Tissue Spheroids Research Articles

Patterning of tissue spheroids biofabricated from human fibroblasts on the surface of electrospun polyurethane matrix using 3D bioprinter

Organ printing is a computer-aided additive biofabrication of functional three-dimensional human tissue and organ constructs according to digital model using the tissue spheroids as building blocks. The fundamental biological principle of organ printing technology is a phenomenon of tissue fusion. Closely placed tissue spheroids undergo tissue fusion driven by surface tension forces. In order to ensure tissue fusion in the course of post-printing, tissue spheroids must be placed and maintained close to each other. We report here that tissue spheroids biofabricated from primary human fibroblasts could be placed and maintained on the surface of biocompatible electrospun polyurethane matrix using 3D bioprinter according to desirable pattern. The patterned tissue spheroids attach to polyurethane matrix during several hours and became completely spread during several days. Tissue constructions biofabricated by spreading of patterned tissue spheroids on the biocompatible electrospun polyurethane matrix is a novel technological platform for 3D bioprinting of human tissue and organs.

Anlotinib potentiates anti-PD1 immunotherapy via transferrin receptor-dependent CD8+ T-cell infiltration in hepatocellular carcinoma.

The therapeutic potential of immune checkpoint blockade (ICB) extends across various cancers; however, its effectiveness in treating hepatocellular carcinoma (HCC) is frequently curtailed by both inherent and developed resistance. This research explored the effectiveness of integrating anlotinib (a broad-spectrum tyrosine kinase inhibitor) with programmed death-1 (PD-1) blockade and offers mechanistic insights into more effective strategies for treating HCC. Using patient-derived organotypic tissue spheroids and orthotopic HCC mouse models, we assessed the effectiveness of anlotinib combined with PD-1 blockade. The impact on the tumour immune microenvironment and underlying mechanisms were assessed using time-of-flight mass cytometry, RNA sequencing, and proteomics across cell lines, mouse models, and HCC patient samples. The combination of anlotinib with an anti-PD-1 antibody enhanced the immune response against HCC in preclinical models. Anlotinib remarkably suppressed the expression of transferrin receptor (TFRC) via the VEGFR2/AKT/HIF-1α signaling axis. CD8+ T-cell infiltration into the tumour microenvironment correlated with low expression of TFRC. Anlotinib additionally increased the levels of the chemokine CXCL14, crucial for attracting CD8+ T cells. CXCL14 emerged as a downstream effector of TFRC, exhibiting elevated expression following the silencing of TFRC. Importantly, low TFRC expression was also associated with a better prognosis, enhanced sensitivity to combination therapy, and a favourable response to anti-PD-1 therapy in patients with HCC. Our findings highlight anlotinib's potential to augment the efficacy of anti-PD-1 immunotherapy in HCC by targeting TFRC and enhancing CXCL14-mediated CD8+ T-cell infiltration. This study contributes to developing novel therapeutic strategies for HCC, emphasizing the role of precision medicine in oncology. Synergistic effects of anlotinib and anti-PD-1 immunotherapy demonstrated in HCC preclinical models. Anlotinib inhibits TFRC expression via the VEGFR2/AKT/HIF-1α pathway. CXCL14 upregulation via TFRC suppression boosts CD8+ T-cell recruitment. TFRC emerges as a potential biomarker for evaluating prognosis and predicting response to anti-PD-1-based therapies in advanced HCC patients.

A three-dimensional lattice-free agent-based model of intracellular ice formation and propagation and intercellular mechanics inliver tissues.

