Culture Platform Research Articles

Abstract 6785: KIYA-PREDICT™ XPDX: An ex vivo 3D spheroid platform for modeling in vivo responses to multiple classes of oncology therapeutics

Abstract Patient-derived xenografts (PDX) are a powerful and commonly used tool in preclinical drug development as they represent an in vivo platform for screening drug response in human tumors that recapitulate many molecular and histological features of the original patient tumor. However, these models are costly and time-intensive, necessitating the development of ex vivo platforms for assessing drug response to PDX-derived tumors to de-risk in vivo studies by aiding in model selection and reducing the number of animals and costs associated with in vivo experimentation. The KIYA-PREDICT™ platform is a clinically validated, ex vivo 3D cell culture platform with demonstrated ability to predict patient response to chemotherapy in high grade glioma and ovarian cancer. Here, we apply this platform to profiling XPDX from XenoSTART’s comprehensive library of well-characterized models as a tool for assessing drug response and mechanism of action ex vivo while also aiding in model selection for in vivo studies. Over 100 XPDX models have been profiled ex vivo for response to several classes of cancer therapeutics, including chemotherapy, targeted therapy, and antibody-drug conjugates (ADCs), demonstrating robust concordance with in vivo drug response. CDK4/6 inhibitors palbociclib and ribociclib evaluated ex vivo in breast XPDX display strong correlation to in vivo response and recapitulate palbociclib resistance. Similarly, G12C inhibitors sotorasib (AMG-510) and adagrasib (MRTX849) screened against a panel of non-small cell lung cancer XPDX ex vivo match in vivo response and drug resistance. Finally, ex vivo and in vivo responses to FDA approved ADC enfortumab vedotin were evaluated in a panel of bladder cancer PDX, with ex vivo and in vivo responses showing good concordance and expected modulation based on Nectin-4 expression levels. The combination of the KIYA-PREDICT™ platform with XenoSTART’s XPDX catalogue is a flexible and powerful tool that can be utilized for streamlining preclinical drug development for a wide range of therapeutic classes. Citation Format: Aaron L. Carlson, Natalie W. Dance, Kimberly J. Burgess, Danielle Y. Nadeau, Melissa Millard, Alyssa Simonson, Armando Diaz, Michael Wick, Teresa M. DesRochers. KIYA-PREDICT™ XPDX: An ex vivo 3D spheroid platform for modeling in vivo responses to multiple classes of oncology therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6785.

Abstract 6777: An ex-vivo organotypic culture platform of mucinous carcinomatosis peritonei identifies CDK4-6 inhibition as a novel treatment

Abstract Concomitant mutations in KRAS and GNAS have been linked to mucinous histology in gastrointestinal neoplasms of the appendix, colon, and pancreas (IPMN). Upon progression, these tumors are characterized by metastasis which favors the peritoneal surface, resulting in mucinous carcinomatosis peritonei (MCP). Recently we have described a novel technique which allows for long term culture and drug intervention of human MCP tumors ex-vivo. With evidence of durable clinical benefit of CDK4-6 inhibition in a single patient with chemo refractory MCP of appendiceal origin, we sought to further investigate anti-tumor responses using CDK4-6 inhibition in ex-vivo tumor slices, in order to identify candidates for a personalized clinical therapy trial. Here, in a comparison of ex-vivo patient tumor slices from 18 individual donors, treatment using CDK4-6 inhibitors revealed a significant reduction in cancer cell specific proliferation in mucinous GNAS-mut tumors compared to non-mucinous cancers. Anti-tumor responses of CDK4-6 were determined to be tumor-cell intrinsic, as CDK4-6 inhibition blocked proliferation of stromal cells in the TME independently of patient tumor mutational and mucin status. Based on these finding a personalized clinical trial was conducted where we report that 13 of 16 patients (81%) enrolled with MCP treated with palbociclib had at least a 10% decrease in CEA, as compared to historical chemotherapy responses rates reported from 14-30%. These results indicate that CDK4/6 inhibition is a novel and efficacious treatment for patients with MCP. Citation Format: Jonathan Weitz, Daisuke Nishizaki, Jay Patel, Isabella Ng, Siming Sun, Dana Ramms, Jingjing Zou, Joel Baumgartner, Kaitlyn Kelly, Hitendra Patel, Rebekah White, Jula Veerapong, Peter Vu, Silvio Gutkind, Herve Tiriac, Shumei Kato, Andrew Lowy. An ex-vivo organotypic culture platform of mucinous carcinomatosis peritonei identifies CDK4-6 inhibition as a novel treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6777.

