Hanging Drop Culture Research Articles

On the design and fabrication of nanoliter-volume hanging drop networks

Hanging drop cultures provide a favorable environment for the gentle, gel-free formation of highly uniform three-dimensional cell cultures often used in drug screening applications. Initial cell numbers can be limited, as with primary cells provided by minimally invasive biopsies. Therefore, it can be beneficial to divide cells into miniaturized arrays of hanging drops to supply a larger number of samples. Here, we present a framework for the miniaturization of hanging drop networks to nanoliter volumes. The principles of a single hanging drop are described and used to construct the fundamental equations for a microfluidic system composed of multiple connected drops. Constitutive equations for the hanging drop as a nonlinear capacitive element are derived for application in the electronic-hydraulic analogy, forming the basis for more complex, time-dependent numerical modeling of hanging drop networks. This is supplemented by traditional computational fluid dynamics simulation to provide further information about flow conditions within the wells. A fabrication protocol is presented and demonstrated for creating transparent, microscale arrays of pinned hanging drops. A custom interface, pressure-based fluidic system, and environmental chamber have been developed to support the device. Finally, fluid flow on the chip is demonstrated to align with expected behavior based on the principles derived for hanging drop networks. Challenges with the system and potential areas for improvement are discussed. This paper expands on the limited body of hanging drop network literature and provides a framework for designing, fabricating, and operating these systems at the microscale.

Therapeutic effect of three-dimensional hanging drop cultured human umbilical cord mesenchymal stem cells on osteoarthritis in rabbits

BackgroundMesenchymal stem cells (MSCs) have shown a positive effect on Osteoarthritis (OA), but the efficacy is still not significant in clinical. Conventional two-dimensional (2D) monolayer culture method is prone to cause MSCs undergoing replication senescence, which may affect the functions of MSCs. Three-dimensional (3D) culture strategy can sustain cell proliferative capacity and multi-differentiation potential. This study aimed to investigate the therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) cultured by 3D hanging drop method on OA.MethodshUC-MSCs were isolated from umbilical cord and cultured by 3D hanging drop method for 48 h. Scanning electron microscopy (SEM) was used to observe gross morphology 2D and 3D hUC-MSCs. Transcriptome comparison of gene expression differences between 2D and 3D hUC-MSCs. GO enrichment analysis, KEGG pathway enrichment analysis and GSEA enrichment analysis were used to analyze the impact of 3D hanging drop culture on the biological functions of hUC-MSCs. Female New Zealand rabbits (n = 12) were divided into 4 groups: Normal group, Model group, 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group. After 8 weeks, the gross and histological appearance of the cartilage was evaluated by safranin O-fast green staining and Mankin scoring system. The expression of type I collagen and type II collagen was detected by immunohistochemistry. The levels of IL-6, IL-7, TNFα, TGFβ1 and IL-10 in the knee joint fluid were tested by ELISA.Results3D hanging drop culture changed cell morphology but did not affect phenotype. The MSCs transcriptome profiles showed that 3D hanging drop culture method enhanced cell-cell contact, improved cell responsiveness to external stimuli and immunomodulatory function. The animal experiment results showed that hUC-MSCs could promote cartilage regeneration compared with Model group. 3D hUC-MSCs treatment group had a higher histological score and significantly increased type II collagen secretion. In addition, 3D hUC-MSCs treatment group increased the expression of anti-inflammatory factors TGFβ1 and IL-10.ConclusionThe above experimental results illustrated that 3D hanging drop culture method could enhance the therapeutic effect of hUC-MSCs, and showed a good clinical application prospect in the treatment of OA.

Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various In Vitro Disease Models: A Narrow Review.

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases.

Reversible chemoresistance of pancreatic cancer grown as spheroids

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

A Novel Ex Vivo Tumor Spheroid-Tissue Model for Investigating Microvascular Remodeling and Lymphatic Blood Vessel Plasticity.

