Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

EnderScope: a low-cost 3D printer-based scanning microscope for microplastic detection.

Low-cost and scalable technologies that allow people to measure microplastics in their local environment could facilitate a greater understanding of the global problem of marine microplastic pollution. A typical way to measure marine microplastic pollution involves imaging filtered seawater samples stained with a fluorescent dye to aid in the detection of microplastics. Although traditional fluorescence microscopy allows these particles to be manually counted and detected, this is a resource- and labour-intensive task. Here, we describe a novel, low-cost microscope for automated scanning and detection of microplastics in filtered seawater samples-the EnderScope. This microscope is based on the mechanics of a low-cost 3D printer (Creality Ender 3). The hotend of the printer is replaced with an optics module, allowing for the reliable and calibrated motion system of the 3D printer to be used for automated scanning over a large area (>20 × 20 cm). The EnderScope is capable of both reflected light and fluorescence imaging. In both configurations, we aimed to make the design as simple and cost-effective as possible, for example, by using low-cost LEDs for illumination and lighting gels as emission filters. We believe this tool is a cost-effective solution for microplastic measurement. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

The use of fluid-phase 3D printing to pattern alginate-gelatin hydrogel properties to guide cell growth and behaviourin vitro.

Three-dimensional (3D) (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is an evolving field and (bio)printing biomimetic constructions for tissue formation is still a challenge. As a new methodology to facilitate the construction of more complex structures, we suggest the use of the fluid-phase 3D printing to pattern the scaffold's properties. The methodology consists of an exchangeable fluid-phase printing medium in which the constructions are fabricated and patterned during the printing process. Using the fluid-phase methodology, the biological and mechanical properties can be tailored promoting cell behaviour guidance and compartmentalization. In this study, we first assessed different formulations of alginate/gelatin to create a stable substrate capable to promote massive cell colonizationin vitroover time. Overall, formulations with lower gelatin content and 2-(N-morpholino)ethanesulfonic acid (MES) buffer as a solvent showed better stability under cell culture conditions and enhanced U2OS cell growth. Next, the fluid-phase showed better printing fidelity and resolution in comparison to air printing as it diminished the collapsing and the spread of the hydrogel strand. In sequence, the fluid-phase methodology was used to create functionalized alginate-gelatin-arginylglycylaspartic acid peptide (RGD) hydrogels via carbodiimides chemistry. The alginate-gelatin-RGD hydrogels showed an increase of 2.97-fold in cell growth and more spread substrate colonization in comparison to alginate-gelatin hydrogel. Moreover, the fluid-phase methodology was used to add RGD molecules to pre-determined parts of the alginate-gelatin substrate during the printing process promoting U2OS cell compartmentalization. In addition, different substrate stiffnesses were also created via fluid-phase by crosslinking the hydrogel with different concentrations of CaCl2during the printing process. As a result, the U2OS cells were also compartmentalized on the stiffer parts of the printings. Finally, our results showed that by combining stiffer hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell metabolism by up to 3.17-fold. This work presents an idea of a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate more faithful replication of thein vivoenvironment.

Structure-dynamics correlations in composite PF127-PEG-based hydrogels; cohesive/hydrophobic interactions determine phase and rheology and identify the role of micelle concentration in controlling 3D extrusion printability.

A library of composite polymer networks (CPNs) were formed by combining Pluronic F127, as the primary gelator, with a range of di-acrylate functionalised PEG polymers, which tune the rheological properties and provide UV crosslinkability. A coarse-grained sol-gel room temperature phase diagram was constructed for the CPN library, which identifies PEG-dependent disruption of micelles as leading to liquefication. Small angle X-ray scattering and rheological measurements provide detailed insight into; (i) micelle-micelle ordering; (ii) micelle-micelle disruption, and; (iii) acrylate-micelle disruption; with contributions that depend on composition, including weak PEG chain length and end group effects. The influence of composition on 3D extrusion printability through modulation of the cohesive/hydrophobic interactions was assessed. It was found that only micelle content provides consistent changes in printing fidelity, controlled largely by printing conditions (pressure and feed rate). Finally, the hydrogels were shown to be UV photo-crosslinkable, which further improves fidelity and structural integrity, and usefully reduces the mesh size. Our results provide a guide for design of 3D-printable CPN inks for future biomedical applications.