A successful cryopreservation of tissues and organs is crucial for medical procedures and drug development acceleration. However, there are only a few instances of successful tissue cryopreservation. One of the main obstacles to successful cryopreservation is intracellular ice damage. Understanding how ice spreads can accelerate protocol development and enable model-based decision-making. Previous models of intracellular ice formation in individual cells have been extended to one-cell-wide arrays to establish the theory of intercellular ice propagation in tissues. The current lattice-based ice propagation models do not account for intercellular forces resulting from cell solidification, which could lead to mechanical disruption of tissue structures during freezing. Moreover, these models have not been expanded to include more realistic tissue architectures. In this article, we discuss the development and validation of a stochastic model for the formation and propagation of ice in small tissues using lattice-free agent-based model. We have improved the existing model by incorporating the mechanical effects of water crystallization within cells. Using information from previous research, we have also created a new model that accounts for ice growth in tissue slabs, spheroids and hepatocyte discs. Our model demonstrates that individual cell freezing can have mechanical consequences and is consistent with earlier findings.

Mouse vascularized adipose spheroids: an organotypic model for thermogenic adipocytes.

Adipose tissues, particularly beige and brown adipose tissue, play crucial roles in energy metabolism. Brown adipose tissues' thermogenic capacity and the appearance of beige cells within white adipose tissue have spurred interest in their metabolic impact and therapeutic potential. Brown and beige fat cells, activated by environmental factors like cold exposure or by pharmacology, share metabolic mechanisms that drive non-shivering thermogenesis. Understanding these two cell types requires advanced, yet broadly applicable in vitro models that reflect the complex microenvironment and vasculature of adipose tissues. Here we present mouse vascularized adipose spheroids of the stromal vascular microenvironment from inguinal white adipose tissue, a tissue with 'beiging' capacity in mice and humans. We show that adding a scaffold improves vascular sprouting, enhances spheroid growth, and upregulates adipogenic markers, thus reflecting increased adipocyte maturity. Transcriptional profiling via RNA sequencing revealed distinct metabolic pathways upregulated in our vascularized adipose spheroids, with increased expression of genes involved in glucose metabolism, lipid metabolism, and thermogenesis. Functional assessment demonstrated increased oxygen consumption in vascularized adipose spheroids compared to classical 2D cultures, which was enhanced by β-adrenergic receptor stimulation correlating with elevated β-adrenergic receptor expression. Moreover, stimulation with the naturally occurring adipokine, FGF21, induced Ucp1 mRNA expression in the vascularized adipose spheroids. In conclusion, vascularized inguinal white adipose tissue spheroids provide a physiologically relevant platform to study how the stromal vascular microenvironment shapes adipocyte responses and influence activated thermogenesis in beige adipocytes.

Advancing Tissue Culture with Light-Driven 3D-Printed Microfluidic Devices.

Three-dimensional (3D) printing presents a compelling alternative for fabricating microfluidic devices, circumventing certain limitations associated with traditional soft lithography methods. Microfluidics play a crucial role in the biomedical sciences, particularly in the creation of tissue spheroids and pharmaceutical research. Among the various 3D printing techniques, light-driven methods such as stereolithography (SLA), digital light processing (DLP), and photopolymer inkjet printing have gained prominence in microfluidics due to their rapid prototyping capabilities, high-resolution printing, and low processing temperatures. This review offers a comprehensive overview of light-driven 3D printing techniques used in the fabrication of advanced microfluidic devices. It explores biomedical applications for 3D-printed microfluidics and provides insights into their potential impact and functionality within the biomedical field. We further summarize three light-driven 3D printing strategies for producing biomedical microfluidic systems: direct construction of microfluidic devices for cell culture, PDMS-based microfluidic devices for tissue engineering, and a modular SLA-printed microfluidic chip to co-culture and monitor cells.

Line-field dynamic optical coherence tomography platform for volumetric assessment of biological tissues.

