3D Platform Research Articles

Mesenchymal Stem Cell Sheet Centrifuge-Assisted Layering Augments Pro-Regenerative Cytokine Production.

A focal advantage of cell sheet technology has been as a scaffold-free three-dimensional (3D) cell delivery platform capable of sustained cell engraftment, survival, and reparative function. Recent evidence demonstrates that the intrinsic cell sheet 3D tissue-like microenvironment stimulates mesenchymal stem cell (MSC) paracrine factor production. In this capacity, cell sheets not only function as 3D cell delivery platforms, but also prime MSC therapeutic paracrine capacity. This study introduces a “cell sheet multilayering by centrifugation” strategy to non-invasively augment MSC paracrine factor production. Cell sheets fabricated by temperature-mediated harvest were first centrifuged as single layers using optimized conditions of rotational speed and time. Centrifugation enhanced cell physical and biochemical interactions related to intercellular communication and matrix interactions within the single cell sheet, upregulating MSC gene expression of connexin 43, integrin β1, and laminin α5. Single cell sheet centrifugation triggered MSC functional enhancement, secreting higher concentrations of pro-regenerative cytokines vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10). Subsequent cell sheet stacking, and centrifugation generated cohesive, bilayer MSC sheets within 2 h, which could not be accomplished within 24 h by conventional layering methods. Conventional layering led to H1F-1α upregulation and increased cell death, indicating a hypoxic thickness limitation to this approach. Comparing centrifuged single and bilayer cell sheets revealed that layering increased VEGF production 10-fold, attributed to intercellular interactions at the layered sheet interface. The “MSC sheet multilayering by centrifugation” strategy described herein generates a 3D MSC-delivery platform with boosted therapeutic factor production capacity.

Effect of the manufacturing parameters on the tensile and fracture properties of FDM 3D-printed PLA specimens

Fused Deposition Modeling (FDM), also known as material extrusion, is currently one of the most popular Additive Manufacturing (AM) technology on the market. In this study, two commercial FDM printers (Prusa i3 MK3 and WN400 3D Platform) were used for layer-by-layer manufacturing of polylactic acid (PLA) Dog Bone (DB) and Single Edge Notched Bend (SENB) specimens, aimed at investigating the influence of the manufacturing parameters on the tensile and fracture properties of PLA elements obtained by FDM technology. The effects of growing direction (horizontal and vertical), building orientation (0°, 45° and 90°), printer type, layer thickness (0.15 and 0.40 mm), specimen thickness (4 and 10 mm) and filament color (purple, white, black, gray, red, orange) are discussed in detail. Tensile tests were performed on DB specimens, while fracture mechanics tests on SENB specimens. Both the tensile and fracture properties of FDM 3D-printed PLA specimens have been found to be dependent on the investigated manufacturing parameters. From the microstructural analyses of the SENB fracture surfaces, it has been observed that the fracture mechanisms and crack propagation is a step-wise process. Finally, the material properties charts (Young’s modulus and mode I fracture toughness versus tensile strength) are plotted.

S-04-02 Development of 3D functional platforms to study peripheral nervous system toxicity

S-04-02 Development of 3D functional platforms to study peripheral nervous system toxicity

Researchon Visual Modeling Technology of 3D Mining Engineering Based on Deep Learning

Deep learning is a study hotspot in the domain of man-made intelligence. It is an inevitable trend to use deep learning to support the study work of man-made intelligence, and it has shown its act advantages in the domains of picture, speech and text. Interpretive approach of deep learning is an interdisciplinary study subject of man-made intelligence, machine learning(ML), cognitive psychics, logic and many other disciplines. It has vital abstract study meaning and actual apply worth in message push, medical study, finance, message security and other domains. Deep learning is a new study direction in the domain of ML. By imitating the structure of human brain, it can efficiently course complicated input data, smartly learn divers knowledge, and availably solve many kinds of complicated smart question. In recent years, with the emergence of efficient learning LRUs for deep learning, the ML community has set off an upsurge of studying the theory and apply of deep learning. The rise of 3D modeling technology has promoted the vigorous expand of computer simulation and virtual reality technology, and various 3D modeling software platforms and simulation systemics have emerged as the times require. These platforms and systemics provide powerful design tools and intellectual support. Through the simulation analysis of real scenes, people can directly start with 3D notions and ideas and make visual design schemes and evaluation systemics.