Role of pH and Crosslinking Ions on Cell Viability and Metabolic Activity in Alginate-Gelatin 3D Prints.

Alginate-gelatin hydrogels are extensively used in bioengineering. However, despite different formulations being used to grow different cell types in vitro, their pH and its effect, together with the crosslinking ions of these formulations, are still infrequently assessed. In this work, we study how these elements can affect hydrogel stability and printability and influence cell viability and metabolism on the resulting 3D prints. Our results show that both the buffer pH and crosslinking ion (Ca2+ or Ba2+) influence the swelling and degradation rates of prints. Moreover, buffer pH influenced the printability of hydrogel in the air but did not when printed directly in a fluid-phase CaCl2 or BaCl2 crosslinking bath. In addition, both U2OS and NIH/3T3 cells showed greater cell metabolic activity on one-layer prints crosslinked with Ca2+. In addition, Ba2+ increased the cell death of NIH/3T3 cells while having no effect on U2OS cell viability. The pH of the buffer also had an important impact on the cell behavior. U2OS cells showed a 2.25-fold cell metabolism increase on one-layer prints prepared at pH 8.0 in comparison to those prepared at pH 5.5, whereas NIH/3T3 cells showed greater metabolism on one-layer prints with pH 7.0. Finally, we observed a difference in the cell arrangement of U2OS cells growing on prints prepared from hydrogels with an acidic buffer in comparison to cells growing on those prepared using a neutral or basic buffer. These results show that both pH and the crosslinking ion influence hydrogel strength and cell behavior.

Remote harm reduction services are key solutions to reduce the impact of COVID-19-like crises on people who use drugs: evidence from two independent structures in France and in the USA.

BACKGROUND: Harm Reduction (HR) policies for People Who Use Drugs (PWUD) have a significant positive impact on their health. Such approaches limit the spread of infections and reduce opioid overdose mortality. These policies have led to the opening of specialized structures located mainly in big cities and urbanized zones. The COVID-19 pandemic reduced access to HR structures in locations undergoing lockdown. Before the pandemic, HR services in France and in the USA were complemented by the development of remote HR programs: HaRePo (Harm Reduction by Post) for France, implemented in 2011, and NEXT Distro for the USA founded in 2017. These programs are free and specifically designed for people who have difficulties accessing HR tools and counseling in-person. PWUD can access HaRePo program by phone and/or email. NEXT Distro users can access the program through its dedicated website. The aim of the study is to test if and possibly how COVID-19 pandemic and the associated lockdowns have impacted the HR services in both countries. METHODS: By using t-test comparing the year 2019 with the year 2020, we analyzed how lockdowns impacted the number of new users entering the programs, as well as the numbers of parcels sent and naloxone distributed, by using records of both structures. RESULTS: We showed that the activity of both programs was significantly impacted by the pandemic. Both show an increase in the number of new users joining the programs (+ 77.6% for HaRePo and + 247.7% for NEXT Distro) as well as for the number of parcels sent per month (+ 42.7% for HaRePo and + 211.3% for NEXT Distro). It shows that remote HR was able to partially compensate for the reduced HR activities due to COVID-19. We also observed that the distribution of naloxone per parcel tends to increase for both structures. CONCLUSION: With the ability to reach PWUD remotely, HaRePo and NEXT Distro were particularly effective at maintaining service continuity and scaling up services to meet the needs of PWUD during the COVID-19 pandemic. By studying two independent structures in France and in the USA sharing similar objectives (remote HR), we showed that this approach can be a key solution to crises that impact classical HR structures despite various differences in operating procedures between countries.

Challenges and advances in optical 3D mesoscale imaging.

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence.

This review discusses the state of the art of an emerging field called mesoscale imaging. Mesoscale imaging refers to the trend towards imaging ever-larger samples that exceed the classic microscopy domain and is also referred to as 'mesoscopic imaging'. In optical imaging, this refers to objects between the microscopic and macroscopic scale that are imaged with subcellular resolution; in practice, this implies the imaging of objects from millimetre up to cm size with µm or nm resolution. As such, the mesoscopy field spans the boundary between classic 'biological' imaging and preclinical 'biomedical' imaging, typically utilising lower magnification objective lenses with a bigger field of view. We discuss the types of samples currently imaged with examples, and highlight how this type of imaging fills the gap between microscopic and macroscopic imaging, allowing further insight into the organisation of tissues in an organism. We also discuss the challenges of imaging such large samples, from sample handling to labelling and optical phenomena that stand in the way of quantitative imaging. Finally, we put the current state of the art into context within the neighbouring fields and outline future developments, such as the use of 'phantom' test samples and artificial intelligence for image analysis that will underpin the quality of mesoscale imaging.