Platforming the past: Nostalgia, video games, and A Hat in Time

Research on nostalgia has shown how media texts can promote ideological visions of the past. The 2017 3D platformer video game A Hat in Time (AHIT) is a case study in this nostalgic construction of values in a contemporary, postmodern, convergence-culture context. The game's marketing and text discursively and materially construct AHIT as a continuation of video games from the 1990s and early 2000s in a way that creates a communal nostalgia. Fans have also used the game itself as a platform by creating a library of more than five thousand modifications, or mods, many of which are based on 1990s and 2000s texts; these mods allow the game to serve as a platform for postmodern remixes of nostalgic texts. AHIT shows how contemporary convergence culture and media platforms allow for communal, customizable nostalgia.

Influence of Remodeled ECM and Co-culture with iPSC-Derived Cardiac Fibroblasts on the Mechanical Function of Micropatterned iPSC-Derived Cardiomyocytes.

In native heart tissue, functions of cardiac fibroblasts (CFs) include synthesis, remodeling, and degradation of the extracellular matrix (ECM) as well as secreting factors that regulate cardiomyocyte (CM) function. The influence of direct co-culture and CF-derived ECM on CM mechanical function are not fully understood. Here we use an engineered culture platform that provides control over ECM geometry and substrate stiffness to evaluate the influence of iPSC-CFs, and the ECM they produce, on the mechanical function of iPSC-CMs. Mechanical analysis was performed using digital image correlation to quantify maximum contractile strain, spontaneous contraction rate, and full-field organization of the contractions. When cultured alone, iPSC-CFs produce and remodel the ECM into fibers following the underlying 15° chevron patterned ECM. The substrates were decellularized and confirmed to have highly aligned fibers that covered a large fraction of the pattern area before reseeding with iPSC-CMs, alone or in co-culture with iPSC-CFs. When seeded on decellularized ECM, larger maximum contractile strains were observed in the co-culture condition compared to the CM Only condition. No significant difference was found in contractile strain between the Matrigel and decellularized ECM conditions; however, the spontaneous contraction rate was lower in the decellularized ECM condition. A methodology for quantifying alignment of cell contraction across the entire field of view was developed based on trajectories approximating the cell displacements during contraction. Trajectory alignment was unaltered by changes in culture or ECM conditions. These combined observations highlight the important role CFs play in vivo and the need for models that enable a quantitative approach to examine interactions between the CFs and CMs, as well as the interactions of these cells with the ECM.

Art in circulation: Creating content and context for digital reproduction of artworks

AbstractThe use of digital museum objects has become an essential part of museums' communication and marketing strategies, research and teaching, and curatorial practices. This new visibility, amplified by the COVID‐19 crisis, has not only revealed the possibilities of digital museum objects but has also underscored significant challenges, including the intricate relationship between digital museum objects and physical objects, the impact of digital museum objects on knowledge creation, and the online interaction with art and cultural heritage. Furthermore, it has drawn attention to the digital platforms that host digital museum objects, ranging from museum collection databases to online encyclopedias, cultural heritage platforms, and social media. The present paper explores these issues by examining how digital platforms are changing the way digital reproductions of artworks are used and reimagined, both inside and outside the institutions that house the artworks. To illuminate these dynamics, it looks at specific case studies, including the Getty Challenge, the online circulation of Delacroix's La liberté guidant le peuple, and the collection databases of Belgian museums and of the Mauritshuis. Theoretically, it combines the art historical concept of circulation with the notion of gray and colored memory drawn from digital memory studies. In doing so, it conceives of museum databases and cultural heritage platforms, as spaces of participation and neglect, of memory and oblivion, with a vast potential for producing new perspectives on art and cultural heritage and telling new (art) histories. In conclusion, the paper advocates for “circulation” as a key concept for revitalizing online collections of digital reproductions of artworks.