Biomimetic tumor microenvironment models bridge the gap between in vitro and in vivo systems and serve as a useful way to address the modeling challenge of how to recreate the cell and system complexity associated with real tissues. Our laboratory has developed an ex vivo rat mesentery culturemodel, which allows for simultaneous investigation of blood and lymphatic microvascular network remodeling in an intact tissue environment. Given that angiogenesis and lymphangiogenesis are key contributors to the progression of cancer, the objective of this study was to combine tissue and tumor spheroid culture methods to establish a novel ex vivo tumor spheroid-tissue model by verifying its use for evaluating the effects of cancer cell behavior on the local microvascular environment. H1299 or A549 tumor spheroids were formed via hanging drop culture and seeded onto rat mesenteric tissues harvested from adult male Wistar rats. Tissues with transplanted spheroids were cultured in serum-free media for 3 to 5days. PECAM, NG2, CD11b, and αSMA labeling identified endothelial cells, pericytes, immune cells, and smooth muscle cells, respectively. Time-lapse imaging confirmed cancer cell type specific migration. In addition to increasing PECAM positive capillary sprouting and LYVE-1 positive endothelial cell extensions indicative of lymphangiogenesis, tumor spheroid presence induced the formation of lymphatic/blood vessel connections and the formation of hybrid, mosaic vessels that were characterized by discontinuous LYVE-1 labeling. The results support the application of a novel tumor spheroid microenvironment model for investigating cancer cell-microvascular interactions.

Abstract 2772: Mechanisms linking STK11 loss with metastatic potential in KRAS-mutated lung adenocarcinoma

Abstract Metastasis is one of the major determinants of worsened patient prognosis and occurs in roughly 50% of lung adenocarcinoma (LUAD) patients at the time of diagnosis. Late-stage diagnosis of LUAD often results in the accumulation of a wide range of mutations in tumor cells, with altered KRAS being the most frequently encountered oncogenic driver. Co-mutation of the tumor suppressor STK11 is also common and occurs in 10-15% of KRAS-driven LUAD. Retrospective patient studies have demonstrated that concurrent oncogenic KRAS and STK11 loss-of-function mutations are associated with metastatic disease, poorer patient survival, and inferior therapy response. The purpose of this study was to identify molecular mechanisms by which STK11 loss results in a greater metastatic potential in KRAS-driven LUAD in hopes of exploiting that knowledge to improve patient outcomes. For our studies, we utilized the KRAS-mutated, STK11-intact human LUAD cell line NCI-H2009. Both parental and ∆STK11 (generated by CRISPR/Cas9) H2009 cells were seeded in Transwell® inserts coated with fibronectin. As expected, H2009 ∆STK11 cells displayed a 1.6-fold increase in trans-well migration compared to the parental line (SEM 14393 ± 877.3 cells, n=6; p<0.0001). To assess the invasive properties of parental and ∆STK11 cells in a three-dimensional environment, spheroids generated by hanging drop culture were embedded in Matrigel and imaged by light microscopy at zero and 24-hours post-embedding. Analysis demonstrates a 2.4-fold increase in invasion in ∆STK11 cells compared to parental cells (SEM 0.03 ± 0.01 mm2, n=19-21; p<0.0001). Three-dimensional LUAD spheroids have been reported to more closely recapitulate in-vivo tumor transcriptomes compared to two-dimensional monolayer cultures. qRT-PCR analyses of our spheroids demonstrate that loss of STK11 promotes a significant increase in the expression of lung cancer stem cell markers CXCR4, NANOG, CD44, and OCT4 (n=3-6; p<0.002). These markers are known to support stem cell maintenance, chemoresistance, as well as metastatic potential, suggesting a mechanistic link between STK11 and their transcriptional regulation. To assess the impact of STK11 loss on early and late-stage metastasis in-vivo, we will use an embryonic zebrafish xenograft model. Zebrafish represent a tractable metastasis model due to the number of conserved human homologs - most notable being the CXCR4/CXCL12 axis which models cross-communication between zebrafish endothelium and cognate human tumor ligands. Current and future studies focus on investigating the role of STK11 in regulating stem cell markers; thereby identifying potentially novel therapeutic targets for STK11 and KRAS co-mutated LUAD patients. Citation Format: Cole M. Royer, Lauren K. Bialek, Hailey M. Sarausky, Shannon M. Prior, Gopika Nandagopal, Paula B. Deming, David J. Seward, Melissa N. Scheiber. Mechanisms linking STK11 loss with metastatic potential in KRAS-mutated lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2772.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.

