Organoid Cultures Research Articles

An Automated Digital Microfluidic System Based on Inkjet Printing

Cellular interactions, such as intercellular communication and signal transduction, can be enhanced within three-dimensional cell spheroids, contributing significantly to cellular viability and proliferation. This is crucial for advancements in cancer research, drug testing, and personalized medicine. The dimensions of the cell spheroids play a pivotal role in their functionality, affecting cell proliferation and differentiation, intercellular interactions, gene expression, protein synthesis, drug penetration, and metabolism. Consequently, different spheroid sizes may be required for various drug sensitivity experiments. However, conventional 3D cell spheroid cultures suffer from challenges such as size inconsistency, poor uniformity, and low throughput. To address these issues, we have developed an automated, intelligent system based on inkjet printing. This system allows for precise control of droplet volume by adjusting algorithms, thereby enabling the formation of spheroids of varying sizes. For spheroids of a single size, the printing pattern can be modified to achieve a coefficient of variation within 10% through a bidirectional compensation method. Furthermore, the system is equipped with an automatic pipetting module, which facilitates the high-throughput preparation of cell spheroids. We have implemented a 3 × 3 spheroid array in a 24-well plate, printing a total of 216 spheroids in just 11 min. Last, we attempted to print mouse small intestinal organoids and cultured them for 7 days, followed by immunofluorescent staining experiments. The results indicate that our equipment is capable of supporting the culture of organoids, which is of great significance for high-throughput drug screening and personalized medicine.

Retinal Ganglion Cell Transplantation: Differentiation and Quantification Strategies.

Glaucoma is a leading cause of irreversible blindness worldwide. Current treatments focus on reducing intraocular pressure but cannot restore lost visual function once it is lost due to retinal ganglion cell (RGC) degeneration and death. Recent advances suggest that transplantation of stem cell-derived RGCs could offer new therapeutic approaches for glaucoma and vision restoration. Here, we present a detailed protocol for differentiating human RGCs from embryonic stem cells using both three-dimensional retinal organoid and two-dimensional culture approaches. Following differentiation, we describe methods for isolating and purifying retinal ganglion cells from these cultures and their subsequent transplantation into the mouse retina, with careful monitoring and postoperative care to ensure successful integration. Finally, we describe a quantitative method for assessing transplantation outcomes involving confocal imaging of retinal flat-mounts and custom ImageJ and MATLAB scripts to map and analyze the spatial distribution of donor RGCs within the host retina. Altogether, this approach provides a robust framework for exploring RGC transplantation as a potential therapy for vision loss in glaucoma and other optic neuropathies.

An Easy-to-Use Arrayed Brain–Heart Chip

Multi-organ chips are effective at emulating human tissue and organ functions and at replicating the interactions among tissues and organs. An arrayed brain–heart chip was introduced whose configuration comprises open culture chambers and closed biomimetic vascular channels distributed in a horizontal pattern, separated from each other by an endothelial barrier based on fibrin matrix. A 300 μm-high and 13.2 mm-long endothelial barrier surrounded each organoid culture chamber, thereby satisfying the material transport requirements. Numerical simulations were used to analyze the construction process of fibrin barriers in order to optimize the structural design and experimental manipulation, which exhibited a high degree of correlation with experiment results. In each interconnective unit, a cerebral organoid, a cardiac organoid, and endothelial cells were co-cultured stably for a minimum of one week. The permeability of the endothelial barrier and recirculating perfusion enabled cross talk between cerebral organoids and cardiac organoids, as well as between organoids and endothelial cells. This was corroborated by the presence of cardiac troponin I (cTnI) in the cerebral organoid culture chamber and the observation of cerebral organoid and endothelial cells invading the fibrin matrix after one week of co-culture. The arrayed chip was simple to manipulate, clearly visible under a microscope, and compatible with automated pipetting devices, and therefore had significant potential for application.

Design and Applications of Supramolecular Peptide Hydrogel as Artificial Extracellular Matrix.

