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An integrated microfluidic device to achieve embryo trapping with orientation selectivity and sequential imaging on multiple protein spatial profiles of Drosophila embryos via n cycles of immunofluorescence staining and de-staining.

Living erythrocyte (red blood cell, RBC) membranes present rich ultrastructural and dynamic details, which require synchronous super-resolution imaging and single-molecule tracking to be revealed. Yet, it poses a serious challenge to achieve these dual functions in a single probe, due to the rigid and conflicting photophysical demands of the different techniques. Herein, we rationally developed a far-red boron dipyrromethene membrane probe with blinking capability and persistent single-molecule emission, and constructed a microfluidic platform for noninvasive trapping and long-term imaging of RBCs. By combining them, multi-dimensional super-resolution reconstructions and single-molecule tracking were achieved at the molecular scale on living human RBC membranes in a high-throughput manner. Our integrated system defines a quantitative method for analyzing RBC membranes under physiological and pathological conditions, improving precision and revealing new perspectives for future disease diagnostics.

Current microfluidic methods for studying multicell strains (e.g., m-types) with multienvironments (e.g., n-types) require large numbers of inlets/outlets (m*n), a complicated procedure or expensive machinery. Here, we developed a novel two-layer-integrated method to combine different PDMS microchannel layers with different functions into one chip by a PDMS through-hole array, which improved the design of a PDMS-based microfluidic system. Using this method, we succeeded in converting 2 × m × n inlets/outlets into m + n inlets/outlets and reduced the time cost of loading processing (from m × n to m) of the device for studying multicell strains (e.g., m-types) in varied multitemporal environments (i.e., n-types). Using this device, the dynamic behavior of the cell-stress-response proteins was studied when the glucose concentration decreased from 2% to a series of lower concentrations. Our device could also be widely used in high-throughput studies of various stress responses, and the new concept of a multilayer-integrated fabrication method could greatly improve the design of PDMS-based microfluidic systems.

Isolation and analysis of circulating tumor cells (CTCs) from peripheral blood provides a potential way to detect and characterize cancer. Existing technologies to separate or capture CTCs from whole blood still have issues with sample throughput, separation efficiency or stable efficiency at different flow rates. Here, we proposed a new concept to capture rare CTCs from blood by integrating a triangular prism array-based capture apparatus with streamline-based focus-separation speed reduction design. The focus-separation design could focus and maintain CTCs, while removing a considerable proportion of liquid (98.9%) containing other blood cells to the outlet, therefore, a high CTC capture efficiency could be achieved in the trap arrays with a high initial flow rate. It is worth mentioning that the new design works well over a wide range of flow rates, so it does not require the stability of the flow rate. The results showed that this novel integrated chip can achieve a sample throughput from 5 to 40 mL h-1 with a stable and high CTC capture efficiency (up to 94.8%) and high purity (up to 4 log white blood cells/WBC depletion). The clinical experiment showed that CTCs including CTC clusters were detected in 11/11 (100.0%) patients (mean = 31 CTCs mL-1, median = 25 CTCs mL-1). In summary, our chip enriches and captures CTCs based on physical properties, and it is simple, cheap, fast, and efficient and has low requirements on flow rate, which is very suitable for large-scale application of CTC testing in clinics.

Circulating tumor cells (CTCs) survive in the bloodstream, seed, and invade to foster tumor metastasis. The arrest of CTCs is favored by permissive flow forces and geometrical constraints. Through the use of high-throughput microfluidic devices designed to mimic capillary-sized vessels, we applied different pressure differences to cancer cells and recorded cell traverse-vessel behaviors. Our results showed that cancer cells would transform from Newtonian droplet state to adhesion/migration state when cancer cells traverse in the artificial vessels. To explain these phenomena, a modified Newtonian droplet model was also proposed. These phenomena and the modified model may reveal how the CTCs in the blood seed and invade in the vessels under suitable conditions.