The biology of imaging.

On the cave wall, a discrete but stunning silhouette runs across the uneven surface. Standing still for more than 45 000 years, this is a witness to the ever-enduring need of mankind to image the world around us. The biological world that feeds us is a primary source of inspiration but also an essential element to creating the imaging systems we use every day. But once obscured by the technological jargon and the thunderstorm of numbers and algorithms, those origins fade away into the background. This small piece is about a few marvellous little stories about the biology of imaging, not the debate about the origin of vision and the eye but rather about plants and animals that open the world to new dimensions of biological imaging to fully image the biological world. An eye for an eye. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Spatiotemporally Resolved Heat Dissipation in 3D Patterned Magnetically Responsive Hydrogels.

Multifunctional nanocomposites that exhibit well-defined physical properties and encode spatiotemporally controlled responses are emerging as components for advanced responsive systems, for example, in soft robotics or drug delivery. Here an example of such a system, based on simple magnetic hydrogels composed of iron oxide magnetic nanoflowers and Pluronic F127 that generates heat upon alternating magnetic field irradiation is described. Rules for heat-induction in bulk hydrogels and the heat-dependence on particle concentration, gel volume, and gel exposed surface area are established, and the dependence on external environmental conditions in "closed" as compared to "open" (cell culture) system, with controllable heat jumps, of ∆T 0-12°C, achieved within ≤10 min and maintained described. Furthermore the use of extrusion-based 3D printing for manipulating the spatial distribution of heat in well-defined printed features with spatial resolution <150 µm, sufficiently fine to be of relevance to tissue engineering, is presented. Finally, localized heat induction in printed magnetic hydrogels is demonstrated through spatiotemporally-controlled release of molecules (in this case the dye methylene blue). The study establishes hitherto unobserved control over combined spatial and temporal induction of heat, the applications of which in developing responsive scaffold remodeling and cargo release for applications in regenerative medicine are discussed.

The incubot: A 3D printer-based microscope for long-term live cell imaging within a tissue culture incubator.

Commercial live cell imaging systems represent a large financial burden to research groups, while current open source incubator microscopy systems lack adaptability and are sometimes inadequate for complex imaging experimentation. We present here a low-cost microscope designed for inclusion within a conventional tissue culture incubator. The build is constructed using an entry level 3D printer as the basis for the motion control system, with Raspberry Pi imaging and software integration, allowing for reflected, oblique, and fluorescence imaging of live cell monolayers. The open source nature of the design is aimed to facilitate adaptation by both the community at large and by individual researchers/groups. The development of an adaptable and easy-to-use graphic user interface (GUI) allows for the scientist to be at the core of experimental design through simple modifications of the base GUI code, or generation of an entirely purpose-built script. This adaptability will allow scientists to adapt this equipment for their experimental needs, as opposed to designing experiments to fit their current equipment. The build can be constructed for a cost of roughly €1000 and thus serves as a low-cost and adaptable addition to the open source microscopy community.

3D imaging of undissected optically cleared Anopheles stephensi mosquitoes and midguts infected with Plasmodium parasites.

Malaria is a life-threatening disease, caused by Apicomplexan parasites of the Plasmodium genus. The Anopheles mosquito is necessary for the sexual replication of these parasites and for their transmission to vertebrate hosts, including humans. Imaging of the parasite within the insect vector has been attempted using multiple microscopy methods, most of which are hampered by the presence of the light scattering opaque cuticle of the mosquito. So far, most imaging of the Plasmodium mosquito stages depended on either sectioning or surgical dissection of important anatomical sites, such as the midgut and the salivary glands. Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) enable imaging fields of view in the centimeter scale whilst providing micrometer resolution. In this paper, we compare different optical clearing protocols and present reconstructions of the whole body of Plasmodium-infected, optically cleared Anopheles stephensi mosquitoes and their midguts. The 3D-reconstructions from OPT imaging show detailed features of the mosquito anatomy and enable overall localization of parasites in midguts. Additionally, LSFM imaging of mosquito midguts shows detailed distribution of oocysts in extracted midguts. This work was submitted as a pre-print to bioRxiv, available at https://www.biorxiv.org/content/10.1101/682054v2.