Supramolecular peptide hydrogels (SPHs) consist of peptides containing hydrogelators and functional epitopes, which can first self-assemble into nanofibers and then physically entangle together to form dynamic three-dimensional networks. Their porous structures, excellent bioactivity, and high dynamicity, similar to an extracellular matrix (ECM), have great potential in artificial ECM. The properties of the hydrogel are largely dependent on peptides. The noncovalent interactions among hydrogelators drive the formation of assemblies and further transition into hydrogels, while bioactive epitopes modulate cell-cell and cell-ECM interactions. Therefore, SPHs can support cell growth, making them ideal biomaterials for ECM mimics. This Review outlines the classical molecular design of SPHs from hydrogelators to functional epitopes and summarizes the recent advancements of SPHs as artificial ECMs in nervous system repair, wound healing, bone and cartilage regeneration, and organoid culture. This emerging SPH platform could provide an alternative strategy for developing more effective biomaterials for tissue engineering.

Suppression of epithelial proliferation and tumourigenesis by immunoglobulin A.

Immunoglobulin A (IgA) is the most abundant antibody isotype produced across mammals and plays a specialized role in mucosal homeostasis 1 . Constantly secreted into the lumen of the intestine, IgA binds commensal microbiota to regulate their colonization and function 2,3 with unclear implications for health. IgA deficiency is common in humans but is difficult to study due to its complex aetiology and comorbidities 4-8 . Using genetically and environmentally controlled mice, here we show that IgA-deficient animals have increased susceptibility to endogenous colorectal tumours. Cellular and molecular analyses revealed that, in the absence of IgA, colonic epithelial cells induce antibacterial factors and accelerate cell cycling in response to the microbiota. Oral treatment with IgA was sufficient to both reduce steady-state proliferation and protect mice from tumours, but this function was due to antibody structure rather than binding specificity. In both organoid and monolayer culture systems, IgA directly suppressed epithelial growth. Co-immunoprecipitation mass spectrometry and a targeted CRISPR screen identified DMBT1 as an IgA-binding epithelial surface protein required for IgA-mediated suppression of proliferation. Together, IgA and DMBT1 regulate Notch signalling and tune the normal cycling of absorptive colonocyte progenitors. In mice, deleting the transmembrane and cytoplasmic signalling portions of DMBT1 or blocking Notch signalling was sufficient to reverse both the increased proliferation and tumour susceptibility of IgA knockouts. These experiments establish a homeostatic function for IgA in tempering physiological epithelial responses to microbiota to maintain mucosal health.

Live-Cell Calcium Imaging in 3D Intestinal Organoids.

Live-cell Ca2+ imaging is an important tool to detect activation of receptors by a putative ligand/drug and complements studies on transport processes, as intracellular Ca2+ changes provide direct evidence for substrate fluxes. Organoid-based systems offer numerous advantages over other in vitro systems such as cell lines, primary cells, or tissue explants, and in particular, intestinal organoid culture has revolutionized research on functional gastrointestinal processes. Calcium imaging using the fluorescent Ca2+ indicator Fura-2-AM can be applied to 3D intestinal organoids, which show an excellent dye-loading efficiency. Here we describe live-cell Ca2+ imaging in intestinal organoids, an important technique to improve research on malabsorption syndromes, secretory diarrhea, and metabolic disorders.

Organoids-On-a-Chip for Personalized Precision Medicine.

The development of personalized precision medicine has become a pivotal focus in modern healthcare. Organoids-on-a-Chip (OoCs), a groundbreaking fusion of organoid culture and microfluidic chip technology, has emerged as a promising approach to advancing patient-specific treatment strategies. In this review, the diverse applications of OoCs are explored, particularly their pivotal role in personalized precision medicine, and their potential as a cutting-edge technology is highlighted. By utilizing patient-derived organoids, OoCs offer a pathway to optimize treatments, create precise disease models, investigate disease mechanisms, conduct drug screenings, and individualize therapeutic strategies. The emphasis is on the significance of this technological fusion in revolutionizing healthcare and improving patient outcomes. Furthermore, the transformative potential of personalized precision medicine, future prospects, and ongoing advancements in the field, with a focus on genomic medicine, multi-omics integration, and ethical frameworks are discussed. The convergence of these innovations can empower patients, redefine treatment approaches, and shape the future of healthcare.