Dynamic optical coherence tomography (dOCT) utilizes time-dependent signal intensity fluctuations to enhance contrast in OCT images and indirectly probe physiological processes in cells. Majority of the dOCT studies published so far are based on acquisition of 2D images (B-scans or C-scans) by utilizing point-scanning Fourier domain (spectral or swept-source) OCT or full-field OCT respectively, primarily due to limitations in the image acquisition rate. Here we introduce a novel, high-speed spectral domain line-field dOCT (SD-LF-dOCT) system and image acquisition protocols designed for fast, volumetric dOCT imaging of biological tissues. The imaging probe is based on an exchangeable afocal lens pair that enables selection of combinations of transverse resolution (from 1.1 µm to 6.4 µm) and FOV (from 250 × 250 µm2 to 1.4 × 1.4 mm2), suitable for different biomedical applications. The system offers axial resolution of ∼ 1.9 µm in biological tissue, assuming an average refractive index of 1.38. Maximum sensitivity of 90.5 dB is achieved for 3.5 mW optical imaging power at the tissue surface and maximum camera acquisition rate of 2,000 fps. Volumetric dOCT images acquired with the SD-LF-dOCT system from plant tissue (cucumber), animal tissue (mouse liver) and human prostate carcinoma spheroids allow for volumetric visualization of the tissues' cellular and sub-cellular structures and assessment of cellular motility.

Ethyl Pyruvate Decreases Collagen Synthesis and Upregulates MMP Activity in Keloid Fibroblasts and Keloid Spheroids.

Keloids, marked by abnormal cellular proliferation and excessive extracellular matrix (ECM) accumulation, pose significant therapeutic challenges. Ethyl pyruvate (EP), an inhibitor of the high-mobility group box 1 (HMGB1) and TGF-β1 pathways, has emerged as a potential anti-fibrotic agent. Our research evaluated EP's effects on keloid fibroblast (KF) proliferation and ECM production, employing both in vitro cell cultures and ex vivo patient-derived keloid spheroids. We also analyzed the expression levels of ECM components in keloid tissue spheroids treated with EP through immunohistochemistry. Findings revealed that EP treatment impedes the nuclear translocation of HMGB1 and diminishes KF proliferation. Additionally, EP significantly lowered mRNA and protein levels of collagen I and III by attenuating TGF-β1 and pSmad2/3 complex expression in both human dermal fibroblasts and KFs. Moreover, metalloproteinase I (MMP-1) and MMP-3 mRNA levels saw a notable increase following EP administration. In keloid spheroids, EP induced a dose-dependent reduction in ECM component expression. Immunohistochemical and western blot analyses confirmed significant declines in collagen I, collagen III, fibronectin, elastin, TGF-β, AKT, and ERK 1/2 expression levels. These outcomes underscore EP's antifibrotic potential, suggesting its viability as a therapeutic approach for keloids.

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.

In vitro evaluation of doxorubicin release from diopside particles on MG-63 and HF spheroids as a 3D model of tumor and healthy tissues

Local drug delivery systems based on bioceramics ensure safe and effective treatment of bone defects and anticancer therapy. A promising drug delivery scaffold material for bone treatment applications is diopside (CaMgSi2O6) which is bioactive, degradable, and possesses drug-release ability. Currently, in vitro assessment of drug release from biomaterials is performed mostly on a 2D cell monolayer. However, to interpret and integrate biochemical signals, cells need a 3D microenvironment that provides cell-cell and cell-extracellular matrix interactions. In this regard, 3D cell models are gaining popularity. In this work, we proposed the protocol for evaluation of the effect of doxorubicin released from diopside on MG-63 cells and primary human fibroblasts in 3D culture conditions. Tissue spheroids with similar diameters were incubated with doxorubicin-loaded diopside for 72 h, the amount of diopside was calculated in accordance with the required doxorubicin concentration. We demonstrated that doxorubicin is gradually released from diopside and exhibits an activity similar to that of the pure drug at the same total concentration. It is important to note that doxorubicin was more potent on MG-63 spheroids compared to HF spheroids, which confirmed the reliability of spheroids as 3D models of tumor and healthy tissues.

