definition and to design the production unit, we expect these definitions/recommendations would improve the quality of data produced in this field and lead to more standardized procedures. In turn, it would allow a better and easier comprehension of scientific and technological communications for specialized readers.

Transient expression of rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells

The transient transfection process has been developed to allow rapid production of recombinant proteins. In this paper, we describe the transient expression of recombinant rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells. Different cell transfection reagents were evaluated, together with the effects of different cell cultivation procedures on RVGP expression. Yields of RVGP in the range 50-90ng/10(7) cells were obtained in multi-well plate transfection experiments, where it was observed that RVGP expression was linked to the DNA concentration. RVGP expression was 1.3 times higher using 10μg rather than 5μg of DNA. Inhibition of RVGP expression was observed at higher concentrations of DNA, with DNA concentrations above 15μg decreasing RVGP expression 1.5-fold for cells transfected with polyethylenimine (PEI) and 1.6-fold for cells transfected with cationic lipid. The results of shake flask transfection indicated that S2 cells were more effectively transfected in suspension than under static conditions. RVGP yields of 182.2ng/10(7) cells (PEI), 201ng/10(7) cells (calcium phosphate), and 215ng/10(7) cells (cationic lipid) were obtained for S2 cell suspension cultures. The highest volumetric RVGP concentration (309ng/mL) was found for cells transfected with cationic lipid. This value was 1.21 and 1.16 times higher, respectively, than for cells transfected with PEI (253.4ng/mL) and calcium phosphate (237.2ng/mL). There was little effect of transfection on the kinetics of cell growth, with growth rates of 1.12 and 1.19d(-1) for transfected and control cells, respectively. In spinner flasks, the expression of RVGP was 150 and 138ng/10(7) cells for transfection using PEI and calcium phosphate, respectively. A comparison of the different transfection reagents (calcium phosphate, cationic lipid, and cationic polymer) showed no significant differences in RVGP expression when shake flasks were used. Overall, the data indicated that transient expression in D. melanogaster S2 cells is a practical way of synthesizing RVGP for use in structural and functional studies.

Evaluating kinetic and physiological features of rCHO-K1 cells cultured on microcarriers for production of a recombinant metalloprotease/disintegrin

We present kinetic and physiological data regarding the culturing of rCHO-K1 cells on various microcarriers, to evaluate the potential of this culture strategy for mass production of these cells and expression of a recombinant disintegrin. Cultures were performed in 500 mL spinner flasks in DMEM culture medium with 10% v/v fetal calf serum, gently shaken at 37oC, pH 7.4, in a 10% v/v CO 2 atmosphere. The following values were obtained, respectively, for the adhesion time-constant Ka (h) and specific growth rate μ max (d -1 ) on each microcarrier: Cytodex 1 (0.91, 0.45), Cultispher S (0.28, 0.34), Immobasil FS (0.85, 0.52) and Pronectin F (5.12, 0.67). Metabolic characteristics showed some variation among the cultures with the four microcarriers, the most significant being the higher production of ammonia with microcarriers coated with adhesive molecules (Cultispher S and Pronectin F) relative to the uncoated carriers (Cytodex 1 and Immobasil FS). Experiments where the DMEM medium was gradually replaced by the serum-free medium (CHO-SFM-II) revealed important advantages over media containing serum, not only for assay purposes, but also for purification of the disintegrin. Altogether these results demonstrate that cultures on microcarriers, especially on Pronectin F, show good potential for larger scale cultures of rCHO-K1 cell.