MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

Epithelial Cells in 2D and 3D Cultures Exhibit Large Differences in Higher-order Genomic Interactions

Recent studies have characterized the genomic structures of many eukaryotic cells,often focusing on their relation to gene expression.However,these studies have largely investigated cells grown in 2D cultures,although the transcriptomes of 3D-cultured cells are generally closer to their in vivo phenotypes.To examine the effects of spatial constraints on chromosome conformation,we investigated the genomic architecture of mouse hepatocytes grown in 2D and 3D cultures using in situ Hi-C.Our results reveal significant differences in higher-order genomic interactions,notably in compartment identity and strength as well as in topologically associating domain(TAD)-TAD interactions,but only minor differences are found at the TAD level.Our RNA-seq analysis reveals up-regulated expression of genes involved in physiological hepatocyte functions in the 3D-cultured cells.These genes are associated with a subset of structural changes,suggesting that differences in genomic structure are critically important for transcriptional regulation.However,there are also many structural differences that are not directly associated with changes in gene expression,whose cause remains to be determined.Overall,our results indicate that growth in 3D significantly alters higher-order genomic interactions,which may be consequential for a subset of genes that are important for the physiological functioning of the cell.

Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of crabs.However,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest.In this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH cells.In 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab serum.Then an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells.These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen.In 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day 103.This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Recent advances in chemically defined and tunable hydrogel platforms for organoid culture

Recent developments in organoid culture technologies have made it possible to closely recapitulate intrinsic characteristics of different tissues under in vitro conditions.These organoids act as a translational bridge between the traditional 2D/3D cultures and the in vivo models for studying the tissue development processes,disease modeling,and drug screening.Matrigel and tissue-specific extracellular matrix have been shown to support organoid development,efficiently;however,their chemically undefined nature,non-tunable properties,and associated batch-to-batch variations often limit reproducibility of the assembly process.In this regard,chemically defined platforms offer wider opportunities to optimize and recreate tissue-specific microenvironment.The present review delineates the current research trends in this sphere,focusing on material perspective and the target tissues(e.g.,neural,liver,pancreatic,renal,and intestinal).The review winds up with a discussion on the current limitations and future perspective to provide a basis for future research.

Production of functional sperm from in vitro-cultured premeiotic spermatogonia in a marine fish

In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture,especially in fish with relatively long generations.Nevertheless,functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish.In this study,we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers(Bostrychus sinensis),which are prone to ovotesticular or sterile testicular development,and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional(3D)culture system.Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae.Furthermore,melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway,and thus increased the efficiency in functional sperm production.The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.

In Vitro Invasive Pattern of Hepatocellular Carcinoma Cell Line HCCLM9 Based on Three-dimensional Cell Culture and Quantum Dots Molecular Imaging

Summary： This study aimed to establish a new in vitro three-dimensional （3D） cell culture and use quantum dots （QDs） molecular imaging to examine the invasive behaviors of hepatocellular carcinoma （HCC） cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C （HRV-C） and human bocavirus （HBoV） cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional （3D） pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR）. Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial （HAE） cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Mechanical stretching of 3D hydrogels for neural stem cell differentiation

While it is known that mechanical dynamics are influential in neural differentiation for critical processes like neurogenesis or neurodegeneration, studies on neural stem cell therapies usually focus on biochemical interactions rather than mechanical aspects, frequently resulting in low efficacy and unfulfilled potential. Therefore, current studies are attempting to elucidate the effect of mechanical stimulus on neural performance using conventional two-dimensional(2D) planar substrates. Yet, these2D substrates fail to capture the defining three-dimensional(3D) characteristics of the in vivo neural stem cell environment.To complete this research gap, we synthesized a series of soft and elastic 3D hydrogels to mimic the neural tissue mechanical environment for 3D cell culture, using long-chain polyethylene glycol diacrylate(PEGDA) and gelatin-methacryloyl(Gel MA).By varying the concentration of the polymer, we obtained biomimicking hydrogels with a tensile modulus as low as 10 k Pa and a compressive modulus as low as 0.8 k Pa. The in vitro results demonstrated that Gel MA-PEGDA hydrogels have the high biocompatibility required to support neural cell growth, proliferation, and differentiation, as well as neurite outgrowth. We then studied the effect of mechanical stretching on the behaviors of neural cells and observed that mechanical stretching could significantly enhance neurite extension and axon elongation. In addition, the neurites were more directionally oriented to the stretching direction. Immunocytochemistry and relative gene expression data also suggested that mechanical tension could upregulate the expression of neural differentiation protein and genes, including GFAP and βIII-Tubulin. Overall, this study shows that in addition to the specific mechanical properties of Gel MA-PEGDA that improve neural differentiation towards specific lineages, hydrogel stretching is also a potentially attractive strategy to improve the therapeutic outcomes of neural stem cell therapies.

