Abstract Formation of new lymphatic vessels, termed lymphangiogenesis, is central for diverse biological processes during development, inflammation and tumor metastasis. However, reliable in vitro ...model is still under demand for detailed elucidation of how sprouting lymphangiogenesis is initiated and coordinated. Here, we describe a microfluidic platform optimized for close reconstitution of lymphangiogenesis, achieved by on-chip integration of salient constituents of lymphatic microenvironment found in vivo . With flexible and precise control over the factors that include biochemical cues, interstitial flow (IF), and endothelial–stromal interactions, we found that orchestrated efforts of multiple environmental factors are necessary for robust lymphatic sprouting in 3D extracellular matrix. Especially, we demonstrate that IF serves as a central regulatory cue which defines lymphangiogenic responses and phenotypes of lymphatic endothelial cells. When synergized with pro-lymphangiogenic factors, IF significantly augmented initiation and outgrowth of lymphatic sprouts toward upstream of the flow while suppressing downstream-directed sprouting. In an appropriate synergism, lymphatic sprouts exhibited structural, molecular signatures and cellular phenotypes that closely approximate sprouting lymphatic neovessels in vivo , and precisely reflected the modulatory effects of pro- and anti-lymphangiogenic stimuli. Our study not only reveals critical but unappreciated role of mechanical cue that regulates lymphangiogenic sprouting, but also provides a novel biomimetic model that may leverage further biological studies as well as phenotypic drug screening.
Metastatic disease remains one of the primary reasons for cancer‐related deaths, yet the majority of in vitro cancer models focus on the primary tumor sites. Here, we describe a metastasis‐on‐a‐chip ...device that houses multiple bioengineered three‐dimensional (3D) organoids, established by a 3D photopatterning technique employing extracellular matrix‐derived hydrogel biomaterials. Specifically, cancer cells begin in colorectal cancer (CRC) organoid, which resides in a single microfluidic chamber connected to multiple downstream chambers in which liver, lung, and endothelial constructs are housed. Under recirculating fluid flow, tumor cells grow in the primary site, eventually enter circulation, and can be tracked via fluorescent imaging. Importantly, we describe that in the current version of this platform, HCT116 CRC cells preferentially home to the liver and lung constructs; the corresponding organs of which CRC metastases arise the most in human patients. We believe that in subsequent studies this platform can be implemented to better understand the mechanisms underlying metastasis, perhaps resulting in the identification of targets for intervention.
Metastatic disease remains one of the primary reasons for cancer‐related deaths, yet the majority of in vitro cancer models focus on the primary tumor sites. Here we describe a metastasis‐on‐a‐chip device, that houses multiple bioengineered three‐dimensional (3D) organoids, established by a 3D photopatterning technique employing extracellular matrix‐derived hydrogel biomaterials.
•TGF-β1 secreted from CAFs promote the migration and invasion of OSCC cells.•CAFs upregulate SOX9 expression of OSCC cells, possibly through inducing EMT.•The presence of CAFs is correlated with SOX9 ...expression in the invasive cancer nests.•The TGF-β/SOX9 axis between CAFs and OSCC cells facilitates cancer progression.•Targeting the TGF-β/SOX9 axis could be a potential novel target for OSCC.
Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.
Recent advances in bone tissue engineering have shown promise for bone repair post osteosarcoma excision. However, conflicting research on mesenchymal stem cells (MSCs) has raised concerns about ...their potential to either promote or inhibit tumor cell proliferation. It is necessary to thoroughly understand the interactions between MSCs and tumor cells. Most previous studies only focused on the interactions between cells within the tumor tissues. It has been challenging to develop an in vitro model of osteosarcoma excision sites replicating the complexity of the bone microenvironment and cell distribution. In this work, we designed and fabricated modular bioceramic scaffolds to assemble into a co-culture model. Because of the bone-like composition and mechanical property, tricalcium phosphate bioceramic could mimic the bone microenvironment and recapitulate the cell-extracellular matrix interaction. Moreover, the properties for easy assembly enabled the modular units to mimic the spatial distribution of cells in the osteosarcoma excision site. Under this co-culture model, MSCs showed a noticeable tumor-stimulating effect with a potential risk of tumor recurrence. In addition, tumor cells also could inhibit the osteogenic ability of MSCs. To undermine the stimulating effects of MSCs on tumor cells, we present the methods of pre-differentiated MSCs, which had lower expression of IL-8 and higher expression of osteogenic proteins. Both in vitro and in vivo studies confirm that pre-differentiated MSCs could maintain high osteogenic capacity without promoting tumor growth, offering a promising approach for MSCs’ application in bone regeneration. Overall, 3D modular scaffolds provide a valuable tool for constructing hard tissue in vitro models.
