Three-dimensional cell culture using CD9-positive cells isolated from marginal cell layer of intermediate lobe of rats sustains in vivo-like primary niche environment.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

Detecting life by behavior, the overlooked sensitivity of behavioral assays.

Detecting life has driven research and exploration for centuries, but recent attempts to compile and generate a framework that summarizes life features, aimed to develop strategies for life detection missions beyond planet Earth, have disregarded a key life feature: behavior. Yet, some behaviors such as biomineralization or motility have occasionally been proposed as biosignatures to detect life. Here, we capitalize on a specific taxis' motility behavior, magnetotaxis, to experimentally provide insights in support of behavior as an unambiguous, sensitive biosignature, and magnetic forces as a prescreening option. Using a magnetotactic bacterial species, Magnetospirillum magneticum, we conducted a lab sensitivity experiment comparing PCR with the hanging drop behavioral assay, using a dilution series. The hanging drop behavioral assay visually shows the motility of MTB toward magnetic poles. Our findings reveal that the behavioral assay exhibits higher sensitivity in the detection of M. magneticum when compared to the established PCR protocol. While both methods present similar detection sensitivities at high concentrations, at ≥ 10-7 fold dilutions, the behavioral method proved more sensitive. The behavioral method can detect bacteria even when samples are diluted at 10-9. Comparable results were obtained with environmental samples from the Hula Valley. We propose behavioral cues as valuable biosignatures in the ongoing efforts of life detection in unexplored aquatic habitats on Earth and to stimulate and support discussions about how to detect extant life beyond Earth. Generic and robust behavioral assays can represent a methodological revolution.

Reconfigurable Hanging Drop Microarray Platform for On-Demand Preparation and Analysis of Spheroid Array.

In response to the increasing demand for spheroid-based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high-throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three-dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK-Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one-step generation of a size-gradient spheroid array. Using the size-gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high-throughput production of cell composition-gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions.

Comparison of three-dimensional cell culture techniques of dedifferentiated liposarcoma and their integration with future research.

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions. Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically. Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response. Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel®. On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis. Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology.

Prostate Cancer Organoids for Tumor Modeling and Drug Screening.

Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.

High throughput generating stable spheroids with tip-refill wafer.

Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210 rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60 rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1% ± 3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.

Principles of Hanging Drop Method (Spheroid Formation) in Cell Culture.

A type of three-dimensional (3D) cell culture models which is simple and easy is hanging drop method. The hanging drop method emerges as a pivotal technique with diverse applications in cancer research and cell biology. This method facilitates the formation of multicellular spheroids, providing a unique environment for studying cell behavior dynamics. The hanging drop method's theoretical underpinning relies on gravity-enforced self-assembly, allowing for cost-effective, reproducible 3D cell cultures with controlled spheroid sizes. The advantages of this approach include its efficiency in producing cellular heterogeneity, particularly in non-adherent 3D cultures, and its ability to create hypoxic spheroids, making it a suitable model for studying cancer. Moreover, the hanging drop method has proven valuable in investigating various aspects such as tissue structure, signaling pathways, immune activation of cancer cells, and notably, cell proliferation. Researchers have utilized the hanging drop method to explore the dynamics of cell proliferation, studying the effects of mesenchymal stem cells (MSC) secretome on cancer cells. The method's application involves co-culturing different cell lines, assessing spheroid formations, and quantifying their sizes over time. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell growth and viability within a three-dimensional co-culture paradigm.

Three-Dimensional Hanging Drop Spheroid Plates for Easy Chimeric Antigen Receptor (CAR) T Cytotoxicity Assay.

Chimeric antigen receptor (CAR) T cell therapy shows a highly effective therapeutic effect on B-cell malignancies. The tumor microenvironment (TME) of solid tumors in vivo poses a great challenge to CAR T cell therapy due to its complexity. Recently, tumor spheroids have attracted much attention because of their ability to recapitulate TME. However, the use of tumor spheroids for the CAR T cytotoxicity assay involves the difficult task of separating unbound T cells and dead tumor cells from the spheroids. Therefore, we developed a three-dimensional hanging spheroid plate (3DHSP) that facilitates spheroid formation and separation of unbound and dead cells from spheroids during cytotoxicity assays. In this work, detailed steps have been described for fabrication and operation of the 3DHSP. This new 3DHSP device is a 96-well plate in which each well consists of a hanging dripper and a spheroid separation plate. A tumor spheroid forms in a droplet hanging in the dripper and is mixed with CAR T cells. The mixture in the droplet is deposited into the spheroid separation plate by pipetting, and unbound and dead CAR T and tumor cells are detached from the spheroid and moved to the waste well in the plate by tilting the 3DHSP at 20°. The size of the spheroid can be used as a readout for CAR T cell cytotoxicity assay, suggesting that the 3DHSP does not require cumbersome fluorescent staining.