Trajectory of hiPSCs derived neural progenitor cells differentiation into dermal papilla-like cells and their characteristics

Dermal papilla cells (DPCs) play roles in key functions of the epidermis such as hair generation. The use of human induced pluripotent cells (hiPSCs) makes it possible to obtain DP-like cells and study the molecular mechanisms of DPC development during embryogenesis. In this work, we studied the phenotypic trajectory of hiPSCs during their differentiation into DP-like cells and evaluated the epithelial-mesenchymal interaction potential of the resulting cell line. Specifically, we differentiated hiPSCs into neural progenitor cells (NPCs) and subsequently into DP-like cells. Analysis of bulk RNA-seq data during this process enabled us to observe gene expression dynamics during five stages of dermal differentiation. Furthermore, functional assays (organoids in both collagen gels and hanging drop cultures and tubulogenesis assays) revealed that the dermal cell lines we generated could interact with epidermal cells.

Thermal inkjet bioprinting drastically alters cell phenotype

Since the first description of inkjet bioprinting of cells in 2003, quantifying the input and measuring the output of the printers has been the hallmark of the field of bioprinting, as it is virtually impossible to characterize cells that are inside the printing orifices or extrusion needles. We will describe here some recent discoveries of cell behavior due to inkjet bioprinting. Primary and immortalized adult dermal fibroblasts were expanded for 2–3 passages upon receiving. The cells were harvested, resuspended in PBS, and bioprinted into a 96-well plate with pluriSTEM media. Cells were then transferred either into precoated 96-well plates or 20 µl drops were pipetted for hanging drop culture. IPC differentiation protocols were applied and the induction was begun approximately 45 min after printing. When differentiating aggregates, the initiation happened 45 min after the aggregates were transferred into the 96 wells. Standard immunostaining and RNA sequencing (RNA-Seq) were used to analyze the cell phenotypes. Preliminary results indicate that all cells expressed the three pluripotency markers oct-4, nanog, and sox-2. After applying a cardiomyocyte differentiation protocol, the cells stained positively for troponin-3. The cells also elongated and became more cardiomyocyte-like in their morphology. We analyzed bulk RNA seq data and our preliminary results show upregulation of some genes that have been implicated as stem cell markers: EPCAM, LEFTY1, ZFP42, and TEX19. In addition, differential expression of genes associated with pluripotency-relevant pathways shows some pathways are off like the MAPK/p38, MAPK/JNK1-3 which is expected for a pluripotent state. We also have data supporting the activation of the hippo pathway with transcriptional co-activator with PDZ binding motif (TAZ) highly upregulated and yes-associated protein staining the cell body. In addition, GSK3B is off and TGFB1, LIF/PIK3, and AKT1 are on as expected for pluripotency. Examining the gene network of upregulated genes, one can clearly distinguish the pivotal role of FOS, FOXO1, and PIK3 all related to pluripotency. Bioprinted fibroblasts will at least temporarily adopt a more primitive or dedifferentiated state, reminiscent of pluripotency. While immunochemistry shows the classic transcription factors required for pluripotency, gene expression shows a more nuanced picture of the transformations that occur upon printing. Understanding these transformations, even if temporary will be crucial when trying to build tissues using bioprinting technologies.

Three-dimensional culture of rat ovarian granulosa cells shows increased SPP1 and FGF7 expression through the PI3K/Akt pathway.