A Three-Dimensional (3D) Environment to Maintain the Integrity of Mouse Testicular Can Cause the Occurrence of Meiosis

Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional （3D） agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum （dpp） mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.

Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development

In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.

In vitro 3D malignant melanoma model for the evaluation of hypericin-loaded oil-in-water microemulsion in photodynamic therapy

Advances in biomimetic three-dimensional(3D) melanoma models have brought new prospects of drug screening and disease modeling, since their physiological relevancy for recapitulating in vivo tumor architectures is more accurate than traditional two-dimensional(2D) cell culture. Gelatin methacryloyl(GelMA) is widely used as a tissue-engineered scaffold hydrogel for 3D cell culture. In the present study, an in vitro 3D malignant melanoma model based on Gel MA was fabricated to evaluate the efficiency of hypericin(Hy)-loaded microemulsion(ME) in photodynamic therapy against melanoma. The ME was produced by the spontaneous emulsification method to enhance the bioavailability of Hy at tumor sites. Hy-loaded MEs were applied to a 3D malignant melanoma model made using 6% Gel MA and the co-culture of B16F10 and Balb/c 3T3 cells,followed by crosslinking using violet light(403 nm). The observation revealed excellent cell viability and the presence of F-actin cytoskeleton network. Hy-loaded MEs exhibited higher phototoxicity and cell accumulation(about threefold) than free Hy, and the cells cultured in the 3D system displayed lower susceptibility(about 2.5-fold) than those in 2D culture.These findings indicate that the developed MEs are potential delivery carriers for Hy;furthermore, Gel MA hydrogel-based modeling in polydimethylsiloxane(PDMS) molds is a user-friendly and cost-effective in vitro platform to investigate drug penetration and provide a basis for evaluating nanocarrier efficiency for skin cancer and other skin-related diseases.

Alginate Matrix of MDA-MB-231 Breast Cancer Cell 3D Culturing Alters CD44 and CD24 mRNA Levels and Induces ALDH1 Expression

CSCs （Cancer stem cells） have been involved in tumor resistance, metastasis and recurrence. In breast cancer, tumor cells are characterized by CD44＋, CD24-/low and ALDH 1 expression represents a subpopulation of BCSC （breast cancer stem cell）. Several three-dimensional （3D） in vitro culturing cancer cells have been used to stimulate BCSC phenotype. The present study aimed to evaluate 3D cell culture in alginate matrix and the CD44, CD24 and ALDH1 mRNA levels of BCSC markers. The 3D culture was performed using MDA-MB-231 breast cancer cell line on alginate matrix 1.2% in RPMI medium. Expression of BCSC markers was evaluated by Real Time PCR （Polymerase Chain Reaction） comparing 3D to 2D culture. The 3D cultures increase of CD44 and CD24 mRNA levels and induce ALDH1 expression comparing to 2D culture. The data suggest that 3D alginate matrix alters the mRNA levels of genes involved in the phenotypic characteristics of BCSC.

Mechanistic Insights of Cells in Porous Scaffolds via Integrated Culture Technologies

This research aimed to combine 3 cell and tissue culture technologies to obtain mechanistic insights of cells in porous scaffolds. When cultivated on 2D （2-dimensional） surfaces, HDFs （human dermal fibroblasts） behaved individually and had no strict requirement on seeding density for proliferation; while HaCat cells relied heavily on initial densities for proliferation and colony formation, which was facilitated when co-cultured with HDFs. Experiments using a 3D CCIS （3-dimensional cell culture and imaging system） indicated that HDFs colonised openpores of varying sizes （125-420 ~tm） on modular substrates via bridge structures; while HaCat cells formed aperture structures and only colonised small pores （125 txm）. When co-cultured, HDFs not only facilitated HaCat attachment on the substrates, but also coordinated with HaCat cells to colonise open pores of varying sizes via bridge and aperture structures. Based on these observations, a 2-stage strategy for the culture of HDFs and HaCat cells on porous scaffolds was proposed and applied successfully on a cellulosic scaffold. This research demonstrated that cell colonisation in scaffolds was dependent on multiple factors; while the integrated 2D＆3D culture technologies and the 3D CCIS was an effective and efficient approach to obtain mechanistic insights of their influences on tissue regeneration.

