3D Cell Structures Research Articles

Insitu magnetic-field-assisted bioprinting process using magnetorheological bioink to obtain engineered muscle constructs

Tissue-engineered anisotropic cell constructs are promising candidates for treating volumetric muscle loss (VML). However, achieving successful cell alignment within macroscale 3D cell constructs for skeletal muscle tissue regeneration remains challenging, owing to difficulties in controlling cell arrangement within a low-viscosity hydrogel. Herein, we propose the concept of a magnetorheological bioink to manipulate the cellular arrangement within a low-viscosity hydrogel. This bioink consisted of gelatin methacrylate (GelMA), iron oxide nanoparticles, and human adipose stem cells (hASCs). The cell arrangement is regulated by the responsiveness of iron oxide nanoparticles to external magnetic fields. A bioprinting process using ring magnets was developed for in situ bioprinting, resulting in well-aligned 3D cell structures and enhanced mechanotransduction effects on hASCs. In vitro analyses revealed upregulation of cellular activities, including myogenic-related gene expression, in hASCs. When implanted into a VML mouse model, the bioconstructs improved muscle functionality and regeneration, validating the effectiveness of the proposed approach.

Visualization of 2D and 3D Tissue Models via Size-Selected Aqueous AgInS/ZnS Quantum Dots.

Three-dimensional (3D) spheroid cell cultures of fibroblast (L929) and tumor mammary mouse (4T1) were chosen as in vitro tissue models for tissue imaging of ternary AgInS/ZnS fraction quantum dots (QDs). We showed that the tissue-mimetic morphology of cell spheroids through well-developed cell-cell and cell-matrix interactions and distinct diffusion/transport characteristics makes it possible to predict the effect of ternary AgInS/ZnS fraction QDs on the vital activity of cells while simultaneously comparing with classical two-dimensional (2D) cell cultures. The AgInS/ZnS fractions, emitting in a wide spectral range from 635 to 535 nm with a mean size from ∼3.1 ± 0.8 to ∼1.8 ± 0.4 nm and a long photoluminescence lifetime, were separated from the initial QD ensemble by using antisolvent-induced precipitation. For ternary AgInS/ZnS fraction QDs, the absence of toxicity at different QD concentrations was demonstrated on 2D and 3D cell structures. QDs show a robust correlation between numerous factors: their sizes in biological fluids over time, penetration capabilities into 2D and 3D cell structures, and selectivity with respect to penetration into cancerous and healthy cell spheroids. A reproducible protocol for the preparation of QDs along with their unique biological properties allows us to consider ternary AgInS/ZnS fraction QDs as attractive fluorescent contrast agents for tissue imaging.

Abstract 4257: A novel 3D co-culture platform for patient-specific immune-oncology treatment

