3D Culture Research Articles

TGF-β effects on adipogenesis of 3T3-L1 cells differ in 2D and 3D cell culture conditions.

The TGF-β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-β, TGF-β-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF-β isoforms, with TGF-β2 having the greater effects. Consistent with these results, treatment with TGF-β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3-L1 cells. However, the inhibitory effect of TGF-β on adipogenesis decreased under 3D spheroid culture conditions and TGF-β isoforms did not affect adipogenesis-induced (a) enlargement and downsizing of 3T3-L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up-regulation of the mRNA expression of adipogenesis-related genes. The findings presented herein suggest that the three TGF-β isoforms have different suppressive effects on adipogenesis-related cellular properties of 2D cultured 3T3-L1 cells and that their effects decrease under 3D spheroid culture conditions.

8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None. Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p>0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model. Presentation: 6/1/2024

Improvement of Mechanical Properties of 3D Bioprinted Structures through Cellular Overgrowth

The common use of hydrogel materials in 3D bioprinting techniques is dictated by the unique properties of hydrogel bioinks, among which some of the most important in terms of sustaining vital cell functions in vitro in 3D cultures are the ability to retain large amounts of liquid and the ability to modify rigidity and mechanical properties to reproduce the structure of the natural extracellular matrix. Due to their high biocompatibility, non-immunogenicity, and the possibility of optimizing rheological properties and bioactivity at the same time, one of the most commonly used hydrogel bioink compositions are polymer solutions based on sodium alginate and gelatin. In 3D bioprinting techniques, it is necessary for hydrogel printouts to feature an appropriate geometry to ensure proper metabolic activity of the cells contained inside the printouts. The desired solution is to obtain a thin-walled printout geometry, ensuring uniform nutrient availability and gas exchange during cultivation. Within this study’s framework, tubular bioprinted structures were developed based on sodium alginate and gelatin, containing cells of the immortalized fibroblast line NIH/3T3 in their structure. Directly after the 3D printing process, such structures are characterized by extremely low mechanical strength. The purpose of this study was to perform a comparative analysis of the viability and spreading ability of the biological material contained in the printouts during their incubation for a period of 8 weeks while monitoring the effect of cellular growth on changes in the mechanical properties of the tubular structures. The observations demonstrated that the cells contained in the 3D printouts reach the ability to grow and spread in the polymer matrix after 4 weeks of cultivation, leading to obtaining a homogeneous, interconnected cell network inside the hydrogel after 6 weeks of incubation. Analysis of the mechanical properties of the printouts indicates that with the increasing time of cultivation of the structures, the degree of their overgrowth by the biological material contained inside, and the progressive degradation of the polymer matrix process, the tensile strength of tubular 3D printouts varies.

3D-Printable Gelatin Methacrylate-Xanthan Gum Hydrogel Bioink Enabling Human Induced Pluripotent Stem Cell Differentiation into Cardiomyocytes.

We describe the development of a bioink to bioprint human induced pluripotent stem cells (hiPSCs) for possible cardiac tissue engineering using a gelatin methacrylate (GelMA)-based hydrogel. While previous studies have shown that GelMA at a low concentration (5% w/v) allows for the growth of diverse cells, its 3D printability has been found to be limited by its low viscosity. To overcome that drawback, making the hydrogel both compatible with hiPSCs and 3D-printable, we developed an extrudable GelMA-based bioink by adding xanthan gum (XG). The GelMA-XG composite hydrogel had an elastic modulus (~9 kPa) comparable to that of cardiac tissue, and enabled 3D printing with high values of printing accuracy (83%) and printability (0.98). Tests with hiPSCs showed the hydrogel's ability to promote their proliferation within both 2D and 3D cell cultures. The tests also showed that hiPSCs inside hemispheres of the hydrogel were able to differentiate into cardiomyocytes, capable of spontaneous contractions (average frequency of ~0.5 Hz and amplitude of ~2%). Furthermore, bioprinting tests proved the possibility of fabricating 3D constructs of the hiPSC-laden hydrogel, with well-defined line widths (~800 μm).

