Disease Modeling Applications Research Articles

Cardiac organoid: multiple construction approaches and potential applications.

The human cardiac organoid (hCO) is three-dimensional tissue model that is similar to an in vivo organ and has great potential on heart development biology, disease modeling, drug screening and regenerative medicine. However, the construction of hCO presents a unique challenge compared with other organoids such as the lung, small intestine, pancreas, liver. Since heart disease is the dominant cause of death and the treatment of such disease is one of the most unmet medical needs worldwide, developing technologies for the construction and application of hCO is a critical task for the scientific community. In this review, we discuss the current classification and construction methods of hCO. In addition, we describe its applications in drug screening, disease modeling, and regenerative medicine. Finally, we propose the limitations of the cardiac organoid and future research directions. A detailed understanding of hCO will provide ways to improve its construction and expand its applications.

Rianú: Multi-tissue tracking software for increased throughput of engineered cardiac tissue screening

Background:The field of tissue engineering has provided valuable three-dimensional species-specific models of the human myocardium in the form of human Engineered Cardiac Tissues (hECTs) and similar constructs. However, hECT systems are often bottlenecked by a lack of openly available software that can collect data from multiple tissues at a time, even in multi-tissue bioreactors, which limits throughput in phenotypic and therapeutic screening applications. Methods:We developed Rianú, an open-source web application capable of simultaneously tracking multiple hECTs on flexible end-posts. This software is operating system agnostic and deployable on a remote server, accessible via a web browser with no local hardware or software requirements. The software incorporates object-tracking capabilities for multiple objects simultaneously, an algorithm for twitch tracing analysis and contractile force calculation, and a data compilation system for comparative analysis within and amongst groups. Validation tests were performed using in-silico and in-vitro experiments for comparison with established methods and interventions. Results:Rianú was able to detect the displacement of the flexible end-posts with a sub-pixel sensitivity of 0.555 px/post (minimum increment in post displacement) and a lower limit of 1.665 px/post (minimum post displacement). Compared to our established reference for contractility assessment, Rianú had a high correlation for all parameters analyzed (ranging from R2=0.7514 to R2=0.9695), demonstrating its high accuracy and reliability. Conclusions:Rianú provides simultaneous tracking of multiple hECTs, expediting the recording and analysis processes, and simplifies time-based intervention studies. It also allows data collection from different formats and has scale-up capabilities proportional to the number of tissues per field of view. These capabilities will enhance throughput of hECTs and similar assays for in-vitro analysis in disease modeling and drug screening applications.

Densified collagen tubular grafts for human tissue replacement and disease modelling applications

There is a significant need across multiple indications for an off-the-shelf bioengineered tubular graft which fulfils the mechanical and biological requirements for implantation and function but does not necessarily require cells for manufacture or deployment. Herein, we present a tissue-like tubular construct using a cell-free, materials-based method of manufacture, utilizing densified collagen hydrogel. Our tubular grafts are seamless, mechanically strong, customizable in terms of lumen diameter and wall thickness, and display a uniform fibril density across the wall thickness and along the tube length. While the method enables acellular grafts to be generated rapidly, inexpensively, and to a wide range of specifications, the cell-compatible densification process also enables a high density of cells to be incorporated uniformly into the walls of the tubes, which we show can be maintained under perfusion culture. Additionally, the method enables tubes consisting of distinct cell domains with cellular configurations at the boundaries which may be useful for modelling aortic disease. Further, we demonstrate additional steps which allow for luminal surface patterning. These results highlight the universality of this approach and its potential for developing the next generation of bioengineered grafts.

