Long-term Cell Culture Research Articles

Acoustic levitation as a tool for cell-driven self-organization of human cell spheroids during long-term 3D culture.

Acoustic levitation, which allows contactless manipulation of micro-objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell-cell interactions, away from the walls of the chip, which leads to the spontaneous self-organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long-term culture of cells in acoustic levitation is feasible. We also demonstrated that this self-organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium-mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix.

Long-term culture of patient-derived mammary organoids in non-biogenic electrospun scaffolds for identifying metalloprotein and motor protein activities in aging and senescence.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotypein tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactonescaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids andpatient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

Reversibly-bonded microfluidic devices for stable cell culture and rapid, gentle cell extraction.

Microfluidic chips have emerged as significant tools in cell culture due to their capacity for supporting cells to adopt more physiologically relevant morphologies in 3D compared with traditional cell culture in 2D. Currently, irreversible bonding methods, where chips cannot be detached from their substrates without destroying the structure, are commonly used in fabrication, making it challenging to conduct further analysis on cells that have been cultured on-chip. Although some reversible bonding techniques have been developed, they are either restricted to certain materials such as glass, or require complex processing procedures. Here, we demonstrate a simple and reversible polydimethylsiloxane (PDMS)-polystyrene (PS) bonding technique that allows devices to withstand extended operations while pressurized, and supports long-term stable cell cultures. More importantly, it allows rapid and gentle live cell extraction for downstream manipulation and characterization after long-term on-chip culturing, and even further subculturing. Our new approach could greatly facilitate microfluidic chip-based cell and tissue cultures, overcoming current analytical limitations and opening up new avenues for downstream uses of on-chip cultures, including 3D-engineered tissue structures for biomedical applications.

PDMS nanoparticles-decorated PDMS substrate promotes adhesion, proliferation and differentiation of skin cells

Polydimethylsiloxane (PDMS) is known to be a common substrate for various cell culture-based applications. However, native PDMS is not very conducive for cell culture and hence, surface modification via cell adhesion moieties is generally needed to make it suitable especially for long-term cell culture. To address this issue, we propose to coat PDMS nanoparticles (NPs) on the surface of PDMS film to improve adhesion, proliferation and differentiation of skin cells. The proposed modification strategy introduces necessary nanotopography without altering the surface chemical properties of PDMS. Due to resemblance in the mechanical properties of PDMS with skin, PDMS NPs can recreate the native extracellular nanoenvironment of skin on the PDMS surface and provide anchoring sites for skin cells to adhere and grow. Human keratinocytes, representing 95% of the epidermal skin cells maintained their characteristic well-spread morphology with the formation of interconnected cell-sheets on this coated PDMS surface. Moreover, our in vitro immunofluorescence studies confirmed expression of distinctive epidermal protein markers on the coated surface indicating close resemblance with the native skin epidermis. Conclusively, our findings suggest that introducing nanotopography via PDMS NPs can be an effective strategy for emulating the native cellular functions of keratinocytes on PDMS based cell culture devices.

Abstract B082: A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC)

