Hanging Drop Plates Research Articles

Three-Dimensional (3D) in vitro cell culture protocols to enhance glioblastoma research.

Three-dimensional (3D) cell culture models can help bridge the gap between in vitro cell cultures and in vivo responses by more accurately simulating the natural in vivo environment, shape, tissue stiffness, stressors, gradients and cellular response while avoiding the costs and ethical concerns associated with animal models. The inclusion of the third dimension in 3D cell culture influences the spatial organization of cell surface receptors that interact with other cells and imposes physical restrictions on cells in compared to Two-dimensional (2D) cell cultures. Spheroids' distinctive cyto-architecture mimics in vivo cellular structure, gene expression, metabolism, proliferation, oxygenation, nutrition absorption, waste excretion, and drug uptake while preserving cell-extracellular matrix (ECM) connections and communication, hence influencing molecular processes and cellular phenotypes. This protocol describes the in vitro generation of tumourspheroids using the low attachment plate, hanging drop plate, and cellusponge natural scaffold based methods. The expected results from these protocols confirmed the ability of all these methods to create uniform tumourspheres.

High-Throughput Screening of Anti-cancer Drugs Using a Microfluidic Spheroid Culture Device with a Concentration Gradient Generator.

Tumor spheroid models are widely used for drug screening as in vitro models of the tumor microenvironment. There are various ways in which tumor spheroid models can be prepared, including the self-assembly of cells using low-adherent plates, micro-patterned plates, or hanging-drop plates. Recently, drug high-throughput screening (HTS) approaches have incorporated the use of these culture systems. These HTS culture systems, however, require complicated equipment, such as robot arms, detectors, and software for handling solutions and data processing. Here, we describe protocols that allow tumor spheroids to be tested with different concentrations of a drug in a parallel fashion using a microfluidic device that generates a gradient of anti-cancer drugs. This microfluidic spheroid culture device with a concentration gradient generator (μFSCD-CGG) enables the formation of 50 tumor spheroids and the testing of drugs at five different concentrations. First, we provide a protocol for the fabrication of the μFSCD-CGG, which has both a culture array in which tumor cells are injected and aggregate to form spheroids and a concentration gradient generator for drug testing. Second, we provide a protocol for tumor spheroid formation and HTS of anti-cancer drugs using the device. Finally, we provide a protocol for assessing the response of tumor spheroids at different drug concentrations. To address the needs of the pharmaceutical industry, this protocol can be used for various cell types, including stem cells, and the number of tumor spheroids and drug concentration ranges that can be tested in the μFSCD-CGG can be increased. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of a microfluidic spheroid culture device with a concentration gradient generator (μFSCD-CGG) Basic Protocol 2: Seeding cells and formation of spheroids in the μFSCD-CGG Basic Protocol 3: Drug treatment and assessment of cell viability in the μFSCD-CGG.

The Role of the Tumor Microenvironment in CSC Enrichment and Chemoresistance: 3D Co-culture Methods.

Cancer stem-like cells (CSC) are responsible for tumor progression, chemoresistance, recurrence, and poor outcomes in many cancers, making them critical research and therapeutic targets. One of the critical components potentiating CSC chemoresistance is the interactions between CSC and the surrounding cells in the tumor microenvironment. Our lab has developed several 3D co-culture models to study ovarian CSC interactions with stromal or immune cells found in ovarian tumor microenvironments. In this chapter, we use ovarian cancer as a model to describe the methodologies developed in our lab; however, these techniques are applicable to a wide range of cancers. First, we discuss our method for isolating CSC from heterogeneous tumors and for creating 3D self-assembled tumoroids in hanging drop plates, in either monoculture or co-culture with mesenchymal stem cells or monocytes/macrophages. We then discuss methods for analyzing these models with a focus on isolating cell-type-specific changes and mechanism investigation. Specifically, we describe lentiviral transduction and flow cytometry as established and robust methods to identify and separate each cell type for downstream analysis. We then describe methods to examine CSC functionality with transwell migration assays and colorimetric MTS-based proliferation assays. Finally, we demonstrate enzyme-linked immunosorbent assays (ELISA ) and quantitative polymerase chain reaction (qPCR) methods as mechanistic investigation tools to decouple paracrine and juxtacrine interactions. These methods have wide-reaching applications in cancer research from basic biological investigations, to drug discovery, and personalized drug screening for precision medicine.