Enhancing biomass production and biochemical compositions of Spirodela polyrhiza through superhydrophobic cultivation platforms at low light intensity

Duckweed, a floating macrophyte, has attracted interest in various fields such as animal feedstocks and bioenergy productions. Its enriched nutritional content and rapid growth rate make it particularly promising. However, common laboratory cultures of duckweed often experience fronds layering, diminishing the efficiency of sunlight capturing due to limited surface area on conventional cultivation platforms. In this work, we aimed to address the issue of fronds layering by introducing a novel cultivation platform - a superhydrophobic coated acrylic sheet. The sheet was prepared by spray-coating a suspension of beeswax and ethanol, and its effectiveness was evaluated by comparing the growth performance of giant duckweed, Spirodela polyrhiza, on this platform with that on a modified version. The superhydrophobic coated acrylic sheet (SHPA) and its variant with a metal mesh added (SHPAM) were employed as growing platforms, with a glass jar serving as the control. The plantlets were grown for 7 days with similar growth conditions under low light stress (25 μmol/m2/s). SHPAM demonstrated superior growth performance, achieving a mass gain of 102.12 ± 17.18 %, surpassing both SHPA (89.67 ± 14.97 %) and the control (39.26 ± 8.94 %). For biochemical compositions, SHPAM outperformed in chlorophyll content, protein content and lipid content. The values obtained were 1.021 ± 0.076 mg/g FW, 14.59 ± 0.58 % DW and 6.21 ± 0.75 % DW respectively. Therefore, this work proved that incorporation of superhydrophobic coatings on a novel cultivation platform significantly enhanced the biomass production of S. polyrhiza. Simultaneously, the biochemical compositions of the duckweeds were well-maintained, showcasing the potential of this approach for optimized duckweed cultivation.

Anthropological Perspectives on the Historical Development of Salar Folk Songs in Qinghai Province, China

Background and Aims: Salar folk songs, an integral part of the cultural heritage of Qinghai Province, China, are a testament to the remarkable diversity within Chinese culture. This research aims to investigate the historical development of Salar folk songs in Qinghai Province, China. Materials and Methods: Result: The research reveals that Salar folk songs have evolved, adapted, and continue to thrive, serving as a testament to the enduring mechanisms of cultural preservation. Recommendations include cultural preservation efforts, education, collaborative research, and international recognition, ensuring the continued vitality of Salar folk songs as a living legacy within the Chinese cultural mosaic. Results: The research reveals that Salar folk songs have evolved, adapted, and continue to thrive, serving as a testament to the enduring mechanisms of cultural preservation. Recommendations include cultural preservation efforts, education, collaborative research, and international recognition, ensuring the continued vitality of Salar folk songs as a living legacy within the Chinese cultural mosaic. Salar folk songs are vital for their cultural heritage, requiring preservation, documentation, and promotion. Collaborating with scholars, incorporating them into festivals, tourism initiatives, and educational curricula, and gaining international recognition can facilitate cross-cultural exchange and contribute to diverse Chinese culture. Conclusion: Safeguarding the rich tapestry of Salar folk songs demands concerted efforts in cultural preservation, documentation, and promotion. Embracing collaborative initiatives, integrating these melodies into various cultural platforms, and securing international recognition will not only ensure the vitality of this living legacy but also foster cross-cultural exchange, enriching the diverse tapestry of Chinese culture.

A miniaturized culture platform for control of the metabolic environment.

The heart is a metabolic "omnivore" and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids, a three-dimensional in vitro model of the heart wall, are a useful tool to study cardiac physiology and pathology. However, cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo, whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media, which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner, we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques, prevents flow blockage issues, and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids, resulting in large fluctuations in this metabolite under static culture. Despite this, neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact, RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together, we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems.