Binary dose level classification of tumour microvascular response to radiotherapy using artificial intelligence analysis of optical coherence tomography images

The dominant consequence of irradiating biological systems is cellular damage, yet microvascular damage begins to assume an increasingly important role as the radiation dose levels increase. This is currently becoming more relevant in radiation medicine with its pivot towards higher-dose-per-fraction/fewer fractions treatment paradigm (e.g., stereotactic body radiotherapy (SBRT)). We have thus developed a 3D preclinical imaging platform based on speckle-variance optical coherence tomography (svOCT) for longitudinal monitoring of tumour microvascular radiation responses in vivo. Here we present an artificial intelligence (AI) approach to analyze the resultant microvascular data. In this initial study, we show that AI can successfully classify SBRT-relevant clinical radiation dose levels at multiple timepoints (t = 2–4 weeks) following irradiation (10 Gy and 30 Gy cohorts) based on induced changes in the detected microvascular networks. Practicality of the obtained results, challenges associated with modest number of animals, their successful mitigation via augmented data approaches, and advantages of using 3D deep learning methodologies, are discussed. Extension of this encouraging initial study to longitudinal AI-based time-series analysis for treatment outcome predictions at finer dose level gradations is envisioned.

Microfluidic 3D platform to evaluate endothelial progenitor cell recruitment by bioactive materials

Most of the conventional in vitro models to test biomaterial-driven vascularization are too simplistic to recapitulate the complex interactions taking place in the actual cell microenvironment, which results in a poor prediction of the in vivo performance of the material. However, during the last decade, cell culture models based on microfluidic technology have allowed attaining unprecedented levels of tissue biomimicry. In this work, we propose a microfluidic-based 3D model to evaluate the effect of bioactive biomaterials capable of releasing signaling cues (such as ions or proteins) in the recruitment of endogenous endothelial progenitor cells, a key step in the vascularization process. The usability of the platform is demonstrated using experimentally-validated finite element models and migration and proliferation studies with rat endothelial progenitor cells (rEPCs) and bone marrow-derived rat mesenchymal stromal cells (BM-rMSCs). As a proof of concept of biomaterial evaluation, the response of rEPCs to an electrospun composite made of polylactic acid with calcium phosphates nanoparticles (PLA+CaP) was compared in a co-culture microenvironment with BM-rMSC to a regular PLA control. Our results show a significantly higher rEPCs migration and the upregulation of several pro-inflammatory and proangiogenic proteins in the case of the PLA+CaP. The effects of osteopontin (OPN) on the rEPCs migratory response were also studied using this platform, suggesting its important role in mediating their recruitment to a calcium-rich microenvironment. This new tool could be applied to screen the capacity of a variety of bioactive scaffolds to induce vascularization and accelerate the preclinical testing of biomaterials. Statement of significanceFor many years researchers have used neovascularization models to evaluate bioactive biomaterials both in vitro, with low predictive results due to their poor biomimicry and minimal control over cell cues such as spatiotemporal biomolecule signaling, and in vivo models, presenting drawbacks such as being highly costly, time-consuming, poor human extrapolation, and ethically controversial. We describe a compact microphysiological platform designed for the evaluation of proangiogenesis in biomaterials through the quantification of the level of sprouting in a mimicked endothelium able to react to gradients of biomaterial-released signals in a fibrin-based extracellular matrix. This model is a useful tool to perform preclinical trustworthy studies in tissue regeneration and to better understand the different elements involved in the complex process of vascularization.

Studies on oxygen availability and the creation of natural and artificial oxygen gradients in gelatin-methacryloyl hydrogel 3D cell culture.

Three-dimensional (3D) cultivation platforms allow the creation of cell models, which more closely resemble in vivo-like cell behavior. Therefore, 3D cell culture platforms have started to replace conventional two-dimensional (2D) cultivation techniques in many fields. Besides the advantages of 3D culture, there are also some challenges: cultivation in 3D often results in an inhomogeneous microenvironment and therefore unique cultivation conditions for each cell inside the construct. As a result, the analysis and precise control over the singular cell state is limited in 3D. In this work, we address these challenges by exploring ways to monitor oxygen concentrations in gelatin methacryloyl (GelMA) 3D hydrogel culture at the cellular level using hypoxia reporter cells and deep within the construct using a non-invasive optical oxygen sensing spot. We could show that the appearance of oxygen limitations is more prominent in softer GelMA-hydrogels, which enable better cell spreading. Beyond demonstrating novel or space-resolved techniques of visualizing oxygen availability in hydrogel constructs, we also describe a method to create a stable and controlled oxygen gradient throughout the construct using a 3D printed flow-through chamber.