MCF‐7 derived cell spheres and clusters generated during the long‐term progression of 3D artificial tumors yield distinctive colonies and monolayer populations potentially reflective of relative malignant stage.

In a continuing effort to establish bio‐physiologically relevant in‐vitro models of tumor biology, our laboratory has shown that MCF‐7 derived 3D artificial tumors undergo a process that closely resembles tumor progression. A significant feature of this process is the release of both single cells and cell clusters as well as spheroids of various sizes. These generally range for clusters of a few cells and perhaps 50um in diameter to large structures which contain hundreds of cells and exceed 300–400um in overall dimension. This progressive release of cell structures continues for extended periods of time once start and easily generates dozens of new clusters per day. Once released these clusters remain suspended in the media of the culture for up to several days, eventually settling and attaching to the plate in most cases. At that point, these spheres/clusters explant onto the plate and form new colonies of tumor cells. Eventually these will cover large portions of the plate, however the individual colonies retain their own distinct structure even when multiple such colonies meet to form large coverage monolayers. In this report we examine the behaviors of these shed cell spheres and clusters using 3D artificial tumors as sources as well as applying two additional methods to generate similar structures. Those additional methods are hanging drop plates and rocking cultures. In all cases, the cultures which result present with distinctly colonial behaviors and in many cases these colonies also form new 3D structures following a period of growth in 2D. Previously we have also shown that these features will attach and invade target artificial stromal tissues as well. Taken together, these data suggest that MCF‐7 derive 3D artificial tumors function as a workable model of in‐vitro metastasis and provides a new tool‐set to examine and better understand this critical phase of cancer biology.

Evaluation of Spheroid 3D Culture Methods to Study a Pancreatic Neuroendocrine Neoplasm Cell Line.

Pancreatic Neuroendocrine Neoplasms (pNEN) are rare tumors which treatment still represent an important clinical problem, due to the paucity of medical treatments. Due to tumor complexity, techniques as 3D cultures are important to study drug activity in a more realistic model. This study aims to compare three different 3D culture methods in order to understand which one can be considered the best option in terms of experimental easiness and reproducibility in studying the efficacy of a target drug on pNEN. The BON1 cell line was used as a pNEN model and the well-known Receptor Tyrosine Kinase inhibitor Sunitinib was used in order to better investigate the different features of each method. The investigated methods are: (1) 96-well hanging drop plates (HD plates), (2) 24-well plates with a cell-repellent surface, and (3) ultra-low attachment 96-well plates with clear round bottom (ULA plates). The evaluated parameters during the study were: cell seeding, easiness in spheroids formation, morphology, culture maintenance, medium change, spheroids monitoring, picture quality, spheroid perimeter measurement reproducibility error, possibility to perform assays into the seeding plate, overall time of the experiment. Moreover, we investigated how culture methods can influence experimental outcomes evaluating perimeter changes, cell viability and immunohistochemistry of spheroids treated with different Sunitinib concentrations. Results showed that each method has weak and strong points but, considering the easiness of spheroids maintenance and reproducibility results, ULA plates method appears to be the best approach to culture BON1 spheroids and, therefore, to study pNEN.

Modeling Tumor Development, Progression and Metastasis in 3D Artificial Tissues: Characterization of Structure and Labeling Patterns in Breast Cancer Hanging‐drop Spheroid Cultures