Abstract 6786: Assessment of immune cell infiltration and cancer metastatic potential in Akura™ Immune Flow Chip - A microfluidic 3D spheroid system

Abstract Evaluation of novel therapeutics often fails to reliably predict severe complications in patients, especially when circulating cells are involved. The reasons may include inter-species differences upon using animal models or the lack of relevant in vitro systems. Moreover, existing in vitro systems mostly rely on static investigations of different cell types leading to overestimation of effects due to non-representative cell-tissue interactions. The incorporation of medium flow may facilitate to mimic physiological conditions more closely. To tackle current limitations of perfused organ-on-chip approaches, we developed a microfluidic chip and operation concept, which prevents undesired sedimentation and accumulation of cells in suspensions. The Akura™ Immune Flow platform was designed to study flow-dependent recruitment of circulating cells to different tissue spheroids via gravity-driven perfusion over several days. The Akura™ Immune Flow platform features standardized formats for co-culturing up to seven spheroids with suspended cells in each microfluidic channel. On-chip staining protocols enabled monitoring of the co-culture. The system allowed for application and extractions of cellular components and liquid for further off-chip analysis. Primary tissue and cell line spheroids (liver and tumor) could be retained in the respective chambers and remained viable over six to 14 days. Circulating cells were kept viable and in suspension for up to six days (PBMCs) and14 days (Prostate Cancer (PCa) cells) respectively. PBMCs were not activated by flow-induced shear forces. Unspecific stimulation of T-cells led to physiological expression of early and late activation markers (CD69 and CD25), which were detected by flow cytometry in periodically collected cells. Multi-photon microscopy imaging confirmed that PBMCs interacted with and infiltrated into 3D tumor spheroids. Application of PCa cells to primary liver spheroids, cultured in Akura™ Immune Flow chips allowed for assessment of their invasive potential. Confocal imaging revealed adhesion of cancer cells to the liver spheroids and offers the potential to study the ability of tissue invasion at later time points. The simple design of the chip facilitates user-friendly operation and high-throughput implementation in cancer research and drug testing. The selected applications support the relevance of using flow solutions for physiologically more complex processes. This work constitutes an important step towards device application as a tool for basic research as well as for efficacy studies of immunotherapeutic drug candidates Citation Format: Tamara Häfeli, Silas Riss, Christian Lohasz, Dzhansu Hasanova, Svenja Lützow, Lisa Hölting, Michal Rudnik, Franziska Linke, Wytske M van Weerden, Laure-Anne Ligeon, Andreas Hierlemann, Mario Modena, Olivier Frey. Assessment of immune cell infiltration and cancer metastatic potential in Akura™ Immune Flow Chip - A microfluidic 3D spheroid system [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 6786.

Highlighting the effects of gamma irradiation of the brain through non-conventional X-ray imaging

Whole brain irradiation is largely used as an alternative radiotherapy of brain tumors that cannot be eliminated by surgery so the effects of ionizing radiation on brain healthy tissue represents an important research domain. The aim of this study is to evaluate the irradiation effects on in vitro brain tissue spheroids models as well as the whole mouse brain using standard cellular biology assays and a multi-energy X-ray imaging technique. The spheroids irradiated with gamma rays (dose between 0–30 Gy) and treated with biocompatible nanoparticles consisting of concentrations between 0–100 μM proved to be morphologically stable and with a high radio-resistance. The reactive oxygen species concentration and the γ-H2AX foci number increase with the irradiation dose, as expected. The X-ray imaging with dual-energy technique method proposed here was able to differentiate between irradiated and control samples (whole brain). Concluding, our results proved the expected effects of ionizing radiation on brain tissue. The dual-energy X-ray imaging method tested here appears as a promising method for characterizing the ionizing radiation effects on the whole brain level.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these invitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and invivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

3D Bioprinted Liver-on-a-Chip for Drug Cytotoxicity Screening.