Abstract Introduction: KRASG12C inhibitors are a new therapeutic approach to treat KRAS mutant-driven cancers, but as with other oncogene directed therapies, adaptive resistance develops quickly and frequently. The predominant resistance mechanisms are 1) secondary mutations in KRAS and/or other mitogen-activated protein kinase (MAPK) signaling genes and 2) amplification of KRAS or MAPK genes. Extrachromosomal DNA (ecDNA) are the predominant site of focal high copy number oncogene or resistance gene amplifications in human cancers. Previous studies have demonstrated KRASG12C amplification on ecDNA to be an acquired resistance mechanism after treatment with approved KRASG12C inhibitors. Cancers enabled by ecDNA remain underserved by current therapeutic options, including targeted therapies, and have poor prognosis. An in silico “loss of function” screen revealed ribonucleotide reductase (RNR) as a critical target required for the survival of ecDNA amplified tumor cells. RNR is the rate-limiting enzyme in the biosynthesis of deoxyribonucleotide triphosphates (dNTP), the building blocks of DNA and ecDNA replication and repair. While non-selective drugs with RNR inhibitory activity (e.g., gemcitabine) indirectly validate RNR as a cancer target, no selective small molecules targeting RNR have been approved. Methods: We have developed BBI-825, a potent, selective, and orally bioavailable RNR inhibitor (RNRi) for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers. Results: BBI-825 inhibits RNR with an IC50 of 20 nM in vitro and reduces deoxyadenosine triphosphate (dATP) levels in ecDNA amplified cancer cells, with an EC50 of less than 100 nM. On target selectivity of BBI-825 was confirmed using SAFETYscan cellular screening assay investigating target selectivity over 78 targets spanning hormone receptors, kinases, calcium, GPCR, ion channel, and transporter signaling. Importantly, BBI-825 prevented resistance-conferring ecDNA-enabled amplification of KRASG12C in KRASG12C mutated colorectal cancer cells treated with the KRAS inhibitor adagrasib in vitro. Prevention of ecDNA amplification correlated with antiproliferation of BBI-825-treated tumor cells in long-term cultures. Treatment of KRASG12C mutated CRC tumor xenograft models with single agent adagrasib resulted in rapid acquired resistance through KRASG12C amplification on ecDNA. In concordance with in vitro observations, co-treatment with BBI-825 delayed the development of adagrasib resistance concurrent with an abrogation of ecDNA-enabled KRASG12C amplification (P=0.001). Furthermore, BBI-825 significantly inhibited the growth of pre-established adagrasib-resistant KRASG12C ecDNA-amplified tumors in vivo (tumor growth inhibition (TGI) = 92%, p<0.002), reinforcing clinical actionability in the KRAS inhibitor-refractory setting. Conclusions: These findings support the advancement of BBI-825 as a selective RNR inhibitor for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers, including KRASG12C mutant CRC. Citation Format: Sudhir Chowdhry, Ryan Hansen, Jacques Mauger, AnneMarie Pferdekamper, Edison Tse, Ben Norman, Jafar Moininazeri, Salvador Garcia, Yen Truong, Debbie Liao, Joshua Lange, Evan Holmes, Anthony Celeste, Dave Solis, Auzon Steffy, Joshua Plum, Julie Wiese, Julius Apuy, Ardalan Ardeshiri, Snezana Milutinovic, Deepti Wilkinson, Anthony Pinkerton, Christian Hassig, Shailaja Kasibhatla. A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B082.

Production of green-natural and “authentic” cultured meat based on proanthocyanidins-dialdehyde chitosan-collagen ternary hybrid edible scaffolds

Cultured meat has the potential to fulfill the meat demand for the growing human population, but cultured meat development will be required to simplify the production process and produce naturally cultured meat, such as no longer stripping off scaffolders and adding artificial dyes. In this study, proanthocyanidins (PC) and dialdehyde chitosan (DAC) were employed as dual crosslinkers with collagen to prepare a hybrid 3D edible scaffold for the production of high-quality cell-cultured meat. The results revealed that the scaffold was biocompatible and could offer robust mechanical support and adhesion sites for bovine myoblasts, enabling long-term cell culture. Meanwhile, the Col-PC-DAC scaffold promoted the myogenic differentiation of bovine myoblasts and extracellular matrix protein secretion, further affecting the texture of cultured meat. After cooking the cultured meat and beef, it was shown that the cultured meat had some similarities to beef in color and flavor. Importantly, our findings demonstrate that cultured meat can acquire a color remarkably similar to that of conventional beef without the need for artificial dyeing. This breakthrough not only simplifies the production process but also ensures a more natural and appealing appearance of cultured meat. In conclusion, the proanthocyanidins-dialdehyde chitosan-collagen hybrid 3D edible scaffolds provide a new option for producing cultured meat that satisfies consumer expectations.

Droplet-based cell-laden microgels for high-throughput analysis

Cell-laden droplet microfluidics has revolutionized bulk biochemical analysis by offering compartmentalized microreactors for individual cells, but downstream operations of regular aqueous droplets are limited. Hydrogel matrix can provide a rigid scaffold for long-term culture of eukaryotic and prokaryotic cells, and can support several manipulations, facilitating subsequent high-throughput analysis of cellular heterogeneity.

Biomimetic alginate/perfluorocarbon microcapsules – the effect on in vitro metabolic activity and long-term cell culture

Biomimetic alginate/perfluorocarbon microcapsules – the effect on in vitro metabolic activity and long-term cell culture

Screening Station, a novel laboratory automation system for physiologically relevant cell-based assays.

Due to their physiological relevance, cell-based assays using human-induced pluripotent stem cell (iPSC)-derived cells are a promising in vitro pharmacological evaluation system for drug candidates. However, cell-based assays involve complex processes such as long-term culture, real-time and continuous observation of living cells, and detection of many cellular events. Automating multi-sample processing through these assays will enhance reproducibility by limiting human error and reduce researchers' valuable time spent conducting these experiments. Furthermore, this integration enables continuous tracking of morphological changes, which is not possible with the use of stand-alone devices. This report describes a new laboratory automation system called the Screening Station, which uses novel automation control and scheduling software called Green Button Go to integrate various devices. To integrate the above-mentioned processes, we established three workflows in Green Button Go: 1) For long-term cell culture, culture plates and medium containers are transported from the automatic CO2 incubator and cool incubator, respectively, and the cell culture medium in the microplates is exchanged daily using the Biomek i7 workstation; 2) For time-lapse live-cell imaging, culture plates are automatically transferred between the CQ1 confocal quantitative image cytometer and the SCALE48W automatic CO2 incubator; 3) For immunofluorescence imaging assays, in addition to the above-mentioned devices, the 405LS microplate washer allows for formalin-fixation and immunostaining of cells. By scheduling various combinations of the three workflows, we successfully automated the culture and medium exchange processes for iPSCs derived from patients with facioscapulohumeral muscular dystrophy, confirmation of their differentiation status by live-cell imaging, and confirmation of the presence of differentiation markers by immunostaining. In addition, deep learning analysis enabled us to quantify the degree of iPSC differentiation from live-cell imaging data. Further, the results of the fully automated experiments could be accessed via the intranet, enabling experiments and analysis to be conducted remotely once the necessary reagents and labware were prepared. We expect that the ability to perform clinically and physiologically relevant cell-based assays from remote locations using the Screening Station will facilitate global research collaboration and accelerate the discovery of new drug candidates.

Next-Generation Modeling of Cancer Using Organoids.

In the last decade, organoid technology has become a cornerstone in cancer research. Organoids are long-term primary cell cultures, usually of epithelial origin, grown in a three-dimensional (3D) protein matrix and a fully defined medium. Organoids can be derived from many organs and cancer types and sites, encompassing both murine and human tissues. Importantly, they can be established from various stages during tumor evolution and recapitulate with high accuracy patient genomics and phenotypes in vitro, offering a platform for personalized medicine. Additionally, organoids are remarkably amendable for experimental manipulation. Taken together, these features make organoids a powerful tool with applications in basic cancer research and personalized medicine. Here, we will discuss the origins of organoid culture, applications in cancer research, and how cancer organoids can synergize with other models of cancer to drive basic discoveries as well as to translate these toward clinical solutions.

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

Long-Term Culturing of FreeStyle 293-F Cells Affects Immunoglobulin G Glycome Composition.

Glycosylation of IgG regulates the effector function of this antibody in the immune response. Glycosylated IgG is a potent therapeutic used for both research and clinical purposes. While there is ample research on how different cell culture conditions affect IgG glycosylation, the data are missing on the stability of IgG glycome during long cell passaging, i.e., cell "aging". To test this, we performed three independent time course experiments in FreeStyle 293-F cells, which secrete IgG with a human-like glycosylation pattern and are frequently used to generate defined IgG glycoforms. During long-term cell culturing, IgG glycome stayed fairly stable except for galactosylation, which appeared extremely variable. Cell transcriptome analysis revealed no correlation in galactosyltransferase B4GALT1 expression with galactosylation change, but with expression of EEF1A1 and SLC38A10, genes previously associated with IgG galactosylation through GWAS. The FreeStyle 293-F cell-based system for IgG production is a good model for studies of mechanisms underlying IgG glycosylation, but results from the present study point to the utmost importance of the need to control IgG galactosylation in both in vitro and in vivo systems. This is especially important for improving the production of precisely glycosylated IgG for therapeutic purposes, since IgG galactosylation affects the inflammatory potential of IgG.

Nanoanalytical Insights into the Stability, Intracellular Fate, and Biotransformation of Metal-Organic Frameworks.

Metal-organic frameworks (MOFs) have found increasing applications in the biomedical field due to their unique properties and high modularity. Although the limited stability of MOFs in biological environments is increasingly recognized, analytical techniques have not yet been harnessed to their full potential to assess the biological fate of MOFs. Here, we investigate the environment-dependent biochemical transformations of widely researched nanosized MOFs (nMOFs) under conditions relevant to their medical application. We assess the chemical stability of antimicrobial zinc-based drug delivery nMOFs (Zn-ZIF-8 and Zn-ZIF-8:Ce) and radio-enhancer candidate nMOFs (Hf-DBA, Ti-MIL-125, and TiZr-PCN-415) containing biologically nonessential group IV metal ions. We reveal that even a moderate decrease in pH to values encountered in lysosomes (pH 4.5-5) leads to significant dissolution of ZIF-8 and partial dissolution of Ti-MIL-125, whereas no substantial dissolution was observed for TiZr-PCN-415 and Hf-DBA nMOFs. Exposure to phosphate-rich buffers led to phosphate incorporation in all nMOFs, resulting in amorphization and morphological changes. Interestingly, long-term cell culture studies revealed that nMOF (bio)transformations of, e.g., Ti-MIL-125 were cellular compartment-dependent and that the phosphate content in the nMOF varied significantly between nMOFs localized in lysosomes and those in the cytoplasm. These results illustrate the delicate nature and environment-dependent properties of nMOFs across all stages of their life cycle, including storage, formulation, and application, and the need for in-depth analyses of biotransformations for an improved understanding of structure-function relationships. The findings encourage the considerate choice of suspension buffers for MOFs because these media may lead to significant material alterations prior to application.

Fabrication of elliptically constructed liquid crystalline elastomeric scaffolds for 3D artificial tissues

Inspired by the orientation and the fibrous structure of human muscle tissues, we fabricated preconstructed porous liquid crystalline (LC) scaffolds through a two-step polymerization and salt leaching method. A novel strategy combining the aligning properties of LCs and the ease of processing of elastomers for the preparation of elliptical scaffolds for muscle cell culture was proposed in this research. Different from the other types of scaffolds, our biocompatible LC scaffold that can be implanted into the human body using a supporting unit to improve the mechanical properties compared with those of natural muscle. To evaluate the synthesized scaffolds, in vitro experiments using normal human dermal fibroblast (NHDF) cells and smooth muscle cells from rats were carried out, and the sample cells were cultured on each sample scaffold. Based on the results of long-term culture of NHDF cells on the LC scaffolds, it can be confirmed that all three kinds of LC scaffolds have good biocompatibility and provide enough space for cell growth. The addition of gelatin can significantly enhance the biocompatibility of the synthesized scaffolds. Evaluation of scaffold morphologies on cell growth indicates that the molecular arrangement on the scaffolds can induce the growth direction of smooth muscle cells to a certain extent, thereby increasing the formation of highly ordered arrangement tissues. The population doubling time of NHDF cells on the different scaffolds suggest that gelatin can improve the attachment and growth of cells. Investigation of cell viability on LC scaffolds shows that the original LC scaffolds already possess excellent biocompatibility. Additionally, the average cell viability of smooth muscle cells was above 90%, showing that the LC scaffolds in this research are suitable for application in muscle tissue engineering. Based on the results, the gelatin-coated scaffolds are more conducive to the growth of cells in this research and provide promising candidates for tissue engineering in biomedical fields and research fields.

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology.

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed byretrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidatethe intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Evaluation of fermented soybean meal and edible insect hydrolysates as potential serum replacement in pig muscle stem cell culture

In this study, we investigated the potential of edible hydrolysates as a replacement for fetal bovine serum (FBS) in pig muscle stem cell culture. Hydrolysates of two fermented soybean meals (fermented with a combination of Aspergillus oryzae and Bacillus subtilis and fermented with Bacillus subtilis) and two edible insects (mealworm and cricket) were prepared (FAB-H, FB-H, TM-H, and GB-H, respectively). All hydrolysates prepared showed antioxidant activity and provided a suitable cell culture environment as the pH of media containing each hydrolysate (at concentrations ranging from 0.01 to 1%) was within the acceptable range. The supplementation with hydrolysate (0.01–5% FAB-H and FB-H, 0.01–1% TM-H, or 0.01–0.1% GB-H) promoted cell proliferation in media containing 10% FBS. Furthermore, 0.01 and 0.1% of FAB-H, FB-H, and TM-H were able to replace up to 50% of FBS while maintaining proliferation and differentiation capacity. Notably, 0.1% of FB-H and TM-H in 50% FBS-reduced media further enhanced differentiation compared to 10% FBS media. However, further investigations are required to improve the effects of these hydrolysates on the long-term culture of pig muscle stem cells. Nevertheless, the partial FBS replacement with edible and cost-effective natural materials (FAB-H, FB-H, and TM-H) could significantly help reduce the cost of cultured meat significantly.

Plasmonic Functional Assay Platform Determines the Therapeutic Profile of Cancer Cells.

Functional assay platforms could identify the biophysical properties of cells and their therapeutic response to drug treatments. Despite their strong ability to assess cellular pathways, functional assays require large tissue samples, long-term cell culture, and bulk measurements. Even though such a drawback is still valid, these limitations did not hinder the interest in these platforms for their capacity to reveal drug susceptibility. Some of the limitations could be overcome with single-cell functional assays by identifying subpopulations using small sample volumes. Along this direction, in this article, we developed a high-throughput plasmonic functional assay platform to identify the growth profile of cells and their therapeutic profile under therapies using mass and growth rate statistics of individual cells. Our technology could determine populations' growth profiles using the growth rate data of multiple single cells of the same population. Evaluating spectral variations based on the plasmonic diffraction field intensity images in real time, we could simultaneously monitor the mass change for the cells within the field of view of a camera with the capacity of > ∼500 cells/h scanning rate. Our technology could determine the therapeutic profile of cells under cancer drugs within few hours, while the classical techniques require days to show reduction in viability due to antitumor effects. The platform could reveal the heterogeneity within the therapeutic profile of populations and determine subpopulations showing resistance to drug therapies. As a proof-of-principle demonstration, we studied the growth profile of MCF-7 cells and their therapeutic behavior to standard-of-care drugs that have antitumor effects as shown in the literature, including difluoromethylornithine (DFMO), 5-fluorouracil (5-FU), paclitaxel (PTX), and doxorubicin (Dox). We successfully demonstrated the resistant behavior of an MCF-7 variant that could survive in the presence of DFMO. More importantly, we could precisely identify synergic and antagonistic effects of drug combinations based on the order of use in cancer therapy. Rapidly assessing the therapeutic profile of cancer cells, our plasmonic functional assay platform could be used to reveal personalized drug therapies for cancer patients.

Simultaneous induction of vasculature and neuronal network formation on a chip reveals a dynamic interrelationship between cell types

BackgroundNeuronal networks receive and deliver information to regulate bodily functions while the vascular network provides oxygen, nutrients, and signaling molecules to tissues. Neurovascular interactions are vital for both tissue development and maintaining homeostasis in adulthood; these two network systems align and reciprocally communicate with one another. Although communication between network systems has been acknowledged, the lack of relevant in vitro models has hindered research at the mechanistic level. For example, the current used in vitro neurovascular models are typically established to be short-term (≤ 7 days) culture models, and they miss the supporting vascular mural cells.MethodsIn this study, we utilized human induced pluripotent stem cell (hiPSC) -derived neurons, fluorescence tagged human umbilical vein endothelial cells (HUVECs), and either human bone marrow or adipose stem/stromal cells (BMSCs or ASCs) as the mural cell types to create a novel 3D neurovascular network-on-a-chip model. Collagen 1–fibrin matrix was used to establish long-term (≥ 14 days) 3D cell culture in a perfusable microphysiological environment.ResultsAprotinin-supplemented endothelial cell growth medium-2 (EGM-2) supported the simultaneous formation of neuronal networks, vascular structures, mural cell differentiation, and the stability of the 3D matrix. The formed neuronal and vascular networks were morphologically and functionally characterized. Neuronal networks supported vasculature formation based on direct cell contacts and by dramatically increasing the secretion of angiogenesis-related factors in multicultures in contrast to cocultures without neurons. Both utilized mural cell types supported the formation of neurovascular networks; however, the BMSCs seemed to boost neurovascular networks to greater extent.ConclusionsOverall, our study provides a novel human neurovascular network model that is applicable for creating in vivo-like tissue models with intrinsic neurovascular interactions. The 3D neurovascular network model on chip forms an initial platform for the development of vascularized and innervated organ-on-chip and further body-on-chip concepts and offers the possibility for mechanistic studies on neurovascular communication both under healthy and in disease conditions.DtMPMfM_nPvk_pFj8pUo8rVideo

Specificity of cancer-related chromosomal translocations is linked to proximity after the DNA double-strand break and subsequent selection.

Most cancer-related chromosomal translocations appear to be cell type specific. It is currently unknown why different chromosomal translocations occur in different cells. This can be due to either the occurrence of particular translocations in specific cell types or adaptive survival advantage conferred by translocations only in specific cells. We experimentally addressed this question by double-strand break (DSB) induction at MYC, IGH, AMLandETO loci in the same cell to generate chromosomal translocations in different cell lineages. Our results show that any translocation can potentially arise in any cell type. We have analyzed different factors that could affect the frequency of the translocations, and only the spatial proximity between gene loci after the DSB induction correlated with the resulting translocation frequency, supporting the 'breakage-first' model. Furthermore, upon long-term culture of cells with the generated chromosomal translocations, only oncogenic MYC-IGH and AML-ETO translocations persisted over a 60-day period. Overall, the results suggest that chromosomal translocation can be generated after DSB induction in any type of cell, but whether the cell with the translocation would persist in a cell population depends on the cell type-specific selective survival advantage that the chromosomal translocation confers to the cell.

De Novo Design of a Versatile Peptide-Based Coating to Impart Targeted Functionality at the Surface of Native Polystyrene.

Peptide sequence periodicity is a simple design tool that can be used to generate functional peptide-based surface coatings. De novo-designed peptide N3-PEG-VK16 is characterized by a hydrophobic periodicity of two that avidly binds to native polystyrene priming its surface for subsequent targeted functionalization via chemical ligation. The peptidic portion of N3-PEG-VK16 is responsible for surface binding, converting polystyrene's hydrophobic surface into a wettable and electrostatically charged environment that facilitates cell attachment. Native polystyrene surfaces are coated by simple peptide adsorption from an aqueous buffered solution, and the resulting primed surface is easily functionalized by cycloaddition chemistry. Herein, we show that ligating a vitronectin-derived peptide to primed polystyrene surfaces enables adhesion, expansion, long-term culture, and phenotype maintenance of human induced pluripotent stem cells. To demonstrate scope, we also show that additional functional ligands can be used, for example, nerve growth factor protein, to control neurite outgrowth.