The BEHAV3D Platform Demonstrates Cellular Immunotherapy Modes of Action.

An organoid-based, 3D imaging-transcriptomic platform was developed to investigate immunotherapy modes of action.

Three-Dimensional Simulation Platform for Optimal Designs of the MEMS Microphone

MEMS microphone has a wide range of application prospects in electronic devices such as mobile phones, headphones, and hearing aids due to its small size, low cost, and reliable performance. Research and development of MEMS microphones involves multiple thermo-electro-mechanical couplings among various physical and electrical fields. Unfortunately, there is not an accurate three-dimensional (3D) MEMS microphone simulation platform, which can be applied for the design of chip parameters and packaging characteristics. Herein, based on commercial COMSOL software, we have established a 3D simulation platform for MEMS microphones, which is used to systematically study the influences of geometric structure and physics parameters on the sensitivity and frequency responses of the microphone and consider the influences of packaging characteristics on the performance of the microphone. The simulation results are consistent with those obtained using a lumped element method, which proves the accuracy of the simulation platform. The platform can be used to design and explore new principles or mechanisms of MEMS microphone devices.

Bioengineered Lab-Grown Meat-like Constructs through 3D Bioprinting of Antioxidative Protein Hydrolysates.

Lab-grown bovine meat analogues are emerging alternatives to animal sacrifices for cultured meat production. The most challenging aspect of the production process is the rapid proliferation of cells and establishment of the desired 3D structure for mass production. In this study, we developed a direct ink writing-based 3D-bioprinted meat culture platform composed of 6% (w/v) alginate and 4% (w/v) gelatin (Alg/Gel)-based hydrogel scaffolds supplemented with naturally derived protein hydrolysates (PHs; 10%) from highly nutritive plants (soybean, pigeon pea, and wheat), and some selected edible insects (beetles, crickets, and mealworms) on in vitro proliferation of bovine myosatellite cells (bMSCs) extracted from fresh meat samples. The developed bioink exhibited excellent shear-thinning behavior (n < 1) and mechanical stability during 3D bioprinting. Commercial proteases (Alcalase, Neutrase, and Flavourzyme) were used for protein hydrolysis. The resulting hydrolysates exhibited lower-molecular-weight bands (12-50 kDa) than those of crude isolates (55-160 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The degree of hydrolysis was higher in the presence of Alcalase for both plant (34%) and insect (62%) PHs than other enzymes. The 3D-printed hydrogel scaffolds displayed excellent bioactivity and stability after 7 days of incubation. The developed prototype structure (pepperoni meat, 20 × 20 × 5 mm) provided a highly stable, nutritious, and mechanically strong structure that supported the rapid proliferation of myoblasts in a low-serum environment during the entire culture period. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay enhanced the free radical reduction of Alcalase- and Neutrase-treated PHs. Furthermore, the bioprinted bMSCs displayed early myogenesis (desmin and Pax7) in the presence of PHs, suggesting its role in bMSC differentiation. In conclusion, we developed a 3D bioprinted and bioactive meat culture platform using Alg/Gel/PHs as a printable and edible component for the mass production of cultured meat.

Position Control of a Mobile Robot through Deep Reinforcement Learning

This article proposes the use of reinforcement learning (RL) algorithms to control the position of a simulated Kephera IV mobile robot in a virtual environment. The simulated environment uses the OpenAI Gym library in conjunction with CoppeliaSim, a 3D simulation platform, to perform the experiments and control the position of the robot. The RL agents used correspond to the deep deterministic policy gradient (DDPG) and deep Q network (DQN), and their results are compared with two control algorithms called Villela and IPC. The results obtained from the experiments in environments with and without obstacles show that DDPG and DQN manage to learn and infer the best actions in the environment, allowing us to effectively perform the position control of different target points and obtain the best results based on different metrics and indices.

