Cell Growth Experiments Research Articles

Macrophages enhance cisplatin resistance in gastric cancer through the transfer of circTEX2.

Cisplatin-based chemotherapy is often used in advanced gastric cancer (GC) treatment, yet resistance to cisplatin may lead to treatment failure. Mechanisms underlying cisplatin resistance remain unclear. Recent evidence highlighted the role of macrophages in cancer chemoresistance. Macrophage-derived exosomes were shown to facilitate intercellular communication. Here, we investigated the cisplatin resistance mechanism based on macrophage-derived exosomes in gastric cancer. Cell growth and apoptosis detection experiments revealed that M2-polarized macrophages increased the resistance of GC cells to cisplatin. qRT-PCR, RNAase R assay, actinomycin D assay and cell nucleo-cytoplasmic separation experiments confirmed the existence of circTEX2 in macrophage cytoplasm, with a higher expression level in M2 macrophages than that in M1 macrophages. Further experiments showed that circTEX2 acted as microRNA sponges for miR-145 and regulated the expression of ATP Binding Cassette Subfamily C Member 1 (ABCC1). Inhibition of the circTEX2/miR-145/ABCC1 axis blocked the cisplatin resistance of gastric cancer induced by M2 macrophages, as evidenced by invitro and invivo experiments. In conclusion, our research suggests that the exosomal transfer of M2 macrophage-derived circTEX2 enhances cisplatin resistance in gastric cancer through miR-145/ABCC1. Additionally, communication between macrophages and cancer cells via exosomes may be a promising therapeutic target for the treatment of cisplatin-resistant gastric cancer.

MiR-598-5p inhibits breast cancer tumor growth and lung metastasis by targeting PPAPDC1A.

This study aimed to explore the effects of PPAPDC1A on the malignant phenotype of breast cancer (BC) in vivo and in vitro. PPAPDC1A expression was examined in BC tissues and cell lines by real-time polymerase chain reaction and Western blot. In this article, cell proliferation was evaluated by Cell Counting Kit-8 assay and colony formation assay, and cell migration and invasion were evaluated by wound healing assay and transwell assays. Furthermore, in vivo cell growth and pulmonary metastasis experiments were also performed using nude mice. The results showed that compared with normal tissues and cells, the PPAPDC1A expression in BC tissues and cell lines were both significantly increased. The PPAPDC1A targeting sequence significantly inhibited the PPAPDC1A expression and cell proliferation, migration, and invasion. The results of xenograft showed that knockdown of PPAPDC1A inhibited tumor growth and lung metastasis of BC. Then, the Dual-Luciferase Reporter Assay confirmed that miR-598-5p targeted the regulation of PPAPDC1A expression. In addition, the miR-598-5p expression in BC tissues was lower than that in the normal tissues. The rescue experiment showed that PPAPDC1A overexpression reversed the inhibitory effect of miR-598-5p mimic on cell proliferation, migration, and invasion. In conclusion, PPAPDC1A was highly expressed in BC tissues and cell lines, and miR-598-5p inhibited the malignant phenotype of BC by targeting PPAPDC1A.

Fabrication of a Patterned Scaffold Using Soft Lithography Technique to be Used in Cell Growth Applications.

In recent years, within tissue engineering, cell growth on patterned surfaces have gained significant attention. Growing cells in patterns is important to manufacture polymeric tissues that can be used within the medical field. For this reason, the main focus of this study was to prepare patterned scaffolds using Titanium (Ti) and polyvinyl chloride (PVC) covered on microscope lamellas and examine their liability for cell growth. A polydimethylsiloxane stamp was initially prepared which was then used to transfer a predefined pattern onto PVC- and Ti-covered surfaces. Cell growth experiments were performed on the prepared materials by seeding L929 mouse fibroblasts. The growth of cells seeded on the surface of the scaffolds were spectroscopically followed using Neutral Red uptake assay. The results showed cell proliferation on both patterned surfaces, however, it was higher on Ti-covered samples. In addition, three different alkanethiols were tested for cell adhesion on patterned surfaces. A higher number of cell proliferation was observed with undecanethiol, which has a shorter alkane group among them. The morphological properties of the samples before and after cell-seeding were analyzed via scanning electron microscope and optical microscopy. Significant amount of cell proliferation was observed on all of the prepared samples.