Development and validation of a human bronchial epithelial spheroid model to study respiratory toxicity in vitro.

The use of laboratory animals in research has been extensively criticized. While most of the critique has been centered around the ethical aspect, also the economic and scientific aspects have been frequently mentioned as points of concern. As a result, the use of alternative methods has gradually become more enticing. The most used alternatives to laboratory animals are the 2D monolayer cell cultures. However, the limited translatability of these monolayer cell cultures to in vivo has led to the development of 3D cell cultures that are believed to better capture the in vivo physiology and pathology. Here we report on the development of a physiologically more relevant 3D cell model (spheroids) comprised of human bronchial epithelial (16HBE14o-) cells, for use in respiratory toxicity research. Culturing 16HBE14o-cells as hanging-drops led to the formation of stable spheroids which showed an increased expression of CLDN1 when compared to 2D monolayer cultured cells. In addition, cell-cycle analysis revealed an increased sub-G0 population and signs of G0/G1 arrest in spheroids. Afterwards, standard operating procedures (SOPs) were established, and existing protocols optimized, for compatibility with spheroids. Spheroids were successfully used to assess cytotoxicity, genotoxicity, apoptosis/necrosis, and oxidative stress after exposure to known cytotoxic or genotoxic compounds. The development of the bronchial epithelial spheroids and the establishment of SOPs can contribute to a more reliable toxicity assessment of chemicals and may aid in bridging the gap between in vivo and in vitro experiments.

A facile method to generate cerebral organoids from human pluripotent stem cells.

Human cerebral organoids (COs) are self-organizing three-dimensional (3D) neural structures that provide a human-specific platform to study the cellular and molecular processes that underlie different neurological events. The first step of CO generation from human pluripotent stem cells (hPSCs) is neural induction, which is an in vitro simulation of neural ectoderm development. Several signaling pathways cooperate during neural ectoderm development and in vitro differentiation of hPSCs toward neural cell lineages is also affected by them. In this study, we considered some of the known sources of these variable signaling cues arising from cell culture media components and sought to modulate their effects by applying a comprehensive combination of small molecules and growth factors for CO generation. Histological analysis demonstrated that these COs recapitulate the neural progenitor zone and early cortical layer organization, containing different types of neuronal and glial cells which was in accordance with single-nucleus transcriptome profiling results. Moreover, patch clamp and intracellular Ca2+ dynamic studies demonstrated that the COs behave as a functional neural network. Thus, this method serves as a facile protocol for generating hPSC-derived COs that faithfully mimic the features of their in vivo counterparts in the developing human brain. See also Figure 1(Fig. 1).

Live Imaging of 3D Hanging Drop Arrays through Manipulation of Light-Responsive Pyroelectric Slippery Surface and Chip Adhesion.

Three-dimensional (3D) hanging drop cell culture is widely used in organoid culture because of its lack of selection pressure and rapid cell aggregation. However, current hanging drop technology has limitations, such as a dependence on complex microfluidic transport channels or specific capillary force templates for drop formation, which leads to unchangeable drop features. These methods also hinder live imaging because of space and complexity constraints. Here, we have developed a hanging drop construction method and created a flexible 3D hanging drop construction platform composed of a manipulation module and an adhesion module. Their harmonious operation allows for the easy construction of hanging drops of varying sizes, types, and patterns. Our platform produces a cell hanging drop chip with small sizes and clear fields of view, thereby making it compatible with live imaging. This platform has great potential for personalized medicine, cancer and drug discovery, tissue engineering, and stem cell research.

Technical report: liquid overlay technique allows the generation of homogeneous osteosarcoma, glioblastoma, lung and prostate adenocarcinoma spheroids that can be used for drug cytotoxicity measurements.

