Facilitate Drug Screening Research Articles

Influence of Microenvironmental Orchestration on Multicellular Lung Alveolar Organoid Development from Human Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have emerged as promising in vitro tools, providing a robust system for disease modelling and facilitating drug screening. Human iPSCs have been successfully differentiated into lung cells and three-dimensional lung spheroids or organoids. The lung is a multicellular complex organ that develops under the symphonic influence of the microenvironment. Here, we hypothesize that the generation of lung organoids in a controlled microenvironment (cmO) (oxygen and pressure) yields multicellular organoids with architectural complexity resembling the lung alveoli. iPSCs were differentiated into mature lung organoids following a stepwise protocol in an oxygen and pressure-controlled microenvironment. The organoids developed in the controlled microenvironment displayed complex alveolar architecture and stained for SFTPC, PDPN, and KRT5, indicating the presence of alveolar epithelial type II and type I cells, as well as basal cells. Moreover, gene and protein expression levels were also increased in the cmO. Furthermore, pathway analysis of proteomics revealed upregulation of lung development-specific pathways in the cmO compared to those growing in normal culture conditions. In summary, by using a controlled microenvironment, we established a complex multicellular lung organoid derived from iPSCs as a novel cellular model to study lung alveolar biology in both lung health and disease.

Novel dual-color fluorescence probe for monitoring the fluctuation of lysosomal polarity and viscosity during apoptosis

Apoptosis is pivotal in the pathogenesis of cancer and various other diseases. Real-time monitoring of apoptosis is crucial for assessing cell viability and facilitating drug screening. The process of apoptosis tends to cause changes in cellular microenvironmental factors. The variations of the microenvironment can serve as an indicator of the degree of apoptosis. Thus, the development of a smart and facile probe responding to cellular microenvironmental factors is significant for visualizing the apoptotic process. In this research, we designed a novel dual-color fluorescent probe, Lyso-DCPV, which incorporates N, N-diethyl coumarin as its polarity-responsive module, and the twisting double bond between N, N-diethyl coumarin and N-propyl quinoline as its viscosity-responsive module. The green fluorescence of Lyso-DCPV is sensitive to polarity changes, diminishing as polarity increases. Conversely, the red fluorescence intensifies with escalating viscosity. The probe specifically targets lysosomes, producing green fluorescence in live cells. Due to Lyso-DCPV’s acute sensitivity to changes in polarity and viscosity, it is well-suited to observe variations in the intracellular lysosomal microenvironment during drug-induced apoptosis. We also employed Lyso-DCPV for the dynamic assessment of polarity and viscosity during peroxide-induced apoptosis, enabling the observation of cell viability through variations in the probe’s emission color and intensity. The distinct probe will pave the way for novel strategies in evaluating apoptosis-related drugs. Moreover, Lyso-DCPV stands as an exemplary molecular instrument to elucidate the interplay between polarity and viscosity, facilitating the exploration of the interrelation in the cellular microenvironment during apoptosis.

Advances in the study of gastric organoids as disease models.

Gastric organoids are models created in the laboratory using stem cells and sophisticated three-dimensional cell culture techniques. These models have shown great promise in providing valuable insights into gastric physiology and advanced disease research. This review comprehensively summarizes and analyzes the research advances in culture methods and techniques for adult stem cells and induced pluripotent stem cell-derived organoids, and patient-derived organoids. The potential value of gastric organoids in studying the pathogenesis of stomach-related diseases and facilitating drug screening is initially discussed. The construction of gastric organoids involves several key steps, including cell extraction and culture, three-dimensional structure formation, and functional expression. Simulating the structure and function of the human stomach by disease modeling with gastric organoids provides a platform to study the mechanism of gastric cancer induction by Helicobacter pylori. In addition, in drug screening and development, gastric organoids can be used as a key tool to evaluate drug efficacy and toxicity in preclinical trials. They can also be used for precision medicine according to the specific conditions of patients with gastric cancer, to assess drug resistance, and to predict the possibility of adverse reactions. However, despite the impressive progress in the field of gastric organoids, there are still many unknowns that need to be addressed, especially in the field of regenerative medicine. Meanwhile, the reproducibility and consistency of organoid cultures are major challenges that must be overcome. These challenges have had a significant impact on the development of gastric organoids. Nonetheless, as technology continues to advance, we can foresee more comprehensive research in the construction of gastric organoids. Such research will provide better solutions for the treatment of stomach-related diseases and personalized medicine.