Alginate‐Laminin Hydrogel Supports Long‐Term Neuronal Activity in 3D Human Induced Pluripotent Stem Cell‐Derived Neuronal Networks

AbstractFor 3D neural cultures durable hydrogels are required, which persist over a long differentiation period and thus enable the maturation of neuronal networks (NN). Here, 3D models based on human induced pluripotent stem cell‐derived neural progenitor cells that are embedded in hydrogels of either pure alginate or alginate functionalized with the extracellular matrix protein laminin 111 (L111) are established. This study analyzes material characteristics such as porosity, L111 distribution and shear viscosity, cell compatibility of hydrogels by measuring viability and cytotoxicity, and neural function by monitoring cell migration, differentiation as well as NN formation and activity on multielectrode arrays. The addition of L111 increases neural migration and enhances differentiation into neurons and astrocytes as well as synaptogenesis in alginate hydrogels. NN formed in hydrogels are electrically active for up to 206 d and L111‐supplementation further increases electrical activity, network maturation, and synchronicity compared to 2D controls and NN grown in pure alginate hydrogels. L111 addition to alginate gels further accelerates recovery of electrical activity after blockage of sodium channels with tetrodotoxin. In conclusion, NN grown in alginate‐L111 hydrogel blends are promising models for future long‐term applications in disease modeling, drug or chemical evaluation.

Single-cell transcriptional and functional analysis of dopaminergic neurons in organoid-like cultures derived from human fetal midbrain.

Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells (PSCs) for application in disease modeling, diagnostics, drug screening, and cell-based therapies for Parkinson's Disease (PD). An increased understanding of the timing and molecular mechanisms promoting the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from PSCs. Here, we used droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11 weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach revealed that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons, which can be used to guide stem cell-based therapies and disease modeling approaches in PD.

Stem cells for organoids.

Organoids are three-dimensional (3D) cell culture systems that simulate the structures and functions of organs, involving applications in disease modeling, drug screening, and cellular developmental biology. The material matrix in organoids can provide a 3D environment for stem cells to differentiate into different cell types and continuously self-renew, thereby realizing the in vitro culture of organs, which has received extensive attention in recent years. However, some challenges still exist in organoids, including low maturity, high heterogeneity, and lack of spatiotemporal regulation. Therefore, in this review, we summarized the culturing protocols and various applications of stem cell-derived organoids and proposed insightful thoughts for engineering stem cells into organoids in view of the current shortcomings, to achieve the further application and clinical translation of stem cells and engineered stem cells in organoid research.

Generation and Differentiation of Induced Pluripotent Stem Cells from Mononuclear Cells in An Age-Related Macular Degeneration Patient.

We aimed to generate induced pluripotent stem cells (iPSCs)-derived retinal pigmented epithelium (RPE) cells from peripheral blood mononuclear cells (PBMCs) and age-related macular degeneration (AMD) patient to provide potential cell sources for both basic scientific research and clinical application. In this experimental study, PBMCs were isolated from the whole blood of a 70-year-old female patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). Flow cytometry, real-time quantitative polymerase chain reaction (qPCR), karyotype analysis, embryoid body (EB) formation, and teratoma detection were performed to confirm that AMD-iPSCs exhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced into RPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence and flow cytometry. The iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphology including roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCs expressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated 99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 and NANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strong differentiation potential and pluripotency. After a series of inductions with differentiation mediums, a monolayer of AMDiPSC- RPE cells was observed on day 50. The AMD-iPSC-RPEs highly expressed specific RPE markers (MITF, ZO-1, Bestrophin, and PMEL17). A high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSC displayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, and therapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE with native RPE characteristics, which might provide potential regenerative treatments for AMD patients.

Cell Seeding on 3D Scaffolds for Tissue Engineering and Disease Modeling Applications.

Scaffold cell seeding is a crucial step for the standardization and homogeneous maturation of tissue engineered constructs. This is particularly critical in the context of additively manufactured scaffolds whereby large pore size and high porosity usually impedes the retention of the seeding solution resulting in poor seeding efficacy and heterogeneous cell distribution. To circumvent this limitation, a simple yet efficient cell seeding technique is described in this chapter consisting of preincubating the scaffold in 100% serum for 1h leading to reproducible seeding. A proof of concept is demonstrated using highly porous melt electrowritten polycaprolactone scaffolds as the cell carrier. As cell density, cell distribution, and differentiation within the scaffold are important parameters, various assays are proposed to validate the seeding and perform quality control of the cellularized construct using techniques such as alizarin red, Sirius red, and immunostaining.