Tissues on a chip are sophisticated three-dimensional (3D) in vitro microphysiological systems designed to replicate human tissue conditions within dynamic physicochemical environments. However, the current fabrication methods for tissue spheroids on a chip require multiple parts and manual processing steps, including the deposition of spheroids onto prefabricated "chips." These challenges also lead to limitations regarding scalability and reproducibility. To overcome these challenges, we employed 3D printing techniques to automate the fabrication process of tissue spheroids on a chip. This allowed the simultaneous high-throughput printing of human liver spheroids and their surrounding polymeric flow chamber "chips" containing inner channels in a single step. The fabricated liver tissue spheroids on a liver-on-a-chip (LOC) were subsequently subjected to dynamic culturing by a peristaltic pump, enabling assessment of cell viability and metabolic activities. The 3D printed liver spheroids within the printed chips demonstrated high cell viability (>80%), increased spheroid size, and consistent adenosine triphosphate (ATP) activity and albumin production for up to 14 days. Furthermore, we conducted a study on the effects of acetaminophen (APAP), a nonsteroidal anti-inflammatory drug, on the LOC. Comparative analysis revealed a substantial decline in cell viability (<40%), diminished ATP activity, and reduced spheroid size after 7 days of culture within the APAP-treated LOC group, compared to the nontreated groups. These results underscore the potential of 3D bioprinted tissue chips as an advanced in vitro model that holds promise for accurately studying in vivo biological processes, including the assessment of tissue response to administered drugs, in a high-throughput manner.

Three Dimensional Models of Endocrine Organs and Target Tissues Regulated by the Endocrine System.

Immortalized cell lines originating from tumors and cultured in monolayers in vitro display consistent behavior and response, and generate reproducible results across laboratories. However, for certain endpoints, these cell lines behave quite differently from the original solid tumors. Thereby, the homogeneity of immortalized cell lines and two-dimensionality of monolayer cultures deters from the development of new therapies and translatability of results to the more complex situation in vivo. Organoids originating from tissue biopsies and spheroids from cell lines mimic the heterogeneous and multidimensional characteristics of tumor cells in 3D structures in vitro. Thus, they have the advantage of recapitulating the more complex tissue architecture of solid tumors. In this review, we discuss recent efforts in basic and preclinical cancer research to establish methods to generate organoids/spheroids and living biobanks from endocrine tissues and target organs under endocrine control while striving to achieve solutions in personalized medicine.

Surface Tension and Neuronal Sorting in Magnetically Engineered Brain-Like Tissue.

Engineered 3D brain-like models have advanced the understanding of neurological mechanisms and disease, yet their mechanical signature, while fundamental for brain function, remains understudied. The surface tension for instance controls brain development and is a marker of cell-cell interactions. Here, 3D magnetic brain-like tissue spheroids composed of intermixed primary glial and neuronal cells at different ratios are engineered. Remarkably, the two cell types self-assemble into a functional tissue, with the sorting of the neuronal cells toward the periphery of the spheroids, whereas the glial cells constitute the core. The magnetic fingerprint of the spheroids then allows their deformation when placed under a magnetic field gradient, at a force equivalent to a 70g increased gravity at the spheroid level. The tissue surface tension and elasticity can be directly inferred from the resulting deformation, revealing a transitional dependence on the glia/neuron ratio, with the surface tension of neuronal tissue being much lower. The results suggest an underlying mechanical contribution to the exclusion of the neurons toward the outer spheroid region, and depict the glia/neuron organization as a sophisticated mechanism that should in turn influence tissue development and homeostasis relevant in the neuroengineering field.

Mathematical and computational models in spheroid-based biofabrication.