Abstract Lung cancer is the leading cause of global cancer-related mortality resulting in ~1.8 million deaths. annually. Over the past decade, the contribution of tumor microenvironment (TME) in lung cancer progression has been highly appreciated and provided significant improvements of survival outcomes for patients. That includes the complexity of the tumor immune microenvironment (TIME) playing a critical role in cancer progression. However, drug resistance usually arises and there is an urgent need for novel immune cell-based therapies. 3D tumor cell structures that recapitulate the architecture and functionality of the original tumor have revolutionized the understanding of disease mechanisms and testing potential therapeutics. However, there are certain drawbacks that need to be overcome. For example, these models mainly consist of only the epithelial compartment and lack signals from the microenvironment. Recent advances with engineering co-culture systems with immune cells at Champions Oncology help to emulate cell-cell and immune cell infiltration. We have successfully established a biobank of TumorGraft3D (CTG3Ds) derived from non-small cell lung cancer (NSCLC) patient-derived xenografts as well as their autologous and allogeneic immune cells (TILs, PBMCs) to establish a proprietary co-culture platform to investigate disease mechanisms and test potential therapeutics. In brief, surgical resections from patients were used to generate patient-derived xenografts organoids, and matched immune cells were expanded using standard rapid expansion protocol. The combination of a comprehensive staining system and high-content imaging as an endpoint allowed for 3D confocal analysis and deep evaluation of cancer-immune cellular interaction at the single cell level. Our pipeline of image analysis not only differentiates cancer-immune cell spatial distribution and cellular death but also allows for the evaluation of tumor infiltration and drug cytotoxicity. The characterization of the platform showed proliferation of the cancer cells and functional immune profiling by flow cytometry. The molecular profile of the organoids composing the platform showed a tight overlap with the original tumor highlighting the clinical relevance of the model. In addition, this novel co-culture platform has been tested with several NSCLC models to test the efficacy of various therapeutics such as immune-checkpoint inhibitors (ICIs) in reproducible, cost-effective, and patient-specific manner. In summary, Champion’s novel co-culture platform allows screening of novel immune targeting agents to determine impacts tumor cytotoxicity. Furthermore, our lung cancer tumorGraft3D platform serves as a powerful tool to facilitate the practice of precision medicine by investigating a variety of therapeutic modalities, alone or in combination, to identify patient-specific treatment strategies. Citation Format: Taylor Light, Samaneh Kamali, Mara Gilardi, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. A novel 3D co-culture platform for patient-specific immune-oncology 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 4257.

Flexible Toolbox of High-Precision Microfluidic Modules for Versatile Droplet-Based Applications.

Although the enormous potential of droplet-based microfluidics has been successfully demonstrated in the past two decades for medical, pharmaceutical, and academic applications, its inherent potential has not been fully exploited until now. Nevertheless, the cultivation of biological cells and 3D cell structures like spheroids and organoids, located in serially arranged droplets in micro-channels, has a range of benefits compared to established cultivation techniques based on, e.g., microplates and microchips. To exploit the enormous potential of the droplet-based cell cultivation technique, a number of basic functions have to be fulfilled. In this paper, we describe microfluidic modules to realize the following basic functions with high precision: (i) droplet generation, (ii) mixing of cell suspensions and cell culture media in the droplets, (iii) droplet content detection, and (iv) active fluid injection into serially arranged droplets. The robustness of the functionality of the Two-Fluid Probe is further investigated regarding its droplet generation using different flow rates. Advantages and disadvantages in comparison to chip-based solutions are discussed. New chip-based modules like the gradient, the piezo valve-based conditioning, the analysis, and the microscopy module are characterized in detail and their high-precision functionalities are demonstrated. These microfluidic modules are micro-machined, and as the surfaces of their micro-channels are plasma-treated, we are able to perform cell cultivation experiments using any kind of cell culture media, but without needing to use surfactants. This is even more considerable when droplets are used to investigate cell cultures like stem cells or cancer cells as cell suspensions, as 3D cell structures, or as tissue fragments over days or even weeks for versatile applications.

Activatable near-infrared fluorescence probe for real-time imaging of PD-L1 expression in tumors.

In clinical practice, determining programmed death-ligand 1 (PD-L1) expression is crucial for selecting patients and monitoring immune checkpoint blockade therapies. Currently, PD-L1 expression is quantified using immunohistochemistry (IHC). However, IHC-based methods do not capture the heterogeneous and dynamic nature of PD-L1 expression. Thus, there is a pressing need for a rapid and efficient method for monitoring PD-L1 expression both in vitro and in vivo, which would considerably aid in prognosis and treatment selection. In this study, we present for the first time an activatable near-infrared (NIR) fluorescence imaging probe (Q-Atezol) for the real-time monitoring of PD-L1 expression in vitro and in vivo. The ability of Q-Atezol to detect PD-L1 expression quickly and in real-time was evaluated in both tumor spheroid and lung cancer xenograft models. An always-on optical probe (ON-Atezol) was synthesized and tested for comparison. In vivo NIR fluorescence imaging studies were conducted on A549 and H1975 tumor-bearing mice, and their tumor-to-background ratios (TBRs) were analyzed. The quenched NIR fluorescence of Q-Atezol is activated upon binding to PD-L1 proteins on the surface of cancer cells, thereby enabling PD-L1 detection in the three-dimensional (3D) tumor spheroids without a washing step. Notably, PD-L1-positive H1975 tumors were clearly visualized with a high TBR 6 hours after Q-Atezol injection, whereas ON-Atezol treatment could not detect H1975 tumors even 24 hours post-injection. The activatable fluorescence probe Q-Atezol demonstrated great potential as an exceptional sensor for assessing PD-L1 expression in 3D cell structures and for in vivo applications.