Long-term effect of sodium selenite on the integrity and permeability of on-chip microvasculature.

Development of the robust and functionally stable three-dimensional (3D) microvasculature remains challenging. One often-overlooked factor is the presence of potential anti-angiogenic agents in culture media. Sodium selenite, an antioxidant commonly used in serum-free media, demonstrates strong anti-angiogenic properties and has been proposed as an anticancer drug. However, its long-term effects on in vitro microvascular systems at the concentrations used in culture media have not been studied. In this study, we used a five-channel microfluidic device to investigate the concentration and temporal effects of sodium selenite on the morphology and functionality of on-chip preformed microvasculature. We found that high concentrations (∼3.0 μM) had adverse effects on microvasculature perfusion, permeability, and overall integrity within the first few days. Moreover, even at low concentrations (∼3.0 nM), a long-term culture effect was observed, resulting in an increase in vascular permeability without any noticeable changes in morphology. A further analysis suggested that vessel leakage may be due to vascular endothelial growth factor dysregulation, disruption of intracellular junctions, or both. This study provides important insight into the adverse effects caused by the routinely present sodium selenite on 3D microvasculature in long-term studies for its application in disease modeling and drug screening.

Development of a high-throughput micropatterned agarose scaffold for consistent and reproducible hPSC-derived liver organoids

Liver organoids represent emerging human-relevant in vitro liver models that have a wide range of biomedical applications in basic medical studies and preclinical drug discovery. However, the generation of liver organoids currently relies on the conventional Matrigel dome method, which lacks precise microenvironmental control over organoid growth and results in significant heterogeneity of the formed liver organoids. Here, we demonstrate a novel high-throughput culture method to generate uniform liver organoids from human pluripotent stem cell-derived foregut stem cells in micropatterned agarose scaffold. By using this approach, more than 8000 uniformly-sized liver organoids containing liver parenchyma cells, non-parenchymal cells, and a unique stem cell niche could be efficiently and reproducibly generated in a 48-well plate with a size coefficient of variation significance smaller than that in the Matrigel dome. Additionally, the liver organoids highly expressed liver-specific markers, including albumin (ALB), hepatocyte nuclear factor 4 alpha (HNF4α), and alpha-fetoprotein (AFP), and displayed liver functions, such as lipid accumulation, glycogen synthesis, ALB secretion, and urea synthesis. As a proof of concept, we evaluated the acute hepatotoxicity of acetaminophen (APAP) in these organoids and observed APAP-induced liver fibrosis. Overall, we expect that the liver organoids will facilitate wide biomedical applications in hepatotoxicity analysis and liver disease modeling.

A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

The human SH-SY5Y neuroblastoma cell line is widely used in neuroscience research as a neuronal cell model. Following differentiation to a neuron-like state, SH-SY5Y cells become more morphologically similar to neurons and form functional synapses. Previous studies have managed to differentiate SH-SY5Y cells towards cholinergic, dopaminergic and adrenergic fates. However, their application in disease modeling remains limited as other neuronal subtypes (e.g., glutamatergic, GABAergic) are also implicated in neurological disorders, and no current protocols exist to generate these subtypes of differentiated SH-SY5Y cells. Our study aimed to evaluate the use of a xeno-free version of B-27, a supplement commonly used in neuronal culture, for SH-SY5Y maintenance and differentiation. To evaluate the proliferative capacity of SH-SY5Y cells cultured in B-27, we performed growth curve analyses, immunocytochemical staining for Ki-67 and qRT-PCR to track changes in cell cycle progression. SH-SY5Y cells cultured in FBS or under serum-starved conditions were used as controls. We observed that SH-SY5Y cells show reduced growth and proliferation rates accompanied by decreased CDK6 and CDK1 expression following 4-day exposure to B-27, suggesting B-27 induces a quiescent state in SH-SY5Y cells. Importantly, this reduced growth rate was not due to increased apoptosis. As cell cycle exit is associated with differentiation, we next sought to determine the fate of SH-SY5Y cells cultured in B-27. B-27-cultured SH-SY5Y cells show changes in cell morphology, adopting pyramidal shapes and extending neurites, and upregulation of neuronal differentiation markers (GAP43, TUBB3, and SYP). B-27-cultured SH-SY5Y cells also show increased expression of glutamatergic markers (GLUL and GLS). These findings suggest that B-27 may be a non-toxic inducer of glutamatergic SH-SY5Y differentiation. Our study demonstrates a novel way of using B-27 to obtain populations of glutamatergic SH-SY5Y cells. As dysregulated glutamatergic signaling is associated with a variety of neuropsychiatric and neurodegenerative disorders, the capability to generate glutamatergic neuron-like SH-SY5Y cells creates endless disease modeling opportunities. The ease of SH-SY5Y culture allows researchers to generate large-scale cultures for high-throughput pharmacological or toxicity studies. Also compatible with the growing popularity of animal-component-free studies, this xeno-free B-27/SH-SY5Y culture system will be a valuable tool to boost the translational potential of preliminary studies requiring glutamatergic neuronal cells of human origin.

