Zonal Patterning of Extracellular Matrix and Stromal Cell Populations Along a Perfusable Cellular Microchannel.

The spatial organization of biophysical and biochemical cues in the extracellular matrix (ECM) in concert with reciprocal cell-cell signaling is vital to tissue patterning during development. However, elucidating the role an individual microenvironmental factor plays using existing in vivo models is difficult due to their inherent complexity. In this work, we have developed a microphysiological system to spatially pattern the biochemical, biophysical, and stromal cell composition of the ECM along an epithelialized 3D microchannel. This technique is adaptable to multiple hydrogel compositions and scalable to the number of zones patterned. We confirmed that the methodology to create distinct zones resulted in a continuous, annealed hydrogel with regional interfaces that did not hinder the transport of soluble molecules. Further, the interface between hydrogel regions did not disrupt microchannel structure, epithelial lumen formation, or media perfusion through an acellular or cellularized microchannel. Finally, we demonstrated spatially patterned tubulogenic sprouting of a continuous epithelial tube into the surrounding hydrogel confined to local regions with stromal cell populations, illustrating spatial control of cell-cell interactions and signaling gradients. This easy-to-use system has wide utility for modeling three-dimensional epithelial and endothelial tissue interactions with heterogeneous hydrogel compositions and/or stromal cell populations to investigate their mechanistic roles during development, homeostasis, or disease.

Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5ß1 integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.

3D layered co-culture model enhances Trastuzumab Deruxtecan sensitivity and reveals the combined effect with G007-LK in HER2-positive non-small cell lung cancer.

Human epidermal growth factor receptor 2 (HER2) aberrations are observed in various cancers. In non-small cell lung cancer, genetic alterations activating HER2, mostly exon 20 insertion mutations, occur in approximately 2-4% of cases. Trastuzumab deruxtecan (T-DXd), a HER2-targeted antibody-drug conjugate has been approved as the first HER2-targeted drug for HER2-mutant lung cancer. However, some cases are not responsive to T-DXd and the primary resistant mechanism remains unclear. In this study, we assessed sensitivity to T-DXd in JFCR-007, a patient-derived HER2-mutant lung cancer cell line. Although JFCR-007 was sensitive to HER2 tyrosine kinase inhibitors, it showed resistance to T-DXd in attachment or spheroid conditions. Accordingly, we established a three-dimensional (3D) layered co-culture model of JFCR-007, where it exhibited a lumen-like structure and became sensitive to T-DXd. In addition, an in-house inhibitor library screening revealed that G007-LK, a tankyrase inhibitor, was effective when combined with T-DXd. G007-LK increased the cytotoxicity of topoisomerase-I inhibitor, DXd, a payload of T-DXd and SN-38. This combined effect was also observed in H2170, an HER2-amplified lung cancer cell line. These results suggest that the proposed 3D co-culture system may help in evaluating the efficacy of T-DXd and may recapitulate the tumor microenvironment.

A Microphysiological Model to Mimic the Placental Remodeling during Early Stage of Pregnancy under Hypoxia-Induced Trophoblast Invasion.

Placental trophoblast invasion is critical for establishing the maternal-fetal interface, yet the mechanisms driving trophoblast-induced maternal arterial remodeling remain elusive. To address this gap, we developed a three-dimensional microfluidic placenta-on-chip model that mimics early pregnancy placentation in a hypoxic environment. By studying human umbilical vein endothelial cells (HUVECs) under oxygen-deprived conditions upon trophoblast invasion, we observed significant HUVEC artery remodeling, suggesting the critical role of hypoxia in placentation. In particular, we found that trophoblasts secrete matrix metalloproteinase (MMP) proteins under hypoxic conditions, which contribute to arterial remodeling by the degradation of extracellular matrix components. This MMP-mediated remodeling is critical for facilitating trophoblast invasion and proper establishment of the maternal-fetal interface. In addition, our platform allows real-time monitoring of HUVEC vessel contraction during trophoblast interaction, providing valuable insights into the dynamic interplay between trophoblasts and maternal vasculature. Collectively, our findings highlight the importance of MMP-mediated arterial remodeling in placental development and underscore the potential of our platform to study pregnancy-related complications and evaluate therapeutic interventions.

Impaired astrocytic synaptic function by peripheral cholesterol metabolite 27-hydroxycholesterol.