S5.3a Unraveling the genetic determinants of virulence in Cryptococcus neoformans

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PM Cryptococcus neoformans is a human pathogenic basidiomycete yeast that can cause cryptococcal meningitis (CM), predominantly in immunocompromised individuals. The patient outcome depends on both host and pathogen-specific factors, including C. neoformans genetics. A groundbreaking 2012 study was the first to show that patient outcome is associated with genetic differences between C. neoformans isolates. Subsequent population-wide sequencing studies have revealed over 100 sequence types (ST) of C. neoformans that are associated with both geographic location and clinical outcome. All these studies have been broad, examining the severity of disease cryptococcal phenotypes in a collection of highly diverse strains. We chose a narrow focus and collected various genotypic and phenotypic data from a single ST: ST93. ST93 is a common sequence type isolated from patients globally and is the most common clinical isolate found in the sub-Saharan African country of Uganda. Previously, we performed whole genome sequencing on 38 ST93 Ugandan clinical isolates. We identified 652 unique SNPs in this ST93 population compared to the H99 reference genome. We also showed that ST93 contained two subpopulations: ST9A and ST93B. In the current study, we further characterized the genotypic, phenotypic, and virulence differences between these 38 clinical isolates. Using Illumina sequence data, we identified a pattern of linkage disequilibrium that suggested that ST93A and ST93B are evolving separately. We performed long-read sequencing on each isolate to investigate chromosomal changes and large structural variations, allowing us to identify a chromosomal translocation event wherein parts of chromosome 11 had recombined with chromosome 3. Additionally, we characterized several in vitro phenotypes for each isolate and identified three distinct phenotypic clusters based on cell wall challenge and growth experiments. Next, we infected mice with 35 isolates and observed eight different disease manifestations, including isolates that caused non-CNS infections. Overall, by working within a single sequence type, we can gain a deeper understanding of how some small genetic changes can impact strain-specific phenotypes while others have no discernable effect. Eventually, these data can be used to provide valuable information about how each clinical isolate impacts patient outcomes.

Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Itraconazole Increases Resolvin E3 Concentration and 12/15-lipoxygenase Inhibitor Attenuates Itraconazole Cytotoxicity in Cervical Cancer Cells

The anticancer mechanism of itraconazole remains unsolved; therefore, we studied itraconazole-induced alterations in specialized pro-resolving mediators (SPMs) in cancer cells. The human cervical squamous carcinoma cell line CaSki was cultured with or without 1 μM itraconazole. Liquid chromatography/mass spectrometry analysis was conducted to identify SPMs that were influenced by itraconazole. Cell growth experiments were conducted using itraconazole and inhibitors targeting the metabolic pathways of candidate SPMs. Resolvin E3, resolvin E2, prostaglandin J2 (PGJ2), delta-12-PGJ2, and maresin 2 were identified as candidate SPMs. The 12/15-lipoxygenase inhibitor, which is involved in the conversion of 18-hydroxy-eicosapentaenoic acid to resolvin E3, attenuated the inhibitory effect of itraconazole. Inhibition of the PGJ2 metabolic pathway did not interfere with itraconazole treatment. The metabolic pathway of SPMs, including resolving E3, could be proposed as an anticancer target of itraconazole.

Upregulation of Long Noncoding RNA_GAS5 Suppresses Cell Proliferation and Metastasis in Laryngeal Cancer via Regulating PI3K/AKT/mTOR Signaling Pathway.