HaRePo (harm reduction by post): an innovative and effective harm reduction programme for people who use drugs using email, telephone, and post service.

BACKGROUND: Despite multiple harm reduction (HR) programmes worldwide, there are still an important number of people who use drugs (PWUD) who do not access those services. Their difficulties to obtain HR tools are due to their inability to reach such services (remoteness and/or limited customer service hours), costs, quantitative restrictions, fear of judgement, lack of confidentiality in pharmacy, and unfamiliarity with HR programmes. We tested an innovative approach using the power of remote online communication and the national postal distribution network to improve HR tool access and counselling. METHODS: Based on these observations, SAFE association created HaRePo in 2011, a free and confidential programme designed for people who have difficulties accessing HR tools and counselling. PWUD can access the programme by phone and/or email. An HR professional delivers HR counselling and HR tools and connects PWUD to other HR services, medical, and social workers. HR tools are prepared and sent according to the person's needs through the French postal service to consumers across Metropolitan France and overseas territories. RESULTS: Since 2011, 1920 PWUD have benefited from HaRePo: 10,450 parcels were sent accounting for more than 1.7 million syringes and 6 million HR-related items. HaRePo receives positive feedback from PWUD who have improved their practices through remote but trusted communication. The percentage of people that, after joining the programme, never reuse and/or share HR tools have significantly increased. On average, 71.5% of beneficiaries never reuse syringes and 81% do not reuse needles. And they are 98.5% consumers who never share syringes and 99% needles any longer. Between 44 and 80% HaRePo beneficiaries have reported that their drug-related practices (injection, inhalation, and snorting) are now safer. Finally, between 39 and 53% HaRePo consumers declared that their overall physical state has improved (e.g. venous condition, the appearance of point of injection, swelling of arms, legs, and hands). CONCLUSION: HaRePo is an innovative HR programme efficient for hard-to-reach PWUD. It shows evidence of a positive feedback loop for PWUD in improving their practices. Finally, HaRePo represents a clear benefit for health authorities in France, who decided to expand the programme in 2016.

Assessing phototoxicity in live fluorescence imaging.

Are the answers to biological questions obtained via live fluorescence microscopy substantially affected by phototoxicity? Although a single set of standards for assessing phototoxicity cannot exist owing to the breadth of samples and experimental questions associated with biological imaging, we need quantitative, practical assessments and reporting standards to ensure that imaging has a minimal impact on observed biological processes and sample health. Here we discuss the problem of phototoxicity in biology and suggest guidelines to improve its reporting and assessment.

Reading the Evolution of Compartmentalization in the Ribosome Assembly Toolbox: The YRG Protein Family.

Reconstructing the transition from a single compartment bacterium to a highly compartmentalized eukaryotic cell is one of the most studied problems of evolutionary cell biology. However, timing and details of the establishment of compartmentalization are unclear and difficult to assess. Here, we propose the use of molecular markers specific to cellular compartments to set up a framework to advance the understanding of this complex intracellular process. Specifically, we use a protein family related to ribosome biogenesis, YRG (YlqF related GTPases), whose evolution is linked to the establishment of cellular compartments, leveraging the current genomic data. We analyzed orthologous proteins of the YRG family in a set of 171 proteomes for a total of 370 proteins. We identified ten YRG protein subfamilies that can be associated to six subcellular compartments (nuclear bodies, nucleolus, nucleus, cytosol, mitochondria, and chloroplast), and which were found in archaeal, bacterial and eukaryotic proteomes. Our analysis reveals organism streamlining related events in specific taxonomic groups such as Fungi. We conclude that the YRG family could be used as a compartmentalization marker, which could help to trace the evolutionary path relating cellular compartments with ribosome biogenesis.

Material- and feature-dependent effects on cell adhesion to micro injection moulded medical polymers.