Drug-Online: an online platform for drug-target interaction, affinity, and binding sites identification using deep learning

BackgroundAccurately identifying drug-target interaction (DTI), affinity (DTA), and binding sites (DTS) is crucial for drug screening, repositioning, and design, as well as for understanding the functions of target. Although there are a few online platforms based on deep learning for drug-target interaction, affinity, and binding sites identification, there is currently no integrated online platforms for all three aspects.ResultsOur solution, the novel integrated online platform Drug-Online, has been developed to facilitate drug screening, target identification, and understanding the functions of target in a progressive manner of “interaction-affinity-binding sites”. Drug-Online platform consists of three parts: the first part uses the drug-target interaction identification method MGraphDTA, based on graph neural networks (GNN) and convolutional neural networks (CNN), to identify whether there is a drug-target interaction. If an interaction is identified, the second part employs the drug-target affinity identification method MMDTA, also based on GNN and CNN, to calculate the strength of drug-target interaction, i.e., affinity. Finally, the third part identifies drug-target binding sites, i.e., pockets. The method pt-lm-gnn used in this part is also based on GNN.ConclusionsDrug-Online is a reliable online platform that integrates drug-target interaction, affinity, and binding sites identification. It is freely available via the Internet at http://39.106.7.26:8000/Drug-Online/.

Cancer Metastasis-on-a-Chip for Modeling Metastatic Cascade and Drug Screening.

Microfluidic chips are valuable tools for studying intricate cellular and cell-microenvironment interactions. Traditional in vitro cancer models lack accuracy in mimicking the complexities of in vivo tumor microenvironment. However, cancer-metastasis-on-a-chip (CMoC) models combine the advantages of 3D cultures and microfluidic technology, serving as powerful platforms for exploring cancer mechanisms and facilitating drug screening. These chips are able to compartmentalize the metastatic cascade, deepening the understanding of its underlying mechanisms. This article provides an overview of current CMoC models, focusing on distinctive models that simulate invasion, intravasation, circulation, extravasation, and colonization, and their applications in drug screening. Furthermore, challenges faced by CMoC and microfluidic technologies are discussed, while exploring promising future directions in cancer research. The ongoing development and integration of these models into cancer studies are expected to drive transformative advancements in the field.

Repair-driven DNA tetrahedral nanomachine combined with DNAzyme for 8-oxo guanine DNA glycosylase activity assay, drug screening and intracellular imaging.

8-oxo guanine DNA glycosylase (8-oxoG DNA glycosylase), a crucial DNA repair enzyme, is essential for maintaining genome integrity and preventing diseases caused by DNA oxidative damage. Imaging 8-oxoG DNA glycosylase in living cells requires a dependable technique. In this study, we designed a DNAzyme-modified DNA tetrahedral nanomachine (DTDN) powered by 8-oxoG restoration. Incorporating a molecular beacon probe (MB), the constructed platform was used for amplified in situ monitoring of 8-oxoG DNA glycosylase. Under normal conditions, duplexing with a complementary strand modified with two 8-oxoG sites inhibited the activity of DNAzyme. The restoration of DNAzyme activity by the repair of intracellular 8-oxoG DNA glycosylase on 8-oxoG bases can initiate a signal amplification reaction. This detection system can detect 8-oxoG DNA glycosylase activity linearly between 0 and 20 U mL-1, with a detection limit as low as 0.52 U mL-1. Using this method, we were able to screen 14 natural compounds and identify 6 of them as 8-oxoG DNA glycosylase inhibitors. In addition, a novel approach was utilized to assess the activity of 8-oxoG DNA glycosylase in living cells. In conclusion, this method provides a universal tool for monitoring the activity of 8-oxoG DNA glycosylase in vitro and in living cells, which holds great promise for elucidating the enzyme's functionality and facilitating drug screening endeavors.