Barrier-free, open-top microfluidic chip for generating two distinct, interconnected 3D microvascular networks

Developing microphysiological cell culture platforms with a three-dimensional (3D) microenvironment has been a significant advancement from traditional monolayer cultures. Still, most of the current microphysiological platforms are limited in closed designs, i.e. are not accessible after 3D cell culture loading. Here, we report an open-top microfluidic chip which enables the generation of two sequentially loaded 3D cell cultures without physical barriers restricting the nurture, gas exchange and cellular communication. As a proof-of-concept, we demonstrated the formation of two 3D vasculatures, one in the upper and the other in the lower compartment, under three distinct flow conditions: asymmetric side-to-center, symmetric side-to-center and symmetric center-to-side. We used computational modelling to characterize initial flow pressures in cell culture compartments. We showed prominent vessel formation and branched vasculatures in upper and lower cell culture compartments with interconnecting, lumenized vessels with in vivo-relevant diameter in all flow conditions. With advanced image processing, we quantified and compared the overall vascular network volume and the total length formed in asymmetric side-to-center, symmetric side-to-center and symmetric center-to-side flow conditions. Our results indicate that the developed chip can house two distinct 3D cell cultures with merging vessels between compartments and by providing asymmetric side-to-center or symmetric center-to-side flow vascular morphogenesis is enhanced in terms of overall network length. The developed open-top microfluidic chip may find various applications in generation of tissue-specific 3D-3D co-cultures for studying cellular interactions in vascularized tissues and organs.

Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

Collagen plays a critical role in regulating breast cancer progression and therapeutic resistance. An improved understanding of both the features and drivers of tumor-permissive and -restrictive collagen matrices are critical to improve prognostication and develop more effective therapeutic strategies. In this study, using a combination of in vitro, in vivo and bioinformatic experiments, we show that type III collagen (Col3) plays a tumor-restrictive role in human breast cancer. We demonstrate that Col3-deficient, human fibroblasts produce tumor-permissive collagen matrices that drive cell proliferation and suppress apoptosis in non-invasive and invasive breast cancer cell lines. In human triple-negative breast cancer biopsy samples, we demonstrate elevated deposition of Col3 relative to type I collagen (Col1) in non-invasive compared to invasive regions. Similarly, bioinformatics analysis of over 1000 breast cancer patient biopsies from The Cancer Genome Atlas BRCA cohort revealed that patients with higher Col3:Col1 bulk tumor expression had improved overall, disease-free, and progression-free survival relative to those with higher Col1:Col3 expression. Using an established 3D culture model, we show that Col3 increases spheroid formation and induces the formation of lumen-like structures that resemble non-neoplastic mammary acini. Finally, our in vivo study shows co-injection of murine breast cancer cells (4T1) with rhCol3-supplemented hydrogels limits tumor growth and decreases pulmonary metastatic burden compared to controls. Taken together, these data collectively support a tumor-suppressive role for Col3 in human breast cancer and suggest that strategies that increase Col3 may provide a safe and effective therapeutic modality to limit recurrence in breast cancer patients.

Impact of perfusion on neuronal development in human derived neuronal networks.

Advanced in vitro models of the brain have evolved in recent years from traditional two-dimensional (2D) ones, based on rodent derived cells, to three-dimensional (3D) ones, based on human neurons derived from induced pluripotent stem cells. To address the dynamic changes of the tissue microenvironment, bioreactors are used to control the in vitro microenvironment for viability, repeatability, and standardization. However, in neuronal tissue engineering, bioreactors have primarily been used for cell expansion purposes, while microfluidic systems have mainly been employed for culturing organoids. In this study, we explored the use of a commercial perfusion bioreactor to control the culture microenvironment of neuronal cells in both 2D and 3D cultures. Namely, neurons differentiated from human induced pluripotent stem cells (iNeurons) were cultured in 2D under different constant flow rates for 72 h. The impact of different flow rates on early-stage neuronal development and synaptogenesis was assessed by morphometric characterization and synaptic analysis. Based on these results, two involving variable flow rates were developed and applied again in 2D culture. The most effective protocol, in terms of positive impact on neuronal development, was then used for a preliminary study on the application of dynamic culturing conditions to neuronal cells in 3D. To this purpose, both iNeurons, co-cultured with astrocytes, and the human neuroblastoma cells SH-SY5Y were embedded into a hydrogel and maintained under perfusion for up to 28 days. A qualitative evaluation by immunocytochemistry and confocal microscopy was carried out to assess cell morphology and the formation of a 3D neuronal network.