Development of Novel 3D Cell Structure and Manufacturing Processes for Highly Efficient, Durable, and Redox Resistant Solid Oxide Electrolysis Cells

A novel cell design and its corresponding manufacturing processes are being evaluated to demonstrate a highly efficient, durable, and reduction-oxidation (redox) resistant solid oxide electrolysis cell (SOEC) technology for hydrogen production. The cell design includes a unique 3D hydrogen electrode configuration composed of two layers ‒ a 3D hydrogen electrode support layer (for improved cell redox resistance) and an exsolved perovskite hydrogen electrode active layer (for enhanced cell performance and increased durability). The hydrogen electrode support layer consists of specifically designed 3D networks of electrode materials; tetragonal zirconia (TZ) and Ni-yttria stabilized zirconia (YSZ). Each phase is topologically connected throughout the electrode with controlled geometry and connectivity. The TZ component plays a role as an outer frame to provide structural support, while the Ni-YSZ component inside the TZ frame imparts electrical conductivity for the hydrogen electrode support layer. The hydrogen electrode active layer, which primarily supports electrochemical reaction, is a nickel-substituted perovskite that will exsolve nickel nanoparticles on the oxide surface when exposed to a reducing environment and is redox-reversible. A fabrication scheme being developed for this cell design incorporates a printing-based additive manufacturing (AM) process for producing the 3D hydrogen electrode. This paper presents the progress to date in developing the printing fabrication process for the 3D hydrogen electrode support and evaluating exsolved nickel-substituted perovskite for the hydrogen electrode active layer.

Methods for assessing the viability of cells cultured in vitro in 2D and 3D structures

Over the past decades, cell viability tests have been an essential research tool in cell biology, tissue engineering, and regenerative medicine. Assessment of cell viability is mandatory in the production and quality control of cell products for biomedical applications.
 Methods of viability assessment can be broadly classified according to the underlying mechanism and how the results obtained are evaluated. This article presents variants of the most commonly used tests and protocols for assessing cell viability. Their advantages and disadvantages are presented, which should be considered when planning experiments, e.g., when developing cell preparations for regenerative medicine.
 The authors point out the main factors influencing the choice of viability assessment method: efficiency, speed, safety, reproducibility, sample integrity, compatibility with biomaterial, and cell line type. Finally, the authors discuss separately cell viability tests that can be applied not only to 2D cell structures but also to 3D cell structures, which have recently become widespread due to more accurate modeling of biological processes.

Development of Novel 3D Cell Structure and Manufacturing Processes for Highly Efficient, Durable, and Redox Resistant Solid Oxide Electrolysis Cells

A novel cell design and its corresponding manufacturing processes are being evaluated to demonstrate a highly efficient, durable, and reduction-oxidation (redox) resistant solid oxide electrolysis cell (SOEC) technology for hydrogen production. The cell design includes a unique 3D hydrogen electrode configuration composed of two layers ‒ a 3D hydrogen electrode support layer (for improved cell redox resistance) and an exsolved perovskite hydrogen electrode active layer (for enhanced cell performance and increased durability). The hydrogen electrode support layer consists of specifically designed 3D networks of electrode materials; tetragonal zirconia (TZ) and Ni-yttria stabilized zirconia (YSZ). Each phase is topologically connected throughout the electrode with controlled geometry and connectivity. The TZ component plays a role as an outer frame to provide structural support, while the Ni-YSZ component inside the TZ frame imparts electrical conductivity for the hydrogen electrode support layer. The hydrogen electrode active layer, which primarily supports electrochemical reaction, is a nickel-substituted perovskite that will exsolve nickel nanoparticles on the oxide surface when exposed to a reducing environment and is redox-reversible. A fabrication scheme being developed for this cell design incorporates a printing-based additive manufacturing (AM) process for producing the 3D hydrogen electrode. This paper presents the progress to date in developing the printing fabrication process for the 3D hydrogen electrode support and evaluating exsolved nickel-substituted perovskite for the hydrogen electrode active layer.