Bone tissue engineering using mesenchymal stem cells (MSCs) and biomaterials has shown promise for bone repair post osteosarcoma excision. However, conflicting researches on MSCs have raised concerns about their potential to either promote or inhibit tumor cell proliferation. It remains challenges to develop in vitro models to investigate cell interactions, especially of osteosarcoma with high hardness and special composition of bone tissue. In this work, modular bioceramic scaffolds were fabricated and assembled to co-culture models. The interactions between MSCs and MG-63 were manifested as tumor-stimulating and osteogenesis-inhibiting, which means potential risk of tumor recurrence. To undermine the stimulating effect, pre-differentiation method was proposed to maintain high osteogenic capacity without tumor-stimulating, offering a promising approach for MSCs’ application in bone regeneration.
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Cell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the human brain ...microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber – fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60–30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the blood-brain barrier functions.
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•Culture under continuous interstitial flow of a 3D in vitro brain micro-vessel model.•A more defined brain microvasculature after 7 days under flow culture.•No significant change in lumen diameters, but a tendency for more numerous lumen.•Flow stress enhanced gene expression of tight junction, transporters and carriers.•Same tendency for protein expression.
Background
Human adult basal stem/progenitor cells (BSCs) obtained from chronic rhinosinusitis with nasal polyps (CRSwNP) when differentiated in an air‐liquid interface (ALI) usually provide a ...pseudostratified airway epithelium with similar abnormalities than original in vivo phenotype. However, the intrinsic mechanisms regulating this complex process are not well defined and their understanding could offer potential new therapies for CRSwNP (incurable disease).
Methods
We performed a transcriptome‐wide analysis during in vitro mucociliary differentiation of human adult BSCs from CRSwNP, compared to those isolated from control nasal mucosa (control‐NM), in order to identify which key mRNA and microRNAs are regulating this complex process in pathological and healthy conditions.
Results
A number of genes, miRs, biological processes, and pathways were identified during mucociliary differentiation of both CRSwNP and control‐NM epithelia, and notably, we have demonstrated for the first time that genetic transcriptional program responsible of ciliogenesis and cilia function is significantly impaired in CRSwNP epithelium, presumably produced by an altered expression of microRNAs, particularly of those miRs belonging to mir‐34 and mi‐449 families.
Conclusions
This study provides for the first time a novel insight into the molecular basis of sinonasal mucociliary differentiation, demonstrating that transcriptome related to ciliogenesis and cilia function is significantly impaired during differentiation of CRSwNP epithelium due to an altered expression of microRNAs.
This study provides an insight into the molecular basis of sinonasal mucociliary differentiation. mRNA and microRNA transcriptome profiling reveals an altered ciliogenesis in CRSwNP compared to control‐NM. Altered ciliogenesis during mucociliary differentiation may be related to the lack of expression of microRNAs from the mir‐34 and mir‐449 families in CRSwNP epithelium.Abbreviations: ALI, air‐liquid interface cell culture system; BSCs, basal stem‐progenitor cells; control‐NM, control nasal mucosa; miR, microRNA; RSwNP, chronic rhinosinusitis with nasal polyps.
There are currently more than 600 diseases characterized as affecting the central nervous system (CNS) which inflict neural damage. Unfortunately, few of these conditions have effective treatments ...available. Although significant efforts have been put into developing new therapeutics, drugs which were promising in the developmental phase have high attrition rates in late stage clinical trials. These failures could be circumvented if current 2D in vitro and in vivo models were improved. 3D, tissue-engineered in vitro systems can address this need and enhance clinical translation through two approaches: (1) bottom-up, and (2) top-down (developmental/regenerative) strategies to reproduce the structure and function of human tissues. Critical challenges remain including biomaterials capable of matching the mechanical properties and extracellular matrix (ECM) composition of neural tissues, compartmentalized scaffolds that support heterogeneous tissue architectures reflective of brain organization and structure, and robust functional assays for in vitro tissue validation. The unique design parameters defined by the complex physiology of the CNS for construction and validation of 3D in vitro neural systems are reviewed here.