Enhanced Osteogenesis in 2D and 3D Culture Systems Using RGD Peptide and α-TCP Phase Transition within Alginate-Based Hydrogel.

Cell-laden hydrogels have been extensively investigated in various tissue engineering fields bytheir potential capacity to deposit numerous types of cells in a specific area. They are largely used in soft-tissue engineering applications because of their low mechanical strength. In addition, sodium alginate is well-known for its encapsulation, loading capacity and for being easily controllable; however, it lacks cell-binding ligands and hence the ability to adhere cells. In this study, it is aimed to enhance osteogenesis in cells encapsulated in alginate and improve its mechanical properties by introducing a synthetic peptide and calcium phosphate phase transition. To increase cell-hydrogel interactions and increasing cell viability, an RGD peptide is added to a photocrosslinkable methacrylate-modified alginate, and alpha-tricalcium phosphate (α-TCP) is added to the hydrogel to increase its mechanical strength via phase transition. Cell proliferation, growth, and differentiation are assessed in both 2D and 3D cell cultures. The addition of α-TCP significantly improved the mechanical properties of the hydrogel. Moreover, the RGD peptide and α-TCP showed a synergistic effect with significantly improved cell adhesion and osteogenesis in both 2D and 3D cell cultures. Therefore, the functional hydrogel developed in this study can potentially be used for bone tissue regeneration.

High-Throughput Assessment of Metabolism-Mediated Neurotoxicity by Co-Culture of Neurospheres and Liver Spheroids.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC50 values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity Basic Protocol 3: Image acquisition, processing, and data analysis.

Interference of FZD2 suppresses proliferation, vasculogenic mimicry and stemness in glioma cells via blocking the Notch/NF‑κB signaling pathway.

Frizzled family protein 2 (FZD2) is widely associated with tumor development and metastasis. The present study aimed to gain an insight into the role and regulatory mechanism of FZD2 in glioma. The expression level of FZD2 in normal astrocyte and glioma cells was determined by reverse transcription-quantitative PCR and western blotting, and cell transfection was conducted for FZD2 expression knockdown. Malignant behaviors including cell proliferation, migration and invasion, vasculogenic mimicry (VM) and cell stemness were determined using Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine (EdU) staining, colony formation, wound healing, Transwell, 3D culturing and sphere formation assays. The expression levels of proteins related to stemness, epithelial-mesenchymal transition (EMT) and Notch/NF-κB signaling were measured by western blotting. Then, the Notch agonist, Jagged-1 (JAG), was adopted for rescue experiments. The results demonstrated that FZD2 was highly expressed in glioma cells. Interference of FZD2 expression suppressed the proliferation of glioma cells, as evidenced by the reduced cell viability and the number of EdU+ cells and colonies. Meanwhile, the reduced sphere formation ability and decreased protein expression of Nanog, Sox2 and Oct4 following FZD2 knockdown confirmed that FZD2 repressed cell stemness in glioma. Additionally, FZD2 knockdown suppressed the migration, invasion, EMT and VM formation capabilities of glioma cells, and also blocked the Notch/NF-κB signaling pathway. Furthermore, activation of Notch by JAG treatment partially reversed the aforementioned FZD2 knockdown-mediated changes in glioma cell malignant behaviors. In conclusion, FZD2 may contribute to glioma progression through activating the Notch/NF-κB signaling pathway, providing a plausible therapeutic target for the treatment of glioma.

Scaffold-free 3D culture systems for stem cell-based tissue regeneration.

