Growing Cell Cultures Research Articles

Hybrid-integrated devices for mimicking malignant brain tumors ("tumor-on-a-chip") for in vitro development of targeted drug delivery and personalized therapy approaches.

Acute and requiring attention problem of oncotheranostics is a necessity for the urgent development of operative and precise diagnostics methods, followed by efficient therapy, to significantly reduce disability and mortality of citizens. A perspective way to achieve efficient personalized treatment is to use methods for operative evaluation of the individual drug load, properties of specific tumors and the effectiveness of selected therapy, and other actual features of pathology. Among the vast diversity of tumor types-brain tumors are the most invasive and malignant in humans with poor survival after diagnosis. Among brain tumors glioblastoma shows exceptionally high mortality. More studies are urgently needed to understand the risk factors and improve therapy approaches. One of the actively developing approaches is the tumor-on-a-chip (ToC) concept. This review examines the achievements of recent years in the field of ToC system developments. The basics of microfluidic chips technologies are considered in the context of their applications in solving oncological problems. Then the basic principles of tumors cultivation are considered to evaluate the main challengers in implementation of microfluidic devices, for growing cell cultures and possibilities of their treatment and observation. The main achievements in the culture types diversity approaches and their advantages are being analyzed. The modeling of angiogenesis and blood-brain barrier (BBB) on a chip, being a principally important elements of the life system, were considered in detail. The most interesting examples and achievements in the field of tumor-on-a-chip developments have been presented.

Genome replication in asynchronously growing microbial populations.

Biological cells replicate their genomes in a well-planned manner. The DNA replication program of an organism determines the timing at which different genomic regions are replicated, with fundamental consequences for cell homeostasis and genome stability. In a growing cell culture, genomic regions that are replicated early should be more abundant than regions that are replicated late. This abundance pattern can be experimentally measured using deep sequencing. However, a general quantitative theory linking this pattern to the replication program is still lacking. In this paper, we predict the abundance of DNA fragments in asynchronously growing cultures from any given stochastic model of the DNA replication program. As key examples, we present stochastic models of the DNA replication programs in budding yeast and Escherichia coli. In both cases, our model results are in excellent agreement with experimental data and permit to infer key information about the replication program. In particular, our method is able to infer the locations of known replication origins in budding yeast with high accuracy. These examples demonstrate that our method can provide insight into a broad range of organisms, from bacteria to eukaryotes.

High purity and integrity RNA from human cell lines stored in liquid nitrogen for over 20 years.

Biobanks are a key resource for obtaining human cell lines for biomedical research, including for drug development projects. Such projects often include comparative RNA-sequencing of large panels of human cell lines from individuals affected by certain disorders and healthy controls, or from individuals with different drug response phenotypes. RNA extractions are typically done from growing cell cultures, a process that may take several weeks. However, maintaining large numbers of cell lines in parallel increases the project workload. Here, we show that extracting RNAs directly from frozen vials of human cell lines stored for over 20 years in a liquid nitrogen freezer yields RNAs with the high purity and integrity parameters that conform to those required for optimal RNA-sequencing and are closely similar to those obtained for RNAs extracted from growing human cell lines.

Topological flow transformations in a universal vortex bioreactor

Research into the flow structure in an aerial vortex bioreactor is relevant for developing the methods to grow cell cultures. It is especially important in the case when, with the culture growth in the bioreactor, such parameters of the medium as density and viscosity can significantly change, accordingly altering the characteristic flow regimes. Since the cultivated culture is not transparent in most cases, it is impossible to visually determine the flow regime. Therefore, a detailed study of the regularities of flow regimes in an aerial vortex reactor is of great fundamental and applied interest. The research was carried out in an 8.5 L universal glass aerial vortex bioreactor, with a washer freely floating on its surface and stabilizing the motion of the working fluid. The regularities of the vortex motion of the culture medium depend on its volume and the rotation intensity of the activator generating vortex motion in the air. The air vortex generated by the impeller (activator) above the liquid surface spins the working fluid and a free-floating washer. The study reveals that despite the complex configuration of the flow stabilizing device (a free-floating washer), the observed vortex structure and its dynamics with increasing flow swirl intensity coincides with the structure of a confined vortex flow in a cylindrical container for both single-fluid and immiscible two-fluid configurations.

Experimental study of the topological flow transformations in an aerial vortex bioreactor with a floating washer.