COX6A2 deficiency leads to cardiac remodeling in human pluripotent stem cell-derived cardiomyocytes

BackgroundCardiac remodeling is the initiating factor for the development of heart failure, which can result from various cardiomyopathies. Cytochrome c oxidase subunit 6A2 (COX6A2) is one of the components of cytochrome c oxidase that drives oxidative phosphorylation. The pathogenesis of myocardial remodeling caused by COX6A2 deficiency in humans remains unclear because there are no suitable research models. In this study, we established a COX6A2-deficient human cardiac myocyte (CM) model that mimics the human COX6A2 homozygous mutation and determined the effects of COX6A2 dysfunction and its underlying mechanism.MethodsA human COX6A2 homozygous knockout cardiomyocyte model was established by combining CRISPR/Cas9 gene editing technology and hiPSC-directed differentiation technology. Cell model phenotypic assays were done to characterize the pathological features of the resulting COX6A2-deficient cardiomyocytes.ResultsCOX6A2 gene knockout did not affect the pluripotency and differentiation efficiency of hiPSCs. Myocardial cells with a COX6A2 gene knockout showed abnormal energy metabolism, increased oxidative stress levels, abnormal calcium transport activity, and decreased contractility. In addition, L-carnitine and trimetazidine significantly improved energy metabolism in the COX6A2-deficient human myocardial model.ConclusionsWe have established a COX6A2-deficient human cardiomyocyte model that exhibits abnormal energy metabolism, elevated oxidative stress levels, abnormal calcium transport, and reduced contractility. This model represents an important tool to gain insight into the mechanism of action of energy metabolism disorders resulting in myocardial remodeling, elucidate the gene-phenotype relationship of COX6A2 deficiency, and facilitate drug screening.

Cell-Laden 3D Printed GelMA/HAp and THA Hydrogel Bioinks: Development of Osteochondral Tissue-like Bioinks.

Osteochondral (OC) disorders such as osteoarthritis (OA) damage joint cartilage and subchondral bone tissue. To understand the disease, facilitate drug screening, and advance therapeutic development, in vitro models of OC tissue are essential. This study aims to create a bioprinted OC miniature construct that replicates the cartilage and bone compartments. For this purpose, two hydrogels were selected: one composed of gelatin methacrylate (GelMA) blended with nanosized hydroxyapatite (nHAp) and the other consisting of tyramine-modified hyaluronic acid (THA) to mimic bone and cartilage tissue, respectively. We characterized these hydrogels using rheological testing and assessed their cytotoxicity with live-dead assays. Subsequently, human osteoblasts (hOBs) were encapsulated in GelMA-nHAp, while micropellet chondrocytes were incorporated into THA hydrogels for bioprinting the osteochondral construct. After one week of culture, successful OC tissue generation was confirmed through RT-PCR and histology. Notably, GelMA/nHAp hydrogels exhibited a significantly higher storage modulus (G') compared to GelMA alone. Rheological temperature sweeps and printing tests determined an optimal printing temperature of 20 °C, which remained unaffected by the addition of nHAp. Cell encapsulation did not alter the storage modulus, as demonstrated by amplitude sweep tests, in either GelMA/nHAp or THA hydrogels. Cell viability assays using Ca-AM and EthD-1 staining revealed high cell viability in both GelMA/nHAp and THA hydrogels. Furthermore, RT-PCR and histological analysis confirmed the maintenance of osteogenic and chondrogenic properties in GelMA/nHAp and THA hydrogels, respectively. In conclusion, we have developed GelMA-nHAp and THA hydrogels to simulate bone and cartilage components, optimized 3D printing parameters, and ensured cell viability for bioprinting OC constructs.

Investigation into Molecular Brain Aging in Senescence-Accelerated Mouse (SAM) Model Employing Whole Transcriptomic Analysis in Search of Potential Molecular Targets for Therapeutic Interventions.