Recent advances in scaffold-free three-dimensional (3D) culture methods have significantly enhanced the potential of stem cell-based therapies in regenerative medicine. This cutting-edge technology circumvents the use of exogenous biomaterial and prevents its associated complications. The 3D culture system preserves crucial intercellular interactions and extracellular matrix support, closely mimicking natural biological niches. Therefore, stem cells cultured in 3D formats exhibit distinct characteristics, showcasing their capabilities in promoting angiogenesis and immunomodulation. This review aims to elucidate foundational technologies and recent breakthroughs in 3D scaffold-free stem cell engineering, offering comprehensive guidance for researchers to advance this technology across various clinical applications. We first introduce the various sources of stem cells and provide a comparative analysis of two-dimensional (2D) and 3D culture systems. Given the advantages of 3D culture systems, we delve into the specific fabrication and harvesting techniques for cell sheets and spheroids. Furthermore, we explore their applications in pre-clinical studies, particularly in large animal models and clinical trials. We also discuss multidisciplinary strategies to overcome existing limitations such as insufficient efficacy, hostile microenvironments, and the need for scalability and standardization of stem cell-based products.

A chemically defined, mechanically tunable, and bioactive hyaluronic acid/alginate double-network hydrogel for liver cancer organoid construction

Liver cancer organoids replicate the pathophysiology of primary tumors, making them ideal for drug screening and efficacy evaluation. However, their growth in complex, variable, animal-derived matrices hinders practical application. Here, we designed an easily accessible, chemically defined, biocompatible double-network hydrogel (HADR) using methacrylated hyaluronic acid (HAMA), sodium alginate (SA), methacrylated dopamine (DMA), and c(RGDFC) for liver cancer organoid culture. By optimizing critical extracellular matrix (ECM) parameters, the HADR hydrogel achieves compatibility with the physiological mechanics of the human liver and fosters the adhesion and proliferation of multiple cell types. In vitro drug efficacy tests showed that HepG2 cell line-derived liver cancer organoids exhibited higher IC50 values than 2D cultures, indicating greater drug resistance. Subcutaneous tumor models in nude mice revealed that HADR hydrogels created a microenvironment for HepG2 cells mirroring the natural tumor ECM, leading to increased tumor volume, denser cell arrangement, and concurrent microvascular development. In vivo drug efficacy evaluations indicated that DOX treatment downregulated Ki-67 and MMP-9 expression, inhibiting HepG2 cell proliferation, invasion, and metastasis. These findings demonstrate the potential of HADR hydrogels for liver cancer organoid culture, offering new strategies for personalized drug screening and efficacy evaluation.

Metallic-based phthalocyanine nanoemulsions for photodynamic purging of ovarian tissue in leukemia patients

For cancer patients with a high risk of ovarian tissue metastasis, ovarian autotransplantation is not advised due to the potential spread of malignant cells. Ex vivo purging of ovarian fragments may offer a more suitable alternative for fertility restoration. Eradicating malignant cells should be done selectively without affecting follicles or ovarian stromal cells (SCs). As a clinically licensed method, photodynamic therapy (PDT) is a minimally invasive treatment to destroy cancer cells. This study evaluates the effectiveness of nanoemulsions (NE) containing two phthalocyanine photosensitizers; aluminum (III) phthalocyanine (AlPc) and zinc (II) phthalocyanine (ZnPc) in eliminating cancer cells. Human leukemic malignant (HL-60) and ovarian stromal cells (SCs) were treated with AlPc/ZnPc loaded NEs with or without diode laser irradiation. HL-60 leukemia cells in 2D culture were eliminated by treatment with 10nM AlPc-NE or 0.1µM ZnPc-NE, while no toxicity was observed in SCs. In 3D culture models, although the cells showed more resistance to the NEs as a result of limited oxygen and photosensitizer penetration, the treatment remained selective for cancer cells. These approaches have the potential to eliminate malignant cells from ovarian tissue fragments.