The Auckland War Memorial Museum Online Cenotaph: community participation, collective memorialisation and social cohesion

Purpose Online/Digital cultural heritage platforms have the potential to serve as empowering sites and tools for democratic participation, and for promoting social cohesion, acting as convergence points for diverse societal groups. They enable the gathering of multiple voices, including those of minorities and groups often marginalised in mainstream cultural heritage documentation. This research paper examines the ways in which these aspirations of cultural heritage platforms as meeting, learning and dialogic spaces for connecting and empowering online communities have been realised. Design/methodology/approach Using a qualitative design, interviews were conducted with users of New Zealand’s Auckland War Memorial Museum’s Online Cenotaph. Participants shared their experiences with the platform, perceptions of it as a collective social history resource and views on its role as a participatory space for online communities. They also discussed their expectations for its development as an online space for collective memorialisation. Findings Interviews revealed that users value Online Cenotaph for placing personal, publicly contributed memories and narratives alongside primary military sources. Participants expressed feelings of civic responsibility, social awareness and a sense of identity and connection through their use and contribution to this online commemorative space. The shift from a one-way flow of information from the Museum towards embracing public contribution embodying a high-trust approach, was a notable finding. Originality/value This research underscores the evolving role of museums and other GLAM institutions in recognising the importance of inclusivity, diversity and community participation. It provides insights into how digital cultural heritage social platforms can contribute towards these goals and promote social cohesion. This research is also a starting point for further studies on crowdsourcing and social Web activities on digital cultural heritage platforms as sites of community building through public participation and engagement in historical/cultural heritage narratives.

Effect of Donor Age and Liver Steatosis on Potential of Decellularized Liver Matrices to be used as a Platform for iPSC-Hepatocyte Culture.

Decellularization of discarded whole livers and their recellularization with patient-specific induced pluripotent stem cells (iPSCs) to develop a functional organ is a promising approach to increasing the donor pool. The effect of extracellular matrix (ECM) of marginal livers on iPSC-hepatocyte differentiation and function has not been shown. To test the effect of donor liver ECM age and steatosis, young and old, as well as no, low, and high steatosis livers, are decellularized. All livers are decellularized successfully. High steatosis livers have fat remaining on the ECM after decellularization. Old donor liver ECM induces lower marker expression in early differentiation stages, compared to young liver ECM, while this difference is closed at later stages and do not affect iPSC-hepatocyte function significantly. High steatosis levels of liver ECM lead to higher albumin mRNA expression and secretion while at later stages of differentiation expression of major cytochrome (CYP) 450enzymes is highest in low steatosis liver ECM. Both primary human hepatocytes and iPSC-hepatocytes show an increase in fat metabolism marker expression with increasing steatosis levels most likely induced by excess fat remaining on the ECM. Overall, removal of excess fat from liver ECM may be needed for inducing proper hepatic function after recellularization.

Biomechanical Remodeling of Aortic Valve Interstitial Cells During Calcified Lesion Formation In Vitro.

Healthy aortic heart valves are essential to the regulation of unidirectional blood flow. Calcific aortic valve disease (CAVD) is an actively progressive disease that involves the disorganization of valve cells and accumulation of calcium deposits on the aortic valve leaflets. CAVD involves disruption of cell environment homeostasis that prior cell culture models have found difficult to portray and model. As it is still poorly understood how tissue stiffening associates with lesion formation, here, we implement a novel 3D culture platform to characterize the relationship between mechanical stress and tissue remodeling and analyze how the application of pro-osteogenic stimulation dysregulates the native ability of valve cells to organize its matrix. Through a temporal study of macroscopic remodeling, we determine that aortic valve interstitial neo-tissues undergo varying stiffness and mechanical stress, demonstrate greater myofibroblastic gene expression, and show greater remodeling activity in the outer surface of the neo-tissue in a banding pattern when cultured in osteogenic growth medium. In human aortic valve interstitial cells cultured in osteogenic growth medium, we observed an increase in stress but significant decreases in myofibroblastic gene expression with the addition of growth factors. In summary, we are able to see the interplay of biochemical and biomechanical stimuli in valvular remodeling by using our platform to model dynamic stiffening of valve interstitial neo-tissues under different biochemical conditions.