Ovarian granulosa cells (OGCs)play an essential role in the regulation of follicular growth and development. However, previous studies of OGCs have concentrated on traditional 2D cultures. In the present study, we used the hanging drop culture method to culture rat OGCs (rOGCs) and assessed the effects of 3D conditions on their proliferation and gene expression profiles. Compared with those grown in 2D conditions, rOGCs grown in 3D cultures showed a significantly different spatial cell distribution and cell alignment under electron microscopy. In particular, rOGCs in 3D cultures showed abundant rough and microvilli-like structures on their cell surface. Here, we showed that these cells grew slowly following 3D culture; the G0/G1-phase increasedand the S- and G2/M-phases decreased. Using whole-transcriptome sequencing analysis, 501 genes were shown to have been significantly upregulated and 502 were shown to have been downregulated. Differentially expressed genes were most enriched in pathways involved in focal adhesion, MAPK, and PI3K/Akt signaling according to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Western blotting revealed that SPP1 and FGF7 in the PI3K/Akt pathway were significantly upregulated following 3D culture. These findings improve our understanding of OGCs in real 3D environments in vivo and provide possible avenues for future research on OGCs.

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.

Sorting and Manipulation of Human PGC-LC Using PDPN and Hanging Drop Cultures.

The generation of oocytes from induced pluripotent stem cells (iPSCs) was proven efficient with mouse cells. However, no human iPSCs have yet been reported to generate cells able to complete oogenesis. Additionally, efficient sorting of human Primordial Germ Cell-like Cells (hPGC-LCs) without genomic integration of fluorescent reporter for their downstream manipulation is still lacking. Here, we aimed to develop a model that allows human germ cell differentiation in vitro in order to study the developing human germline. The hPGC-LCs specified from two iPS cell lines were sorted and manipulated using the PDPN surface marker without genetic modification. hPGC-LCs obtained remain arrested at early stages of maturation and no further differentiation nor meiotic onset occurred when these were cultured with human or mouse fetal ovarian somatic cells. However, when cultured independently of somatic ovarian cells, using BMP4 and the hanging drop-transferred EBs system, early hPGC-LCs further differentiate efficiently and express late PGC (DDX4) and meiotic gene markers, although no SYCP3 protein was detected. Altogether, we characterized a tool to sort hPGC-LCs and an efficient in vitro differentiation system to obtain pre-meiotic germ cell-like cells without using a gonadal niche.

Prolonged Maintenance of Ex Vivo Functional Vk*MYC Multiple Myeloma Cells Allows for in Vitro Manipulation and Subsequent In Vivo Analysis