Novel repurposing of sulfasalazine for the treatment of adrenocortical carcinomas, probably through the SLC7A11/xCT-hsa-miR-92a-3p-OIP5-AS1 network pathway

BackgroundRecent multigenomic analysis of adrenocortical carcinomas (ACCs) identified SLC7A11/xCT as a novel biomarker. The Food and Drug Administration–approved anti-inflammatory drug, sulfasalazine (SAS), induces ferroptosis by blocking SLC7A11 expression. We hypothesize that SAS could be repurposed to target ACC cells. MethodsExpression of SLC7A11 and its association with ACC survival was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA). The validated ACC cell lines NCI-H295R, ACC1, and ACC2 were grown in 2D culture. In vitro studies included the CellTiter-Glo assay to calculate viability, Western blot (WB) analysis for apoptosis and other target protein changes, reverse transcriptase polymerase chain reaction for steroidogenic enzyme changes, C11BODIPY for lipid peroxidation, and mass spectrometry for changes in lipids. ResultsThe Cancer Genome Atlas Program database analysis in GEPIA showed that SLC7A11 and linked long noncoding RNA OAP5-AS1 are highly expressed in ACC tumors versus normal adrenals (n = 77 vs 128; P < .05). This was associated with poor overall and disease-free survival with hazard ratios of 4.3 and 5.2 for SLC7A11 and 4.8 and 2.7 for OAP5-AS1, respectively. ACC cell line half-inhibitory maximum concentration values after 72-hour SAS treatment ranged from 412 nM (ACC1) to 799 nM (ACC2), and all showed cleavage of poly (ADP-ribose) polymerase, upregulation of p-Akt and p-ERK, and downregulation of GPX4 and SLC7A11 (P < .05) by WB analysis. Sphere formation, migration, and invasion assay showed inhibition, and lipid peroxidation using C11BODIPY, increase in intracellular iron, induction of oxidative stress, and significant upregulation of oxidized polyunsaturated fatty acid phospholipids (P < .05 each) by mass spectrometry suggests induction of ferroptosis. ConclusionSAS downregulates tSLC7A11 in ACCs, targets the Akt/ERK pathway and lipid metabolism, and induces cell death in vitro, warranting additional translational studies to define its therapeutic potential in ACC.

Dual scalable osteogenic microtissue engineering via GelMA microsphere-inspired mechanical training and autonomous assembling of dental pulp stem cell

Large bone tissue defects present a significant clinical challenge due to the lack of stem cells and an osteogenic microenvironment, leading to fibrotic healing and impaired bone regeneration. Microsphere-based cell-on three-dimensional (3D) culture systems show great promise for constructing osteogenic microtissues. However, the underlying mechanisms require further investigation. In this study, we propose a simple, scalable framework for highly efficient osteogenic microtissue construction, utilizing gelatin methacryloyl (GelMA) microspheres and dental pulp stem cells (DPSCs). The GelMA microspheres provide an extensive, scalable 3D framework for the autonomous adhesion, migration, and proliferation of DPSCs. Within the enormous 3D space created by the microspheres, DPSCs anchor to the microspheres and neighboring cells, inducing intrinsic tensile stress and simulating a mechanical force akin to “rock climbing training”. Transcriptomic sequencing results reveal that the 3D spatial and mechanical microenvironment modulates biological processes involved in cell adhesion, extracellular matrix organization, and the positive regulation of cell migration. Further investigations demonstrate that triggering the FAK/YAP pathway mediate mechanical driven differentiation of DPSCs into the osteoblastic lineage in the excellent osteogenic microtissues. Moreover, this simple scalable 3D framework strategy is expected to enable the efficient and large-scale preparation of stem cell-based microtissues.

Modeling tumor-immune interactions using hybrid spheroids and microfluidic platforms for studying tumor-associated macrophage polarization in melanoma