Research into the flow structure in an aerial vortex bioreactor is relevant for developing the methods for growing cell cultures. Determining the optimal cultivation condition for a certain process is especially important in the case when such parameters of the medium as density and viscosity significantly change with the culture growth in the bioreactor. The research of the flow dynamic was carried out in an 8.5 L universal aerial vortex bioreactor, with a washer freely floating on its surface and stabilizing the motion of the working fluid. The regularities of the vortex motion of the culture medium have been determined by Particle Image Velocimetry depending on its volume and the intensity of rotation of the activator, generating vortex motion in the air. The observed vortex structure and its dynamics at increasing flow swirl intensity are established to coincide with the structure of a confined vortex flow in a cylindrical container with no washer for both single and two-fluid configurations. This novel methodology of the flow optimization shows that an ascending swirling jet forms in the vicinity of the bioreactor axis, and a bubble-like vortex breakdown forms in the axial region. This article is protected by copyright. All rights reserved.

Abstract 192: A novel and rapid patient-derived organoid breast cancer platform for precision medicine

Abstract An increasing number of studies performing correlative drug screens on patient-derived organoids (PDO) are revealing enormous potential for these models in predicting patient response to therapy. Despite this, their future use in a clinical setting is hindered by intrinsic limitations of PDO models, namely low success rates in establishing growing cell cultures from tumor tissue samples and long return times for drug response data that fall outside timescales of clinical actionability. To address these limitations, we leveraged technological advances in emulsion-based microfluidics and droplet generators to develop MicroOrganoSpheres (MOS) - microscale environments containing tumorspheres that retain structural, cellular, and genetic properties of an individual patient’s diseased tissue. Here, we focused on generating MOS from breast cancer tissue biopsies and resections across different subtypes of breast cancer. We established over 30 unique patients’ breast MOS samples at an average of 80% success, with the majority of these samples being hormone receptor positive (ER+/PR+) - the most clinically lethal and challenging subtype to establish. We evaluated MOS dose-responses across several first line standard of care chemotherapies for breast cancer. For this, we created a semi-automated workflow paired with high-content imaging for quantifying dose responses and tracking longitudinal cell dynamics during dosing. We generated dose response data for MOS samples, being able to move from primary tissue samples to MOS to drug dosing data within a clinically relevant timescale of 2-3 weeks. We conclude that our platform demonstrates the feasibility of 1) efficiently establishing MOS from breast cancer patient tumor samples from different subtypes and 2) performing drug dosing studies on these samples with improved turnaround times that enable clinical actionability for the patient. Together, these findings provide a foundation for evaluating this technology as a diagnostic tool in future clinical settings. Citation Format: David M. Graham, Gabrielle Rupprecht, Eric D. Bankaitis, Jeremy M. Force, Wylie Watlington, Steven W. Metzger, Xiling Shen, David Hsu. A novel and rapid patient-derived organoid breast cancer platform for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 192.

Preclinical Models of Low-Grade Gliomas.

Diffuse infiltrating low-grade glioma (LGG) is classified as WHO grade 2 astrocytoma with isocitrate dehydrogenase (IDH) mutation and oligodendroglioma with IDH1 mutation and 1p/19q codeletion. Despite their better prognosis compared with glioblastoma, LGGs invariably recur, leading to disability and premature death. There is an unmet need to discover new therapeutics for LGG, which necessitates preclinical models that closely resemble the human disease. Basic scientific efforts in the field of neuro-oncology are mostly focused on high-grade glioma, due to the ease of maintaining rapidly growing cell cultures and highly reproducible murine tumors. Development of preclinical models of LGG, on the other hand, has been difficult due to the slow-growing nature of these tumors as well as challenges involved in recapitulating the widespread genomic and epigenomic effects of IDH mutation. The most recent WHO classification of CNS tumors emphasizes the importance of the role of IDH mutation in the classification of gliomas, yet there are relatively few IDH-mutant preclinical models available. Here, we review the in vitro and in vivo preclinical models of LGG and discuss the mechanistic challenges involved in generating such models and potential strategies to overcome these hurdles.

Growing cell cultures of some rare local species of medicinal plants

Growing cell cultures of some rare local species of medicinal plants

Structural Characterization of Polycrystalline Titania Nanoparticles on C. striata Biosilica for Photocatalytic POME Degradation.