The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1.

Lineage plasticity is a hallmark of cancer progression that impacts therapy outcomes, yet the mechanisms mediating this process remain unclear. Here, we introduce a versatile in vivo platform to interrogate neuroendocrine lineage transformation throughout prostate cancer progression. Transplanted mouse prostate organoids with human-relevant driver mutations (Rb1-/-; Trp53-/-; cMyc+ or Pten-/-; Trp53-/-; cMyc+) develop adenocarcinomas, but only those with Rb1 deletion advance to aggressive, ASCL1+ neuroendocrine prostate cancer (NEPC) resistant to androgen receptor signaling inhibitors. Notably, this transition requires an in vivo microenvironment not replicated by conventional organoid culture. Using multiplexed immunofluorescence and spatial transcriptomics, we reveal that ASCL1+ cells arise from KRT8+ luminal cells, progressing into transcriptionally heterogeneous ASCL1+;KRT8- NEPC. Ascl1 loss in established NEPC causes transient regression followed by recurrence, but its deletion before transplantation abrogates lineage plasticity, resulting in castration-sensitive adenocarcinomas. This dynamic model highlights the importance of therapy timing and offers a platform to identify additional lineage plasticity drivers.

Evidence for a lipofibroblast-to- Cthrc1 + myofibroblast reversible switch during the development and resolution of lung fibrosis in young mice.

Fibrosis is often associated with aberrant repair mechanisms that ultimately lead to organ failure. In the lung, idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease (ILD) to which there is currently no curative therapy. From the cell biology point of view, the cell of origin and eventual fate of activated myofibroblasts (aMYFs) have taken center stage as these cells are believed to drive structural remodeling and lung function impairment. While aMYFs are now widely believed to originate from resident fibroblasts, the heterogeneity of aMYFs and ultimate fate during fibrosis resolution remain elusive. We have previously shown that aMYFs dedifferentiation and acquisition of a lipofibroblast (LIF)-like phenotype represent a route of fibrosis resolution. In this study, we combined genetic lineage tracing and single-cell transcriptomics in mice, and data mining of human IPF datasets to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis resolution. Furthermore, organoid cultures were utilized as a functional readout for the alveolar mesenchymal niche activity during various phases of injury and repair in mice. Our data demonstrate that aMYFs consist of four subclusters displaying unique pro-alveologenic versus profibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrogenesis and resolution respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature. Our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.

Rediscovering the human thymus through cutting-edge technologies.

Recent technological advances have transformed our understanding of the human thymus. Innovations such as high-resolution imaging, single-cell omics, and organoid cultures, including thymic epithelial cell (TEC) differentiation and culture, and improvements in biomaterials, have further elucidated the thymus architecture, cellular dynamics, and molecular mechanisms underlying T cell development, and have unraveled previously unrecognized levels of stromal cell heterogeneity. These advancements offer unprecedented insights into thymic biology and hold promise for the development of novel therapeutic strategies for immune-related disorders.

AP-1B regulates interactions of epithelial cells and intraepithelial lymphocytes in the intestine

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system.

Cellular liquid biopsy provides unique chances for disease monitoring, preclinical model generation and therapy adjustment in rare salivary gland cancer patients.