Synthetic Biology aims to create predictable biological circuits and fully operational biological systems. Although there are methods to create more stable oscillators, such as repressilators, orthogonally controlling the oscillation of reporter genes in terms of their amplitude and period is only on theoretical level. Here, we introduce a new oscillator circuit that can be orthogonally controlled by two inducers in Escherichia coli. Some control components, including σECF11 and NahR, were added to the circuit. By systematically tuning the concentration of the inducers, salicylate and IPTG, the amplitude and period can be modulated independently. Furthermore, we constructed a quantitative model to forecast the regulation results. Under the guidance of the model, the expected oscillation can be regulated by choosing the proper concentration combinations of inducers. In summary, our work achieved orthogonal control of the oscillator circuit, which allows the oscillator to be modularized and used in more complex circuit designs.

Microbial communities can survive in complex and variable environments by using different cooperative strategies. However, the behaviors of these mutuality formed communities remain poorly understood, particularly with regard to the characteristics of spatial cooperation. Here, we selected two Escherichia coli strains, designated as the nutrition provider and the antibiotic protector, respectively, for construction of a mutually beneficial bacterial community that could be used to study these behaviors. We found that in addition to the functional mutualism, the two strains also cooperated through their spatial distribution. Under antibiotic pressure, the bacterial distribution changed to yield different spatial distributions, which resulted in community growth advantages beyond functional cooperation. The mutualistic behavior of these two strains suggested that similar communities could also use variations in spatial distribution to improve their survival rates in a natural environment or under the action of antibiotics., Lingjun Li et al. show that antibiotic pressure provides a growth advantage to a mutually beneficial bacterial community, in which one strain is a nutrient provider and the other is an antibiotic protector, by changing their spatial distribution. This study presents spatial coordination as a strategy that drives mutually beneficial symbiosis.

The mechanical properties of cells are of great significance to their normal physiological activities. The current methods used for the measurement of a cell’s mechanical properties have the problems of complicated operation, low throughput, and limited measuring range. Based on micropipette technology, we designed a double-layer micro-valve-controlled microfluidic chip with a series of micropipette arrays. The chip has adjustment pressure ranges of 0.03–1 and 0.3–10 kPa and has a pressure stabilization design, which can achieve a robust measurement of a single cell's mechanical properties under a wide pressure range and is simple to operate. Using this chip, we measured the mechanical properties of the cells treated with different concentrations of paraformaldehyde (PFA) and observed that the viscoelasticity of the cells gradually increased as the PFA concentration increased. Then, this method was also used to characterize the changes in the mechanical properties of the differentiation pathways of stem cells from the apical papilla to osteogenesis.

The causes and consequences of aging have always been a concern. In recent studies, changes in the stress response capacity of cells during aging were quantitatively analyzed. It was found that aging was accompanied by a decline in response capacity. When the response capacity decreased to a critical value, which we assumed was the internal noise level, the cell soon died. To survive, the response capacity should be, at minimum, sufficiently strong to resist intracellular noise. Here, we discuss the role of stress response capacity in aging and conjecture that lifespan might be extended by enhancing stress response capacity.

The aging process is regarded as the progressive loss of physiological integrity, leading to impaired biological functions and the increased vulnerability to death. Among various biological functions, stress response capacity enables cells to alter gene expression patterns and survive when facing internal and external stresses. Here, we explored changes in stress response capacity during the replicative aging of Saccharomyces cerevisiae. To this end, we used a high-throughput microfluidic device to deliver intermittent pulses of osmotic stress and tracked the dynamic changes in the production of downstream stress-responsive proteins, in a large number of individual aging cells. Cells showed a gradual decline in stress response capacity of these osmotic-related downstream proteins during the aging process after the first 5 generations. Among the downstream stress-responsive genes and unrelated genes tested, the residual level of response capacity of Trehalose-6-Phosphate Synthase (TPS2) showed the best correlation with the cell remaining lifespan. By monitor dynamics of the upstream transcription factors and mRNA of Tps2, it was suggested that the decline in downstream stress response capacity was caused by the decline of translational rate of these proteins during aging.

