-
1.
Production of Monascus pigments as extracellular crystals by cell suspension culture.
Lu, F, Liu, L, Huang, Y, Zhang, X, Wang, Z
Applied microbiology and biotechnology. 2018;(2):677-687
Abstract
It is generally accepted that Monascus pigments are predominantly cell-bound, including both intracellular and surface-bound pigments. This long-term misconception was corrected in the present work. Production of extracellular crystal pigments by submerged culture of Monascus sp. was confirmed by microscopic observation and collection of Monascus pigments from extracellular broth by direct membrane filtration. Following up the new fact, the bioactivity of mycelia as whole-cell biocatalyst for biosynthesis and biodegradation of Monascus pigments had been detailedly examined in both an aqueous solution and a nonionic surfactant micelle aqueous solution. Based on those experimental results, cell suspension culture in an aqueous medium was developed as a novel strategy for accumulation of high concentration of Monascus pigments. Thus, glucose feeding during submerged culture in the aqueous medium was carried out successfully and high orange Monascus pigments concentration of near 4 g/L was achieved.
-
2.
Millifluidic culture improves human midbrain organoid vitality and differentiation.
Berger, E, Magliaro, C, Paczia, N, Monzel, AS, Antony, P, Linster, CL, Bolognin, S, Ahluwalia, A, Schwamborn, JC
Lab on a chip. 2018;(20):3172-3183
Abstract
Human midbrain-specific organoids (hMOs) serve as an experimental in vitro model for studying the pathogenesis of Parkinson's disease (PD). In hMOs, neuroepithelial stem cells (NESCs) give rise to functional midbrain dopaminergic (mDA) neurons that are selectively degenerating during PD. A limitation of the hMO model is an under-supply of oxygen and nutrients to the densely packed core region, which leads eventually to a "dead core". To reduce this phenomenon, we applied a millifluidic culture system that ensures media supply by continuous laminar flow. We developed a computational model of oxygen transport and consumption in order to predict oxygen levels within the hMOs. The modelling predicts higher oxygen levels in the hMO core region under millifluidic conditions. In agreement with the computational model, a significantly smaller "dead core" was observed in hMOs cultured in a bioreactor system compared to those ones kept under conventional shaking conditions. Comparing the necrotic core regions in the organoids with those obtained from the model allowed an estimation of the critical oxygen concentration necessary for ensuring cell vitality. Besides the reduced "dead core" size, the differentiation efficiency from NESCs to mDA neurons was elevated in hMOs exposed to medium flow. Increased differentiation involved a metabolic maturation process that was further developed in the millifluidic culture. Overall, bioreactor conditions that improve hMO quality are worth considering in the context of advanced PD modelling.
-
3.
Three-Dimensional Cell Culture Conditions Affect the Proteome of Cancer-Associated Fibroblasts.
Tölle, RC, Gaggioli, C, Dengjel, J
Journal of proteome research. 2018;(8):2780-2789
Abstract
In vitro cell culture systems are an invaluable tool for cell biological research to study molecular pathways and to characterize processes critical in human pathophysiology. However, the experimental conditions in two-dimensional (2D) cell cultures often differ substantially from the in vivo situation, which continuously raises concerns about the reliability and conferrability of the obtained results. Three-dimensional (3D) cell cultures have been shown to closer mimic in vivo conditions and are commonly employed, for example, in pharmacological screens. Here, we introduce a 3D cell culture system based on a mixture of collagen I and matrigel amenable to stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics analyses. We study the extra- and intracellular proteomic response of skin fibroblast isolated from healthy volunteers in comparison to cancer-associated fibroblasts (CAF) on 3D culture conditions. Both, control cells and CAF, change their proteomic composition based on the culture conditions. Critically, cell type differences observed in 2D are often not preserved in 3D, which commonly closer resemble phenotypes observed in vivo. Especially, extracellular matrix and plasma membrane proteins are differentially regulated in 2D versus 3D.
-
4.
NAD Metabolome Analysis in Human Cells Using ¹H NMR Spectroscopy.
Shabalin, K, Nerinovski, K, Yakimov, A, Kulikova, V, Svetlova, M, Solovjeva, L, Khodorkovskiy, M, Gambaryan, S, Cunningham, R, Migaud, ME, et al
International journal of molecular sciences. 2018;(12)
Abstract
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.
-
5.
Binary culture of microalgae as an integrated approach for enhanced biomass and metabolites productivity, wastewater treatment, and bioflocculation.