In this report our laboratory presents the results of a series of studies being conducted to explore the application of 3D artificial tumor tissues as in‐vitro models of breast cancer development, progression and metastasis. In this case, we have employed MCF‐7 breast ductal adenocarcinoma cells and 3D Biomatrix hanging drop plates to evaluate these tumor properties within the context of staged tumor spheroids. A consistent observation of ours in nearly a decade of modeling breast cancer using various natural 3D scaffolds and hydrogel matrices has been that solid tumor models grown in long‐term culture predictably begin to shed both single cells and cell clusters in the form of small groupings of cells as well as progressively larger spheroids. Although we have shown that at least some of these structures have properties consistent with metastasis, including attachment and invasion of target tissues, spontaneously generated versions of cancer clusters also present as pleiomorphic structures and in relatively small numbers which complicates their study. Therefore, we sought to use a system that generates identical clusters and spheres from specific numbers of suspended single cells. The hanging drop system we employ comes in a 96 well format that allows us to generate literally as many spheres as we need for each study. Furthermore, these spheres can be grown out to match any of the sizes commonly found in our tumor models thereby allowing us to study each stage for structure, cell populations and markers. In addition these spheres are also assisting us in systematically studying the process of cancer cell cluster attachment and invasion and to test our working hypothesis that this is controlled through activation of EMT‐like responses through CD44 binding of hyaluronate on the target tissue. Using direct labeling of intact spheres coupled to embedding and sectioning followed by immunolabeling, these studies are demonstrating that over a period of 144 hours our spheroid cultures progress from simple clusters of a few cells to aggregated spheres containing thousands of cells and measuring up to ~600um in diameter. By this later stage, many spheres actually begin to generate their own smaller spheres and clusters as well. In our view this reflects the concept that past 48 hours in culture these spheres transition into being actual tumor organoids themselves. This is supported by development of a complex CD24+ internal structure distinct from an outer shell of CD44+ cells. These studies are ongoing and support our view that 3D artificial tumors are effective tools to study cancer biology in‐vitro.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Minimally Manipulative Method for the Expansion of Human Bone Marrow Mesenchymal Stem Cells to Treat Osseous Defects.

Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates®. Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix; Acellular bone composite discs; Allowash®, cancellous bone cubes; PLGA (poly lactic-co-glycolic acid); and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body.

Characterization and reproducibility of HepG2 hanging drop spheroids toxicology in vitro

Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating a reproducible phenotype in HepG2 hanging drop spheroids.

Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer.

Pancreatic cancer 3D in vitro models including multicellular tumor spheroid (MCTS), single cell-derived tumor spheroid (SCTS), tissue-derived tumor spheroid, and organotypic models provided powerful platforms to mimic in vivo tumor. Recent work supports that circulating tumor cell (CTC) clusters are more efficient in metastasis seeding than single CTCs. The purpose of this study is to establish 3D culture models which can mimic single CTC, monoclonal CTC clusters, and the expansion of macrometastases. Seven pancreatic ductal adenocarcinoma cell lines were used to establish MCTS and SCTS using hanging drop and ultra-low attachment plates. Spheroid immunofluorescence staining, spheroid formation assay, immunoblotting, and literature review were performed to investigate molecular biomarkers and the morphological characteristics of pancreatic tumor spheroids. Single cells experienced different growth patterns to form SCTS, like signet ring-like cells, blastula-like structures, and solid core spheroids. However, golf ball-like hollow spheroids could also be detected, especially when DanG and Capan-1 cells were cultivated with fibroblast-conditioned medium (p<0.05). The size of golf ball-like hollow spheroids hardly grew after getting matured. Only DanG and Capan-1 could establish SCTS- and MCTS-derived hollow spheroids using hanging drop plates and ultra-low attachment plates. Other PDA cell lines could also establish tumor spheroid with hanging drop plates by adding methylated cellulose. Tumor spheroids derived from pancreatic cancer cell line DanG possessed asymmetrically distributed proliferation center, immune-checkpoint properties. ß-catenin, Ki-67, and F-actin were active surrounding the crater-like structure distributing on the inner layer of viable rim cover of the spheroids, which was relevant to well-differentiated tumor cells. It is possible to establish 3D CTC cluster models from homogenous PDA cell lines using hanging drop and ultra-low attachment plates. PDA cell line displays its own intrinsic properties or heterogeneity. The mechanism of formation of the crater-like structure as well as golf ball-like structure needs further exploration.

Abstract 4265: Automation-enabled 3D tumor chemosensitivity screening by imaging and flow cytometry analysis