Background:Laryngeal cancer is one of the most common malignant tumors among head and neck cancers. Accumulating studies have indicated that long noncoding RNAs (lncRNAs) play an important role in laryngeal cancer occurrence and progression, however, the functional roles and relative regulatory mechanisms of lncRNA growth arrest-specific transcript 5 (GAS5) in laryngeal cancer progression remain unclear.Methods:The expression of lncRNA GAS5 in both laryngeal cancer tissues and cell lines was evaluated using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. The relationships between lncRNA GAS5 expression and clinical parameters were also analyzed. To determine the biological function of lncRNA GAS5, a lncRNA GAS5-specific plasmid was first transfected into laryngeal cancer cells using lentiviral technology. Cell counting kit-8 assay, flow cytometry, and Transwell assays were used to detect in vitro cell proliferation, apoptosis, cycle distribution, and metastasis abilities, respectively. Furthermore, in vivo cell growth experiments were also performed using nude mice. Additionally, western blotting was performed to identify the underlying regulatory mechanism.Results:In the current study, lncRNA GAS5 was downregulated in laryngeal cancer tissues and its low expression was closely associated with poor tumor differentiation, advanced TNM stage, lymph node metastasis, and shorter overall survival time. In addition, lncRNA GAS5 upregulation significantly inhibited laryngeal cancer cell proliferation both in vitro and in vivo. Moreover, in response to lncRNA GAS5 overexpression, more laryngeal cancer cells were arrested at the G2/M stage, accompanied by increased cell apoptosis rates and suppressed migration and invasion capacities. Mechanistically, our data showed that the overexpression of lncRNA GAS5 significantly regulated the PI3K/AKT/mTOR signaling pathway.Conclusion:LncRNA GAS5 might act as a suppressor gene during laryngeal cancer development, as it suppressed cell proliferation and metastasis by regulating the PI3K/AKT/mTOR signaling pathway; thus, lncRNA GAS5 is a promising therapeutic biomarker for the treatment of laryngeal cancer.

Simulations Studies of Protein Kinases that are Molecular Targets in Cancer

AbstractProtein kinases are enzymes with partially overlapping specificities, many of which are important clinical targets. In this article, we give some background on protein kinases and discuss in more depth four such enzymes that have been studied in our labs using computer simulations. The combination of molecular dynamics simulations and enzyme or cell growth experiments was instrumental to explain why certain mutations lead (or do not lead) to resistance to targeted therapy aimed at these proteins. Stochastic network simulations were used to study protein‐protein interactions and suggest points for intervention against tumour growth.

In Situ High-Resolution AFM Imaging and Force Probing of Cell Culture Medium-Forming Nanogranular Surfaces for Cell Growth.

Utilizing cell culture medium to grow cells in vitro has been widely studied in the past decades and has been recognized as an acknowledged way for investigating cell activities. However, due to the lack of adequate observation tools, the detailed mechanisms regulating cell growth in cell culture medium are still not fully understood. In this work, atomic force microscopy (AFM), a powerful tool for observing native biological systems under near-physiological conditions with high resolution, was applied to reveal the nanogranular surfaces formed in cell culture medium in situ for promoting cell growth. First, AFM imaging of glass slides (glass slides were previously incubated in cell culture medium) in aqueous environment clearly visualized the cell culture medium-forming nanogranular surfaces on glass slides. By altering the incubation time of glass slides in cell culture medium, the dynamic formation of nanogranular surfaces was remarkably observed. Next, fluorescent labeling experiments of the cell culture medium-treated glass slides showed that bovine serum proteins were contained in the nanogranular surfaces. Further, the adhesive interactions between cells and nanogranular surfaces probed by AFM force spectroscopy and the cell growth experiments showed that cell culture medium-forming nanogranular surfaces promote cell attachment and growth. The study provides novel insights into nanotopography-regulated molecular mechanisms in cell growth and demonstrates the outstanding capabilities of AFM in addressing biological issues with unprecedented spatial resolution under aqueous conditions, which will have potential impacts on the studies of cell behaviors and cell functions.

Cell growth on electrospun nanofiber mats from polyacrylonitrile (PAN) blends

Nanofiber mats can be produced by electrospinning from diverse polymers and polymer blends as well as with embedded ceramics, metals, etc. The large surface-to-volume ratio makes such nanofiber mats a well-suited substrate for tissue engineering and other cell growth experiments. Cell growth, however, is not only influenced by the substrate morphology, but also by the sterilization process applied before the experiment as well as by the chemical composition of the fibers. A former study showed that cell growth and adhesion are supported by polyacrylonitrile/gelatin nanofiber mats, while both factors are strongly reduced on pure polyacrylonitrile (PAN) nanofibers. Here we report on the influence of different PAN blends on cell growth and adhesion. Our study shows that adding ZnO to the PAN spinning solution impedes cell growth, while addition of maltodextrin/pea protein or casein/gelatin supports cell growth and adhesion.