Different priming strategies improve distinct therapeutic capabilities of mesenchymal stromal/stem cells:Potential implications for their clinical use

Mesenchymal stromal/stem cells(MSCs)have shown significant therapeutic potential,and have therefore been extensively investigated in preclinical studies of regenerative medicine.However,while MSCs have been shown to be safe as a cellular treatment,they have usually been therapeutically ineffective in human diseases.In fact,in many clinical trials it has been shown that MSCs have moderate or poor efficacy.This inefficacy appears to be ascribable primarily to the heterogeneity of MSCs.Recently,specific priming strategies have been used to improve the therapeutic properties of MSCs.In this review,we explore the literature on the principal priming approaches used to enhance the preclinical inefficacy of MSCs.We found that different priming strategies have been used to direct the therapeutic effects of MSCs toward specific pathological processes.Particularly,while hypoxic priming can be used primarily for the treatment of acute diseases,inflammatory cytokines can be used mainly to prime MSCs in order to treat chronic immune-related disorders.The shift in approach from regeneration to inflammation implies,in MSCs,a shift in the production of functional factors that stimulate regenerative or anti-inflammatory pathways.The opportunity to fine-tune the therapeutic properties of MSCs through different priming strategies could conceivably pave the way for optimizing their therapeutic potential.

3D （Three-Dimensional） Caco-2 Spheroids： Optimized in vitro Protocols to Favor Their Differentiation Process and to Analyze Their Cell Growth Behavior

3D （Three-dimensional） Caco-2 spheroids closely recapitulating in vivo physiological organization of intestinal epithelial cells, provide an excellent in vitro model system to study their pathophysiology and their response to stressful stimuli. The objective of this technical note is to provide optimized in vitro experimental protocols for culturing 3D Caco-2 spheroids and for analyzing their cell growth features. An optimized 3D Caco-2 spheroid culturing technique based on a new configuration of the culture medium is provided A methodological approach to determine the distribution of the cell cycle phases in disaggregated Caco-2 spheroids by using cytofluorimetric analysis is also described. The optimized culturing protocol favors 3D Caco-2 spheroid differentiation process, as evaluated by the number of well-differentiated spheroids with a single hollow lumen. The cytofluorimetric analysis allows rapid collection of cell cycle phase data from high numbers of spheroid samples, thus, permitting to estimate their growth dynamics in a relatively short time. The optimized technical approaches described here can be applied in systematic manner to a variety of research activities utilizing 3D Caco-2 spheroids. Ease of use, time and economic saving advantages deriving from these protocols further highlight their potential.

Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro

Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2′-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3 D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3 D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3 D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3 D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3 D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.

Emerging roles of 3D-culture systems in tackling tumor drug resistance

Drug resistance that affects patients universally is a major challenge in cancer therapy.The development of drug resistance in cancer cells is a multifactor event,and its process involves numerous mechanisms that allow these cells to evade the effect of treatments.As a result,the need to understand the molecular mechanisms underlying cancer drug sensitivity is imperative.Traditional 2D cell culture systems have been utilized to study drug resistance,but they often fail to mimic the 3D milieu and the architecture of real tissues and cell-cell interactions.As a result of this,3D cell culture systems are now considered a comprehensive model to study drug resistance in vitro.Cancer cells exhibit an in vivo behavior when grown in a three-dimensional environment and react to therapy more physiologically.In this review,we discuss the relevance of main 3D culture systems in the study of potential approaches to overcome drug resistance and in the identification of personalized drug targets with the aim of developing patient-specific treatment strategies that can be put in place when resistance emerges.

Effects of simulated zero gravity on adhesion,cell structure,proliferation,and growth behavior,in glioblastoma multiforme

All life on Earth has evolved under the influence of continuous gravity,and methods have been developed to balance this influence with the biological evolution of organisms at the cellular and system levels.However,when exposed to zero gravity in space,the balance between cell structure and external forces is destroyed,resulting in changes at the cellular level(e.g.,cell morphology,adhesion,viability,apoptosis,etc.),and understanding the molecular mechanism of cell response to zero gravity will help to cope with diseases that rely on mechanical response.Therefore,biological research in space and zero gravity is a unique step in developing the best anti-cancer treatments,which is a great challenge to humanity.In this study,multicellular glioma cancer cells from a brain tumor in a 72-year-old Iraqi patient were subjected to simulated zero gravity for 24 h,and the results showed that most of the cells lost their adhesion,which is considered to be the first step toward cell apoptosis.In addition to the formation of multicellular spheroids,the results also showed that the inhibition rate for cell death was 32%in comparison to the control cells.Moreover,the cells showed a clear change in their cellular morphology and growth behavior.These results give new hope for fighting cancer distinctively,and such a treatment method has no side effects in comparison to traditional chemical and radiological ones.

Development of a miniaturized 3D organoid culture platform for ultra-high-throughput screening

The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening(HTS)-based drug discovery due to technical difficulties.Using genetically engineered human colon organoids as a model system,here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS.We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library.We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening.Our miniaturized organoid culturing method may be adapted to other types of organoids.By leveraging the power of 3D organoid culture in a high-density plate format,we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.