Ubiquitous in embryonic development, tissue fusion is of interest to tissue engineers who use tissue spheroids or organoids as building blocks of three-dimensional (3D) multicellular constructs. This review presents mathematical models and computer simulations of the fusion of tissue spheroids. The motivation of this study stems from the need to predict the post-printing evolution of 3D bioprinted constructs. First, we provide a brief overview of differential adhesion, the main morphogenetic mechanism involved in post-printing structure formation. It will be shown that clusters of cohesive cells behave as an incompressible viscous fluid on the time scale of hours. The discussion turns then to mathematical models based on the continuum hydrodynamics of highly viscous liquids and on statistical mechanics. Next, we analyze the validity and practical use of computational models of multicellular self-assembly in live constructs created by tissue spheroid bioprinting. Finally, we discuss the perspectives of the field as machine learning starts to reshape experimental design, and modular robotic workstations tend to alleviate the burden of repetitive tasks in biofabrication. STATEMENT OF SIGNIFICANCE: Bioprinted constructs are living systems, which evolve via morphogenetic mechanisms known from developmental biology. This review presents mathematical and computational tools devised for modeling post-printing structure formation. They help achieving a desirable outcome without expensive optimization experiments. While previous reviews mainly focused on assumptions, technical details, strengths, and limitations of computational models of multicellular self-assembly, this article discusses their validity and practical use in biofabrication. It also presents an overview of mathematical models that proved to be useful in the evaluation of experimental data on tissue spheroid fusion, and in the calibration of computational models. Finally, the perspectives of the field are discussed in the advent of robotic biofabrication platforms and bioprinting process optimization by machine learning.

OTHR-04. DEVELOPMENT OF A FUNCTIONAL PLATFORM FOR REAL-TIME PERSONALIZED DRUG SENSITIVITY PROFILING OF PATIENT-DERIVED 3D FRESH TUMOR TISSUE CULTURES IN THE PEDIATRIC PRECISION ONCOLOGY PROGRAM INFORM

Abstract The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75-78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs.

An Eulerian Nonlinear Elastic Model for Compressible and Fluidic Tissue with Radially Symmetric Growth

Cell proliferation, apoptosis, and myosin-dependent contraction can generate elastic stress and strain in living tissues, which may be dissipated by internal rearrangement through cell topological transition and cytoskeletal reorganization. Moreover, cells and tissues can change their sizes in response to mechanical cues. The present work demonstrates the role of tissue compressibility and internal rearranging activities on its size and mechanics regulation in the context of differential growth induced by a field of growth-promoting chemical factors. We develop a mathematical model based on finite elasticity and growth theory and the reference map techniques to describe the coupled tissue growth and mechanics in the Eulerian frame. We incorporate the tissue rearrangement by introducing a rearranging rate to the reference map evolution, leading to elastic-energy dissipation when tissue growth and deformation are in radial symmetry. By linearizing the model, we show that the stress follows the Maxwell-type viscoelastic relaxation. The rearrangement rate, which we call tissue fluidity, sets the stress relaxation time, and the ratio between the shear modulus and the fluidity sets the tissue viscosity. By nonlinear simulation of growing tissue spheroids and discs with graded growth rates along the radius, we find that the tissue compressibility and fluidity influence their equilibrium size. By comparing the nonlinear simulations with the linear analytical solutions, we show the size change as a nonlinear effect due to the advection of the tissue density flow, which only occurs when both tissue compressibility and fluidity are small. We apply the model to study tumor spheroid growth and epithelial disc growth when a reaction-diffusion process determines the growth-promoting factor field.

Drug sensitivity profiling of 3D tumor tissue cultures in the pediatric precision oncology program INFORM

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75–78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs.

Design, Fabrication, and Application of Mini-Scaffolds for Cell Components in Tissue Engineering

The concept of "lockyballs" or interlockable mini-scaffolds fabricated by two-photon polymerization from biodegradable polymers for the encagement of tissue spheroids and their delivery into the desired location in the human body has been recently introduced. In order to improve control of delivery, positioning, and assembly of mini-scaffolds with tissue spheroids inside, they must be functionalized. This review describes the design, fabrication, and functionalization of mini-scaffolds as well as perspectives on their application in tissue engineering for precisely controlled cell and mini-tissue delivery and patterning. The development of functionalized mini-scaffolds advances the original concept of "lockyballs" and opens exciting new prospectives for mini-scaffolds' applications in tissue engineering and regenerative medicine and their eventual clinical translation.