Abstract Three-dimensional (3D) cancer cell cultures are being utilized for drug screening with increasing frequency since they provide a more physiologically relevant solid tumor model than two-dimensional cell cultures. Spheroids are a well characterized, in vitro tumor model system that has been utilized with multiple cancer cell lines. Growth and manipulation of these cultures in hanging drop plates (HDPs) can be challenging to manipulate and these challenges are amplified as sample throughput increases. We automated the culture and compound treatment of cancer spheroids in HDPs, as well as the preparation of these spheroids for analysis by high content imaging and flow cytometry. Automated cancer spheroid cultures showed excellent consistency in size and shape, providing higher confidence in the screening results. Creating single cell suspensions from the cancer spheroids for flow cytometry analysis was another significant challenge overcome through automation. Imaging provided information such as the location of drug-responding cells within the cancer spheroid while flow cytometry removed the potential for spheroid exclusion of staining reagents leading to false negatives. Automation enabled the use of these complementary analyses, thereby achieving a more complete understanding of compound sensitivity in solid tumors and illustrating how spheroids can be utilized to screen for novel cancer therapeutics. Citation Format: Michael Kowalski, Vipat Raksakulthai, Kristin Prasauckas, Tara Jones-Roe. Automation-enabled 3D tumor chemosensitivity screening by imaging and flow cytometry analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4265.

연골세포와 중간엽줄기세포의 3차원 Co-culture를 통한 연골화 향상

Two-dimensional cultivation is typically used for cell growth, but the method reduces the characteristics of chondrocytes and stem cells, and limits culture area. Therefore, development of three-dimensional culture method is needed to mimic in vivo environment, improve quality of cells and scale-up efficiently. Improving proliferation and chondrogenesis is available by co-culture of chondrocytes and mesenchymal stem cells (MSCs) that leads to interaction between two kinds of cells. However, the co-culture has problems that permeability of sphere diminishes as aggregate size increased and ratio of two kinds of cells composing each spheres is different. In this work, co-cultivation method using controlled sphere composed of chondrocytes and MSCs was established and enhanced chondrogenesis. Periosteum-derived progenitor cells (PDPCs) that are appropriate for cell therapy source of articular cartilage were used as MSCs. Controlled spheres were formed in the hanging-drop plates and shifted for being induced chondrogenesis in 35-mm non-adhesive culture dishes at a rotation rate of 60 rpm. After inducing chondrogenesis, gene expressions related with chondrogenesis were found to be improved and it was apparent that the utilization of controlled spheres promoted chondrogenesis. As a result, available numbers of cells per unit area were increased and chondrogenic differentiation ability was improved compared to typical two-dimensional culture. This approach shows the potential in cartilage regeneration as it can provide sufficient numbers of chondrocytes.

Abstract 4942: Activation of Phosphatase toward the Retinoblastoma protein in breast and colorectal cancer cell spheroids

Abstract Excessive phosphorylation of the Retinoblastoma protein is found in most cancer tumor types. Several cyclin dependent kinase (cdk) inhibitors are in development or in clinical trials at the present time. Our previous studies have focused on the regulation of Rb phosphorylation by the phosphatase, PP1. Specificity toward substrates is imparted onto PP1 by many different interacting proteins. In proliferating cells, PP1 is associated with a regulatory protein called PNUTS (Phosphatase Nuclear Targeting Subunit). Our previous experiments have shown that PNUTS inhibits PP1 activity toward specific Rb phosphorylation sites. Further we have demonstrated that siRNA mediated knockdown of PNUTS in several cancer cell types (colon, breast, ovarian) leads to an increase in Rb specific PP1 activity, Rb dephosphorylation on several sites, and a dramatic induction of apoptosis. Furthermore, the ability of PNUTS siRNA to induce apoptosis was shown to be dependent on Rb expression. Interestingly, further studies showed that PNUTS knockdown had no effect on non-transformed breast and colorectal cells, in which Rb is not hyperphosphorylated. Thus we have developed a method to activate the Rb phosphatase in cells by knockdown of the PP1 binding protein, PNUTS. The current study was undertaken to analyze the effect of PNUTS knockdown in cancer cells grown in three-dimensional spheroids grown by incubation of cells in hanging drop plates. The effect of PNUTS knockdown on Rb phosphorylation, cell proliferation and cell viability using this model system will be presented. Citation Format: Jacklynn Egger, Lisa Antonucci, Maria Lane, Nancy A. Krucher. Activation of Phosphatase toward the Retinoblastoma protein in breast and colorectal cancer cell spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4942. doi:10.1158/1538-7445.AM2015-4942

Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays.

Recent guidance/guidelines for industry recommend that cytochrome P450 induction can be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug- drug interactions. To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (>2-fold) by the treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture. After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8x10(4) HepaRG cells per well was lower than that for 7.2x10(4) cells. CYP1A2 induction levels at 24 h were higher than those after 48 h. In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well. To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates. Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2x10(4) cells per well. Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1'- hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates. These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.