While cell-free liquid biopsy (cfLB) approaches provide simple and inexpensive disease monitoring, cell-based liquid biopsy (cLB) may enable additional molecular genetic assessment of systemic disease heterogeneity and preclinical model development. We investigated 71 blood samples of 62 patients with various advanced cancer types and subjected enriched circulating tumor cells (CTCs) to organoid culture conditions. CTC-derived tumoroid models were characterized by DNA/RNA sequencing and immunohistochemistry, as well as functional drug testing. Results were linked to molecular features of primary tumors, metastases, and CTCs; CTC enumeration was linked to disease progression. Of 52 samples with positive CTC counts (≥1) from eight different cancer types, only CTCs from two salivary gland cancer (SGC) patients formed tumoroid cultures (P = 0.0005). Longitudinal CTC enumeration of one SGC patient closely reflected disease progression during treatment and revealed metastatic relapse earlier than clinical imaging. Multiomics analysis and functional invitro drug testing identified potential resistance mechanisms and drug vulnerabilities. We conclude that cLB might add a functional dimension (to the genetic approaches) in the personalized management of rare, difficult-to-treat cancers such as SGC.

GZMA suppressed GPX4-mediated ferroptosis to improve intestinal mucosal barrier function in inflammatory bowel disease

BackgroundOur previous study has demonstrated a decreased colonic CD8+CD39+ T cells, enrichment of granzyme A (GZMA), was found in pediatric-onset colitis and inflammatory bowel disease (IBD) characterized by impaired intestinal barrier function. However, the influence of GZMA on intestinal barrier function remains unknown.MethodsWestern blotting(WB), real-time PCR (qPCR), immunofluorescence (IF) and in vitro permeability assay combined with intestinal organoid culture were used to detect the effect of GZMA on intestinal epithelial barrier function in vivo and in vitro. Luciferase, immunoprecipitation (IP) and subcellular fractionation isolation were performed to identify the mechanism through which GZMA modulated intestinal epithelial barrier function.ResultsHerein, we, for the first time, demonstrated that CD8+CD39+ T cells promoted intestinal epithelial barrier function through GZMA, leading to induce Occludin(OCLN) and Zonula Occludens-1(ZO-1) expression, which was attributed to enhanced CDX2-mediated cell differentiation caused by increased glutathione peroxidase 4(GPX4)-induced ferroptosis inhibition in vivo and in vitro. Mechanically, GZMA inhibited intestinal epithelial cellular PDE4B activation to trigger cAMP/PKA/CREB cascade signaling to increase CREB nuclear translocation, initiating GPX4 transactivity. In addition, endogenous PKA interacted with CREB, and this interaction was enhanced in response to GZMA. Most importantly, administration of GZMA could alleviate DSS-induced colitis in vivo.ConclusionThese findings extended the novel insight of GZMA contributed to intestinal epithelial cell differentiation to improve barrier function, and enhacement of GZMA could be a promising strategy to patients with IBD.

Establishment of goat mammary organoid cultures modeling the mammary gland development and lactation.

Although several cell culture systems have been developed to investigate the function of the mammary gland in dairy livestock, they have potential limitations, such as the loss of alveolar structure or genetic and phenotypic differences from their native counterparts. Overcoming these challenges is crucial for lactation research. Development of protocols to establish lactating organoid of livestock represents a promising goal for the future. In this study, we developed a protocol to establish a culture system for mammary organoids in dairy goats to model the mammary gland development and lactation process. The organoids cultured within an extracellular matrix gel maintained a bilayer structure that closely resembled the native architecture of mammary tissue. The expansion of mammary organoids was significantly promoted by growth factors containing epidermal growth factor and fibroblast growth factor 2 whereas the proliferative index of the organoids was significantly inhibited by the treatment with WNT inhibitors. Upon stimulation with a lactogenic medium containing prolactin, the mammary organoids exhibited efficient lactation, characterized by the accumulation of lipid droplets in the lumen space. The lactation could be sustained for more than 3 weeks. Importantly, the expression patterns of genes related to fatty acid synthesis and milk proteins in lactating organoids closely mirrored those observed in mammary tissues. These observations were confirmed by data from proteomic analysis that the bulk of milk proteins was produced in the lactating organoids. This study is the first to establish a mammary organoid culture system modeling the mammary gland development and lactation process in ruminants. The efficient induction of lactation in ruminant mammary organoids holds promises for advancing the field of cell-based milk bio-manufacture in the food industry.

NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage.

The gene encoding nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), a nicotinamide adenine dinucleotide synthetase localized in the cell nucleus, is a causative factor in Leber's congenital amaurosis, which is the earliest onset type of inherited retinal degeneration. We sought to investigate the roles of NMNAT1 in early retinal development. We used human induced pluripotent stem cells (hiPSCs) and established NMNAT1-knockout (KO) hiPSCs using CRISPR/cas9 technology to reveal the roles of NMNAT1 in human retinal development. NMNAT1 was not essential for the survival and proliferation of immature hiPSCs; therefore, we subjected NMNAT1-KO hiPSCs to retinal organoid (RO) differentiation culture. The expression levels of immature hiPSC-specific genes decreased in a similar manner after organoid culture initiation up to 2 weeks in the control and NMNAT1-KO. Neuroectoderm-specific genes were induced in the control and NMNAT1-KO organoids within a few days after starting the organoid culture; PAX6 and TUBB3 were higher in NMNAT1-KO organoids up to 7days than in the control organoids. However, the induction of genes involving retinal early development, such as RAX, which was induced at around day 10 in this culture, was considerably reduced in NMNAT1-KO organoids. Morphological examination also showed failure of retinal primordial structure formation, which became visible at around 2 weeks of the control culture, in the NMNAT1-KO organoids. Decreased intracellular NAD levels and poly(ADP-ribosyl)ation were observed in NMNAT1-KO organoids at 7 to 10days of the culture. Mass spectrometry analysis of inhibited proteins in the poly(ADP-ribosyl)ation pathway identified poly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase 1 (PARP1) as a major protein. These results indicate that NMNAT1 was dispensable for neural lineage differentiation but essential for the commitment of retinal fate differentiation in hiPSCs. The NMNAT1-NAD-PARP1 axis may play a critical role in the appropriate development of human retinal lineage differentiation.

P53 mutation biases squamocolumnar junction progenitor cells towards dysplasia rather than metaplasia in Barrett’s oesophagus

BackgroundWhile p53 mutations occur early in Barrett’s oesophagus (BE) progression to oesophageal adenocarcinoma (EAC), their role in gastric cardia stem cells remains unclear.ObjectiveThis study investigates the impact of p53 mutation...

A chemically defined, mechanically tunable, and bioactive hyaluronic acid/alginate double-network hydrogel for liver cancer organoid construction

Liver cancer organoids replicate the pathophysiology of primary tumors, making them ideal for drug screening and efficacy evaluation. However, their growth in complex, variable, animal-derived matrices hinders practical application. Here, we designed an easily accessible, chemically defined, biocompatible double-network hydrogel (HADR) using methacrylated hyaluronic acid (HAMA), sodium alginate (SA), methacrylated dopamine (DMA), and c(RGDFC) for liver cancer organoid culture. By optimizing critical extracellular matrix (ECM) parameters, the HADR hydrogel achieves compatibility with the physiological mechanics of the human liver and fosters the adhesion and proliferation of multiple cell types. In vitro drug efficacy tests showed that HepG2 cell line-derived liver cancer organoids exhibited higher IC50 values than 2D cultures, indicating greater drug resistance. Subcutaneous tumor models in nude mice revealed that HADR hydrogels created a microenvironment for HepG2 cells mirroring the natural tumor ECM, leading to increased tumor volume, denser cell arrangement, and concurrent microvascular development. In vivo drug efficacy evaluations indicated that DOX treatment downregulated Ki-67 and MMP-9 expression, inhibiting HepG2 cell proliferation, invasion, and metastasis. These findings demonstrate the potential of HADR hydrogels for liver cancer organoid culture, offering new strategies for personalized drug screening and efficacy evaluation.