With the progression of an aging society, cognitive aging has emerged as a pressing concern necessitating attention. The senescence-accelerated mouse-prone 8 (SAMP8) model has proven instrumental in investigating the early stages of cognitive aging. Through an extensive examination of molecular changes in the brain cortex, utilizing integrated whole-genome transcriptomics, our principal aim was to uncover potential molecular targets with therapeutic applications and relevance to drug screening. Our investigation encompassed four distinct conditions, comparing the same strain at different time points (1 year vs. 16 weeks) and the same time point across different strains (SAMP8 vs. SAMR1), namely: physiological aging, accelerated aging, early events in accelerated aging, and late events in accelerated aging. Focusing on key functional alterations associated with aging in the brain, including neurogenesis, synapse dynamics, neurometabolism, and neuroinflammation, we identified candidate genes linked to these processes. Furthermore, employing protein-protein interaction (PPI) analysis, we identified pivotal hub genes involved in interactions within these functional domains. Additionally, gene-set perturbation analysis allowed us to uncover potential upstream genes or transcription factors that exhibited activation or inhibition across the four conditions. In summary, our comprehensive analysis of the SAMP8 mouse brain through whole-genome transcriptomics not only deepens our understanding of age-related changes but also lays the groundwork for a predictive model to facilitate drug screening for cognitive aging.

Organotypic culture model of mouse meibomian gland as a screening platform for risk factors related to meibomian gland dysfunction

PurposeMeibomian glands (MGs) are crucial for maintaining tear film stability and ocular surface health. Here, we aim to establish a novel organotypic culture model of MGs and explore the risk factors of MG dysfunction (MGD). MethodsWe developed a novel organotypic culture model for MGs at the air-liquid interface. The viability and cell proliferation of MGs were assessed using CCK-8, immunofluorescence, and qPCR. Lipid accumulation was evaluated by Nile red staining and microscopic examination. Protein expression levels were evaluated by immunofluorescence and Western blot assay. EdU assay was employed to track the proliferation of acinar cells. The validity of the model was confirmed through culturing MGs from mice of different ages and incorporating certain drugs (Dex) into the culture system. ResultsUtilizing the novel culture model, the MG tissue exhibited sustained viability, cellular division, and continuous production of lipids for a duration of 7 days. Lipid droplets formed were directly visualized using light field microscopy. Through the cultivation of aged mice's MGs, it was discovered that aging resulted in diminished proliferation and lipid synthesis, along with an aberrant increase in Krt10 expression. Further application of this model showed that Dex treatment diminished MG's proliferation and lipid synthesis. Finally, an in vivo study was conducted to provide additional confirmation of the phenomenon of Dex-induced abnormalities. ConclusionsIn this study, a stable organotypic culture model of the MGs was established. The organotypic culture model offers a valuable tool to investigate the pathophysiological mechanisms and facilitate drug screening for MG-related diseases.

Precision Cut Lung Slices as a Preclinical Model for Non-Small Cell Lung Cancer Chemoprevention.

PCLS could be a new model for premalignancy and chemoprevention research, and this work evaluates the model with tissue from prevention-relevant genetic and carcinogen exposed in vivo mouse models, in addition to evaluating chemoprevention agents.

Quantification of bacterial adhesion to tissue in high-throughput kinetics.

Bacterial adhesion to tissue is the starting point for many pathogenic processes and beneficial interactions. The dynamics and speed of adhesion (minutes) make high-resolution temporal kinetic data important, but this capability is absent from the current toolset. We present a high-throughput method with a second-to-minute kinetic resolution, testing the adhesion of Pseudomonas aeruginosa PAO1 wild-type, flagella-, pili-, and quorum-sensing mutants to human embryonic kidney (HEK293) cells. Adhesion rates were in good correlation with HEK293 confluence, and the ways in which various bacterial mutations modified adhesion patterns are in agreement with the published literature. This simple assay can facilitate drug screening and treatment development as well as provide a better understanding of the interactions of pathogenic and probiotic bacteria with tissues, allowing the design of interventions and prevention treatments.

Single-cell thermometry with a nanothermocouple probe.

Herein, a nanopipette-based thermocouple probe that possesses high temperature resolution, rapid response, good reversibility and stability was constructed and successfully applied for single-cell temperature sensing. Different intracellular temperatures were observed in diverse types of cells, which reveals differences in their metabolism levels. Temperature responses of cancer and normal cells against various exogenous drugs were also demonstrated. The spatially resolved temperature sensing of three-dimensional cell culture models unveils the existence of their inner temperature gradients. This work would facilitate drug screening and disease diagnosis.