The multi-organ chip--a microfluidic platform for long-term multi-tissue coculture.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Bottom‐Up Engineering of Well‐Defined 3D Microtissues Using Microplatforms and Biomedical Applications

During the last decades, the engineering of well-defined 3D tissues has attracted great attention because it provides in vivo mimicking environment and can be a building block for the engineering of bioartificial organs. In this Review, diverse engineering methods of 3D tissues using microscale devices are introduced. Recent progress of microtechnologies has enabled the development of microplatforms for bottom-up assembly of diverse shaped 3D tissues consisting of various cells. Micro hanging-drop plates, microfluidic chips, and arrayed microwells are the typical examples. The encapsulation of cells in hydrogel microspheres and microfibers allows the engineering of 3D microtissues with diverse shapes. Applications of 3D microtissues in biomedical fields are described, and the future direction of microplatform-based engineering of 3D micro-tissues is discussed.

Pseudoislet of Hybrid Cellular Spheroids From Commercial Cell Lines

Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 μm. Their morphology was similar to naïve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy.

Transfer, Imaging, and Analysis Plate for Facile Handling of 384 Hanging Drop 3D Tissue Spheroids

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated high-fidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type-dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.

Guardian angels in the graveyard: B1 B cells promote thymic Treg survival (P1386)

Abstract The thymus is the primary site for T cell development. Within the medulla of the thymus, negative selection of immature T cells causes a large number of cells to undergo apoptosis, appropriately deeming this thymic region a “grave yard.” In observing the thymus medulla of BXSB mice, we noted a significant increase of B cells in lupus-symptomatic males as compared to female controls or lupus- asymptomatic males. Composition analysis of these thymic B cells revealed that both B1 and B2 cells are present. Immunohistochemistry analysis suggests possible chemo-attraction for B cells in the medullar of the thymus due to the expression of CXCL13 in lupus male mice, but not control females. In vivo tracking of peritoneal B cells over a two-week time period indicated that some of the thymic B cells originated from the peritoneal cavity. To test the possible influence of B cells on thymic T cell development, we co-cultured peritoneal and spleen B cells with thymocytes in a 3D hanging drop plate. Our results suggest a “guardian angel” effect by B1 B cells, depicting an enhanced central tolerance by thymic-migration and promoting regulatory T cell survival.

Plastic pillar inserts for three-dimensional (3D) cell cultures in 96-well plates

Abstract We have developed a plastic pillar insert to facilitate miniaturized three-dimensional (3D) cell cultures in 96-well plates by forming 3D hydrogel droplets containing cells (about 1 μL) on the tip of the pillar insert. Hemispherical 3D droplets containing cells were formed simply by immersing the tip of the pillar insert into a mixture of poly- l -lysine (PLL) and BaCl 2 in a cell-encapsulation apparatus, followed by dipping it into cell suspension in alginate. Compared to traditional 3D cell culture platforms such as polymer scaffolds in 96 wells and 3D hanging drop plates, it allows us to easily change cell growth media or expose 3D cells to reagents by immersing the tip of the pillar inserts in different 96 wells filled with growth media or reagents. As a proof of concept, A549 and PC9 cell lines from human non-small cell lung cancer (NSCLC) were grown on the pillar insert and tested for cytotoxicity with Erlotinib. Both cells on the tip of the pillar insert grew over time, forming 3D structures unlike traditional 2D cell monolayer cultures and mimicking in vivo -like cellular microenvironment. The number of cells in alginate droplets was linearly proportional to the cell seeding density. The doubling time of A549 and PC9 cells were 15.9 h and 16.1 h, respectively, which were similar to those obtained from traditional 2D cell cultures. IC 50 values from A549 and PC9 cells exposed to Erlotinib for 3 days were 15.2 ± 7.0 μM and 1.7 ± 0.4 nM, respectively, indicating that PC9 cells with EGFR mutation are highly sensitive to Erlotinib. Interestingly, the IC 50 value of 3D PC9 cells grown on the pillar insert was 6 times lower than those obtained from 2D PC9 cells grown on the surface of 96-wells, whereas the IC 50 value of 3D A549 cells were 5 times higher than those from 2D A549 cells. The result may represent that A549 and PC9 cells grown on the pillar insert are better mimicking what happens in humans.