Nanoparticle penetration through tumor tissue after extravasation is considered as a key issue for tumor distribution and therapeutic effects. Most tumors possess abundant stroma, a fibrotic tissue ...composed of cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM), which acts as a barrier for nanoparticle penetration. There is however a lack of suitable in vitro systems to study the tumor stroma penetration of nanoparticles. In the present study, we developed and thoroughly characterized a 3D co-culture spheroidal array to mimic tumor stroma and investigated the penetration of silica and PLGA nanoparticles in these spheroids. First, we examined human breast tumor patient biopsies to characterize the content and organization of stroma and found a high expression of alpha-smooth muscle actin (α-SMA; 40% positive area) and collagen-1 (50% positive area). Next, we prepared homospheroids of 4T1 mouse breast cancer cells or 3T3 mouse fibroblasts alone as well as heterospheroids combining 3T3 and 4T1 cells in different ratios (1:1 and 5:1) using a microwell array platform. Confocal live imaging revealed that fibroblasts distributed and reorganized within 48h in heterospheroids. Furthermore, immunohistochemical staining and gene expression analysis showed a proportional increase of α-SMA and collagen in heterospheroids with higher fibroblast ratios attaining 35% and 45% positive area at 5:1 (3T3:4T1) ratio, in a good match with the clinical breast tumor stroma. Subsequently, we studied the penetration of high and low negatively charged fluorescent silica nanoparticles (30nm; red and 100 or 70nm; green; zeta potential: −40mV and −20mV) and as well as Cy5-conjugated pegylated PLGA nanoparticles (200nm, −7mV) in both homo- and heterospheroid models. Fluorescent microscopy on spheroid cryosections after incubation with silica nanoparticles showed that 4T1 homospheroids allowed a high penetration of about 75–80% within 24h, with higher penetration in case of the 30nm nanoparticles. In contrast, spheroids with increasing fibroblast amounts significantly inhibited NP penetration. Silica nanoparticles with a less negative zeta potential exhibited lesser penetration compared to highly negative charged nanoparticles. Subsequently, similar experiments were conducted using Cy5-conjugated pegylated PLGA nanoparticles and confocal laser scanning microscopy; an increased nanoparticle penetration was found in 4T1 homospheroids until 48h, but significantly lower penetration in heterospheroids. Furthermore, we also developed human homospheroids (MDA-MB-231 or Panc-1 tumor cells) and heterospheroids (MDA-MB-231/BJ-hTert and Panc-1/pancreatic stellate cells) and performed silica nanoparticle (30 and 100nm) penetration studies. As a result, heterospheroids had significantly a lesser penetration of the nanoparticles compared to homospheroids. In conclusion, our data demonstrate that tumor stroma acts as a strong barrier for nanoparticle penetration. The 30-nm nanoparticles with low zeta potential favor deeper penetration. Furthermore, the herein proposed 3D co-culture platform that mimics the tumor stroma, is ideally suited to systematically investigate the factors influencing the penetration characteristics of newly developed nanomedicines to allow the design of nanoparticles with optimal penetration characteristics.
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3-dimensional (3D)
in vitro
models were developed in order to mimic the complexity of real organ/tissue in a dish. They offer new possibilities to model biological processes in more physiologically ...relevant ways which can be applied to a myriad of applications including drug development, toxicity screening and regenerative medicine. Hydrogels are the most relevant tissue-like matrices to support the development of 3D
in vitro
models since they are in many ways akin to the native extracellular matrix (ECM). For the purpose of further improving matrix relevance or to impart specific functionalities, composite hydrogels have attracted increasing attention. These could incorporate drugs to control cell fates, additional ECM elements to improve mechanical properties, biomolecules to improve biological activities or any combinations of the above. In this Review, recent developments in using composite hydrogels laden with cells as biomimetic tissue- or organ-like constructs, and as matrices for multi-cell type organoid cultures are highlighted. The latest composite hydrogel systems that contain nanomaterials, biological factors, and combinations of biopolymers (e.g., proteins and polysaccharide), such as Interpenetrating Networks (IPNs) and Soft Network Composites (SNCs) are also presented. While promising, challenges remain. These will be discussed in light of future perspectives toward encompassing diverse composite hydrogel platforms for an improved organ environment
in vitro
.
Embryo implantation is a complex and highly coordinated process that involves an intricate network of factors establishing intimate contact at the maternal-fetal interface. Knowledge of the human ...implantation process is compromised by both ethical issues, which do not allow the study of this process in vivo, and by the accuracy and reproducibility of in vitro models of human endometrium. Effective and reliable embryo implantation models are, therefore, necessary to mimic the molecular event cascade that occurs in vivo. 3D models are considered a new step to foster precision medicine and an advanced tool for the study of endometrial biology, endometrium associated diseases and to understand the complex mechanisms surrounding endometrium-embryo crosstalk. In this review we explore the various methods by which 3D cultures of endometrium and trophoblast can be created, exploring targets and applications of these in vitro models.
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