Introduction: The mechanisms involved in cancer initiation, progression, drug resistance, and disease recurrence are traditionally investigated through in vitro adherent monolayer (2D) cell models. However, solid malignant tumor growth is characterized by progression in three dimensions (3D), and an increasing amount of evidence suggests that 3D culture models, such as spheroids, are suitable for mimicking cancer development. The aim of this report was to reaffirm the relevance of simpler 3D culture methods to produce highly reproducible spheroids, especially in the context of drug cytotoxicity measurements. Methods: Human A549 lung adenocarcinoma, LnCaP prostate adenocarcinoma, MNNG/HOS osteosarcoma and U251 glioblastoma cell lines were grown into spheroids for 20 days using either Liquid Overlay Technique (LOT) or Hanging Drop (HD) in various culture plates. Their morphology was examined by microscopy. Sensitivity to doxorubicin was compared between MNNG/HOS cells grown in 2D and 3D. Results: For all cell lines studied, the morphology of spheroids generated in round-bottom multiwell plates was more repeatable than that of those generated in flat-bottom multiwell plates. HD had no significant advantage over LOT when the spheroids were cultured in round-bottom plates. Finally, the IC50 of doxorubicin on MNNG/HOS cultured in 3D was 18.8 times higher than in 2D cultures (3D IC50 = 15.07 ± 0.3 µM; 2D IC50 = 0.8 ± 0.4 µM; *p < 0.05). Discussion: In conclusion, we propose that the LOT method, despite and because of its simplicity, is a relevant 3D model for drug response measurements that could be scaled up for high throughput screening.

Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

Initial Characterization of 3D Culture of Yolk Sac Tissue.

The role of the yolk sac (YS) in miscarriage is not yet clear, largely due to ethical reasons that make in vivo studies difficult to conduct. However, 3D cultures could provide a solution to this problem by enabling cells to be arranged in a way that more closely mimics the structure of the YS as it exists in vivo. In this study, three domestic species (porcine, canine, and bovine) were chosen as models to standardize 3D culture techniques for the YS. Two techniques of 3D culture were chosen: the Matrigel® and Hanging-Drop techniques, and the 2D culture technique was used as a standardized method. The formed structures were initially characterized using scanning electron microscopy (SEM), immunohistochemistry (IHC), and quantitative real-time PCR (RT-qPCR). In general, the 3D culture samples showed better organization of the YS cells compared to 2D cultures. The formed structures from both 3D methods assemble the mesothelial layer of YS tissue. Regarding the IHC assay, all in vitro models were able to express zinc and cholesterol transport markers, although only 3D culture techniques were able to generate structures with different markers pattern, indicating a cell differentiation process when compared to 2D cultures. Regarding mRNA expression, the 3D models had a greater gene expression pattern on the Hemoglobin subunit zeta-like (HBZ) gene related to the YS tissue, although no significant expression was found in Alpha-fetoprotein (AFP), indicating a lack of endodermal differentiation in our 3D model. With the initial technique and characterization established, the next step is to maintain the cultures and characterize the diversity of cell populations, stemness, functions, and genetic stability of each 3D in vitro model.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Polystyrene-Based Slippery Surfaces Enable the Generation and Easy Retrieval of Tumor Spheroids.

Multicellular tumor spheroids are the most well-characterized organotypic models for cancer research. Generally, scaffold-based and scaffold-free techniques are widely used for culturing spheroids. In scaffold-free techniques, the hanging drop (HD) method is a more versatile technique, but the retrieval of three-dimensional (3D) cell spheroids in the hanging drop method is usually labor-intensive. We developed oil-coated polystyrene nanofiber-based reusable slippery surfaces for the generation and easy retrieval of 3D spheroids. The developed slippery surfaces facilitated the rolling and gliding of the cell medium drops as well as holding the hydrophilic drops for more than 72 h by the virtue of surface tension as in the hanging drop method. In this study, polystyrene nanofibers were developed by the facile technique of electrospinning and the morphological evaluation was performed by scanning electron microscopy (SEM) and cryo-FESEM. We modeled the retrieval process of 3D spheroids with the ingredients of 3D spheroid generation, such as water, cell culture media, collagen, and hyaluronic acid solution, demonstrating the faster and easy retrieval of 3D spheroids within a few seconds. We created MCF-7 spheroids as a proof of concept with a developed slippery surface. 3D spheroids were characterized for their size, homogeneity, reactive oxygen species, proliferative marker (Ki-67), and hypoxic inducing factor 1ά (HIF-1ά). These 3D tumor spheroids were further tested for evaluating the cellular toxicity of the doxorubicin drug. Hence, the proposed slippery surfaces demonstrated the potential alternative of culturing 3D tumor spheroids with an easy retrieval process with intact 3D spheroids.

