Continuous Cell Growth Monitoring Research Articles

Integration of melt electrowritten microfibers with magnetoelastic sensors for continuous monitoring of cell growth

In this study, melt electrowriting (MEW) is used to define and integrate specific topographies on the surface of magnetoelastic sensors, which enable custom control of attachment, distribution, and alignment of cells along the sensor surface.

Using Raman spectroscopy and chemometrics to identify the growth phase of Lactobacillus casei Zhang during batch culture at the single-cell level

BackgroundAs microbial cultures are comprised of heterogeneous cells that differ according to their size and intracellular concentrations of DNA, proteins, and other constituents, the detailed identification and discrimination of the growth phases of bacterial populations in batch culture is challenging. Cell analysis is indispensable for quality control and cell enrichment.MethodsIn this paper, we report the results of our investigation on the use of single-cell Raman spectrometry (SCRS) for real-time analysis and prediction of cells in different growth phases during batch culture of Lactobacillus (L.) casei Zhang. A targeted analysis of defined cell growth phases at the level of the single cell, including lag phase, log phase, and stationary phase, was facilitated by SCRS.Results Spectral shifts were identified in different states of cell growth that reflect biochemical changes specific to each cell growth phase. Raman peaks associated with DNA and RNA displayed a decrease in intensity over time, whereas protein-specific and lipid-specific Raman vibrations increased at different rates. Furthermore, a supervised classification model (Random Forest) was used to specify the lag phase, log phase, and stationary phase of cells based on SCRS, and a mean sensitivity of 90.7% and mean specificity of 90.8% were achieved. In addition, the correct cell type was predicted at an accuracy of approximately 91.2%.Conclusions To conclude, Raman spectroscopy allows label-free, continuous monitoring of cell growth, which may facilitate more accurate estimates of the growth states of lactic acid bacterial populations during fermented batch culture in industry.

Noninvasive online biomass detector system for cultivation in shake flasks

A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake flasks is described. The measurement principle is based on turbidity measurement by detecting 180°‐scattered light and correlation to OD by nonlinear calibration models. The sensor system was integrated into a commercial shaking tablar to read out turbidity from below the shake flasks bottom. The system was evaluated with two model microorganisms, Escherichia coli K12 as prokaryotic and Saccharomyces cerevisiae as eukaryotic model. The sensor allowed an accurate monitoring of turbidity and correlation with OD600 ≤ 30. The determination of online OD showed relative errors of about 7.5% for E. coli K12 and 12% for S. cerevisiae. This matches the errors of the laborious offline OD and thus facilitates to overcome the drawbacks of the classical method as risk of contamination and decreasing volumes through sampling. One major challenge was to ensure a defined, nonvarying measurement zone as the rotating suspension in the shake flask forms a liquid sickle which circulates round the flasks inner bottom wall. The resulting alteration of liquid height above the sensor could be compensated by integration of an acceleration sensor into the tablar to synchronize the sensor triggering.

Differential cytotoxic effects of gold nanoparticles in different mammalian cell lines

Gold nanoparticles (AuNPs) possess unique properties that have been exploited in several medical applications. However, a more comprehensive understanding of the environmental safety of AuNPs is imperative for use of these nanomaterials. Here, we describe the impacts of AuNPs in various mammalian cell models using an automatic and dye-free method for continuous monitoring of cell growth based on the measurement of cell impedance. Several well-established cytotoxicity assays were also used for comparison. AuNPs induced a concentration-dependent decrease in cell growth. This inhibitory effect was associated with apoptosis induction in Vero cells but not in MRC-5 or NIH3T3 cells. Interestingly, cDNA microarray analyses in MRC-5 cells supported the involvement of DNA damage and repair responses, cell-cycle regulation, and oxidative stress in AuNP-induced cytotoxicity and genotoxicity. Moreover, autophagy appeared to play a role in AuNPs-induced attenuation of cell growth in NIH3T3 cells. In this study, we present a comprehensive overview of AuNP-induced cytotoxicity in a variety of mammalian cell lines, comparing several cytotoxicity assays. Collectively, these assays offer convincing evidence of the cytotoxicity of AuNPs and support the value of a systematic approach for analyzing the toxicology of nanoparticles.

Fed‐batch microbioreactor platform for scale down and analysis of a plasmid DNA production process

The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well.

Abstract 4536: Expression and cellular activation of peroxisome proliferator-activated receptor γ in granulosa cell tumors

Abstract Granulosa cell tumors (GCT) of the ovary are rare, hormonally-active neoplasms characterized by endocrine manifestations, an indolent course and late relapse. Chemotherapy and hormonal therapy have proved to be of limited efficacy. Nuclear receptors (NR) have a central pathogenic role in endocrine malignancy and present as potential targets for therapeutic intervention. NR have established roles in granulosa cell biology but their roles in GCT remain largely unexplored. In order to systematically examine the NR family in GCT, we used ABI Taqman® Low Density Array microfluidic cards to analyse 14 GCT and two GCT-derived cell lines for expression of the 48 NR. The levels of expression were remarkably consistent across the GCT. We found that peroxisome proliferator-activated receptor gamma (PPARγ) had greater than ten-fold absolute expression when compared with either the NCBI tumour or brain reference RNA pools. PPARγ agonists are regarded as potential therapeutic agents in the treatment of inflammatory diseases and certain cancers. Given the high expression levels of PPARγ in GCT, we investigated whether the use of PPARγ and/or retinoid × receptor (RXR) agonists or antagonists have an effect on the GCT-derived cell lines, KGN and COV434. We used real-time cell analysis (RTCA) to investigate the cytotoxicity of the PPARγ agonist, troglitazone, and the RXR agonist, 9-cis-retinoic acid, by continuous monitoring of cell growth, proliferation and viability in the GCT-derived cell lines in real-time. We observed that a combination of troglitazone and 9-cis-retinoic acid significantly inhibited cell proliferation in the cell lines (56% compared to control). Because we have previously reported that steroid receptors are transrepressed in these GCT cells due to constitutive activation of the NF-κB signalling pathway, we tested the effect of troglitazone and 9-cis-retinoic acid in conjunction with a NF-κB inhibitor, BAY-117082. We observed a further decrease in cell proliferation (85% compared to control). This was a PPARγ-mediated effect as it was reversed by co-administration with the PPARγ antagonist, GW9662. The decrease in proliferation was due to cellular apoptosis. We also investigated whether PPARγ is transcriptionally active in these cells using a reporter construct, specific for PPARγ, and observed that GCT cells were not responsive to either PPARγ agonists or antagonists in vitro. This was found to be due to NF-κB transrepression; when NF-κB was inactivated, PPARγ signaling was restored. We conclude that, while the use of PPARγ agonists may have potential as therapeutic agents for treating GCT, a combination of therapies involving the abrogation of NF-κB signaling may be of greater efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4536. doi:10.1158/1538-7445.AM2011-4536