The biosilica shell of marine diatoms has emerged as a unique matrix for photocatalysis, owing to its sophisticated architecture with hierarchical nanopores and large surface area. Although the deposition of titania nanoparticles on diatom biosilica has been demonstrated previously, their photocatalytic activity has been tested only for degradation of pure compounds, such as dyes, nitrogen oxide, and aldehydes. The efficiency of such photocatalysts for degradation of mixtures, for instance, industrial wastewaters, is yet to be investigated. Furthermore, reports on the lattice structures and orientation of nanotitania crystals on biosilica are considerably limited, especially for the underexplored tropical marine diatoms. Here, we report an extensive characterization of titania-loaded biosilica from the tropical Cyclotella striata diatom, starting from freshly grown cell cultures to photodegradation of wastewaters, namely, the palm oil mill effluent (POME). As Indonesia is the largest palm oil producer in the world, photocatalytic technology could serve as a sustainable alternative for local treatment of POME. In this study, we achieved a 54% loading of titania on C. striata TBI strain biosilica, as corroborated by XRF analyses, which was considerably high compared to previous studies. Through visualization using HR-TEM, supported by SAED and XRD analyses, nanocrystal TiO2 appeared to be trapped in an anatase phase with polycrystalline characteristics and distinct crystallographic orientations. Importantly, the presence of C. striata biosilica lowered the band gap of titania from 3.41 eV to around 3.2 eV upon deposition, enabling photodegradation of POME using a broad-range xenon lamp as the light source, mimicking the sunlight. Kinetic analyses revealed that POME degradation using the photocatalysts followed quasi-first-order kinetics, in which the highest titania content resulted in the highest photocatalytic activity (i.e., up to 47% decrease in chemical oxygen demand) and exhibited good photostability throughout the reaction cycles. Unraveling the structure and photoactivity of titania-biosilica catalysts allows transforming marine diatoms into functional materials for wastewater photodegradation.

Characterisation of induced Malus x domestica ‘Royal Gala’ cell differentiation by using different hormones in cell cultures

ABSTRACT Fruit development, following pollination, involves a cell division stage, followed by cell expansion . A major challenges to study each individual stages is the limitations imposed by analyses of fruit development on the plant. Rapid growing cell culture systems that differentiate could be used to identify and understand the molecular control of each of these processes in vitro. In this study, an apple cell culture was initiated from the widely characterised ‘Royal Gala’ cultivar. The established apple cell culture was used to develop a screening system to assess the effect of different hormones on cell differentiation, which then were characterised at molecular and cellular levels. Three different hormone pairs were screened to determine the optimal for cell expansion on actively dividing cells . Using this strategy, we were able to reproduce, in vitro, the first two stages of apple fruit cell differentiation, cell division and expansion, which was correlated with changes in genes expression of specific markers for those two stages, a cyclin-dependent kinase gene (CDKB2;2) and an expansion gene (EXP3). The cell culture provides a unique tool and opportunity to study the effect of plant hormones on cell differentiation and will allow to decipher the molecular mechanism of apple fruit development irrespective of y.

Green synthesized nano-cellulose polyethylene imine-based biological membrane

In hemodialysis process, membrane serves as a barrier between blood and the dialysate. The barrier when contacted by blood accompanied activation of coagulation, immunity, and cellular passageways. In the recent years, hemodialysis membrane's biocompatibility has become a issue which leads to reduce the performance during the separation process. In previous work, we developed and evaluated a cellulose-based membrane blended with polyaziridine or polyetyleneimine in formic acid for hydrophilicity, pure water flux, surface morphology, and permeation efficiency. Biocompatibility was accessed, by conducting cellular viability and cellular attachments tests. In this study, the membrane compared to a non-treated control, and cell viability revealed active and growing cell cultures after 14 days. During the cellular attachment experiment, cell cultures attached to the fabricated membrane simulated the formation of cell junctions, proving that the membrane is non-toxic and biocompatible. CA + PEI + FA membrane tested with a blood mimic fluid having density identical to renal patient's blood. The BSA concentration in the feed solution was the same as that in the blood of the renal patient. The results revealed that the CA + PEI + FA membrane was able to reject 99% bovine serum albumin (BSA) and 69.6% urea. Therefore, from biocompatibility and blood mimic fluid testing, it is confirmed that the CA + PEI + FA membrane is the finest implant for dialysis applications.

Isolation of Aged Yeast Cells Using Biotin-Streptavidin Affinity Purification.

Yeast (Saccharomyces cerevisiae) has been used as one of the main model systems for studying molecular mechanisms underlying cellular aging. A major technical challenge in studying aging in yeast is the isolation of aged cells from exponentially growing cell cultures, since aged cells in such cultures are rare. Several methods for isolating aged cells have been developed to achieve this. Here, we describe a biotin-streptavidin affinity purification protocol for isolating aged yeast cells. It consists of three main steps: biotinylation of yeast cells, culturing cells to the desired age, and harvesting the aged cells using streptavidin-coated magnetic microbeads. The isolated aged cells can be used for microscopy, biochemistry, or molecular biology analysis.