Microparticle Manipulation Based on the Bulk Acoustic Wave Combined with the Liquid Crystal Backflow Effect Driving in 2D/3D Platforms

Microparticle manipulation has been widely used in clinical diagnosis, cell separation, and biochemical analysis via optics, electronics, magnetics, or acoustic wave driving. Among them, the bulk acoustic wave (BAW) driving method has been increasingly adopted because of non-contact, easy control, and precise manipulation. However, its low manipulation efficiency limits the usage of the BAW driving in high viscosity solutions. Therefore, in order to obtain larger driving force and more flexible manipulation of microparticles, both two-dimensional (2D) and three-dimensional (3D) platforms based on the BAW and liquid crystal backflow effect (LCBE) driving in liquid crystal (LC) solutions are proposed. The driving forces applied on the microparticles allow for the change of microparticle moving direction, which is also ascertained through theory analysis combined with various driving methods. Specifically, the maximum moving speed (68.78 μm/s) of the polystyrene particles is obtained by the BAW (13 Vpp) combined with LCBE (30 V) at a low frequency of 7.2 kHz in the 2D platform. Precise position manipulation in 3D is also fulfilled through a programmable logic control model using polystyrene particles as a demonstration. In addition, red blood cells mixed with LC solutions are arranged in a line or gathered in the pressure nodes of the BAW forces along with sinusoid signals generated by various transducer combinations. Therefore, it is approved that the LC solution that induces the LCBE force could increase the microparticle manipulation efficiency in both 2D and 3D platforms. The proposed method will open up new avenues in particle manipulation and benefit a variety of applications in cell separation, drug synthesis, analytical chemistry, and others.

3D printing of heterogeneous microfibers with multi-hollow structure via microfluidic spinning.

Tissues with tubular structures play important roles in the human bodies, such as mass transport, nutrition exchange, and waste filtration. However, it remains a challenge to generate micro-scaffolds with well-defined luminal structure in biomedical field. In this study, we proposed a novel method to fabricate multi-component microfibers with multi-hollow structure via microfluidic spinning, which can subsequently be integrated with 3D printing for tissue-like block assembling. To achieve this goal, we fabricated a microchip using a 3D printed template with adjustable heights. Utilizing this microchip, we successfully generated the Calcium alginate microfibers with multi-components and defined hollow structures in a controllable manner. Then this microfluidic spinning method was integrated with a 3D mobile platform to assemble the microfibers into a grid-like 3D architecture. The resulted 3D scaffolds exhibited good organization and maintained the hollow structure of the fibers. Furthermore, we successfully developed a bronchus model utilizing this strategy by loading pulmonary bronchial epithelium cells and endothelial cells into microfibers with two hollow structures. The present strategy provides a potential platform to rebuild the lumen-like tissues using microfibers.

Bioinspired CS@PAN Electrospun Fibrous Aerogels with Vertical Capsule Vessels for Self-Feedback Temperature Sensing

Unconventional sensors with characteristics of portability, safety, remote manipulation, and high spatiotemproal resolution are highly desirable to detect varied temperature under specific conditions such as local temperature fields of the microenvironment. Herein, a 3D high-performance optical thermometry platform driven by the up-conversion green fluorescence of Er3+/Yb3+ co-doped electrospun fibrous aerogels is developed. The system simply consists of polycrystalline-encapsulated (Y1–x–yErxYby)2O2S/polyacrylonitrile (YOS-EY/PAN) electrospun fibers and chitosan (CS). The flexible fibers form a hierarchically ordered bionic cellular structure through bio-based cross-linking, which avoids toxic chemical cross-linking. Additionally, the intense green emission is ascribed to 2H11/2/4S3/2 → 4I15/2 transitions, as the main indicator to determine the relative sensitivity of 1.22% K–1 at 303 K. More importantly, multipoint dispersed lanthanide (Ln) between cell walls confers stronger temperature sensing capability to aerogels. The successful strategy provides insights for the development of luminescence thermometry and opens a window for multidimensional temperature sensing.