Manufacturing and Control of a Robotic Device for Time-averaged Simulated Micro and Partial Gravity of a Cell Culture Environment

Gravity is omnipresent for all objects on Earth. However, in an environment of different gravitational stress (e.g., microgravity or partial gravity), cells and organs show different biological responses. So, researchers have attempted to achieve micro- or partial gravity on Earth through various approaches, such as parabolic flight or free fall. However, the duration of such ground experiments is highly limited, making it very difficult to conduct time-consuming tasks, such as cell culture. Thus, a three-dimensional (3D) clinostat is utilized as an alternative for experiments on the International Space Station. It provides time-averaged simulated micro- and partial gravity by using mechanical frames with two rotating actuators. This study proposes novel control algorithms for simulating micro- and partial gravity and validates them by applying it to the control of a manufactured 3D clinostat. First, the novel algorithm for time-averaged simulated microgravity (taSMG) provided a more uniformly distributed gravity field by reducing two poles the gravity-concentrated areas. The taSMG with reduced poles provides isotropic gravitational patterns, from which it is possible to minimize the unnecessary effect due to nonuniformity of the gravity vector direction. Second, the other suggested novel algorithm for time-averaged simulated partial gravity (taSPG) controls the pole sizes asymmetrically to generate the intended size of partial gravity. The suggested algorithms are based on mathematical models rather than totally randomized motions. Therefore, the convergence of gravity values, in the rotating frame over time, can be analytically predicted with improved accuracy compared with previously reported algorithms. The developed 3D clinostat hardware and algorithms will effectively provide well-validated taSMG and taSPG for cell growth experiments in future studies for space medicine.

N-isopropyl chitosan. A pH- and thermo-responsive polysaccharide for gel formation

The present contribution deals with the synthesis and characterization of N-isopropyl chitosan in which the introduction of hydrophobic groups leads to an increased flexibility of the polysaccharide backbone. The isopropyl groups extend the solubility of the modified-chitosan samples and render the modified chitosan a pH- and thermo-sensitive system for hydrogel formation. Indeed, upon varying the pH of the system and/or its temperature within a range compatible with biological applications, a non-reversible sol-gel transition occurs, as determined through extended rheological analyses. The modified chitosan samples show a very good biocompatibility as determined through preliminary viability and cell growth experiments.

Laser-assisted surface processing for functionalization of polymers on micro- and nano-scale

Rapid manufacturing of functionalized polymers enable a cost-efficient method for many applications in the biological, and medical sector. Laser structuring techniques differ for fast fabrication processes of thermoplastics. Here we report on direct UV laser ablation of polystyrene and polydimethylsiloxane to control wettability and bio-compatibility. For this purpose contact angle measurements and L929 cell growth experiments were performed as function of laser and process parameters. This work also describes the potential of laser-assisted hot embossing which has been introduced to replicate pre-fabricated micro- and nano-sized structures onto polymers proving it to be a cost-efficient and flexible method for surface functionalization. Therefore, pre-fabricated laser-induced periodic surface structures have been manufactured as mould inserts using ultrafast laser radiation near the ablation threshold. These mould inserts can also be made of nickel, silicon or glass proving laser-assisted hot embossing to be a versatile tool for the functionalization of thermoplastics and meet the requirements for modern manufacturing with regard to quality and production flexibility.

POLYMERIC SCAFFOLDS FOR TISSUE ENGINEERING APPLICATIONS

Polymeric materials are commonly used for many purposes in Tissue Engineering Applications. In particular, they are used as scaffolds which are specially prepared in cell growth studies as well as drug loading and release systems. In drug delivery and controlled release systems functional, scaffolds are widely used in order to send the drug to its target region. On the other hand, in cell growth systems patterned polymeric scaffolds are prepared and used to allow the cells to grow at a certain region with a particular form. For this purpose, different techniques are used for the adhesion of cells onto the material surfaces. In this study, patterned scaffolds from various polymers as Polymethylacrylate (PAM), Polystyrene (PS) and polyvinylchloride (PVC) were prepared using micro contact printing with the soft Lithographic Technique. The prepared materials were morphologically analyzed and cell growth was followed by using electron scanning microscope (SEM). Poly (dimethylsiloxane) (PDMS) molds were prepared in different shapes and used as stamp materials to transfer the designed patterns. The cell growth on these patterned surfaces was followed after seeding L929 mouse fibroblasts cells. Neutral Red Uptake Assay was applied to observe cell growth. The cell growth experiments showed that the cells were attached to the patterned surfaces and a significant increase in cell growth on the surfaces were observed.