The quantitative studies of cell proliferation and migration under different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a new device to pattern several types of cells in 24-well-plate and demonstrated its’ application in cancer cell proliferation and migration assay. The new device combined 3D-printed-silica-part for multi cell types loading with PDMS-through-hole-layer-part for cell micro-patterning which was matched with commercial 24-well-plate. This 24-well-plate based device is flexible and feasible in many applications and can be used in one piece or multi pieces. Besides the application for two types of cells proliferation and migration assay in one chemical condition, as a demonstration, the migration behaviors of four types of cells under 24 types of EGF + bFGF combinatorial conditions were studied. We believed this device could be widely used in clinical searching for new anti-cancer therapeutics and other related studies.

As a reaction-diffusion system strongly affected by temperature, the early fly embryos surprisingly show highly reproducible and accurate developmental patterns during embryogenesis under temperature perturbations. To reveal the underlying temperature compensation mechanism, it is important to overcome the challenge in quantitative imaging on fly embryos under temperature perturbations. Inspired by a microfluidics generating temperature steps on fly embryos, here we design a microfluidic device capable of ensuring the normal development of multiple fly embryos as well as achieving real-time temperature control and fast temperature jumps for quantitative live imaging with a home-built two-photon microscope. We apply this system to quantify the temperature compensation of the morphogen Bicoid (Bcd) gradient in fly embryos. The length constant of the exponential Bcd gradient reaches the maximum at 25 °C within the measured temperatures of 18-29 °C and gradually adapts to the corresponding value at new temperatures upon a fast temperature switch. Such an adaption decreases to a less degree if temperature is switched in a later developmental stage. This age-dependent temperature compensation could not be explained with the traditional synthesis-diffusion-degradation (SDD) model assuming the static parameters but an extended SDD model incorporating the dynamic change of the parameters controlling the formation of Bcd gradients.SIGNIFICANCEThermal robustness is important for biological systems experiencing temperature fluctuations. To reveal the temperature compensation mechanism, the fruit fly embryo is an ideal model system. It is intriguing how the early fly embryo achieves highly reproducible and accurate patterning despite it is a reaction-diffusion system strongly affected by temperature. However, it has been challenging to quantitatively measure the developmental patterns in fly embryos under temperature perturbations. To overcome this problem, we construct a fast temperature tunable microfluidic device for fly embryos. Combining quantitative imaging with this device and mathematical modeling, we successfully quantify the temperature response of the morphogen Bicoid (Bcd) gradient and reveal that the temperature compensation for the Bcd gradient is stronger in the later developmental stage.

Summary We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC., Graphical Abstract, Highlights • Low-adhesion substrates reveal two different subtypes co-exist in the hPSC population • SRF-based regulatory loop-coupled adhesion, cell morphology, and KLF4/5 expression • The low-adhesion substrates are more suitable for high-pluripotency cell culture, When culturing hPSCs on low-adhesion substrate (gelatin nanofiber), Dr. Liu Li and her colleagues found two subtypes with different colony morphologies. The dome-like cells showed higher proliferation capacity and KLF4/5 and NANOG expression than the monolayer cells. A serum response factor-based regulatory double loop was proposed to explain how cell-matrix adhesion mediates the interaction between cell morphology and pluripotency genes.

Air pollution has become one of major environmental challenges facing mankind in this century. However, current atmospheric toxicity studies are most offline with low sensitivity and limited understanding of collective biological responses to ambient stimuli on the single cell level. Here, we developed an online analysis system named as SLEPTor (Single Living yEast PM Toxicity Sensor) which allows us to analyze aerosol particle toxicity in vivo. The SLEPTor is composed of an automated air sampling, microfluidics, GFP(green fluorescent protein)-tagged Saccharomyces cerevisiae yeast sensor as well as multiplexing automated fluorescence imaging system. In testing the SLEPTor, 63 yeast genes were screened simultaneously here for their responses to both man-made reactive oxygen species (ROS) and also Beijing aerosol samples. For ROS samples, MRS3, MRS4, BSD2 and ZRC1 proteins were detected in increased abundances, and the BSD2 protein expression, related to oxidative stress, was more pronounced, followed by MRS4 (for DNA repair) with a peak expression after 20 min exposure. In contrast, HSP60 (oxidative stress), SSA1 (DNA repair) and MSB1 (MAPK pathway) proteins were observed to have elevated expressions visualized with time-lapsed movies when challenged with Beijing air samples (150 µg/m 3 ) for 100 min. The same experiments were repeated twice involving 63 genes using the SLEPTor and further confirmed by a spectra method. Among the screened proteins, SSA1 was found to be sensitive to ambient air. Using the SLEPTor developed, we have shown that PM exposure resulted in oxidative stress and DNA repair on living yeast cells. The demonstrated proof-of-concept technology here pioneers a new arena for investigating health impacts of aerosol particles in an interactive manner. Yet, its sensitivity and repeatability need to be further explored using a large set of different functional proteins.

Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or 60Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

The separation or purification of bacterial samples from a mixed cell suspension is critical in a variety of biomedical applications, such as sputum diagnostics and cell biology studies. We propose a streamline-based microfluidic filtration device for highly efficient purification of bacterial samples from a mixed cell suspension. The device is composed of tens of repeated streamline-based separation units that continuously filter the solution. By injecting a liquid sample such as liquefied human sputum solution through the device, approximately 50% of the injected sample solution can be collected from the filtration collection channels, which filter approximately 99.9% of the mammalian cells but retain approximately 60% to 90% of the bacteria. Different injection rates (0.2 ml h-1 to 30 ml h-1), different sample viscosities, and different initial bacterial densities were tested and confirmed that our separation method was robust. The easy operation, robustness and high efficiency indicate that our method may be useful for the separation or purification of bacterial samples from a mixed cell suspension, such as bacterial samples for sputum diagnostics.

The airway microbial community, which takes important pathogenic roles for respiratory diseases, is far from clear in terms of taxonomy and gene functions. One of the critical reasons is the heavy contamination of host cell/DNA in airway samples, which hinders the subsequent sequencing of the whole genomic contents of the microbial community—the metagenome. Here, we describe a protocol for airway sample preparation which couples a microbe enrichment microfluidic device and a DNA amplification method performed in numerous droplets. When evaluated with mock and clinical sputum samples, the microfluidics-based enrichment device and emulsion-based whole-genome amplification (MEEA) procedure efficiently removes host cells, amplifies the microbial genome, and shows no obvious bias among microbes. The efficiency of MEEA makes it a promising method in research of respiratory microbial communities and their roles in diseases., Dysbiosis of airway microbiomes has been found in various respiratory diseases, but its molecular details in terms of taxonomic profile, metabolic characteristics, defensive function, and inhabit adaption are far from clear. Shotgun metagenome sequencing provides detailed information for microbes, whereas its application is rather limited in airways due to host DNA contaminants that overwhelm a minute amount of microbial content. Here, we describe a microfluidics-based enrichment device and an emulsion-based whole-genome amplification procedure (MEEA) for the preparation of DNA from sputa for shotgun sequencing in a metagenomics study. The two protocols coupled in MEEA are first separately assayed with mock samples and are both promising in efficiency and bias. The efficiency and consistency of MEEA are further evaluated in six clinical sputum samples against direct sequencing without enrichment, and MEEA enables 2 to 14 times enrichment for microbial reads, which take 14.68% to 33.52% of total reads. The dominant pathogens detected in MEEA are in excellent agreement with those from clinical etiological tests. Meanwhile, MEEA presents much more microbiome complexity and genome information at a strain level than direct sequencing, exhibiting high sensitivity for identifying prophages and DNA viruses. MEEA provides better microbiome profiling than direct sequencing without a preference for specific microorganisms. The more detailed functional and taxonomic characterization of their species constituents, including both bacterium and virus, facilitates metagenomics studies on the pathogenesis of respiratory microbiomes. IMPORTANCE The airway microbial community, which takes important pathogenic roles for respiratory diseases, is far from clear in terms of taxonomy and gene functions. One of the critical reasons is the heavy contamination of host cell/DNA in airway samples, which hinders the subsequent sequencing of the whole genomic contents of the microbial community—the metagenome. Here, we describe a protocol for airway sample preparation which couples a microbe enrichment microfluidic device and a DNA amplification method performed in numerous droplets. When evaluated with mock and clinical sputum samples, the microfluidics-based enrichment device and emulsion-based whole-genome amplification (MEEA) procedure efficiently removes host cells, amplifies the microbial genome, and shows no obvious bias among microbes. The efficiency of MEEA makes it a promising method in research of respiratory microbial communities and their roles in diseases.