CellRemorph: A Toolkit for Transforming, Selecting, and Slicing 3D Cell Structures on the Road to Morphologically Detailed Astrocyte Simulations

Understanding functions of astrocytes can be greatly enhanced by building and simulating computational models that capture their morphological details. Novel computational tools enable utilization of existing morphological data of astrocytes and building models that have appropriate level of details for specific simulation purposes. In addition to analyzing existing computational tools for constructing, transforming, and assessing astrocyte morphologies, we present here the CellRemorph toolkit implemented as an add-on for Blender, a 3D modeling platform increasingly recognized for its utility for manipulating 3D biological data. To our knowledge, CellRemorph is the first toolkit for transforming astrocyte morphologies from polygonal surface meshes into adjustable surface point clouds and vice versa, precisely selecting nanoprocesses, and slicing morphologies into segments with equal surface areas or volumes. CellRemorph is an open-source toolkit under the GNU General Public License and easily accessible via an intuitive graphical user interface. CellRemorph will be a valuable addition to other Blender add-ons, providing novel functionality that facilitates the creation of realistic astrocyte morphologies for different types of morphologically detailed simulations elucidating the role of astrocytes both in health and disease.

Acoustic and Magnetic Stimuli-Based Three-Dimensional Cell Culture Platform for Tissue Engineering.

In a conventional two-dimensional (2D) culture method, cells are attached to the bottom of the culture dish and grow into a monolayer. These 2D culture methods are easy to handle, cost-effective, reproducible, and adaptable to growing many different types of cells. However, monolayer 2D cell culture conditions are far from those of natural tissue, indicating the need for a three-dimensional (3D) culture system. Various methods, such as hanging drop, scaffolds, hydrogels, microfluid systems, and bioreactor systems, have been utilized for 3D cell culture. Recently, external physical stimulation-based 3D cell culture platforms, such as acoustic and magnetic forces, were introduced. Acoustic waves can establish acoustic radiation force, which can induce suspended objects to gather in the pressure node region and aggregate to form clusters. Magnetic targeting consists of two components, a magnetically responsive carrier and a magnetic field gradient source. In a magnetic-based 3D cell culture platform, cells are aggregated by changing the magnetic force. Magnetic fields can manipulate cells through two different methods: positive magnetophoresis and negative magnetophoresis. Positive magnetophoresis is a way of imparting magnetic properties to cells by labeling them with magnetic nanoparticles. Negative magnetophoresis is a label-free principle-based method. 3D cell structures, such as spheroids, 3D network structures, and cell sheets, have been successfully fabricated using this acoustic and magnetic stimuli-based 3D cell culture platform. Additionally, fabricated 3D cell structures showed enhanced cell behavior, such as differentiation potential and tissue regeneration. Therefore, physical stimuli-based 3D cell culture platforms could be promising tools for tissue engineering.

Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments.

Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis.

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed.