Simulating 3D Human Postural Stabilization in Vibration and Dynamic Driving

In future automated vehicles we will often engage in non-driving tasks and will not watch the road. This will affect postural stabilization and may elicit discomfort or even motion sickness in dynamic driving. Future vehicles will accommodate this with properly designed seats and interiors, whereas comfortable vehicle motion will be achieved with smooth driving styles and well-designed (active) suspensions. To support research and development in dynamic comfort, this paper presents the validation of a multi-segment full-body human model, including visuo-vestibular and muscle spindle feedback, for postural stabilization. Dynamic driving is evaluated using a “sickening drive”, including a 0.2 Hz 4 m/s2 slalom. Vibration transmission is evaluated with compliant automotive seats, applying 3D platform motion and evaluating 3D translation and rotation of pelvis, trunk and head. The model matches human motion in dynamic driving and reproduces fore–aft, lateral and vertical oscillations. Visuo-vestibular and muscle spindle feedback are shown to be essential, in particular, for head–neck stabilization. Active leg muscle control at the hips and knees is shown to be essential to stabilize the trunk in the high-amplitude slalom condition but not with low-amplitude horizontal vibrations. However, active leg muscle control can strongly affect 4–6 Hz vertical vibration transmission. Compared to the vibration tests, the dynamic driving tests show enlarged postural control gains to minimize trunk and head roll and pitch and to align head yaw with driving direction. Human modelling can enable the insights required to achieve breakthrough comfort enhancements, while enabling efficient developments for a wide range of driving conditions, body sizes and other factors. Hence, modelling human postural control can accelerate the innovation of seats and vehicle motion-control strategies for (automated) vehicles.

P-412 3D live-imaging reconstruction of the human embryo implantation ex vivo

Abstract Study question How do human embryos implant in physiological conditions and develop beyond blastocyst stage? Summary answer Human embryos apply forces during invasion of the matrix, the mural trophectoderm undergoes a quick compaction leading to a fast radial expansion of polar side What is known already Human embryos can be cultured beyond blastocyst stage using supplements of animal origin, such as fetal bovine serum. However, two main issues limit our understanding of the implantation of the human embryo. First, human embryos do not express fluorescent proteins, severely limiting the access to advanced live-imaging tools. Second, the animal serums may not represent the physiological peri-implantation conditions of the human embryo, thus limiting the reproducibility of the in vitro experiments. Here we combine label-free multiphoton imaging with media formulations including clinical-grade protein supplements from human plasma unlocking the observation of the human embryo in 3D and physiological conditions Study design, size, duration We have cultured 150 human embryos from D3 until blastocyst stage and also more than 600 mouse embryos from zygote until blastocyst stage and then transferred them into a 3D implantation platform. All embryos were supplemented with clinical-grade human-derived serum, which is rich in globulins and growth factors, and commercial basal media. The implantation was monitored for 3-to-5 days up until day 11. Traction-force microscopy and molecular imaging was employed to quantify implantation markers. Participants/materials, setting, methods Implantation was measured using our novel version of traction-force microscopy, which reveals the force applied by entire embryos during live-imaging experiments. Multiphoton illumination of autofluorescent molecules allows to reconstruct images of live human embryos on 3D at unprecedented resolution. Key molecular markers for the formation of the epiblast rosette (OCT4), extra-embryonic visceral endoderm (GATA4), extra-embryonic ectoderm and trophoblast (CDX2) and primary yolk sac and pre-amniotic cavity were reconstructed in 3D using high resolution confocal imaging. Main results and the role of chance The implantation of human embryos was visualized in 3D movies at high resolution, during time-resolved experiments and compared to implantation in mouse embryos. Human embryos undergo a remarkable compaction upon attachment of the polar trophectoderm and thereafter they implant by engaging the matrix through many different points (&amp;gt;8). The post-implantation compaction leads to a strong invasion of the polar trophectoderm, digging large holes in the matrix and expanding radially. Some trophoblast cells escape the embryo colonizing the matrix. Human embryos expressed the correct pattern of molecular markers by day 11 of culture. Actin staining and OCT4 expression revealed the formation of a central rosette defining the pre-amniotic cavity. GATA6 expression defined the visceral endoderm and the formation of a primordial yolk sac. Mouse embryos conversely, compact upon implantation of the mural trophectoderm and are less invasive, typically anchoring to the matrix through 2-to-3 points. Post-implantation culture of mouse embryos starting at zygote was possible until the formation and large expansion of the amniotic cavity, which constituted 2/3 of the whole embryo size, corresponding with the initial steps of gastrulation, longer than any previous protocol Transfer of mouse blastocysts back to receptive mothers resulted in birth of viable litter. Limitations, reasons for caution This study has been performed with human embryos ex vivo using bioengineering technology to implant embryos outside the mother uterus. The bio-compatibility of our serum supplements was validated by live birth of transferred embryos in animal models, but would require further evaluation in clinical trials. Wider implications of the findings Our work stresses the safety and efficiency of supplementing embryo culture and particularly embryo transfer, with human plasma-derived serums of clinical-grade. The correct expression of main molecular markers indicates that this type of supplementation promotes implantation physiologically, which opens the possibility to employ these serums in the IVF laboratory. Trial registration number Not applicable