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures - temperature and osmolarity - across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

Preclinical Validation Studies Support Causal Machine Learning Based Identification of Novel Drug Targets for High-Risk Multiple Myeloma

IntroductionRegardless of significant advances in the therapy of multiple myeloma (MM) there is still a lack of effective treatment options for patients with high-risk disease. In this context, we recently developed a network of high-risk disease based on more than 30 000 genomic and clinical variables from 645 patients of the CoMMpass dataset (Gruber et al., ASH 2016). Validation of these findings has been performed in the IFM/DFCI 2009 trial dataset (Furchtgott et al., ASH 2017). This comprehensive computational approach revealed a network of 17 genes driving high-risk (defined as progression or myeloma-related death within 18 months). Here, we performed preclinical validation of potential novel drug targets to confirm the utility of in silico guided target discovery in high-risk MM.MethodsTTK (CFI402257, BAY-1217389), PLK4 (CFI400945, Centrinone), MELK (OTSSP167) and CDK1 (CPG71514) inhibitors were studied in a panel of human MM cell lines (n=11) for their activity in cell viability, cell growth, cell cycle, apoptosis, colony formation, drug combination and co-culture experiments. PKMYT1, TTK and PLK4 were targeted with doxycycline-inducible shRNAs. Analysis of gene expression (GEP) data (GSE24080) was used to link candidate genes to certain MM subgroups.ResultsThe network of 17 genes driving high-risk disease contained eight kinases that serve as attractive drug targets (AURKA, NEK2, CDK1, BUB1B, MELK, TTK, PKMYT1, and PLK4), all of them involved in cell cycle regulation. Accordingly, expression levels of all kinases (except PKMYT1) were enriched in the GEP-defined proliferation associated subgroup of MM and thus linked to poor outcome. To study the interconnectedness of the individual network genes we first investigated the impact of previously reported CDK1 and MELK inhibitors on other network members. This demonstrated rapid loss of CDK1, NEK2, MELK, PKMYT1 and FOXM1 protein levels.We then selected TTK and PLK4 as putative novel MM targets with available inhibitors undergoing clinical testing in solid tumors. Protein and mRNA expression of both genes was confirmed in all MM cell lines. Two selective compounds per gene were used for preclinical studies. All four inhibitors significantly reduced MM cell viability and single dose IC70 treatment impaired cell growth up to 10 days (60-98% reduction, P<0.01). This growth inhibitory effect was confirmed with inducible shRNAs. Mechanistically, growth impairment was linked to G2M cell cycle arrest followed by the accumulation of polyploid cells (15-90% of cells 72h post treatment) which is in line with the role of both genes in chromosome segregation. The formation of aberrant mitoses led to the induction of apoptosis 3-5 days post treatment (≤20% viable cells at day 5 post treatment with 3/4 inhibitors) and was accompanied by the presence of active caspase 3 and cleaved PARP. Importantly, the activity of these drugs persisted in the presence of BMSCs and showed potent activity in colony formation assays (DMSO: 168±15 and 131±57, BAY-1217389: 39±29 and 41±3, CFI-400945: 19±26 and 30±6 colonies in KMS12BM and OPM2 cells at day 14, P<0.05). Drug combination studies pointed to favorable activity in combination with dexamethasone and lenalidomide. Furthermore, confirmatory TTK knockdown with two independent shRNAs sensitized MM cells to dexamethasone.Finally, PKMYT1 was chosen as putative target based on its role as major driver of high-risk in our model. We transduced MM cells with three doxycycline-inducible PKMYT1-targeting shRNAs and observed an impressive impact on myeloma cell growth upon doxycycline induction compared to non-targeting control shRNA (up to 85% reduction at day 10, P<0.01). Furthermore, PKMYT1 knockdown led to the induction of apoptosis in all MM cell lines tested. Based on these encouraging results we currently perform in-depth in silico and in vitro analyses of the underlying PKMYT1 signaling network. Detailed results of these sub-studies will be presented at the meeting.ConclusionsOur results confirm the utility of computational based modelling of high-risk disease. This strategy not only revealed a genetic network closely associated to adverse prognosis, but also enabled the identification of so far unnoticed drug targets. Importantly, inhibitors of TTK and PLK4 are already in clinical testing and thus enable rapid clinical translation of our findings to MM patients in need of alternative therapeutic options. DisclosuresGruber:GNS Healthcare: Employment. Furchtgott:GNS Healthcare: Employment. Raut:GNS Healthcare: Employment. Wuest:GNS Healthcare: Employment. Runge:GNS Healthcare: Employment. Khalil:GNS Healthcare: Employment. Munshi:OncoPep: Other: Board of director. Hayete:GNS Healthcare: Employment. Ludwig:Amgen: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Cilag-Janssen: Speakers Bureau; Celgene: Speakers Bureau.