Comparison of Lugol’s solution and Fe3O4 nanoparticles as contrast agents for tumor spheroid imaging using microcomputed tomography

Abstract Background Lugol’s solution is well known for its unique contrasting properties to biological samples in in microcomputed tomography imaging. On the other hand, iron oxide nanoparticles (IONPs), which have much lower attenuation capabilities to X-ray radiation show decent cell penetration and accumulation properties, are increasingly being used as quantitative contrast agents in biology and medicine. In our research, they were used to stain 3D cell structures called spheroids. Aim In this study, the micro computed tomography (µCT) technique was used to visualize and compare the uptake and accumulation of two contrast agents, Lugol’s solution and iron (II, III ) oxid e nanoparticles (IONPs) in the in vitro human spheroid tumour model. Methods The metastatic human melanoma cell line WM266-4 was cultured, first under standard 2D conditions, and after reaching 90% confluence cells was seeded in a low adhesive plate, which allows spheroid formation. On the 7th day of growth, the spheroids were transferred to the tubes and stained with IONPs or Lugol’s solution and subjected to µCT imaging. Results Our research allows visualization of the regions of absorption at the level of single cells, with relatively short incubation times - 24h - for Lugol’s solution. IONPs proved to be useful only in high concentrations (1 mg/ml) and long incubation times (96h). Conclusions When comparing the reconstructed visualizations of the distribution of these stating agents, it is worth noting that Lugol’s solution spreads evenly throughout the spheroids, whereas IONPs (regardless of their size 5 and 30 nm) accumulate only in the outer layer of the spheroid structure.

Efficiency of accelerated electron beam sterilization of a hydrogel for 3D cultivation of mesenchymal multipotent cells

Background. Hydrogels are promising for use in tissue engineering for the restoration and regeneration of various tissues, since they are able to perform the functions of bulk scaffolds, providing the formation of 3D cell structures. Population of such scaffolds with autologous or heterogeneous mesenchymal multipotent stromal cells in vitro makes it possible to localize these cells in the area of target tissues after implantation in a patient. One of the difficult tasks is the choice of the method and mode of sterilization of the hydrogel, which does not change its properties.Aim. Study of the effectiveness of hydrogel sterilization by an accelerated electron beam in various modes, changes in the structure and biocompatibility of the scaffold, to assess the prospects for its use for medical purposes, including as a platform for mesenchymal stromal cells.Materials and methods. We used a hydrogel based on 4 % solutions of sodium alginate and sodium salt of carboxymethyl cellulose, cross-linked with calcium chloride, which was developed, obtained and provided for our research by the team of the Research and Educational Center for Biomedical Engineering of the National University of Science and Technology “MISIS”. Hydrogel samples loaded with Escherichia coli, Lactobacillus acidophilus, Saccharomyces cerevisiae were subjected to electron beam treatment in the range of 5–100 kGy. After electron beam treatment of hydrogel, the presence of living microorganisms and its structure were evaluated by IR-Fourier spectroscopy, as well as the phenotype and formation of 3D structures by mesenchymal multipotent cells.Results. It was found that the treatment of hydrogels with an electron beam at a mode of 25 kGy ensures the death of microorganisms, but does not destroy the structure of the hydrogel and does not inhibit the ability to form capillary-like structures by mesenchymal multipotent cells.Conclusion. Treatment with an accelerated electron beam at a 25 kGy can be used to sterilize hydrogels to obtain bulk scaffolds for cell engineering implants.

3D Cell Spheroids as a Tool for Evaluating the Effectiveness of Carbon Nanotubes as a Drug Delivery and Photothermal Therapy Agents

Cell spheroids (CSs) are three-dimensional models in vitro that have a microenvironment similar to tissues. Such three-dimensional cellular structures are of great interest in the field of nano biomedical research, as they can simulate information about the characteristics of nanoparticles (NPs) by avoiding the use of laboratory animals. Due to the development of areas such as bioethics and tissue engineering, it is expected that the use of such 3D cell structures will become an even more valuable tool in the hands of researchers. We present an overview of carbon nanotubes (CNTs) research on CSs in order to determine the mechanism of their incorporation into CSs, drug delivery, and photothermal therapy. We will look at such areas as the application of CNTs for medical purposes, the advantages of spheroids over classical 2D cell culture, the ways in which CNTs pass into the intercellular space, and the ways in which they are absorbed by cells in a three-dimensional environment, the use of the spheroid model for such studies as drug delivery and photothermal therapy. Thus, CSs are suitable models for obtaining additional information on the required properties of CNTs in their application in nanobiomedicine.