Human multiple myeloma (MM) cell lines are easy to culture but only partially reflect the heterogeneity of primary MM samples as most cell lines are derived from primary human plasma-cell leukemia samples and have often lost their dependence on the marrow microenvironment. In contrast, maintaining primary human MM samples ex vivo and in vivo is very challenging. To bridge this gap, the immunocompetent Vk*MYC transgenic MM model was developed. It has high biological fidelity to the human disease characteristics and features easy in vivo engraftment and serial transplantation. However, Vk*MYC cells are considered notoriously difficult to grow and manipulate in vitro, limiting their usage for functional studies. Aim To overcome these limitations, we aimed to develop a robust culture system which promotes survival and function of primary Vk*MYC MM cells in culture as a research tool for in vitro manipulation and subsequent in vivo transplantation. Method Splenocytes from tVk*MYC (Vk12598 clone) engrafted mice were routinely maintained ex vivo and conditioned media collected for M-spike quantification. Analysis of cultures by flow cytometry allowed for Vk*MYC population quantification over time. Our novel plating method includes the following components to support Vk*MYC cell survival: 3D plating as hanging drops, low (6.5%) oxygen culture, high density cultures and a cytokine cocktail. Results The culture method described here routinely results in maintenance or slight enrichment of Vk*MYC (CD138+B220-) splenocyte fraction. Monoclonal immunoglobulin (M spike) accumulation in conditioned culture media was assessed after 7 days culture as a clinically relevant, functional output marker. Culture media displayed the same immunoglobulin isotype as parent Vk*MYC (Vk12598 clone). Figure 1 shows previously frozen donor Vk*MYC splenocytes (day 0) assessed by flow cytometry and either injected directly, or maintained 7 days ex vivo, prior to IV injection into naive C57BL/6 recipient mice. Culture-recipient mice developed Vk*MYC MM disease with the same serum M-spike isotype band, similar engraftment ratio, similar time to evidence of biochemical disease and similar overall survival time as mice transplanted with freshly defrosted Vk*MYC splenocytes. To exemplify in vitro manipulation, Vk*MYC splenocytes were cultured as described and treated with Cy3-labelled liposomal nanoparticles (LNP), a non-toxic way to deliver treatment loads e.g. siRNAs, then maintained in culture for up to 7 days after treatment. Flow analysis showed >95% of viable CD138+ cells were Cy3 positive as a marker of LNP uptake. In addition, Vk*MYC splenocytes were transduced with GFP-encoding lentivirus, then maintained in culture for up to 7 days. Flow analysis showed viable, CD138+B220- cells expressing GFP as a marker of lentiviral transduction, highlighting the potential of this culture method to allow manipulation of Vk*MYC cells in vitro. Discussion This innovative culture method allows for survival and enrichment, but not expansion of Vk*MYC splenocytes in culture. Our seven-day culture protocol allows in vitro manipulation with liposomal nanoparticles as well as viral vectors and subsequent in vivo investigations. Figure 1. Experimental setup. A) (Day 0) donor tVk*MYC spleen was defrosted and assessed for MM burden by flow (CD138+ B220-). Splenocytes were maintained for 7 days ex vivo in 3D hanging drop cultures. At day 7, conditioned media was assessed by modified SPEP method for evidence of biochemical disease, and cultured cells assessed by flow or injected into naive C57BL/6 recipient mice. B) After 4 weeks, evidence of biochemical disease (M-spike) was confirmed by SPEP and mouse spleen Vk*MYC burden assessed by flow cytometry. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In vitro reconstitution 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 vitro organoids hold unprecedented promise for this purpose; however, the development of organoids with tissue architecture similar to that of the testis in vivo remains 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.

Extended longevity geometrically-inverted proximal tubule organoids

This study reports the cellular self-organization of primary human renal proximal tubule epithelial cells (RPTECs) around a minimal Matrigel scaffold to produce basal-in and apical-out proximal tubule organoids (tubuloids). These tubuloids are produced and maintained in hanging drop cultures for 90+ days, the longest such culture of any kind reported to date. The tubuloids upregulate maturity markers, such as aquaporin-1 (AQP1) and megalin (LRP2), and exhibit less mesenchymal and proliferation markers, such as vimentin and Ki67, compared to 2D cultures. They also experience changes over time as revealed by a comparison of gene expression patterns of cells in 2D culture and in day 31 and day 67 tubuloids. Gene expression analysis and immunohistochemistry reveal an increase in the expression of megalin, an endocytic receptor that can directly bind and uptake protein or potentially assist protein uptake. The tubuloids, including day 90 tubuloids, uptake fluorescent albumin and reveal punctate fluorescent patterns, suggesting functional endocytic uptake through these receptors. Furthermore, the tubuloids release kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, when exposed to albumin in both dose- and time-dependent manners. While this study focuses on potential applications for modeling proteinuric kidney disease, the tubuloids may have broad utility for studies where apical proximal tubule cell access is required.

Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels

Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell–cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testing in vitro (organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapies in vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells in poly-N-isopropylacrylamid (PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes and bone marrow-derived mesenchymal stem cells were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32 °C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 h, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

Novel Brain-Stiffness-Mimicking Matrix Gel Enables Comprehensive Invasion Analysis of 3D Cultured GBM Cells.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults, which is fast growing and tends to invade surrounding normal brain tissues. Uncovering the molecular and cellular mechanisms of GBM high invasion potential is of great importance for the treatment and prognostic prediction. However, the commonly used two-dimensional (2D) cell culture and analysis system suffers from lack of the heterogeneity and in vivo property of brain tissues. Here, we established a three-dimensional (3D) cell culture-based analysis system that could better recapitulate the heterogeneity of GBM and mimic the in vivo conditions in the brain. The GBM cell lines, DBTRG and U251, were cultured by hanging drop culture into the GBM multicellular spheroids, which were embedded in the optimized 3D brain-stiffness-mimicking matrix gel (0.5 mg/ml Collagen Ⅰ + 3 mg/ml Matrigel+ 3.3 mg/ml Hyaluronic Acid (HA)). The biochemical composition of the optimized matrix gel is similar to that of the brain microenvironment, and the elastic modulus is close to that of the brain tissue. The dynamics of the GBM spheroids was examined using high-content imaging for 60 h, and four metrics including invasion distance, invasion area, single-cell invasion velocity, and directionality were employed to quantify the invasion capacity. The result showed that DBTRG cells possess higher invasion capacity than U251 cells, which was consistent with the results of the classic transwell test. Transcriptome analysis of both cell lines was performed to explore the underlying molecular mechanisms. Our novel brain-stiffness-mimicking matrix gel enables comprehensive invasion analysis of the 3D cultured GBM cells and provides a model basis for in-depth exploration of the mechanisms regulating GBM invasion including the interaction between GBM cells and brain stroma.

Incorporation of Tumor Spheroids into an Ex‐vivo Tissue Culture Model for Investigating Cancer Cell–Microvascular Interactions

A challenge in cancer research is the lack of biomimetic models that incorporate cancer cells, blood vessels, lymphatic vessels, and immune cells. This challenge highlights the gap between an in vivo system and common in vitro assays, which do not recapitulate the tumor microenvironment. To bridge this gap, our lab has introduced the rat mesentery culture model, which provides a complex and physiologically relevant culture system that enables the real‐time tracking of multiple cell types during angiogenesis and lymphangiogenesis. The objective of this study was to demonstrate the feasibility of culturing transplanted tumor spheroids on rat mesenteric tissues for investigating cancer cell‐angiogenesis interactions. Two lung carcinoma cell lines, A549 and H1299, were labeled with DiI and suspended in a hanging drop culture system consisting of MEM + 10% FBS for 4 days to form fluorescent spheroids. These spheroids were then seeded onto excised rat mesentery tissues (approximately 20 spheroids per tissue). Tissues with transplanted spheroids were cultured for 3 days. To identify blood vessels during various culture timepoints, tissues were labeled with FITC‐conjugated lectin and imaged on Day 2 and Day 3. On Day 3, tissues were labeled for PECAM, NG2, and CD11b to identify endothelial cells, pericytes, and macrophages respectively. Labeling a subset of tissues for Calcien AM enabled the detection of live cells. Observations confirmed that tumor spheroids remained viable and intact during culture. The apparent increase in individual cells along the periphery of the spheroids suggests radial cell migration into the tissue. Spheroids were observed in the same regions of angiogenic microvascular networks characterized by increased sprouting and vessel density. Angiogenesis was also confirmed by comparing time‐lapse lectin labeling of vessels. For some spheroids, directed sprouting towards spheroids was also observed. The presence of lymphatic vessels (identified by PECAM labeling morphology), NG2+ pericytes, and CD11b+ macrophages confirmed a multi‐cellular environment. These results support the feasibility of co‐culturing tumor spheroids and rat mesenteric tissues as well as a new approach for exploring how cancer cells interact with a surrounding microvasculature and their local environment.