Once upon a dish: the next frontier in engineering multicellular systems.

In June 2022, the second meeting on 'Engineering Multicellular Systems', organized by the European Molecular Biology Laboratory and the Institute of Bioengineering of Catalonia, took place in Barcelona. Stem cell and systems biologists, physicists and engineers from all over the world gathered to discuss how recent breakthroughs in organoid technologies, engineering and mechanobiology are boosting our understanding of early morphogenesis, organogenesis and organ function with applications in tissue engineering, disease modeling and drug screening. The meeting was organized with sustainability in mind, and included an ethics session and an outreach public activity.

A Microfluidic Device for Long-Term Maintenance of Organotypic Liver Cultures.

Liver cultures may be used for disease modeling, testing therapies and predicting drug-induced injury. The complexity of the liver cultures has evolved from hepatocyte monocultures to co-cultures with non-parenchymal cells and finally to precision-cut liver slices. The latter culture format retains liver's native biomolecular and cellular complexity and therefore holds considerable promise for in vitro testing. However, liver slices remain functional for ~72 h in vitro and display limited utility for some disease modeling and therapy testing applications that require longer culture times. This paper describes a microfluidic device for longer-term maintenance of functional organotypic liver cultures. Our microfluidic culture system was designed to enable direct injection of liver tissue into a culture chamber through a valve-enabled side port. Liver tissue was embedded in collagen and remained functional for up to 31 days, highlighted by continued production of albumin and urea. These organotypic cultures also expressed several enzymes involved in xenobiotic metabolism. Conversely, matched liver tissue embedded in collagen in a 96-well plate lost its phenotype and function within 3-5 days. The microfluidic organotypic liver cultures described here represent a significant advance in liver cultivation and may be used for future modeling of liver diseases or for individualized liver-directed therapies.

Tissue engineered drug delivery vehicles: Methods to monitor and regulate the release behavior.

Tissue engineering is a rapidly evolving, multidisciplinary field that aims at generating or regenerating 3D functional tissues for in vitro disease modeling and drug screening applications or for in vivo therapies. A variety of advanced biological and engineering methods are increasingly being used to further enhance and customize the functionality of tissue engineered scaffolds. To this end, tunable drug delivery and release mechanisms are incorporated into tissue engineering modalities to promote different therapeutic processes, thus, addressing challenges faced in the clinical applications. In this review, we elaborate the mechanisms and recent developments in different drug delivery vehicles, including the quantum dots, nano/micro particles, and molecular agents. Different loading strategies to incorporate the therapeutic reagents into the scaffolding structures are explored. Further, we discuss the main mechanisms to tune and monitor/quantify the release kinetics of embedded drugs from engineered scaffolds. We also survey the current trend of drug delivery using stimuli driven biopolymer scaffolds to enable precise spatiotemporal control of the release behavior. Recent advancements, challenges facing current scaffold-based drug delivery approaches, and areas of future research are discussed.