MEDB-24. Tumour secreted extracellular matrix predicts survival and influences migration and cell death in SHH medulloblastoma 3D models

Medulloblastoma is the most common malignant paediatric brain tumour. Four molecular sub-groups exist (WNT, Sonic hedgehog (SHH), Group 3 and Group 4), each associated with different patterns of metastasis and chemoresistance. We have shown that within these sub-groups further clinically relevant sub-types exist, characterised by differential expression of extracellular matrix (ECM) proteins. For example, good overall survival in two-thirds of SHH sub-group patients is associated with the expression of specific ECM proteins. Our aim here is to further characterise these ECM components in SHH medulloblastoma and to determine how they confer better overall survival in this sub-group. Using a combination of 3D OrbiSIMs and immunohistochemical staining we have identified, that when grown in 3D hyaluronic acid hydrogels or as spheroids, SHH medulloblastoma cell lines form an ECM shell-like structure composed of laminin, collagen and lumican. In addition, scRNAseq of SHH hydrogel nodules revealed sub-group specific clusters of cells with high levels of ECM interaction and adhesion. We therefore hypothesise that ECM interaction restricts SHH tumour invasion and metastasis through this shell-like structure. To understand how this ECM shell-like structure potentiates better survival in SHH medulloblastoma we have created a CRISPRcas9 laminin knockout SHH medulloblastoma cell line. 3D culture of the laminin knockout cell line demonstrated that laminin is essential for the formation of 3D cell structures as well as migration in SHH medulloblastoma. Furthermore, we have identified that apoptosis is increased in the laminin knockout SHH cell line, suggesting that apoptosis-targeted therapeutics may represent a beneficial treatment option for SHH patients whose tumours exhibit this ECM shell. In summary, tumour-secreted ECM plays a major role in SHH medulloblastoma progression. Expression of laminin, collagen or lumican can be used to classify SHH patients into low and high-risk groups with different therapeutic outcomes and treatment options.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Whole-cell quantitative imaging of structural changes induced by SARS-CoV-2 using soft X-ray tomography

Quantitative analysis of three-dimensional (3D) cell structure and composition is crucial for unveiling mechanisms of virus-host interactions. To study the interaction of virus particles with cells at the sub-cellular ultra-structural level, we are using soft x-ray tomography (SXT) to image the whole cell with minimal sample preparation. SXT takes the advantage of the “water window” property to acquire 3D tomographic data non-invasively and free of labeling. Here, we employ soft x-ray tomography (SXT) with a full-rotation stage to examine organelle remodeling induced by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the whole-cell level with high spatial resolution and throughput.

Perfusion System for Modification of Luminal Contents of Human Intestinal Organoids and Realtime Imaging Analysis of Microbial Populations.

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.

Organoid Transplantation Can Improve Reproductive Prognosis by Promoting Endometrial Repair in Mice

Objective: Intrauterine adhesion (IUA) is one of the major causes of refractory secondary infertility, especially in regions and countries with high abortion rates. In this study, we used the mouse IUA model to evaluate the feasibility of the organoids, a 3D cell structure derived from endometrial tissue, as grafts for the treatment of post-traumatic endometrial regeneration disorders. Methods: The isolated and cultured endometrial organoid was transplanted into the model IUA uterus by the hydrogel scaffold method. Results: The cultured endometrial organoids were transplanted into the basal layer of the damaged endometrium for 28 days. They were completely implanted and grew normally. They not only reconstructed the structural integrity of the endometrial epithelium but also realized the functional repair of the endometrium through differentiation cultures and secretory functions. Conclusion: For severe IUA, this method may be better than stem cell transplantation. These findings provide useful insights into the use of endometrial organoid regeneration in the treatment of injury repair.