Abstract P5-02-02: Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer organoids

Abstract Background Patient-derived cancer organoids, which reliably conserve original features of tumors, are emerging as an excellent model for predicting therapy response and drug screening. Developing optimized 3D high-throughput drug screening platform to establish patient-derived cancer organoids and simultaneously perform drug screening is essential for personalized medicine. Methods We established normal breast organoids (n=4) and breast cancer organoids (n=10) from 20 fresh surgical specimen (normal 7, tumor 13 cases). A number (500-2000) of normal and cancer cells were automatically dispensed with the ASFA™ Spotter ST and organoids were generated by hydrogel hanging-drop culture on Cellvitro™ Pillar platform (Medical & Bio Decision, South Korea). Organoids were subjected to drug screening for 17 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 5 days. Drug cytotoxic effect was assessed by calcein AM staining. Acquisition and analysis of high-content 3D organoid images were peformed using ASFA™ SCANNER (Medical & Bio Decision, South Korea). The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. We analyzed a drug response index (DRI) using a prediction alogorithm to evalute drug sensitivity (DRI<-0.5) and resistance (DRI>0.5). Results We summarized the DRI value of patient-derived breast organoids of 7 drugs (Table 1). 6 tumor organoids (2T, 6T, 8T, 10T, 13T, 15T) showed high sensitivity to Docetaxel, Doxorubicin, Paclitaxel, and Gemcitabin while 4 tumor organoids (1T, 9T, 14T, 16T) were less sensitive and resistant. 2 tumor organoids (10T, 16T) were sensitive to Tamoxifen and 2 tumor organoids (6T, 8T) show high sensitivity to palbociclib and erlotinib. Normal organoids show less sensitivity and resistance to chemotherapeutic drugs. Drug response index >0.5 : resistancy top 30%, Drug response index <-0.5 : sensitivity top 30%. Conclusions Herein, we developed the hydrogel hanging-drop culture on Cellvitro™ Pillar platform for easily and rapidly high-throughput drug screening in patient-derived organoids using a small number of cells by testing clinically actionable drugs at different concentrations. There were different drug response indeces for each individual organoids to chemoreagents and targeted drugs. We anticipate that 3D high-throughput drug screenings platform based on patient-derived organoids can provide the information to predict drug response and allow for finding more appropriate therapy for individual patients. Table 1.DRI values of patient-derived breast organoids of 7 drugs.Patient No.Tumor/NormalDocetaxelPaclitaxelDoxorubicinTamoxifenGemcitabinePalbociclibErlotinib1TTumor1.051.261.080.060.590.26-2TTumor-0.64-0.72-0.60-0.08-0.79--6TTumor-0.98-0.99-2.330.00-1.44-2.31-1.608TTumor-0.32-0.50-0.250.64-1.07-1.47-0.669TTumor1.050.980.610.870.430.610.4410TTumor-0.84-0.77-0.78-1.680.790.610.0913TTumor-0.87-0.430.560.70-0.510.611.1714TTumor0.861.261.180.481.81--15TTumor-1.59-1.45-1.41--0.71-0.13-16TTumor1.051.261.18-1.70-1.22-2NNormal1.051.260.650.741.810.610.7810NNormal1.05-0.910.810.870.880.610.084NNormal-0.31-0.25-0.530.25-0.190.61-1.575NNormal1.051.130.380.75-0.630.610.10 Citation Format: Jungeun Kim, Hoe Suk Kim, Ga Yeon Kim, Kyung hyeun Park, Seung yeon Ryu, Sangeun Lee, Dong Woo Lee, Bosung Ku, Han-Byoel Lee, Wonshik Han. Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-02.

High-throughput production of liver parenchymal microtissues and enrichment of organ-specific functions in gelatin methacrylamide microenvironment.

Liver parenchymal microtissues (LPMTs) are three-dimensional (3D) aggregates of hepatocytes that recapitulate in vivo-likecellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural-functional integrity over the long term. Here, we present a high-throughput methodology to produce LPMTsin a novel, economic, and reusable Hanging-drop Culture Chamber (HdCC). A drop-on-demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200-300 μm. Tissue size, cell viability, cellular arrangement and polarity, and insulin-mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate-like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver-specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ-specific functions of LPMTs in vitro.