It is commonly believed that bacterial chemotaxis helps cells find food. However, not all attractants are nutrients, and not all nutrients are strong attractants. Here, by using microfluidic experiments, we studied Escherichia coli chemotaxis behavior in the presence of a strong chemoattractant (e.g., aspartate or methylaspartate) gradient and an opposing gradient of diluted tryptone broth (TB) growth medium. Our experiments showed that cells initially accumulate near the strong attractant source. However, after the peak cell density ([Formula: see text]) reaches a critical value [Formula: see text] , the cells form a “escape band” (EB) that moves toward the chemotactically weaker but metabolically richer nutrient source. By using various mutant strains and varying experimental conditions, we showed that the competition between Tap and Tar receptors is the key molecular mechanism underlying the formation of the escape band. A mathematical model combining chemotaxis signaling and cell growth was developed to explain the experiments quantitatively. The model also predicted that the width [Formula: see text] and the peak position [Formula: see text] of EB satisfy two scaling relations: [Formula: see text] and [Formula: see text] , where [Formula: see text] is the channel length. Both scaling relations were verified by experiments. Our study shows that the combination of nutrient consumption, population growth, and chemotaxis with multiple receptors allows cells to search for optimal growth condition in complex environments with conflicting sources.

Microfluidics, in combination with time-lapse microscopy, is a transformative technology that significantly enhances our ability to monitor and probe biological processes in living cells. However, high-throughput microfluidic devices mostly require sophisticated preparatory and setup work and are thus hard to adopt by non-experts. In this work, we designed an easy-to-use microfluidic chip, which enables tracking of 48 GFP-tagged yeast strains, with each strain under two different stimulus conditions, in a single experiment. We used this technology to investigate the dynamic pattern of protein expression during the yeast mating differentiation response. High doses of pheromone induce cell cycle arrest and the shmoo morphology, whereas low doses of pheromone lead to elongation and chemotrophic growth. By systematically analyzing the protein dynamics of 156 pheromone-regulated genes, we identified groups of genes that are preferentially induced in response to low-dose pheromone (elongation during growth) or high-dose pheromone (shmoo formation and cell cycle arrest). The protein dynamics of these genes may provide insights into the mechanisms underlying the differentiation switch induced by different doses of pheromone.

Synthetic biologists are dedicated to designing genetic systems from the bottom up to understand how living systems work. To date, a variety of genetic circuits exhibiting bistability have been designed, greatly expanding our understanding of the biological multistability in natural systems. However, the study of more complex forms of biological multistability using synthetic methods is still limited. In this report, we describe the engineering of a genetic circuit with regulatable multistability. A novel genetic toggle switch exhibiting inducible bistability and a self-activation circuit were individually designed and characterized, after which they were assembled to create a circuit that presents tristability. In bacteria, this synthetic circuit enables cells to differentiate spontaneously into three different states of gene expression. Moreover, the multistability of the circuit can be modulated by external inputs. This work provides a synthetic biology framework for the study of biological multistability and may help to understand natural multistability phenomena.

Enterococcus faecalis(E. faecalis) is a common microorganism could be isolated from the infected canals, especially in the case of refractory apical periodontitis. Due to its ability to invade the dentinal tubules and highly resistant to antimicrobial strategies, the thorough debridement of E.faecalis is hard to achieve. And that may be one of the reasons to cause reinfection and therapeutic failure. According to the anatomy of dentinal tubules published before and the results of our team previous work, we designed six types of microtubes with different sizes. By using the method of centrifugation and incubation, a standard infected model mimicking dentinal tubules was established. Sodium hypochlorite (NaClO) is the most popular irrigant applied in root canal treatment. We used three different concentrations with four distinct irrigation duration to observe the antibacterial process of E. faecalis colonies within microtubes dynamically. We concluded that the role of NaClO in the microtubes is concentration dependent and duration dependent. And the interpretation of the results has a certain reference value for clinicians.