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems. Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.

Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

Application of Big Data in Tourism Destination Management: A Case Study of Changsha City

In the era of information technology, the utilization of big data technology is rapidly growing, leading to significant changes in the tourism industry. Big data not only creates more business opportunities for the industry but also drives the transformation and enhancement of tourist destinations and the implementation of efficient management. This study employs two research methods: literature review and case analysis. Firstly, by reviewing relevant literature, the latest research findings and trends in big data technology for tourism destination management are summarized. Secondly, through case analysis, a comprehensive understanding of the current situation and challenges in the application of big data technology in tourism destination management in Changsha is obtained. Leveraging the Changsha cultural and tourism data platform, this study retrieves information such as tourist reception data of tourism destinations in Changsha and assesses the impact of Changsha’s big data technology on tourism destination management. The research reveals limitations and challenges in the application of big data technology in Changsha’s tourism destination management, including data privacy protection and technical security, which require further exploration in future practices. The goal of this study is to offer insights for the application of big data in tourism destination management in Changsha and provide guidance for destination managers in similar cities.

Nanopore sequencing of infectious fluid is a promising supplement for gold-standard culture in real-world clinical scenario.

Infectious diseases are major causes of morbidity and mortality worldwide, necessitating the rapid identification and accurate diagnosis of pathogens. While unbiased metagenomic next-generation sequencing (mNGS) has been extensively utilized in clinical pathogen identification and scientific microbiome detection, there is limited research about the application of nanopore platform-based mNGS in the diagnostic performance of various infectious fluid samples. In this study, we collected 297 suspected infectious fluids from 10 clinical centers and detected them with conventional microbiology culture and nanopore platform-based mNGS. The objective was to assess detective and diagnostic performance of nanopore-sequencing technology (NST) in real-world scenarios. Combined with gold-standard culture and clinical adjudication, nanopore sequencing demonstrated nearly 100% positive predictive agreements in microbial-colonized sites, such as the respiratory and urinary tracts. For samples collected from initially sterile body sites, the detected microorganisms were highly suspected pathogens, and the negative predictive agreements were relatively higher than those in the microbial-colonized sites, particularly with 100% in abscess and 95.7% in cerebrospinal fluid. Furthermore, consistent performance was also observed in the identification of antimicrobial resistance genes and drug susceptibility testing of pathogenic strains of Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii. Rapid NST is a promising clinical tool to supplement gold-standard culture, and it has the potential improve patient prognosis and facilitate clinical treatment of infectious diseases.

Construction and characterisation of a structured, tuneable, and transparent 3D culture platform for soil bacteria.

We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the carbon catabolism of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms, Bacillus subtilis and Streptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as competition and warfare.

Electroactive 4D Porous Scaffold Based on Conducting Polymer as a Responsive and Dynamic In Vitro Cell Culture Platform.

In vivo, cells reside in a 3D porous and dynamic microenvironment. It provides biochemical and biophysical cues that regulate cell behavior in physiological and pathological processes. In the context of fundamental cell biology research, tissue engineering, and cell-based drug screening systems, a challenge is to develop relevant in vitro models that could integrate the dynamic properties of the cell microenvironment. Taking advantage of the promising high internal phase emulsion templating, we here designed a polyHIPE scaffold with a wide interconnected porosity and functionalized its internal 3D surface with a thin layer of electroactive conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) to turn it into a 4D electroresponsive scaffold. The resulting scaffold was cytocompatible with fibroblasts, supported cellular infiltration, and hosted cells, which display a 3D spreading morphology. It demonstrated robust actuation in ion- and protein-rich complex culture media, and its electroresponsiveness was not altered by fibroblast colonization. Thanks to customized electrochemical stimulation setups, the electromechanical response of the polyHIPE/PEDOT scaffolds was characterized in situ under a confocal microscope and showed 10% reversible volume variations. Finally, the setups were used to monitor in real time and in situ fibroblasts cultured into the polyHIPE/PEDOT scaffold during several cycles of electromechanical stimuli. Thus, we demonstrated the proof of concept of this tunable scaffold as a tool for future 4D cell culture and mechanobiology studies.