Two polymers, polymethylmethacrylate (PMMA) and cyclic olefin copolymer (COC), containing a range of nano- to micron- roughness surfaces (Ra 0.01, 0.1, 0.4, 1.0, 2.0, 3.2 and 5.0µm) were fabricated using electrical discharge machining (EDM) and replicated using micro injection moulding (µIM). Polymer samples were characterized using optical profilometry, atomic force microscopy (AFM) and water surface contact angle. Cell adhesion tests were carried out using bacterial Pseudomonas fluorescens and mammalian Madin-Darby Canine Kidney (MDCK) cells to determine the effect of surface hydrophobicity, surface roughness and stiffness. It is found that there are features which gave insignificant differences (feature-dependent effect) in cell adhesion, albeit a significant difference in the physicochemical properties (material-dependent effect) of substrata. In bacterial cell adhesion, the strongest feature-dependence is found at Ra 0.4µm surfaces, with material-dependent effects strongest at Ra 0.01µm. Ra 0.1µm surfaces exhibited strongest feature-dependent effects and Ra 5.0µm has strongest material-dependent effects on mammalian cell adhesion. Bacterial cell adhesion is found to be favourable to hydrophobic surfaces (COC), with the lowest adhesion at Ra 0.4µm for both materials. Mammalian cell adhesion is lowest in Ra 0.1µm and highest in Ra 1.0µm, and generally favours hydrophilic surfaces (PMMA). These findings can be used as a basis for developing medical implants or microfluidic devices using micro injection moulding for diagnostic purposes, by tuning the cell adhesion on different areas containing different surface roughnesses on the diagnostic microfluidic devices or medical implants.

Using hydrogels in microscopy: A tutorial.

Sample preparation for microscopy is a crucial step to ensure the best experimental outcome. It often requires the use of specific mounting media that have to be tailored to not just the sample but the chosen microscopy technique. The media must not damage the sample or impair the optical path, and may also have to support the correct physiological function/development of the sample. For decades, researchers have used embedding media such as hydrogels to maintain samples in place. Their ease of use and transparency has promoted them as mainstream mounting media. However, they are not as straightforward to implement as assumed. They can contain contaminants, generate forces on the sample, have complex diffusion and structural properties that are influenced by multiple factors and are generally not designed for microscopy in mind. This short review will discuss the advantages and disadvantages of using hydrogels for microscopy sample preparation and highlight some of the less obvious problems associated with the area.

A 3-D cell culture system to study epithelia functions using microcarriers.

In vitro cell culture models used to study epithelia and epithelial diseases would benefit from the recognition that organs and tissues function in a three-dimensional (3D) environment. This context is necessary for the development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3D models of epithelium. We engineered 3D epithelia cultures using a kidney epithelia cell line (MDCK) and spherical polymer scaffolds. These kidney epithelia were characterized by live microscopy, immunohistochemistry and transmission electron microscopy. Strikingly, the epithelial cells displayed increased physiological relevance; they were extensively polarized and developed a more differentiated phenotype. Using such a growth system allows for direct transmission and fluorescence imaging with few restrictions using wide-field, confocal and Light Sheet Fluorescence Microscopy. We also assessed the wider relevance of this 3D culturing technique with several epithelial cell lines. Finally, we established that these 3D micro-tissues can be used for infection as well as biochemical assays and to study important cellular processes such as epithelial mesenchymal transmission. This new biomimetic model could provide a broadly applicable 3D culture system to study epithelia and epithelia related disorders.

End to End Digitisation and Analysis of Three-Dimensional Coral Models, from Communities to Corallites.

Coral reefs hosts nearly 25% of all marine species and provide food sources for half a billion people worldwide while only a very small percentage have been surveyed. Advances in technology and processing along with affordable underwater cameras and Internet availability gives us the possibility to provide tools and softwares to survey entire coral reefs. Holistic ecological analyses of corals require not only the community view (10s to 100s of meters), but also the single colony analysis as well as corallite identification. As corals are three-dimensional, classical approaches to determine percent cover and structural complexity across spatial scales are inefficient, time-consuming and limited to experts. Here we propose an end-to-end approach to estimate these parameters using low-cost equipment (GoPro, Canon) and freeware (123D Catch, Meshmixer and Netfabb), allowing every community to participate in surveys and monitoring of their coral ecosystem. We demonstrate our approach on 9 species of underwater colonies in ranging size and morphology. 3D models of underwater colonies, fresh samples and bleached skeletons with high quality texture mapping and detailed topographic morphology were produced, and Surface Area and Volume measurements (parameters widely used for ecological and coral health studies) were calculated and analysed. Moreover, we integrated collected sample models with micro-photogrammetry models of individual corallites to aid identification and colony and polyp scale analysis.