Tumor-associated macrophages (TAMs), as key components of tumor microenvironment (TME), exhibit phenotypic plasticity in response to environmental cues, causing polarization into either pro-inflammatory M1 phenotypes or immunosuppressive M2 phenotypes. Although TAM has been widely studied for its crucial involvement in the initiation, progression, metastasis, and immune regulation of cancer cells, there have been limited attempts to understand how the metastatic potentials of cancer cells influence TAM polarization within TME. Here, we developed a miniaturized TME model using a 3D hybrid system composed of murine melanoma cells and macrophages, aiming to investigate interactions between cancer cells exhibiting various metastatic potentials and macrophages within TME. The increase in spheroid size within this model was associated with a reduction in cancer cell viability. Examining macrophage surface marker expression and cytokine secretion indicated the development of diverse TMEs influenced by both spheroid size and the metastatic potential of cancer cells. Furthermore, a high-throughput microfluidic platform equipped with trapping systems and hybrid spheroids was employed to simulate the tumor-immune system of complex TMEs and for comparative analysis with traditional 3D culture models. This study provides insight into TAM polarization in melanoma with different heterogeneities by modeling cancer-immune systems, which can be potentially employed for immune-oncology research, drug-screening, and personalized-therapy. STATEMENT OF SIGNIFICANCE: This study presents the development of a 3D hybrid spheroid system designed to model tumor-immune interactions, providing a detailed analysis of how melanoma cell metastatic potential influences tumor-associated macrophage (TAM) polarization. By utilizing a microfluidic platform, we are able to replicate and investigate the complex tumor-immune system of the tumor microenvironments (TMEs) under continuous flow conditions. Our model holds significant potential for high-throughput drug screening and personalized medicine applications, offering a versatile tool for advancing cancer research and treatment strategies.

Engineered autologous nasal cartilage for repair of nasal septal perforations - a case series.

This phase I clinical trial assessed the use of autologous nasal chondrocyte tissue-engineered cartilage (N-TEC) for functional repair of nasal septal perforations (NSP). The most widely used technique to treat NSP, namely interposition grafting with a polydioxanone (PDS) plate combined with a deep temporal fascia (DTF) graft, is still suboptimal towards patient satisfaction and revision rates. Patients (n=5, all female, age range: 23-54 years) had a 0.5-2.0cm diameter NSP. N-TEC was manufactured by expansion and 3D culture of autologous nasal septum chondrocytes into Chondro-Gide® collagen membranes. N-TEC was then shaped intraoperatively and enveloped in the harvested DTF before suturing it into the NSP. Safety (primary outcome) was assessed by the number of serious adverse reactions (SAR) until 12 months. Secondary outcomes included feasibility, assessed by surgical graft manipulation, and efficacy, assessed using subjective scoring (Nasal Obstruction Symptom Evaluation, NOSE, and Visual Analogue Scale, VAS, scores) and objective breathing function tests. Structural closure of NSP after 12 months was defined using endoscopy and computed tomography (CT) scans. NSP treatment by N-TEC implantation was safe and feasible, as no SAR and no challenge in graft manipulation was recorded for any of the patients. One year postoperative, subjective scoring improved in all patients, unless already optimal (average improvement of 23 and 28.6 points out of 100 respectively for NOSE and VAS scores). Objective respiratory function overall confirmed - with the exception of one case - the observations above (average improvement of 172 ml/s). NSP were closed and the mucosae completely healed in three patients. Autologous N-TEC is a valid treatment for NSP and warrants further clinical tests.

Construction of a lung cancer 3D culture model based on alginate/gelatin micro-beads for drug evaluation

Construction of a lung cancer 3D culture model based on alginate/gelatin micro-beads for drug evaluation

Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay

Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.

Photo/thermo-sensitive chitosan and gelatin-based interpenetrating polymer network for mimicking muscle tissue extracellular matrix

The dynamic interplay between extracellular matrix (ECM), its 3D architecture and resident cells plays a pivotal role in cell behavior influencing essential processes like proliferation, migration, and differentiation. Matrix-based 3D culture systems have emerged as valuable tools to model organ and tissue interactions in vitro. A 3D matrix analog must possess high biocompatibility and fully reproduce the characteristics of the native tissue in terms of mechanical properties. In this regard, interpenetrating polymer networks (IPNs) are particularly attractive because of the high tunability of their physicochemical properties. In this study, a chitosan (Ch) and modified gelatin (GelMA) IPN with a sol-gel transition triggered by two external physical stimuli, UV light and temperature, was designed to mimic the muscle tissue ECM in terms of mechanical stiffness. The system was deeply characterized demonstrating to support not only the growth and viability of muscle cells embedded within the hydrogel, but also cell differentiation toward muscle phenotype.