Assessment of Antitumor and Antiproliferative Efficacy and Detection of Protein-Protein Interactions in Cancer Cells from 3D Tumor Spheroids.

When compared to two-dimensional (2D) cell cultures, 3D spheroids have been considered suitable in vitro models for drug discovery research and other studies of drug activity. Based on different 3D cell culture procedures, we describe procedures we have used to obtain 3D tumor spheroids by both the hanging-drop and ultra-low-attachment plate methods and to analyze the antiproliferative and antitumor efficacy of different chemotherapeutic agents, including a peptidomimetic. We have applied this method to breast and lung cancer cell lines such as BT-474, MCF-7, A549, and Calu-3. We also describe a proximity ligation assay of the cells from the spheroid model to detect protein-protein interactions of EGFR and HER2. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of 3D spheroids using the hanging-drop method Basic Protocol 2: Growth of spheroids using ultra-low-attachment plates Support Protocol 1: Cell viability assay of tumor spheroids Support Protocol 2: Antiproliferative and antitumor study in 3D tumor spheroids Support Protocol 3: Proximity ligation assay on cells derived from 3D spheroids.

Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

In vitroreconstitution of the hormone-responsive testicular organoids from murine primary testicular cells.

Increasing rates of male infertility require more experimental models to understand the mechanisms underlying male infertility.In vitroorganoids hold unprecedented promise for this purpose; however, the development of organoids with tissue architecture similar to that of the testisin vivoremains a challenge. Here, we generated testicular organoids derived from testicular cells by combining a hanging drop culture and a rotation culture system. Our results indicated that testicular cells could self-assemble into spheroid organoids with tubule-like structures in hanging drop culture. The organoids can subsequently be cultured and maintained in a rotation culture system. These established organoids have gene expression profiles similar to those of adult testis tissue, produce testosterone with preserved gonadotropin responsiveness, and exhibit sensitivity to reproductive toxicants. More importantly, each testicular organoid can be generated from only 2000 cells, and they maintain their proliferative ability after freezing and thawing. These features make it possible to obtain fresh primary testis cells from testicular biopsies taken from patients or endangered wild species, and to build individual-specific biobanks. These findings will help enable the exploration of self-organization process of testicular cells and provide an experimental model for reproductive biology research, pharmacotoxicology testing, and regenerative medicine.

3D Printed Solutions for Spheroid Engineering and Cancer Research.

In multicellular organisms, cells are organized in a 3-dimensional framework and this is essential for organogenesis and tissue morphogenesis. Systems to recapitulate 3D cell growth are therefore vital for understanding development and cancer biology. Cells organized in 3D environments can evolve certain phenotypic traits valuable to physiologically relevant models that cannot be accessed in 2D culture. Cellular spheroids constitute an important aspect of in vitro tumor biology and they are usually prepared using the hanging drop method. Here a 3D printed approach is demonstrated to fabricate bespoke hanging drop devices for the culture of tumor cells. The design attributes of the hanging drop device take into account the need for high-throughput, high efficacy in spheroid formation, and automation. Specifically, in this study, custom-fit, modularized hanging drop devices comprising of inserts (Q-serts) were designed and fabricated using fused filament deposition (FFD). The utility of the Q-serts in the engineering of unicellular and multicellular spheroids-synthetic tumor microenvironment mimics (STEMs)-was established using human (cancer) cells. The culture of spheroids was automated using a pipetting robot and bioprinted using a custom bioink based on carboxylated agarose to simulate a tumor microenvironment (TME). The spheroids were characterized using light microscopy and histology. They showed good morphological and structural integrity and had high viability throughout the entire workflow. The systems and workflow presented here represent a user-focused 3D printing-driven spheroid culture platform which can be reliably reproduced in any research environment and scaled to- and on-demand. The standardization of spheroid preparation, handling, and culture should eliminate user-dependent variables, and have a positive impact on translational research to enable direct comparison of scientific findings.