Mechanical, physical–chemical and biological properties of the new Ti–30Nb–13Ta–5Zr alloy

A technology was developed to obtain homogeneous Ti–30Nb–13Ta–5Zr alloy. The alloy was shown to consist of a pure β-crystalline phase and possesses requisite mechanical properties. The alloy surface exhibits micro- and nanoscale structures. In terms of the ability to generate reactive oxygen species and long-lived reactive protein species, Ti–30Nb–13Ta–5Zr alloy is more favorable compared to nitinol. A high mitotic index (2%) and low content of non-viable cells (< 5%) were observed when growing cell cultures on Ti–30Nb–13Ta–5Zr alloy scaffolds. Cells readily attach and spread over the alloy surface. The biocompatibility of Ti–30Nb–13Ta–5Zr was confirmed by the results of implantation of the alloy in the form of plates or looped wire. Surface morphology of the samples showed no significant changes after biological testing. It can be assumed that Ti–30Nb–13Ta–5Zr is a material potentially suitable for use in medicine.

Increasing Biomanufacturing Yield with Bleed-Feed: Optimal Policies and Insights

Problem definition: Bleed-feed is a novel technology that allows biomanufacturers to skip intermediary bioreactor setups. However, the specific time at which bleed-feed is performed is critical for success. The process is stringently regulated, and its implementation involves unique trade-offs in operational decision-making. Our analysis formalizes the operational challenges related to bleed-feed decisions, and our results inform biomanufacturers and policy-makers on the potential impact of this technology on current practice. Academic/Practical Relevance: Operations Management (OM) methodologies have not yet been widely adopted in the biomanufacturing industry. This research presents one of the first attempts to demonstrate how OM can complement biomanufacturing to improve operational decisions. We present a practically-relevant problem and a rigorous solution approach that is relevant to both OM and biomanufacturing communities. Methodology: We develop a finite-horizon, discrete-time Markov decision process, and analyze the structural characteristics of optimal bleed-feed policies. Results: We show that optimal bleed-feed policies have a two-way control-limit structure under mild conditions that were validated with industry data. Unlike common industry opinions, our analysis reveals that regulatory requirements do not necessarily limit the potential benefits of bleed-feed. Managerial Implications: Our analysis (supported with an industry case study) shows that bleed-feed implementation can provide benefits. Slow growing and/or high-risk cell cultures benefit the most from the use of the bleed-feed technology. Real-world test runs at MSD (using one bleed--feed) resulted in 85% improvement in the batch yield.

Near-Physiological Cell Cycle Synchronization with Countercurrent Centrifugal Elutriation.

The bioreactor conditions and cell diversity in mammalian cell cultures are often regarded as homogeneous. Recently, the influence of various kinds of heterogeneities on production rates receives increasing attention. Besides spatial gradients within the cultivation system, the variation between cell populations and the progress of the cells through the cell cycle can affect the dynamics of the cultivation process. Strong metabolic up- and down-regulations leading to variable productivities, even in exponentially growing cell cultures, have been identified in CHO cell cultivations. Consequently, scientific studies of cell cycle-related effects and metabolic regulations require experiments utilizing cell cycle-enriched subpopulations. Importantly, the enrichment procedure itself must not strongly interfere with the cell culture under investigation. Such subpopulations can be generated by near-physiological countercurrent centrifugal elutriation, which is described in the following chapter. At first, a brief overview regarding the cell cycle, currently identified effects and commonly used methods, and their applicability is outlined. Then, the experimental setup and the synchronization itself are explained.

The Sustained Induction of c-MYC Drives Nab-Paclitaxel Resistance in Primary Pancreatic Ductal Carcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. IMPLICATIONS: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal.

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell-cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an "enhanced" strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 μM AI-2 to growing cell cultures, we determine that this "enhanced" AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.

Isolation of Bacterial RNA from Foods Inoculated with Pathogens.