Pre-glycation impairs gelation of high concentration collagen solutions.

There remains a need for stiffer collagen hydrogels for tissue engineering and disease modeling applications. Pre-glycation, or glycation of collagen in solution prior to gelation, has been shown to increase the mechanics of collagen hydrogels while maintaining high viability of encapsulated cells. The stiffness of glycated collagen gels can be increased by increasing the collagen concentration, sugar concentration, and glycation time. However, previous studies on pre-glycation of collagen have used low collagen concentrations and/or low sugar concentrations and have not investigated the effect of glycation time. Therefore, the objective of this study was to determine the effects of pre-glycation with high sugar concentrations (up to 500 mM) and extended glycation times (up to 21 days) on high concentration collagen (8mg/ml). The addition of sugar to the collagen and the formation of advanced glycation end products (AGEs) were quantified. The ability to gel successfully and rheological properties were determined and correlated with biochemical characterizations. Successful collagen gelation and rheological properties of pre-glycated collagen were found to be strongly dependent on the ratio of added sugars to added AGEs with high ratios impairing gelation and low ratios resulting in optimal storage moduli. There is likely a competing effect during pre-glycation of the formation of AGEs resulting in crosslinking of collagen and the formation of Amadori intermediates acting to increase collagen solubility. Overall, this study shows that collagen glycation can be optimized by increasing the formation of AGEs while maintaining a low ratio of added sugar to added AGEs.

In vivo toxicological evaluation of barium-doped bioactive glass in rats

Bioactive glasses (BGs) are inorganic biomaterials with several FDA-approved marketed products, mainly for orthopedic and dental applications. However, in the last few decades, the soft tissue regenerative potential of BGs has also been established preclinically, and its clinical translation requires elaborate in vivo toxicity studies for future biomedical applications. Thus, in the present study, we evaluated the comparative acute and sub-acute toxicity of orally administered barium-doped BG (BaBG) with 45S5 in rats. The lethal dose (LD50) of BaBG and 45S5 was more than 2000 mg/kg body weight (b.w.), with no mortality at the highest dose tested. The oral acute toxicity study was performed at doses of 300 and 2000 mg/kg b.w. according to OECD 423. The organ coefficients of vital organs and histological analysis affirmed the safety profile of BaBG. Moreover, various biochemical indices like alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine, and CK-MB confirmed that BaBG did not cause organ toxicity at all doses tested. Similarly, the repeated dose 28-days sub-acute toxicity study was performed according to OECD 407 at doses 50, 500, and 1000 mg/kg b.w. There was no alteration in the hematological parameters, which ascertains that BaBG had no toxic effects on the hematopoietic centers like 45S5. Furthermore, there was no observed neuro-behavioral toxicity of 45S5 and BaBG. Thus, these findings confirm that BaBG is non-toxic and can be used for therapeutic applications in different disease models.

Abstract P1136: Amino Acid Availability Regulates Differentiation Of HIPSC-derived Cardiomyocytes In A Protein-free System

Traditional protocols for the differentiation of hiPSC-derived cardiomyocytes (hiPSC-CMs) rely on complex media and the use of animal or recombinant proteins either directly or in supplements, such as B27, to obtain tissue monolayers. This fact decreases their reproducibility, while potentially increasing their costs and limiting higher-throughput production of cardiomyocytes for applications in both regenerative medicine, disease modeling, and pharmacological screenings. Albumin/serum-free protocols have been developed but produce cells with limited functionality and at lower yields, despite them being TNNT2-positive. We previously demonstrated it is possible to simplify the differentiation protocol utilizing a stepwise approach and combining time-point interventions with small molecules or nutritional inputs. Here, we performed a thorough screening of commercial media and their suitability for differentiation of hiPSC-CMs. We identified suitable basal media formulations and developed a novel protein-free differentiation protocol (PFDM) for generating hiPSC-CMs that preserves purity, yield, morphology, and functionality while reducing the cost of the differentiation process. PFDM also enabled the identification of nutritional requirements essential for cardiac differentiation and allowed a detailed investigation into how some common components supplemented during hiPSC-CM differentiation impact tissue morphology and contractility. We showed that non-essential amino acids regulate differentiation in a time-dependent manner. Similarly, antioxidant agents such as sodium pyruvate and ascorbic acid, while essential for differentiation, are potentially harmful during early cardiac mesoderm induction. Lastly, we validated this PFDM methodology in 35 control and patient-derived hiPSC lines, with monolayer differentiations in 6- and 12-well plates, 15 cm dishes, in addition to differentiations in 3D using 100- and 250-mL spinner flasks.