Enhancing Retinal Regeneration through Dental Pulp Stem Cells -Based 3D Organoid Culture, Cytokine Regulation and Gene Editing Technologies.

Enhancing Retinal Regeneration through Dental Pulp Stem Cells -Based 3D Organoid Culture, Cytokine Regulation and Gene Editing Technologies.

CSF1 is expressed by the intestinal epithelial cells to regulate Mφ macrophages and maintain epithelial homeostasis and is downregulated in neonates with necrotizing enterocolitis

BackgroundColony stimulating factor 1 (CSF1) is generally expressed by immune cells in response to pro-inflammatory stimuli. The CSF1 receptor (CSFR) is activated by CSF1, and plays a key role in macrophage homeostasis. Furthermore, the CSF1R+ macrophages maintain homeostasis in the intestinal epithelium. The aim of this study was to explore the functions of CSF1-expressing and CSF1R+ macrophages in necrotizing enterocolitis (NEC), which commonly affects the ileum of neonates.MethodsIn-situ CSF1 expression in the intestines of neonates with NEC or intestinal atresia (n = 4 each) was detected by immunofluorescence staining. The CSF1 levels in the intestinal crypt-derived organoid cultures were measured by ELISA. Peripheral blood monocyte-derived Mφ macrophages were co-cultured with the organoids and stimulated with lipopolysaccharide (LPS) to mimic the inflamed state of the ileum in NEC patients.ResultsCSF1 was expressed in the intestinal epithelial cells of the fetal and neonatal samples, but suppressed in the NEC samples. Furthermore, CSF1 expression was downregulated in the intestinal crypt-derived organoids by LPS. CSF1R+ macrophages were detected near the intestinal crypts in the non-inflamed intestines but were absent in tissues obtained from pediatric NEC patients. Peripheral blood monocyte-derived macrophages promoted intestinal organoid proliferation in vitro following CSF1 stimulation. Finally, low concentrations of LPS slightly enhanced the proliferation of organoids co-cultured with the macrophages, whereas higher doses had a significant inhibitory effect.ConclusionsIntestinal epithelial cells express CSF1 to regulate the resident macrophages, maintain epithelial homeostasis, and resist infection. The abundant CSF1R+ macrophages in the fetal intestine may overexpress TNF-α upon activation of the TLR4/NF-κB pathway, resulting in epithelial damage and NEC induction.

Regenerative capacity of alveolar type 2 cells is proportionally reduced following disease progression in idiopathic pulmonary fibrosis-derived organoid cultures.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease that leads to respiratory failure and death due to irreversible scarring of the distal lung. While historically considered a chronic inflammatory disorder, the aberrant function of the alveolar epithelium is now recognized to play a central role in IPF pathophysiology. This study aimed to investigate the regenerative capacity of AT2 cells using IPF-derived alveolar organoids and to examine the effects of disease progression on this capacity. Lung tissues from 3 pneumothorax patients and 6 IPF patients (early and advanced stages) were obtained by VATS and lung transplantation. HTII-280+ cells were isolated from CD31-CD45-EpCAM+ cells in the distal lungs of IPF and pneumothorax patients using fluorescence-activated cell sorting (FACS) and resuspended in 48-well plates to establish IPF-derived alveolar organoids. Immuno-staining was used to confirm the presence of AT2 cells. FACS sorting yielded approximately 1% AT2 cells of the total cells in early IPF tissue, and the number decreased as the disease progressed, compared with 2.7% in pneumothorax. Additionally, the cultured organoids in the IPF groups were smaller in size and fewer in number compared to those from pneumothorax patients. The colony-forming efficiency decreased as the disease progressed. In immuno-staining results, the IPF organoids showed lower expression of SFTPC compared to the pneumothorax group and contained KRT5+ cells. This study confirmed that the regenerative capacity of AT2 cells in IPF decreases as the disease progresses, and IPF AT2 cells inherently exhibit functional abnormalities and altered differentiation plasticity.