Biomimetic integrated gustatory and olfactory sensing array based on HL-1 cardiomyocyte facilitating drug screening for tachycardia treatment

The ectopic co-expression of taste and olfactory receptors in cardiomyocytes provides not only possibilities for the construction of biomimetic gustatory and olfactory sensors but also promising novel therapeutic targets for tachycardia treatment. Here, bitter taste and olfactory receptors endogenously expressed in HL-1 cells were verified by RT-PCR and immunofluorescence staining. Then HL-1 cardiomyocyte-based integrated gustatory and olfactory sensing array coupling with the microelectrode array (MEA) was first constructed for drugs screening and evaluation for tachycardia treatment. The MEA sensor detected the extracellular field potentials and reflected the systolic-diastolic properties of cardiomyocytes in real time in a label-free and non-invasive way. The in vitro tachycardia model was constructed using isoproterenol as the stimulator. The proposed sensing array facilitated potential drug screening for tachycardia treatment, such as salicin, artemisinin, xanthotoxin, and azelaic acid which all activated specific receptors on HL-1 cells. IC50 values for four potential drugs were calculated to be 0.0036 μM, 309.8 μM, 14.68 μM, and 0.102 μM, respectively. Visualization analysis with heatmaps and PCA cluster showed that different taste and odorous drugs could be easily distinguished. The mean inter-class Euclidean distance between different bitter drugs was 1.681, which was smaller than the distance between bitter and odorous drugs of 2.764. And the inter-class distance was significantly higher than the mean intra-class Euclidean distance of 1.172. In summary, this study not only indicates a new path for constructing novel integrated gustatory and olfactory sensors but also provides a powerful tool for the quantitative evaluation of potential drugs for tachycardia treatment.

A Bioluminescence-Based Drug Screen Identifies Activities of Fexinidazole and Its Metabolites against Helicobacter pylori.

Helicobacter pylori is responsible for a wide range of gastric diseases, including gastric cancer and gastritis. With half of the world's population infected by H. pylori and the current standard of care associated with suboptimal outcomes, a search for more effective drugs is critical. To facilitate drug screening for H. pylori, we developed a microtiter plate-based compound screening method that is faster and can screen multiple compounds. We identified activities of fexinidazole and its sulfoxide and sulfone metabolites against H. pylori. Both fexinidazole and its metabolites exhibited equipotency against SS1, 60190, and G27 strains, which were about 3-6-fold more potent than the currently used metronidazole. We also determined the minimal inhibitory concentration (MIC) of metronidazole, fexinidazole, and its metabolites against these strains by a traditional agar plate-based method. While MIC values of fexinidazole and metronidazole were similar against all the strains, both sulfoxide and sulfone showed lower MIC values than metronidazole against SS1 and 60190. Given the recent FDA approval of fexinidazole, our data on the in vitro antibacterial activities of fexinidazole and its metabolites support further evaluation of this drug with the goal of producing an alternative nitro-based antimicrobial with good safety profiles for the treatment of H. pylori infection.

Adipocyte-based high throughput screening for anti-obesity drug discovery: Current status and future perspectives.

Drug discovery for obesity treatment, particularly bodily slimming, is a topic of timely importance that requires continued investigation, as the current therapies have limited efficacy with many adverse effects. Obesity is associated with adipose tissue expansion, where the size and number of adipocytes increase. Over the past few decades, high-throughput/content screening (HTS/HCS) has been carried out on morphological changes in adipose tissues and adipocytes for the development of anti-obesity therapies. Increased understating of current adipocyte-based HTS/HCS technology will facilitate drug screening for obesity and weight control.

Using 2D and 3D pluripotent stem cell models to study neurotropic viruses.