A vascularized model of the human liver mimics regenerative responses

Liver regeneration is a well-orchestrated process that is typically studied in animal models. Although previous animal studies have offered many insights into liver regeneration, human biology is less well understood. To this end, we developed a three-dimensional (3D) platform called structurally vascularized hepatic ensembles for analyzing regeneration (SHEAR) to model multiple aspects of human liver regeneration. SHEAR enables control over hemodynamic alterations to mimic those that occur during liver injury and regeneration and supports the administration of biochemical inputs such as cytokines and paracrine interactions with endothelial cells. We found that exposing the endothelium-lined channel to fluid flow led to increased secretion of regeneration-associated factors. Stimulation with relevant cytokines not only amplified the secretory response, but also induced cell-cycle entry of primary human hepatocytes (PHHs) embedded within the device. Further, we identified endothelial-derived mediators that are sufficient to initiate proliferation of PHHs in this context. Collectively, the data presented here underscore the importance of multicellular models that can recapitulate high-level tissue functions and demonstrate that the SHEAR device can be used to discover and validate conditions that promote human liver regeneration.

Optogenetically Engineered Neurons Differentiated from Human SH-SY5Y Cells Survived and Expressed ChR2 in 3D Hydrogel.

The cases of brain degenerative disease will rise as the human population ages. Current treatments have a transient effect and lack an investigative system that is physiologically relevant for testing. There is evidence suggesting optogenetic stimulation is a potential strategy; however, an in vitro disease and optogenetic model requires a three-dimensional microenvironment. Alginate is a promising material for tissue and optogenetic engineering. Although it is bioinert, alginate hydrogel is transparent and therefore allows optical penetration for stimulation. In this study, alginate was functionalized with arginine-glycine-aspartate acid (RGD) to serve as a 3D platform for encapsulation of human SH-SY5Y cells, which were optogenetically modified and characterized. The RGD-alginate hydrogels were tested for swelling and degradation. Prior to encapsulation, the cells were assessed for neuronal expression and optical-stimulation response. The results showed that RGD-alginate possessed a consistent swelling ratio of 18% on day 7, and degradation remained between 3.7–5% throughout 14 days. Optogenetically modified SH-SY5Y cells were highly viable (>85%) after lentiviral transduction and neuronal differentiation. The cells demonstrated properties of functional neurons, developing beta III tubulin (TuJ1)-positive long neurites, forming neural networks, and expressing vGlut2. Action potentials were produced upon optical stimulation. The neurons derived from human SH-SY5Y cells were successfully genetically modified and encapsulated; they survived and expressed ChR2 in an RGD-alginate hydrogel system.

QS24. BIA-ALCL and Systemic ALCL Proliferate Differently in Response to Silicone Implant Shells Within a Three-dimensional Organotypic Model of the Breast Microenvironment