One strategy bacteria use to acclimate to changing environmental conditions is modulation of gene expression. Alterations in gene expression are indicative of activation or repression of certain physiological responses. In order to understand which genetic responses are utilized to cope with various environmental conditions by analyzing transcriptomes, obtaining RNA of high quality, yield, and integrity is paramount. Here, we describe an acid phenol-chloroform method employed to extract RNA from laboratory grown cell cultures, as well as cultures inoculated onto complex matrices such as lettuce and cold-smoked salmon. The method results in high-quality RNA, which can be used for various downstream processes such as cDNA library construction, RNA sequencing, real-time quantitative PCR, and northern analysis. Extraction of RNA from bacterial foodborne pathogens in conjunction with transcriptome sequencing is a useful technique to elucidate pathogens' transcriptional responses.

Differential gene expression of a feed-spiked super-producing CHO cell line

Feed supplements are concentrated cell culture media that contain a variety of nutrients, which can be added during a bioprocess. During fed-batch cultivation, feed media are typically added to a growing cell culture to maximize cell and product concentrations. In this study, only a single shot of feed medium was added on day 0 to a basal cell culture medium and compared to non-supplemented basal medium (feed-spiked at day 0 versus control experiments) by cultivation of a recombinant mAb expressing CHO cell line in batch mode under controlled conditions in a bioreactor. Since the feed-spike at day 0 was based on existing medium components without introducing additional supplements, a desirable process with decreased complexity was generated. Unlike cells in basal medium, feed-spiked cultures reached almost 2× higher peak cell concentrations (10 × 106 c/mL vs. 18 × 106 c/mL) and 3× higher antibody concentrations (0.8 g/L vs. 2.4 g/L). Batch process time and the integral over the viable cell count were similar for both process types. Constantly high cell-specific production rates in feed-spiked cultures (70 pg/cell/day) compared to continuously declining rates in basal medium (from 70 to 10 pg/cell/day) were responsible for an overall 70% higher cell-specific production rate and the higher product concentrations. To associate gene expression patterns to different process proceedings, transcriptome analysis was performed using microarrays. Several transcripts that are involved with glutamine de novo synthesis and citric acid cycle were significantly upregulated on several days in feed-spiked cultures. The top identified gene ontology (GO) terms related well to cell cycle and primary metabolism, cellular division as well as nucleobase formation or regulation, which indicated a more active proliferative state for feed-spiked cultures. KEGG biochemical pathway analysis and Gene set enrichment analysis (GSEA) further confirmed these findings from a complementary perspective. Moreover, several interesting gene targets, which have not yet been associated with recombinant protein expression, were identified that related to a higher proliferative state, growth, protein synthesis, cell-size control, metabolism, cell survival as well as genes that are associated with the control of the mammalian target of rapamycin (mTOR) in feed-spiked cultures. Analysis of critical product quality attributes (i.e. glycosylation, charge variants and size distribution) showed that feed-spiking did not change antibody quality.

Impacts of hormonal elicitors and photoperiod regimes on elicitation of bioactive secondary volatiles in cell cultures of Ajuga bracteosa

Light is an important physical factor necessary for the growth, morphogenesis and production of bioactive compounds in plants. In this study, effects of different photoperiod regimes and hormonal elicitors were investigated on the accumulation of biomass, antioxidant potential and biosynthesis of secondary volatiles in the cell cultures of Ajuga bracteosa. Maximum accumulation of biomass (13.2 g/L) was recorded in cell cultures established at 1.0 mg/L benzylaminopurine (BA) in exposure to continuous dark. Biochemical assays showed that in the presence of 0.5 methyl jasmonate (Me-J), cell cultures grown under continuous dark had the higher activities of superoxide dismutase (SOD: 4.5 U/mg), peroxidase (POD: 3.1 U/mg), total phenolic content (TPC: 8.1 mg GAE/g of DW) and total flavonoid content (TFC: 5.2 mg QE/g of DW) respectively. Nonetheless, the free radical scavenging activity (FRSA) was found correlated with the phenyl ammonia lyase (PAL) activity in the dark grown cell cultures. Analysis through gas chromatography-mass spectrometry (GC–MS) showed, biosynthesis of 29 compounds in the in vitro raised cell cultures. The major identified compounds consisted of monoterpene hydrocarbons such as β-pinene (2.1–9.5%), β-ocimene (1.4–8.3%), 1-terpinene-4-ol (5.8–9.6%), caryophyllene (1.3–6.2%), β-farnesene (0.82–7.8), oxygenated monoterpenes including myrtenal (2.2–8.4%), citronellyl acetate (2.1–7.3%) and sesquiterpenes such as caryophyllene oxide (1.5–5.5) and β-elemene (2.2–8.8%). This protocol has the potential for commercial production of important secondary volatiles.