Understanding the impact of viral pathogens on the human central nervous system (CNS) has been challenging due to the lack of viable human CNS models for controlled experiments to determine the causal factors underlying pathogenesis. Human embryonic stem cells (ESCs) and, more recently, cellular reprogramming of adult somatic cells to generate human induced pluripotent stem cells (iPSCs) provide opportunities for directed differentiation to neural cells that can be used to evaluate the impact of known and emerging viruses on neural cell types. Pluripotent stem cells (PSCs) can be induced to neural lineages in either two- (2D) or three-dimensional (3D) cultures, each bearing distinct advantages and limitations for modeling viral pathogenesis and evaluating effective therapeutics. Here we review the current state of technology in stem cell-based modeling of the CNS and how these models can be used to determine viral tropism and identify cellular phenotypes to investigate virus-host interactions and facilitate drug screening. We focus on several viruses (e.g., human immunodeficiency virus (HIV), herpes simplex virus (HSV), Zika virus (ZIKV), human cytomegalovirus (HCMV), SARS-CoV-2, West Nile virus (WNV)) to illustrate key advantages, as well as challenges, of PSC-based models. We also discuss how human PSC-based models can be used to evaluate the safety and efficacy of therapeutic drugs by generating data that are complementary to existing preclinical models. Ultimately, these efforts could facilitate the movement towards personalized medicine and provide patients and physicians with an additional source of information to consider when evaluating available treatment strategies.

TdT-dUTP DSB End Labeling (TUDEL), for Specific, Direct In Situ Labeling of DNA Double Strand Breaks.

The genome of a living cell is continuously damaged by various exogenous and endogenous factors yielding multiple types of DNA damage including base damage and damage to the sugar-phosphate backbone of DNA. Double Strand Breaks (DSBs) are the most severe form of DNA damage and if left unchecked, may precipitate genomic rearrangements, cell death or contribute to malignancy. In clinical contexts, radiation is often used to induce DSBs as a form of genotoxic therapy. Despite the importance of DSBsand their repair, as yetthere is no facile assay to detect DSBs in situ or to quantify their location or proximity to other cellular constituents. Such an assay would help to disentangle DDR signaling pathways and identify new molecular players involved in DSB repair. These efforts, in turn, may facilitate drug screening and accelerate the discovery of novel, more effective genotoxic agents. We have developed such an assay, presented here, and term it TdT-dUTP DSB End Labeling (TUDEL).TUDEL makes use of Terminal Deoxynucleotidyl Transferase (TdT), a template-independent DNA polymerase. TdT is commonly used in TUNEL assays to yielda binary output of DNA damage. We haveadapted this approach, usingTdT and EdUTP tolabel individual DNA double strand breaks in irradiated cellsand detecting the incorporated EdU with fluorescent probes via Click chemistry.This tool complements and is compatible with existing, indirect methods to track DSBssuch as immunofluorescent detection of γH2AX. TUDEL is also sufficientlyspecific, sensitive,quantitative,and robustto replace the neutral Comet assay for routine measurement of DSB formation and repair. Here we present a protocol for TUDEL.

Commentary: GPR160 De-Orphanization Reveals Critical Roles in Neuropathic Pain in Rodents (Finally, a Receptor for CART Peptide).

1. Abstract The discovery of the CART transcript and peptides implicated CART peptide (CARTp) as a neurotransmitter involved in the action of psychostimulants and several other physiological processes, buttressing the importance of the CARTp system. While there is evidence that a receptor(s) for CARTp exists, the receptor(s) has/have not been cloned. To understand how CARTp functions, it is important to identify its receptor(s). Given the evidence that CARTp receptor is a GPCR, a reasonable approach to searching for a CARTp receptor is to closely examine GPCR orphan receptors, receptors for which there are no known neurotransmitters. One of such GPCR orphan receptors that may be linked directly to CARTp function is GPR160. While studying neuropathic pain, Yosten et al (2020) identified CARTp effects were related to GPR160 expression. While studying food and water intake, Haddock et al (2021) also determined that the effects of CARTp were blocked by immuno-neutralization of GPR160. These findings suggest that the orphan receptor GPR160 may be the elusive CARTp receptor, a welcome observation after so many years of searching. This important discovery will advance the understanding of the CARTp system, including facilitating drug screening for small molecule agonists and antagonists and the identification of therapeutic compounds.

Generation of 2.5D lung bud organoids from human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70% confluence (SC_70%_hom) or a clump seeding group with heterogeneously distributed cells at 90% confluence (CL_90%_het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.