Massive technological advances enabled high-throughput measurements of proteomic changes in biological processes. However, retrieving biological insights from large-scale protein dynamics data remains a challenging task. Here we used the mating differentiation in yeast Saccharomyces cerevisiae as a model and developed integrated experimental and computational approaches to analyze the proteomic dynamics during the process of cell fate determination. When exposed to a high dose of mating pheromone, the yeast cell undergoes growth arrest and forms a shmoo-like morphology; however, at intermediate doses, chemotropic elongated growth is initialized. To understand the gene regulatory networks that control this differentiation switch, we employed a high-throughput microfluidic imaging system that allows real-time and simultaneous measurements of cell growth and protein expression. Using kinetic modeling of protein dynamics, we classified the stimulus-dependent changes in protein abundance into two sources: global changes due to physiological alterations and gene-specific changes. A quantitative framework was proposed to decouple gene-specific regulatory modes from the growth-dependent global modulation of protein abundance. Based on the temporal patterns of gene-specific regulation, we established the network architectures underlying distinct cell fates using a reverse engineering method and uncovered the dose-dependent rewiring of gene regulatory network during mating differentiation. Furthermore, our results suggested a potential crosstalk between the pheromone response pathway and the target of rapamycin (TOR)-regulated ribosomal biogenesis pathway, which might underlie a cell differentiation switch in yeast mating response. In summary, our modeling approach addresses the distinct impacts of the global and gene-specific regulation on the control of protein dynamics and provides new insights into the mechanisms of cell fate determination. We anticipate that our integrated experimental and modeling strategies could be widely applicable to other biological systems., Author summary A systematic characterization of the proteomic changes during the process of cell differentiation is critical for understanding the underlying molecular mechanisms. However, protein expression can be largely affected by changes in cell physiological state, which hampers the detection of regulatory interactions. Here we proposed an integrated experimental and computational framework to reconstruct regulatory circuits in mating differentiation of budding yeast Saccharomyces cerevisiae, in which distinct cell fates are triggered by alteration in pheromone concentration. A modeling approach was developed to decouple gene-specific regulation from growth-dependent global regulation of protein expression, allowing us to reverse engineering the gene regulatory circuits underlying distinct cell fates. Our work highlights the importance of model-based analysis of proteomic data and delivers new insight into dose-dependent differentiation behavior of budding yeast.

Motorized fluorescence microscopy combined with high-throughput microfluidic chips is a powerful method to obtain information about different biological processes in cell biology studies. Generally, to observe different strains under different environments, high-throughput microfluidic chips require complex preparatory work. In this study, we designed a novel and easily operated high-throughput microfluidic system to observe 96 different GFP-tagged yeast strains in one switchable culture condition or 24 different GFP-tagged yeast strains in four parallel switchable culture conditions. A multi-pipette is the only additional equipment required for high-throughput patterning of cells in the chip. Only eight connections are needed to control 96 conditions. Using these devices, the proteomic dynamics of the yeast stress response pathway were carefully studied based on single-cell data. A new method to characterize the proteomic dynamics using a single cell’s data is proposed and compared to previous methods, and the new technique should be useful for studying underlying control networks. Our method provides an easy and systematic way to study signaling pathways at the single-cell level.

We developed a gel-embossing technique which could be used to fabricate gelatin nanopatterns for cell culture studies. A gel of gelatin solution was dropped on the surface of a glass slide and embossed using a gas permeable stamp made of polydimethylsiloxane (PDMS). After drying in a desiccator, nanostructure patterns could be replicated from the stamp into the gelatin layer. After crosslinking in a 10% formaldehyde solution in phosphate buffered saline (PBS), the embossed gelatin layer could be used for cell culture studies. The gel-embossed patterns before and after crosslinking were characterized by atomic force microscopy, revealing significant changes of the pattern morphology introduced by both gel-emboss and crosslink processes. Nonetheless, strong effects of cell elongation and fast cell growth were observed for the proof-of-concept using NIH-3T3 cells cultured on gelatin nanogrooves.