Genetic and Epigenetic Regulation of Brain Organoids.

Revealing the mechanisms of neural development and the pathogenesis of neural diseases are one of the most challenging missions in life science. Pluripotent stem cells derived brain organoids mimic the development, maturation, signal generation, and function of human brains, providing unique advantage for neurology. Single-cell RNA sequencing (scRNA-Seq) and multielectrode array independently revealed the similarity between brain organoids and immature human brain at early developmental stages, in the context of gene transcription and dynamic network of neuronal signals. Brain organoids provided the unique opportunity to investigate the underlying mechanism of neural differentiation, senescence, and pathogenesis. In this review, we summarized the latest knowledge and technology in the brain organoid field, the current and potential applications in disease models and pre-clinic studies, with emphasizing the importance of transcriptional and epigenetic analysis.

Tissue-engineered heart chambers as a platform technology for drug discovery and disease modeling.

Current drug screening approaches are incapable of fully detecting and characterizing drug effectiveness and toxicity of human cardiomyocytes. The pharmaceutical industry uses mathematical models, cell lines, and in vivo models. Many promising drugs are abandoned early in development, and some cardiotoxic drugs reach humans leading to drug recalls. Therefore, there is an unmet need to have more reliable and predictive tools for drug discovery and screening applications. Biofabrication of functional cardiac tissues holds great promise for developing a faithful 3D in vitro disease model, optimizing drug screening efficiencies enabling precision medicine. Different fabrication techniques including molding, pull spinning and 3D bioprinting were used to develop tissue-engineered heart chambers. The big challenge is to effectively organize cells into tissue with structural and physiological features resembling native tissues. Some advancements have been made in engineering miniaturized heart chambers that resemble a living pump for drug screening and disease modeling applications. Here, we review the currently developed tissue-engineered heart chambers and discuss challenges and prospects.

Evaluation of different methodologies for primary human dermal fibroblast spheroid formation: automation through 3D bioprinting technology

Cell spheroids have recently emerged as an effective tool to recapitulate native microenvironments of living organisms in an in vitro scenario, increasing the reliability of the results obtained and broadening their applications in regenerative medicine, cancer research, disease modeling and drug screening. In this study the generation of spheroids containing primary human dermal fibroblasts was approached using the two-widely employed methods: hanging-drop and U-shape low adhesion plate (LA-plate). Moreover, extrusion-based three-dimensional (3D) bioprinting was introduced to achieve a standardized and scalable production of cell spheroids, decreasing considerably the possibilities of human error. This was ensured when U-shape LA-plates were used, showing an 85% formation efficiency, increasing up to a 98% when it was automatized using the 3D bioprinting technologies. However, sedimentation effect within the cartridge led to a reduction of 20% in size of the spheroid during the printing process. Hyaluronic acid (HA) was chosen as viscosity enhancer to supplement the bioink and overcome cell sedimentation within the cartridge due to the high viability values exhibited by the cells—around 80%—at the used conditions. Finally, (ANCOVA) of spheroid size over time for different printing conditions stand out HA 0.4% (w/v) 60 kDa as the viscosity-improved bioink that exhibit the highest cell viability and spheroid formation percentages. Besides, not only did it ensure cell spheroid homogeneity over time, reducing cell sedimentation effects, but also wider spheroid diameters over time with less variability, outperforming significantly manual loading.