Rashid, N, Park, WK, Selvaratnam, T
Chemosphere. 2018;:67-75
Abstract
Ecological studies of microalgae have revealed their potential to co-exist in the natural environment. It provides an evidence of the symbiotic relationship of microalgae with other microorganisms. The symbiosis potential of microalgae is inherited with distinct advantages, providing a venue for their scale-up applications. The deployment of large-scale microalgae applications is limited due to the technical challenges such as slow growth rate, low metabolites yield, and high risk of biomass contamination by unwanted bacteria. However, these challenges can be overcome by exploring symbiotic potential of microalgae. In a symbiotic system, photosynthetic microalgae co-exist with bacteria, fungi, as well as heterotrophic microalgae. In this consortium, they can exchange nutrients and metabolites, transfer gene, and interact with each other through complex metabolic mechanism. Microalgae in this system, termed as a binary culture, are reported to exhibit high growth rate, enhanced bio-flocculation, and biochemical productivity without experiencing contamination. Binary culture also offers interesting applications in other biotechnological processes including bioremediation, wastewater treatment, and production of high-value metabolites. The focus of the study is to provide a perspective to enhance the understanding about microalgae binary culture. In this review, the mechanism of binary culture, its potential, and limitations are briefly discussed. A number of queries are evolved through this study, which needs to be answered by executing future research to assess the real potential of binary culture.
-
6.
Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol.
Naschberger, E, Regensburger, D, Tenkerian, C, Langheinrich, M, Engel, FB, Geppert, C, Hartmann, A, Grützmann, R, Schellerer, VS, Stürzl, M
Journal of visualized experiments : JoVE. 2018;(134)
-
-
Free full text
-
Abstract
Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.
-
7.
Challenge in particle delivery to cells in a microfluidic device.
Moghadas, H, Saidi, MS, Kashaninejad, N, Nguyen, NT
Drug delivery and translational research. 2018;(3):830-842
Abstract
Micro and nanotechnology can potentially revolutionize drug delivery systems. Novel microfluidic systems have been employed for the cell culture applications and drug delivery by micro and nanocarriers. Cells in the microchannels are under static and dynamic flow perfusion of culture media that provides nutrition and removes waste from the cells. This exerts hydrostatic and hydrodynamic forces on the cells. These forces can considerably affect the functions of the living cells. In this paper, we simulated the flow of air, culture medium, and the particle transport and deposition in the microchannels under different angles of connection inlet. It was found that the shear stress induced by the medium culture flow is not so high to damage the cells and that it is roughly uniform in the cell culture section (CCS). However, the local shear stresses in the other parts of the microchip differ by changing the angles of the connection inlet. The results showed that the particle deposition was a function of the particle size, the properties of the fluid, and the flow rate. At a lower air flow rate, both small and large particles deposited in the entrance region and none of them reached the CCS. Once the airflow rate increased, the drag of the flow could overcome the diffusion of the small particles and deliver them to the CCS so that more than 88% of the 100 nm and 98% of the 200 nm particles deposited in the CCS. However, larger particles with average diameters in micrometers could not reach the CCS by the airflow even at high flow rate. In contrast, our findings indicated that both small and large particles could be delivered to the CCS by liquid flow. Our experimental data confirm that microparticles (with diameters of 5 and 20 μm) suspended in a liquid can reach the CCS at a well-adjusted flow rate. Consequently, a liquid carrier is suggested to transport large particles through microchannels. As a powerful tool, these numerical simulations provide a nearly complete understanding of the flow field and particle patterns in microchips which can significantly lower the trial and error in the experiment tests and accordingly save researchers considerable cost and time for drug delivery to the cell in the microchip by micro/nanocarriers.
-
8.
A vascularized and perfused organ-on-a-chip platform for large-scale drug screening applications.
Phan, DTT, Wang, X, Craver, BM, Sobrino, A, Zhao, D, Chen, JC, Lee, LYN, George, SC, Lee, AP, Hughes, CCW
Lab on a chip. 2017;(3):511-520
-
-
Free full text
-
Abstract
There is a growing awareness that complex 3-dimensional (3D) organs are not well represented by monolayers of a single cell type - the standard format for many drug screens. To address this deficiency, and with the goal of improving screens so that drugs with good efficacy and low toxicity can be identified, microphysiological systems (MPS) are being developed that better capture the complexity of in vivo physiology. We have previously described an organ-on-a-chip platform that incorporates perfused microvessels, such that survival of the surrounding tissue is entirely dependent on delivery of nutrients through the vessels. Here we describe an arrayed version of the platform that incorporates multiple vascularized micro-organs (VMOs) on a 96-well plate. Each VMO is independently-addressable and flow through the micro-organ is driven by hydrostatic pressure. The platform is easy to use, requires no external pumps or valves, and is highly reproducible. As a proof-of-concept we have created arrayed vascularized micro tumors (VMTs) and used these in a blinded screen to assay a small library of compounds, including FDA-approved anti-cancer drugs, and successfully identified both anti-angiogenic and anti-tumor drugs. This 3D platform is suitable for efficacy/toxicity screening against multiple tissues in a more physiological environment than previously possible.
-
9.
Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches.
Singh, P, Guleri, R, Angurala, A, Kaur, K, Kaur, K, Kaul, SC, Wadhwa, R, Pati, PK
BioMed research international. 2017;:3278494
Abstract
Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.
-
10.
Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates.
Kijlstra, JD, Hu, D, van der Meer, P, Domian, IJ
Current protocols in stem cell biology. 2017;:1F.20.1-1F.20.9
-
-
Free full text
-
Abstract
Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc.