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The separation of circulating tumor cells (CTCs) that originate from tumor or cancer tissue plays an important role in cancer diagnostics, progression analyses, and treatment proficiency. Cancer metastasis occurs when CTCs spread throughout the body and invade healthy tissues, which leads to new tumors in that area. Although a dramatic rate of death begins from CTCs spreading around the body, valuable measures can be taken to control their development. A first step is separating these harmful cells from the bloodstream and then investigating their features to introduce complementary treatments that can affect the cancerous cells without damaging healthy cells. Numerous techniques have been developed for continuous and fast separation of CTCs. Over the last two decades, the reduction in reagent demand, sample volume, analysis time, and patient safety are just a few of the motivations that encourage researchers to study microfluidic instruments for CTC separation from other blood cells. Among them, inertial microfluidic devices are promising due to their simple structure and setup. However, one shortcoming of this technique is the need for pumps to drive fluid flow, a low ability to control cell movement, and the possibility of clogging the channel. One technique that may potentially overcome these shortcomings is the so-called rotational micro-fluidic platform. However, this technique alone is still not sufficient. In this paper, a detailed analysis of each technique that emphasizes both strengths and shortcomings is presented. Subsequently, a new approach that combines microfluidics with magnetic nanoparticles and is based on the antibody binding principle is proposed. The feasibility of implementing this combined technique will also be discussed.

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to ninety percent of a targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division, and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

The development of therapeutic regenerative medicine and accurate drug discovery cell-based products requires effective, with respect to obtaining sufficient numbers of viable, proliferative, and functional cell populations, cell expansion ex vivo. Unfortunately, traditional cell culture systems fail to recapitulate the multifaceted tissue milieu in vitro, resulting in cell phenotypic drift, loss of functionality, senescence, and apoptosis. Substrate-, environment-, and media-induced approaches are under intense investigation as a means to maintain cell phenotype and function while in culture. In this context, herein, the potential of macromolecular crowding, a biophysical phenomenon with considerable biological consequences, is discussed.

Background In our previous study, missense mutations in the Notch1 gene were found in chemotherapy-resistant esophageal squamous cell cancer (ESCC) patients. In this study, we explored changes in the interaction between Notch1 and DLL4 resulting from missense mutations. Methods Bioinformatics analysis was performed to assess and compare the different biological structures and functions of wild type (WT) and mutation type (MT) sequences of Notch1. A genetic information search was performed, and the results were analyzed using in silico modeling. Homology modeling of the Notch1 protein was carried out using Swiss-Model software, and modeling of site-directed mutations was carried out using PyMOL software to observe the protein structure. The Notch1-DLL4 ligand-receptor complex protein model was constructed, Wincoot software was used to determine site-directed mutations, and a protein-ligand interaction profiler (PLIP) was used to calculate the noncovalent interactions in the complex. Results The mutation site was located in the region where Notch1 binds to DLL4. A careful examination of the in silico structural model revealed that the mutation caused an alteration in the surface charge, and the water-bridge bonds of the interaction between Notch1-DLL4 increased in number from 5 to 7. Conclusions Notch1 gene missense mutation leads to an increase in the number of water-bridge bonds, thus enhancing the Notch1-DLL4 interaction, which may lead to tighter Notch1-DLL4 binding, either making the pathway easier to activate or increasing the length of time it is active.

We studied the dynamic of proliferative activity of cultured mouse transformed fibroblast-like L-929 cells in the logarithmic growth phase. During a long period (December 5-23, 2020), we revealed a 4-day rhythm of daily increase in the number of L-929 cells with an amplitude not lower than in a culture of embryonic fibroblast-like cells. Hence, the formation of the 4-day rhythm is not associated with the molecular mechanisms of inhibition of proliferation, which are absent in transformed cells. Daily thawing of samples of one culture over 17 days showed the presence of a 4-day rhythm synchronous between all thawed samples and the control cell culture. As deep freezing leads to the cessation of all life processes in cells, the formation of a 4-day rhythm of proliferative activity of cell culture is determined by an exogenous mechanism.

In recent years, the inputs from magnetically assisted strategies have been contributing to the development of more sensitive screening methods and precise means of diagnosis to overcome existing and emerging treatment challenges. The features of magnetic materials enabling in vivo traceability, specific targeting and space- and time-controlled delivery of nanomedicines have highlighted the resourcefulness of the magnetic toolbox for biomedical applications and theranostic strategies. The breakthroughs in magnetically assisted technologies for contact-free control of cell and tissue fate opens new perspectives to improve healing and instruct regeneration reaching a wide range of diseases and disorders. In this review, the contribution of magnetic nanoparticles (MNPs) will be explored as sophisticated and versatile nanotriggers, evidencing their unique cues to probe and control cell function. As cells detect and engage external magnetic features, these approaches will be overviewed considering molecular engineering and cell programming perspectives as well as cell and tissue targeting modalities. The therapeutic relevance of MNPs will be also emphasized as key components of nanostructured systems to control the release of nanomedicines and in the context of new therapy technologies., ERC CoG grant MagTendon (No. 772817), H2020 Twinning project Achilles (No. 810850), FCT Project MagTT PTDC/CTM-CTM/29930/2017 (POCI-01-0145-FEDER29930), and project Norte-01-0145-FEDER-02219015 supported by Norte Portugal Regional Operational Programme (NORTE 2020). Ana F. Almeida is funded by Fundação para a Ciência e Tecnologia (FCT) – Doctoral Grant SFRD/BD/144816/2019

Hydrogels possessing the ability to control cell functions have great potential as artificial substrates for cell culture. Herein, we report dual-functionalizable protein-polymer hybrid hydrogels prepared by thiol oxidation catalyzed by horseradish peroxidase and a phenolic molecule. A chimera protein of streptavidin (SA) and the SpyCatcher protein, with a cysteine residue at its N-terminus, (C-SA-SC) was constructed and co-cross-linked with thiol-functionalized four-arm polyethylene glycol (PEG-SH) to obtain hydrogels possessing two orthogonal conjugation moieties. Hydrogel formation using C-SA-SC conjugated with biotinylated or SpyTagged functional molecules (premodification strategy) resulted in the formation of hydrogels with a uniform distribution of the functional molecules. Postmodification of the functional molecules of the C-SA-SC hydrogel with biotin or SpyTag could alter the three-dimensional (3D) spatial distribution of the functional molecules within the hydrogels depending on the mode of conjugation (SA/biotin or SpyCatcher/SpyTag), the size of the functional molecules, and the length of time of the modification. NIH-3T3 cells cultured on a C-SA-SC hydrogel, dual-functionalized with a biotinylated-Arg-Gly-Asp-Ser (RGDS) peptide and a basic fibroblast growth factor (bFGF) with SpyTag, showed cell adhesion to the PEG-SH-based hydrogels and cell morphological changes in response to the immobilized RGDS peptide and the bFGF. Moreover, the cells showed higher proliferation on the dual-functionalized C-SA-SC hydrogel than the cells cultured on hydrogels without either the RGDS peptide or the bFGF, demonstrating the benefits of dual-functionalizable hydrogels. The C-SA-SC hydrogel presented in this study is capable of being orthogonally functionalized by two different functional molecules with different 3D distributions of each molecule within the hydrogel and thus has the potential for use as a cell culturing scaffold for creating artificial cellular microstructures.

Summary Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are commercially available, and cardiac differentiation established routine. Systematic evaluation of several control hiPSC-CM is lacking. We investigated 10 different control hiPSC-CM lines and analyzed function and suitability for drug screening. Five commercial and 5 academic hPSC-CM lines were casted in engineered heart tissue (EHT) format. Spontaneous and stimulated EHT contractions were analyzed, and 7 inotropic indicator compounds investigated on 8 cell lines. Baseline contractile force, kinetics, and rate varied widely among the different lines (e.g., relaxation time range: 118-471 ms). In contrast, the qualitative correctness of responses to BayK-8644, nifedipine, EMD-57033, isoprenaline, and digoxin in terms of force and kinetics varied only between 80% and 93%. Large baseline differences between control cell lines support the request for isogenic controls in disease modeling. Variability appears less relevant for drug screening but needs to be considered, arguing for studies with more than one line., Graphical Abstract, Highlights • EHTs are stable from all tested hPSC-CM control lines • EHT baseline contractility shows high levels of variability between cell lines • Batch-to-batch variability is a large confounder of contractile parameters • Despite baseline variability, canonical drug responses were seen in all lines, In this article, Mannhardt and colleagues show that a systematic evaluation of 10 control hPSC-CM lines (commercial and academic) revealed high levels of variability regarding baseline contractility between the lines. In contrast, inotropic drug effects showed less prominent variability in canonical responses, arguing for a smaller relevance in drug screening.

Transcription factors (TFs) coordinate the on-and-off states of gene expression typically in a combinatorial fashion. Studies from embryonic stem cells and other cell types have revealed that a clique of self-regulated core TFs control cell identity and cell state. These core TFs form interconnected feed-forward transcriptional loops to establish and reinforce the cell-type-specific gene-expression program; the ensemble of core TFs and their regulatory loops constitutes core transcriptional regulatory circuitry (CRC). Here, we summarize recent progress in computational reconstitution and biologic exploration of CRCs across various human malignancies, and consolidate the strategy and methodology for CRC discovery. We also discuss the genetic basis and therapeutic vulnerability of CRC, and highlight new frontiers and future efforts for the study of CRC in cancer. Knowledge of CRC in cancer is fundamental to understanding cancer-specific transcriptional addiction, and should provide important insight to both pathobiology and therapeutics.

The significant progress in supramolecular chemistry since the end of last century includes the development of supramolecular gels. In particular, the spatiotemporal self-assembly of synthetic small gelator molecules has attracted increasing attention owing to their ability to achieve certain functional properties in a designated space at a designated time. Peptides conjugated with hydrophobic moieties are typical examples of supramolecular gelators (low molecular weight gelators, LMWGs), which can be designed or programmed to self-assemble to form nanofibers/nanosheets in response to a broad range of stimuli or microenvironments. In the last decade, several groups have reported that the self-assembly of small gelator molecules achieved inside living cells or on the surfaces of living cells induced selective cell death, which would lead to a novel therapeutic approach or a novel cell selection tool. This focused review outlines the self-assembly of small gelator molecules inside living cells that control cell fate. Peptides conjugated with hydrophobic moieties are typical examples of supramolecular gelators (low molecular weight gelators, LMWGs), which can be designed or programmed to self-assemble to form nanofibers/nanosheets in response to stimuli or microenvironments. In the last decade, several groups have reported that the self-assembly of small gelator molecules achieved inside living cells or on the surfaces of living cells induced selective cell death. This focused review outlines the self-assembly of small gelator molecules inside or around living cells that control cell fate.

This study presents a new approach for enabling the development of high-performance lithium-sulfur (Li–S) cells by simply inserting a sulfur-infused separator (SIS) between a common S cathode and a Li metal anode. All solid sulfur electrically isolated from the cathode is electrochemically reduced to polysulfides during the first discharge. Notably, scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) studies have demonstrated that the S in the separator disappears completely even when the cell is discharged to 2.1 V in the first cycle. The combination of a SIS with a typical S cathode results in the doubling of the areal capacity with superior cycling stability upon comparison with the control cell. This result demonstrates that the introduction of additional active materials, such as elemental sulfur, to a separator is a highly effective method for the fabrication of Li–S cells with a high areal capacity.

Purpose To identify the aberrantly expressed long non‐coding RNAs (lncRNAs) in ovarian high‐grade serous carcinoma (HGSC). Methods Total RNA was isolated in HGSC cell lines, ovarian surface epithelial cells, and normal ovaries. Aberrantly expressed lncRNAs in HGSC were identified by PCR array, which analyzes 84 kinds of lncRNAs. To infer their functions, HGSC cell lines with different levels of expression of the identified lncRNAs were established, and then, activities of proliferation, migration, and apoptosis were examined. Expression levels of the identified lncRNAs were also examined in multiple ovarian HGSC tissues. Results Ten aberrantly expressed lncRNAs, six upregulated and four downregulated, were identified in the HGSC cell lines. The authors established four HGSC cell lines: in two of the cell lines, one of the upregulated lncRNAs was knocked down, and in two other cell lines, one of the downregulated lncRNAs (MEG3 and POU5F1P5) was overexpressed. Migration activities were inhibited in the HGSC cell lines overexpressing MEG3 or POU5F1P5 while there were no differences in proliferation and apoptosis between the established and control cell lines. The four lncRNAs downregulated in the HGSC cell lines were also observed to be downregulated in ovarian HGSC tissues. Conclusion The authors identified four downregulated lncRNAs in ovarian HGSC.

Plant cells have a powerful capacity in their propagation to adapt to environmental change, given that a single plant cell can give rise to a whole plant via somatic embryogenesis without the need for fertilization. The reprogramming of somatic cells into totipotent cells is a critical step in somatic embryogenesis. This process can be induced by stimuli such as plant hormones, transcriptional regulators and stress. Here, we review current knowledge on how the identity of totipotent cells is determined and the stimuli required for reprogramming of somatic cells into totipotent cells. We highlight key molecular regulators and associated networks that control cell fate transition from somatic to totipotent cells. Finally, we pose several outstanding questions that should be addressed to enhance our understanding of the mechanisms underlying plant cell totipotency.

Cell detachment is a routine process to recover seeded cells from culture systems. However, traditional enzymatic methods might easily induce senescence or apoptosis of cells during the routine passage process due to an improper handing process. The pH-responsive characteristic of chitosan has been demonstrated to control cell attachment/detachment by precise adjustment of medium pH within 1 h without additional enzymatic treatment and extensive washing steps. But the time 1 h is not satisfying to all applications to recover adherent cells from chitosan surface. The purpose of this study is to regulate pH-responsive characteristics of chitosan by blending with non-pH-responsive biomaterials, polycaprolactone (PCL) and polyvinyl alcohol (PVA) to provide a gentle rapid method for cell detachment. In this study, PCL could delay the pH-responsive characteristics of chitosan to reduce cell detachment ratio ( 95%) from the chitosan/PVA blended surface within 30 min after raising medium pH to 7.65. We further discuss the related mechanism among cell detachment, fibronectin, and chitosan-based blends. These results indicate non-pH-responsive PCL and PVA could be added into pH-responsive chitosan to develop a rapid non-enzymatic strategy to control cell detachment rate for cell passage and various tissue engineering applications.

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies., Protein & Cell, 13 (7), ISSN:1674-800X, ISSN:1674-8018

Summary Phase separation of proteins regulates transcription. Here, we present a protocol to manipulate phase separation capacity of a protein. We use this protocol to disrupt phase separation by mutating residues at intrinsically disordered regions (IDRs). Further, we rescue the disabled phase separation by fusing an IDR known to drive phase separation. Phase separation promotes cell fate transitions, whereas disruption of phase attenuates the transitions. The major challenge is how to effectively predict mutation residues. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021)., Graphical abstract, Highlights • Phase separation with liquid-like behavior can be verified in vitro and in vivo • Residue mutation at IDR can disrupt phase separation capacity of a protein • The disabled phase separation of a protein can be rescued by fusing an IDR • Phase separation regulates cell fate transitions, Phase separation of proteins regulates transcription. Here, we present a protocol to manipulate phase separation capacity of a protein. We use this protocol to disrupt phase separation by mutating residues at intrinsically disordered regions (IDRs). Further, we rescue the disabled phase separation by fusing an IDR known to drive phase separation. Phase separation promotes cell fate transitions, whereas disruption of phase attenuates the transitions. The major challenge is how to effectively predict mutation residues.

Entry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 initiates cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that play roles in control of cell growth and size. Together, the data are consistent with models in which Cln3 serves as the crucial link between the cell cycle and signals that control cell growth and size.

Advances in mechanistic understanding of integrin-mediated adhesion highlight the importance of precise control of ligand presentation in directing cell migration. Top-down nanopatterning limited the spatial presentation to sub-micron. To enhance it to molecular level, we propose a bottom-up nanofabrication strategy. Via self-assembly and co-assembly, precise control of ligand presentation is succeeded by varying the proportions of assembling ligand and nonfunctional peptide. Assembled nanofilaments fulfill multi-functions exerting enhancement to suppression effect on cell migration with tunable amplitudes. Self-assembled nanofilaments possessing super high ligand density selectively suppress cancer cell migration by preventing integrin/actin disassembly at cell rear, which provides new insights to ligand-density-dependent-modulation, revealing valuable inputs to therapeutic innovations in tumor metastasis.One-Sentence SummaryEngineering integrin ligand assembly from bottom-up offers a generalized tool to selectively control cell migration with tunable amplitudes.

Advances in molecular biology continue to help us uncover how information written into genes provides a blueprint for the initiation and orchestration of programs and processes that control cell division, cell-type specification, cell migration, cellular differentiation, and cell death. Clearly, molecular elements into which information is written must "talk to one another" and "work with each other" to generate appropriate cellular responses to cues and signals.

The intricate molecular and cellular interactions between spermatogonial stem cells (SSCs) and their cognate niche form the basis for life-long sperm production. To maintain long-term fertility and sustain sufficiently high levels of spermatogenesis, a delicate balance needs to prevail between the different niche factors that control cell fate decisions of SSCs by promoting self-renewal, differentiation priming or spermatogenic commitment of undifferentiated spermatogonia (Aundiff). Previously the SSC niche was thought to be formed primarily by Sertoli cells. However, recent research has indicated that many distinct cell types within the testis contribute to the SSC niche including most somatic cell populations and differentiating germ cells. Moreover, postnatal testis development involves maturation of somatic supporting cell populations and onset of cyclic function of the seminiferous epithelium. The stochastic and flexible behavior of Aundiff further complicates the definition of the SSC niche. Unlike in invertebrate species, providing a simple anatomical description of the SSC niche in the mouse is therefore challenging. Rather, the niche needs to be understood as a dynamic system that is able to serve the long-term reproductive function and maintenance of fertility both under steady-state and during development plus regeneration. Recent data from us and others have also shown that Aundiff reversibly transition between differentiation-primed and self-renewing states based on availability of niche-derived cues. This review focuses on defining the current understanding of the SSC niche and the elements involved in its regulation.

Aim: This study assesses the technical performance and cost of a targeted next-generation sequencing (NGS) multigene pharmacogenetic (PGx) test. Materials & methods: A genetic test was developed for 21 PGx genes using molecular inversion probes to generate library fragments for NGS. Performance of this test was assessed using 53 unique reference control cell lines from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results: 93.7% of variants were successfully called and the repeatability rate was 99.9%. Reference calls were available for 78.4% of diplotype calls resulting from PGx testing, and concordance for the test was 85.7%. Cost per sample was $32–$56. Conclusion: A targeted NGS assay using molecular inversion probe technology is able to characterize the pharmacogenome efficiently.

Plant extracts and pharmacopoeias represent an exceptional breeding ground for the discovery of new antioxidants. Until recently, the antioxidant activity was only measured by chemical hydrogen atom transfer (HAT) and single-electron transfer (SET) cell-free assays that do not inform about the actual effect of antioxidants in living systems. By providing information about the mode of action of antioxidants at the subcellular level, recently developed live cell assays are now changing the game. The idea of this review is to present the different cell-based approaches allowing a quantitative measurement of antioxidant effects of plant extracts. Up to date, only four different approaches have reached a certain degree of standardization: (1) the catalase-like assay using H2O2 as a stressor, (2) the cell antioxidant assay (CAA) using AAPH as a stressor and DCFH-DA as a readout, (3) the AOP1 assay which uses photoinduction to monitor and control cell ROS production, and (4) the Nrf2/ARE gene reporter system. The molecular aspects of these assays are presented in detail along with their features, drawbacks, and benefits. The Nrf2/ARE gene reporter system dedicated to indirect antioxidant effect measurement currently represents the most standardized approach with high-throughput applications. AOP1, the first technology linking a fine-tuning of cell ROS production with a quantitative signal, appears to be the most promising tool for the assessment of direct cellular ROS-scavenging effects at an industrial scale.

Bioreactors are essential tools for the development of efficient and high-quality cell therapy products. However, their application is far from full potential, holding several challenges when reconciling the complex biology of the cells to be expanded with the need for a manufacturing process that is able to control cell growth and functionality towards therapy affordability and opportunity. In this review, we discuss and compare current bioreactor technologies by performing a systematic analysis of the published data on automated lymphocyte expansion for adoptive cell therapy. We propose a set of requirements for bioreactor design and identify trends on the applicability of these technologies, highlighting the specific challenges and major advancements for each one of the current approaches of expansion along with the opportunities that lie in process intensification. We conclude on the necessity to develop targeted solutions specially tailored for the specific stimulation, supplementation and micro-environmental needs of lymphocytes’ cultures, and the benefit of applying knowledge-based tools for process control and predictability.

RAS oncogenes are amongst the most commonly mutated proteins in human cancers. They regulate a wide range of effector pathways that control cell proliferation, survival, differentiation, migration and metabolic status. Including aberrations in these pathways, RAS dependent signaling is altered in more than half of human cancers. Targeting mutant RAS proteins and their downstream oncogenic signaling pathways has been elusive. However, recent results comprising detailed molecular studies, large scale omics studies and computational modeling have painted a new and more comprehensive portrait of RAS signaling that helps us to understand the intricacies of RAS, how its physiological and pathophysiological functions are regulated, and how we can target them. Here, we review these efforts particularly trying to relate the detailed mechanistic studies with global functional studies. We highlight the importance of computational modeling and data integration to derive an actionable understanding of RAS signaling that will allow us to design new mechanism based therapies for RAS mutated cancers.

Latour and McGuigan highlight work from the Derivery laboratory that describes a new method for micropatterning proteins while maintaining their activity using fibrinogen anchors., Micropatterning is a process to precisely deposit molecules, typically proteins, onto a substrate of choice with micrometer resolution. Watson et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202009063) describe an innovative yet accessible strategy to enable the reproducible micropatterning of virtually any protein while maintaining its biological activity.

Biomaterials engineered with specific cell binding sites, tunable mechanical properties, and complex architectures are essential to control cell adhesion and proliferation. The influence of the local properties, such as the local hardness and stability on the interaction with cells, has not been yet fully understood and exploited. This is particularly relevant for hydrogels, very promising materials with, unfortunately, poor cell adhesion properties, attributed mostly to their softness. Here, we propose a new approach for producing hybrid hydrogels by functionalizing them with particles and performing a thermal treatment. Exploring the interaction of cells with these materials we introduce a new concept, cells-grabbing-onto-particles, a facilitation of the cell adhesion through modulation of local properties. The approach is implemented on alginate hydrogels typically unsuitable for cell growth by turning them into a very effective cell culture growth platform. Specifically, alginate hydrogels are bio-mineralized with calcium carbonate (CaCO

Selection often favours large bodies, visible as Cope’s rule over macroevolutionary time — but size increases are not inevitable. One understudied cost of large bodies is the high number of cell divisions and the associated risk of oncogenic mutations. Our elasticity analysis shows that selection against a proportional increase in size becomes ever more intense with increasing body size if cancer is the sole selective agent. Thus cancer potentially halts body size increases even if no other constraint does so. We then provide multicellular realism with potentially sexually dimorphic body sizes and traits that control cell populations from zygote to maturity and beyond (ontogenetic management). This shows sexual conflict to extend to ontogeny; sexual dimorphism in mortalities and other life history measures may evolve even in the absence of any ecological causes underlying size- or sex-dependent mortality. Coadaptation of ontogenetic management and body size is required for substantial increases in size.

KCNQ-Kv7 channels are found at the axon initial segment of pyramidal neurons where they control cell firing and membrane potential. In oriens lacunosum moleculare (O-LM) interneurons, these channels are mainly expressed in the dendrites, suggesting a peculiar function of Kv7 channels in these neurons. The physiology of Kv7 channels is well characterized today but the precise contribution of these channels to neuronal plasticity is still unknown. Here, we show that Kv7 channel activity is up-regulated following induction of presynaptic long-term synaptic depression (LTD) in O-LM interneurons, thus resulting in a synergistic long-term depression of intrinsic neuronal excitability (LTD-IE). Both LTD and LTD-IE involve endocannabinoid (eCB) biosynthesis for their induction. Molecular modeling shows strong interaction of eCBs with Kv7.2/3 channel, suggesting a persistent action of these lipids on Kv7 channel activity. Our data thus unveil a major role for eCB synthesis in triggering both synaptic and intrinsic depression in O-LM interneurons.

Growth factors (GFs) are important for tissue engineering and regenerative medicine because of their interactions with cells and the extracellular matrix. Immobilization of GFs on a matrix is one way to control cell behavior. This review focuses on GF immobilization from the standpoint of chemical immobilization and design of GF proteins. In addition, the total design of environmental factors surrounding cells is discussed for future development of tissue engineering and regenerative medicine.

Three dimensional (3D) printing of heart patches usually provides the ability to precisely control cell location in 3D space. Here, one‐step 3D printing of cardiac patches with built‐in soft and stretchable electronics is reported. The tissue is simultaneously printed using three distinct bioinks for the cells, for the conducting parts of the electronics and for the dielectric components. It is shown that the hybrid system can withstand continuous physical deformations as those taking place in the contracting myocardium. The electronic patch is flexible, stretchable, and soft, and the electrodes within the printed patch are able to monitor the function of the engineered tissue by providing extracellular potentials. Furthermore, the system allowed controlling tissue function by providing electrical stimulation for pacing. It is envisioned that such transplantable patches may regain heart contractility and allow the physician to monitor the implant function as well as to efficiently intervene from afar when needed., The one‐step 3D printing of cardiac patches with built‐in soft and stretchable electronics is reported. The tissue is printed using three distinct bioinks for the cells and the conducting and dielectric parts of the electronics. The electrodes within the printed patch can monitor the function of the engineered tissue, and control tissue function by providing electrical stimulation for pacing.

Hydrogels are promising candidates for recapitulation of the native extracellular matrix (ECM), yet recreating molecular and spatiotemporal complexity within a single network remains a challenge. Double network (DN) hydrogels are a promising step towards recapitulating the multicomponent ECM and have enhanced mechanical properties. Here, we investigate DNs based on dynamic covalent and covalent bonds to mimic the dynamicity of the ECM and enable biofabrication. We also investigate the spatiotemporal molecular attachment of a bioactive adhesive peptide within the networks. Using oxidized alginate (dynamic network, Schiff base) and polyethylene glycol diacrylate (static network, acrylate polymerization) we find an optimized procedure, where the dynamic network is formed first, followed by the static network. This initial dynamically cross-linked hydrogel imparts self-healing, injectability, and 3D printability, while the subsequent DN hydrogel improves the stability of the 3D gels and imparts toughness. Rheology and compression testing show that the toughening is due to the combination of energy dissipation (dynamic network) and elasticity (static network). Furthermore, where we place adhesive sites in the network matters; we find distinct differences when an adhesive peptide, Arg-Gly-Asp (RGD), is attached to the different networks. This DN strategy bring us closer to understanding and recreating the complex multicomponent ECM—pushing us past a materials view of cell adhesion— while enabling injectabiltiy and printing of tough hydrogels.

In order to gain a greater understanding of the factors that drive spatial organization in multicellular aggregates of cancer cells, we investigate the segregation patterns of 6 breast cell lines of varying degree of mesenchymal character during formation of mixed aggregates. Cell sorting is considered in the context of available adhesion proteins and cellular contractility. It is found that the primary compaction mediator (cadherins or integrins) for a given cell type in isolation plays an important role in compaction speed, which in turn is the major factor dictating preference for interior or exterior position within mixed aggregates. In particular, cadherin-deficient, invasion-competent cells tend to position towards the outside of aggregates, facilitating access to extracellular matrix. Reducing actomyosin contractility is found to have a differential effect on spheroid formation depending on compaction mechanism. Inhibition of contractility has a significant stabilizing effect on cell-cell adhesions in integrin-driven aggregation and a mildly destabilizing effect in cadherin-based aggregation. This differential response is exploited to statically control aggregate organization and dynamically rearrange cells in pre-formed aggregates. Sequestration of invasive cells in the interior of spheroids provides a physical barrier that reduces invasion in three-dimensional culture, revealing a potential strategy for containment of invasive cell types.

Cells sense a variety of different mechanochemical stimuli and promptly react to such signals by reshaping their morphology and adapting their structural organization and tensional state. Cell reactions to mechanical stimuli arising from the local microenvironment, mechanotransduction, play a crucial role in many cellular functions in both physiological and pathological conditions. To decipher this complex process, several studies have been undertaken to develop engineered materials and devices as tools to properly control cell mechanical state and evaluate cellular responses. Recent reports highlight how the nucleus serves as an important mechanosensor organelle and governs cell mechanoresponse. In this review, we will introduce the basic mechanisms linking cytoskeleton organization to the nucleus and how this reacts to mechanical properties of the cell microenvironment. We will also discuss how perturbations of nucleus–cytoskeleton connections, affecting mechanotransduction, influence health and disease. Moreover, we will present some of the main technological tools used to characterize and perturb the nuclear mechanical state.

Cell patterning holds significant implications for cell-based analysis and high-throughput screening. The challenge and key factor for formation of cell patterns is to precisely modulate the interaction between cells and substrate surfaces. Many nanosubstrates have been developed to control cell adhesion and patterning, however, requirements of complicated fabrication procedures, harsh reaction conditions, and delicate manipulation are not routinely feasible. Here, we developed a hierarchical polydimethylsiloxane nanosubstrate (HPNS) coated with mussel-inspired polydopamine (PDA) micropatterns for effective cell patterning, depending on both surface topography and chemistry. HPNSs obtained by facile template-assisted replication brought enhanced topographic interaction between cells and substrates, but they were innately hydrophobic and cell-repellent. The hydrophobic nanosubstrates were converted to be hydrophilic after PDA coatings formed via spontaneous self-polymerization, which greatly facilitated cell adhesion. As such, without resorting to any external forces or physical constraints, cells selectively adhered and spread on spatially defined PDA regions with high efficiency, and well-defined cell microarrays could be formed within 20 min. Therefore, this easy-to-fabricate nanosubstrate with no complex chemical modification will afford a facile yet effective platform for rapid cell patterning.

Collagens provide the building blocks for diverse tissues and organs. Furthermore, these proteins act as signaling molecules that control cell behavior during organ development, growth, and repair. Their long half-life, mechanical strength, ability to assemble into fibrils and networks, biocompatibility, and abundance from readily available discarded animal tissues make collagens an attractive material in biomedicine, drug and food industries, and cosmetic products. About three decades ago, pioneering experiments led to recombinant human collagens’ expression, thereby initiating studies on the potential use of these proteins as substitutes for the animal-derived collagens. Since then, scientists have utilized various systems to produce native-like recombinant collagens and their fragments. They also tested these collagens as materials to repair tissues, deliver drugs, and serve as therapeutics. Although many tests demonstrated that recombinant collagens perform as well as their native counterparts, the recombinant collagen technology has not yet been adopted by the biomedical, pharmaceutical, or food industry. This paper highlights recent technologies to produce and utilize recombinant collagens, and it contemplates their prospects and limitations.

Living cells interact with the extracellular matrix (ECM) in a complex and reciprocal manner. Much has been learned over the past few decades about cell-ECM interactions from targeted studies in which a specific matrix parameter (e.g. stiffness, adhesivity) has been varied across a few discrete values, or in which the level or activity of a protein is controlled in an isolated fashion. As the field moves forward, there is growing interest in addressing cell-matrix interactions from a systems perspective, which has spurred a new generation of matrix platforms capable of interrogating multiple ECM inputs in a combinatorial and parallelized fashion. Efforts are also actively underway to integrate specialized, synthetic ECM platforms with global measures of cell behaviors, including at the transcriptomic, proteomic and epigenomic levels. Here we review recent advances in both areas. We describe how new combinatorial ECM technologies are revealing unexpected crosstalk and nonlinearity in the relationship between cell phenotype and matrix properties. Similarly, efforts to integrate "omics" measurements with synthetic ECM platforms are illuminating how ECM properties can control cell biology in surprising and functionally important ways. We expect that advances in both areas will deepen the field's understanding of cell-ECM interactions and offer valuable insight into the design of biomaterials for specific biomedical applications.

Imposed deformations play an important role in morphogenesis and tissue homeostasis, both in normal and pathological conditions. To perceive mechanical perturbations of different types and magnitudes, tissues need appropriate detectors, with a compliance that matches the perturbation amplitude. By comparing results of selective osmotic compressions of cells within multicellular aggregates with small osmolites and global aggregate compressions with big osmolites, we show that global compressions have a strong impact on the aggregates growth and internal cell motility, while selective compressions of same magnitude have almost no effect. Both compressions alter the volume of individual cells in the same way but, by draining the water out of the extracellular matrix, the global one imposes a residual compressive mechanical stress on the cells while the selective one does not. We conclude that, in aggregates, the extracellular matrix is as a sensor which mechanically regulates cell proliferation and migration in a 3D environment.

Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures.Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures.

Summary Cells often communicate by the secretion, transport, and perception of molecules. Information conveyed by molecules is encoded, transmitted, and decoded by cells within the context of the prevailing microenvironments. Conversely, in electronics, transmission reliability and message validation are predictable, robust, and less context dependent. In turn, many transformative advances have resulted by the formal consideration of information transfer. One way to explore this potential for biological systems is to create bio-device interfaces that facilitate bidirectional information transfer between biology and electronics. Redox reactions enable this linkage because reduction and oxidation mediate communication within biology and can be coupled with electronics. By manipulating redox reactions, one is able to combine the programmable features of electronics with the ability to interrogate and modulate biological function. In this review, we examine methods to electrochemically interrogate the various components of molecular communication using redox chemistry and to electronically control cell communication using redox electrogenetics., Graphical Abstract, Bio-Electrochemistry; Bioelectronics; Cell Biology

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice, and is now routinely used in cell biology and in vitro reconstitution. However, a drawback of current technology is that micropatterning efficiency can be variable between proteins, and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, Fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard to micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

Asymmetric cell division (ACD) is an evolutionarily conserved mechanism used by prokaryotes and eukaryotes alike to control cell fate and generate cell diversity. A detailed mechanistic understanding of ACD is therefore necessary to understand cell fate decisions in health and disease. ACD can be manifested in the biased segregation of macromolecules, the differential partitioning of cell organelles, or differences in sibling cell size or shape. These events are usually preceded by and influenced by symmetry breaking events and cell polarization. In this Review, we focus predominantly on cell intrinsic mechanisms and their contribution to cell polarization, ACD and binary cell fate decisions. We discuss examples of polarized systems and detail how polarization is established and, whenever possible, how it contributes to ACD. Established and emerging model organisms will be considered alike, illuminating both well-documented and underexplored forms of polarization and ACD.

3-dimensional (3D) in vitro models were developed in order to mimic the complexity of real organ/tissue in a dish. They offer new possibilities to model biological processes in more physiologically relevant ways which can be applied to a myriad of applications including drug development, toxicity screening and regenerative medicine. Hydrogels are the most relevant tissue-like matrices to support the development of 3D in vitro models since they are in many ways akin to the native extracellular matrix (ECM). For the purpose of further improving matrix relevance or to impart specific functionalities, composite hydrogels have attracted increasing attention. These could incorporate drugs to control cell fates, additional ECM elements to improve mechanical properties, biomolecules to improve biological activities or any combinations of the above. In this Review, recent developments in using composite hydrogels laden with cells as biomimetic tissue- or organ-like constructs, and as matrices for multi-cell type organoid cultures are highlighted. The latest composite hydrogel systems that contain nanomaterials, biological factors, and combinations of biopolymers (e.g., proteins and polysaccharide), such as Interpenetrating Networks (IPNs) and Soft Network Composites (SNCs) are also presented. While promising, challenges remain. These will be discussed in light of future perspectives toward encompassing diverse composite hydrogel platforms for an improved organ environment in vitro. Agency for Science, Technology and Research (A*STAR) Published version This research was supported by the Agency for Science, Technology and Research (A*STAR) under its Acne and Sebaceous Gland Program & Wound Care Innovation for the Tropics IAF-PP (H17/01/a0/008 and H17/01/a0/0L9).