A Modified Kulka Micromethod for the Rapid and Safe Analysis of Fructose and 1-Deoxy-d-xylulose-5-phosphate.

The Kulka resorcinol assay (Kulka, R.G., Biochemistry 1956, 63, 542–548) for ketoses has been widely used in the literature but suffers from two major disadvantages: (a) it employs large amounts of potentially harmful reagents for a general biology laboratory environment; and (b) in its original formulation, it is unsuited for modern high-throughput applications. Here, we have developed a modified Kulka assay, which contains a safer formulation, employing approx. 5.4 M HCl in 250 µL aliquots, and is suitable for use in high-throughput systems biology or enzymatic applications. The modified assay has been tested extensively for the measurement of two ketoses—fructose (a common substrate in cell growth experiments) and 1-deoxy-d-xylulose-5-phosphate (DXP), the product of the DXP-synthase reaction—which until now has only been assayable using time-consuming chromatographic methods or radioactivity. The Kulka microassay has a sensitivity of 0–250 nmol fructose or 0–500 nmol DXP. The assay is suitable for monitoring the consumption of fructose in bacterial growth experiments but is too insensitive to be used directly for the measurement of DXP in in vitro enzyme assays. However, we show that after concentration of the DXP-enzyme mix by butanol extraction, the Kulka resorcinol method can be used for enzyme assays.

Femtosecond laser-induced modification of PLLA/hydroxyapatite composite

In the article, we present the surface modification method of poly(l-lactide)/hydroxyapatite hybrid material using ultrashort pulse/femtosecond laser for tissue engineering application. Using femtosecond laser ablation we obtained 3D grooved structures at the composite surface with precisely controlled dimensions having 50–100 μm in width and a depth of 20 μm with porous bottom. Differential scanning calorimetry and XRD showed no significant influence of laser process on the supramolecular structure of the polymer in composite after modification. ATR analysis revealed partial surface “amorphisation” due to extremely high temperature gradient provided by laser pulses and relatively long crystallization time of PLLA. GPC revealed decrease in molecular weight of PLLA in the composite after laser modification. In addition biological tests were conducted. Human osteoblasts ATCC CRL-11372 were cultured on the laser-modified surface. Cytotoxicity and real time cell growth experiments showed no toxic effects of laser treated material on cells. This implies that femtosecond laser surface treatment is a promising method which potentially can be used in tissue engineering for scaffold modification and facilitating integration of bioresorbable implant and bone.

Development of an impedance spectroscopy device for on‐line cell growth monitoring

The impedance and dielectric properties of biological samples are often used to estimate the number of cells growing in a culture, and monitor the cell growth in real time. For these type of studies, high-cost and complex commercial equipment is often required. The design and implementation of a standalone device for impedance spectroscopy of cell samples is presented, featuring low cost, portability and easy reconfigurability of frequency and voltage. The device can generate sinusoidal, square and triangular signals with frequencies up to 125 kHz, at voltages in the range from 0 V to 100 mV. Cell growth experiments were carried out with yeast cultures, and the results are compared with a commercial Agilent 4284A LCR meter. The error percentages for magnitude and phase measurements are <5%.

Nanocomposite (Janus) paper as 3D cell culture system

Nanocomposite (NC) papers using bifunctional nanoparticles are prepared for 3D cell growth experiments. Cells show better affinity to NC papers than to paper itself and differentiate the chemical group on the external surface of the nanoparticles. Cells possess spherical shaped morphology and form agglomerates onto the (NC) papers like as observed by cell growth in 3D materials. Furthermore, the preparation of “Janus” paper and the differentiation of its two distinct faces by cells is presented.