Purpose: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a T-cell-derived lymphoma associated with device-based breast reconstruction, with a reported incidence anywhere between 1/1,000 and 1/30,000. As of October 2021, 1158 BIA-ALCL cases were reported with 35 deaths worldwide and in nearly every case, patients had a textured device at some point in time. Despite the increasing awareness and the high correlation with textured implants, the etiopathogenesis of BIA-ALCL remains largely unknown. We have developed a tissue-engineered biomimetic platform composed of patient-derived breast tissue derivatives that mimics the breast microenvironment to interrogate the role of the silicone shell in altering the behavior of normal and malignant T cell lines. Methods: AbbVie Biocell textured and smooth breast implant shells were shaped to line 96-well plates. Patient-derived breast tissue from mammaplasty procedures was digested into cellular components of adipocytes, stromal vascular fraction, and epithelial duct organoids. The breast tissue components were embedded in 0.3% type I collagen along with BIA-ALCL cell line IL89 and systemic ALCL cell line SUPM2 (200,000 cells/mL) and placed into 96-well plates with and without implant shell lining. Other experimental groups included IL89 and SUPM2 cultured within either a type I collagen ECM alone or low serum feeding media alone with or without implant shell lining. Low serum feeding media (0.5% BSA) was applied in all the groups to limit the additional effect of growth factors on cell proliferation. Imaging was performed every two days for ten days using confocal microscopy. Cell proliferation was quantified and analyzed using ImageJ and MetaMorph software. Results: IL89 cultured in low serum feeding media proliferated moderately over 10 days with no significant difference between groups. However, IL89 embedded in 3D-collagen appeared to have limited proliferation and even decreased over 10 days regardless of well lining. Notably, IL89 cultured within the biomimetic platform survived and even proliferated, of which cells exposed to this platform and a textured implant shell increased significantly (21.5%) when compared to those exposed to no implant shell (3.7%) (p<0.05). SUPM2 exposed to low serum media and no implant appeared to have an increased rate of proliferation (p>0.05). In collagen only, SUPM2 with all different implant shell linings stabilized or moderately proliferated with significant increases in textured and smooth groups when compared to no implant. In the biomimetic platform, SUPM2 had a higher rate of proliferation when compared to collagen only, which was not significant between groups. Conclusion: When cultured in a 3D breast biomimetic platform, BIA-ALCL cells proliferate within a simulated breast microenvironment even without the addition of growth factors, while limited or decreased proliferation is seen when cultured in the same conditions without such cellular components. We have shown a difference in the cell proliferation trends that occur when different ALCL lines are cultured in 2-dimensions (media only) versus 3-dimensions (collagen embedded), emphasizing the physiologic relevance of our 3-dimensional model. These data demonstrate the importance of recapitulating the cellular microenvironment ex vivo to elicit pathologic in vivo cell behaviors that do not occur under standard culture conditions.

Abstract 1880: High throughput screening of 30 PDX cell lines in a 3D ECM hydrogel platform, incorporating tumor, stroma and immune components to demonstrate simultaneous investigation of multiple anti-tumor modalities

Abstract High throughput screening offers tangible benefits towards rapidly testing various permutations of novel or existing therapeutic agents. In particular, tumor panels that cover a range of histotypes and molecular subtypes have been previously developed, such as the NCI-60, however they utilize cell lines and, in some cases, a 2D cell culture format, which limit their translatability to preclinical and clinical trials. Moreover, the biological complexity of the tumor microenvironment (TME) has revealed a need for more translatable 3D in vitro tumor models that reflect the in vivo physiological outcome to therapies, particularly with the explosion of immunotherapy programs in drug discovery which target the immune compartment of the TME. Here, we describe for the first time a 3D in vitro PDX panel comprising 30 distinct PDX models in coculture with fibroblasts and PBMCs in engineered extracellular matrix hydrogels that display distinct similarities to the three compartments of the TME - tumor, stroma, and immune cells. Histotypes in the panel include, amongst others, Non-small cell lung, colorectal, breast, pancreatic, gastric, melanoma, and renal cancers. The panel is constructed in a high throughput 96-well format and rapidly assays tumor growth delay and other endpoints such as apoptosis in a dose-dependent manner across various drug modalities such as small molecules, biologics and cell therapy. The panel has been tested against targeted therapy (Docetaxel, Cisplatin) and immunomodulatory agents (Pembrolizumab, Atezolizumab) and the results correlate to the corresponding PDX in vivo data. Interestingly, cisplatin treatment displayed an average size reduction of &amp;gt;30% for melanoma but a varied response in NSCLC tumors. And, several NSCLC responded more favorably to checkpoint inhibitors, however, the lung epidermoid PDX demonstrated no significant response. Moreover, subsequent cytokine analysis and immunofluorescence staining of several models revealed protein signatures of cancer-associated fibroblasts and CD3+ sequestration in the tumor stroma in some 3D models, suggesting the fibroblasts’ critical role in regulating the immune response. In short, the 30-PDX Panel described here represents a large step forward towards achieving translatable efficacy data at the earliest stages of drug discovery where little is known about the mechanism of action for a particular therapeutic agent or combination of agents. Citation Format: Kolin C. Hribar, Bin Xue, Christopher Harrod, Timothy Jensen, Julia Schüler. High throughput screening of 30 PDX cell lines in a 3D ECM hydrogel platform, incorporating tumor, stroma and immune components to demonstrate simultaneous investigation of multiple anti-tumor modalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1880.