Probing T-cell activation in nanoliter tumor co-cultures using membrane displacement trap arrays.

Immune responses against cancer are inherently stochastic, with small numbers of individual T cells within a larger ensemble of lymphocytes initiating the molecular cascades that lead to tumor cytotoxicity. A potential source of this intra-tumor variability is the differential ability of immune cells to respond to tumor cells. Classical microwell co-cultures of T cells and tumor cells are inadequate for reliably culturing and analyzing low cell numbers needed to probe this variability, and have failed in recapitulating the heterogeneous small domains observed in tumors. Here we leverage a membrane displacement trap array technology that overcomes limitations of conventional microwell plates for immunodynamic studies. The microfluidic platform supports on-demand formation of dense nanowell cultures under continuous perfusion reflecting the tumor microenvironment, with real-time monitoring of T cell proliferation and activation within each nanowell. The system enables selective ejection of cells for profiling by fluorescence activated cell sorting, allowing observed on-chip variability in immune response to be correlated with off-chip quantification of T cell activation. The technology offers new potential for probing the molecular origins of T cell heterogeneity and identifying specific cell phenotypes responsible for initiating and propagating immune cascades within tumors. Insight Box Variability in T cell activation plays a critical role in the immune response against cancer. New tools are needed to unravel the mechanisms that drive successful anti-tumor immune response, and to support the development of novel immunotherapies utilizing rare T cell phenotypes that promote effective immune surveillance. To this end, we present a microfluidic cell culture platform capable of probing differential T cell activation in an array of nanoliter-scale wells coupled with off-chip cell analysis, enabling a high resolution view of variable immune response within tumor / T cell co-cultures containing cell ensembles orders of magnitude smaller than conventional well plate studies.

The Development and Characterisation of a P(3HB-co-4HB)-Bioactive Glass-Graphene Hydrogel as a Potential Formulation for Biomedical and Therapeutical Translation.

The clinical management of wounds is known to be a significant challenge: not only does the dressing need to ensure and provide the appropriate barrier and healing characteristics, but consideration of patient compliance concerning comfort, functionality, and practicality also needs to be included. The poly(3-hydroxybutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) copolymer, isolated from Cupriavidus malaysiensis USM1020 (C. malaysiensis USM1020), was produced in the presence of excess carbon sources (1,4-butanediol and 1,6-hexanediol) using either a shake flask cultivation process or a bioreactor fermentation system. P(3HB-co-4HB) is widely known to be biodegradable and highly biocompatible and contains a tuneable 4HB monomer molar fraction, which is known to affect the final physicochemical properties of the intracellular copolymer. In this paper, we describe not only the fabrication of the polymeric gel but also its optimised profiling using a range of physical and mechanical techniques, i.e., SEM, FTIR, DMA, DSC, and WCA. The further enhancement of the gel through additional functionalisation with sol-gel-derived bioactive glass and liquid-exfoliated graphene was also investigated. The biocompatibility and biological characterisation of the substrates was assessed using murine osteoblasts (MC3T3), human primary dermal fibroblasts (HDFs), human fibroblast (BJ) cells, and standard cell culture assays (i.e., metabolic activity, LDH release, and live/dead staining). In short, P(3HB-co-4HB) was successfully isolated from the bacteria, with the defined physico-chemical profiles dependent on the culture substrate and culturing platform used. The additional enhancement of the copolymer with bioactive glass and/or graphene was also demonstrated by varying the combination loading of the materials, i.e., graphene resulted in an increase in tensile strength (~11 MPa) and the wettability increased following the incorporation of bioactive glass and 0.01 wt% graphene (WCA ~46.3°). No detrimental effects in terms of biocompatibility were noticed during the 7 days of culture in the primary and established cell lines. This study demonstrates the importance of optimising each of the individual components within the biocomposite and their relationship concerning the fine-tuning of the material's properties, thus targeting and impacting the endpoint application.