Astrocytes represent the most abundant cell type in the brain, where they play critical roles in synaptic transmission, cognition, and behavior. Recent discoveries show astrocytes are involved in synaptic dysfunction during Alzheimer's disease (AD). AD patients have imbalanced cholesterol metabolism, demonstrated by high levels of side-chain oxidized cholesterol known as 27-hydroxycholesterol (27-OH). Evidence from our laboratory has shown that elevated 27-OH can abolish synaptic connectivity during neuromaturation, but its effect on astrocyte function is currently unclear. Our results suggest that elevated 27-OH decreases the astrocyte function in vivo in Cyp27Tg, a mouse model of brain oxysterol imbalance. Here, we report a downregulation of glutamate transporters in the hippocampus of CYP27Tg mice together with increased GFAP. GLT-1 downregulation was also observed when WT mice were fed with high-cholesterol diets. To study the relationship between astrocytes and neurons, we have developed a 3D co-culture system that allows all the cell types from mice embryos to differentiate in vitro. We report that our 3D co-cultures reproduce the effects of 27-OH observed in 2D neurons and in vivo. Moreover, we found novel degenerative effects in astrocytes that do not appear in 2D cultures, together with the downregulation of glutamate transporters GLT-1 and GLAST. We propose that this transporter dysregulation leads to neuronal hyperexcitability and synaptic dysfunction based on the effects of 27-OH on astrocytes. Taken together, these results report a new mechanism linking oxysterol imbalance in the brain and synaptic dysfunction through effects on astrocyte function.

Three-dimensional Co-culture Model for Live Imaging of Pancreatic Islets, Immune Cells, and Neurons in Agarose Gel.

During the onset of autoimmune diabetes, nerve-immune cell interactions seem to play an important role; however, there are currently no models to follow and interfere with these interactions over time in vivo or in vitro. Two-dimensional in vitro models provide insufficient information and microfluidics or organs on a chip are usually challenging to work with. We present here what we believe to be the first simple model that provides the opportunity to co-culture pancreatic islets with sympathetic nerves and immune cells. This model is based on our stamping device that can be 3D printed (STL file provided). Due to the imprint in the agarose gel, sympathetic neurons, pancreatic islets, and macrophages can be seeded in specific locations at a level that allows for confocal live-cell imaging. In this protocol, we provide the instructions to construct and perform live cell imaging experiments in our co-culture model, including: 1) design for the stamping device to make the imprint in the gel, 2) isolation of sympathetic neurons, pancreatic islets, and macrophages, 3) co-culture conditions, 4) how this can be used for live cell imaging, and 5) possibilities for wider use of the model. In summary, we developed an easy-to-use co-culture model that allows manipulation and imaging of interactions between sympathetic nerves, pancreatic islets, and macrophages. This new co-culture model is useful to study nerve-immune cell-islet interactions and will help to identify the functional relevance of neuro-immune interactions in the pancreas. Key features • A novel device that allows for 3D co-culture of sympathetic neurons, pancreatic islets, and immune cells • The device allows the capture of live interactions between mouse sympathetic neurons, pancreatic islets, and immune cells in a controlled environment after six days of co-culturing. • This protocol uses cultured sympathetic neurons isolated from the superior cervical ganglia using a previously established method (Jackson and Tourtellotte, 2014) in a 3D co-culture. • This method requires 3D printing of our own designed gel-stamping device (STL print file provided on SciLifeLab FigShare DOI: 10.17044/scilifelab.24073062).

NF-κB pathway affects silica nanoparticle-induced fibrosis via inhibited inflammatory response and epithelial-mesenchymal transition in 3D co-culture.

Long-term inhalation of silica nanoparticles (SiNPs) can induce pulmonary fibrosis (PF), nevertheless, the potential mechanisms remain elusive. Herein, we constructed a three-dimensional (3D) co-culture model by using Matrigel to investigate the interaction among different cells and potential regulatory mechanisms after SiNPs exposure. Methodologically, we dynamically observed the changes in cell morphology and migration after exposure to SiNPs by co-culturing mouse monocytic macrophages (RAW264.7), human non-small cell lung cancer cells (A549), and medical research council cell strain-5 (MRC-5) in Matrigel for 24 h. Subsequently, we detected the expression of nuclear factor kappa B (NF-κB), inflammatory factor and epithelial-mesenchymal transition (EMT) markers. The results showed that SiNPs produced toxic effects on cells. In the 3D co-culture state, the cell's movement velocity and displacement increased, and the cell migration ability was enhanced. Meanwhile, the expression of inflammatory factor tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) were upregulated, the epithelial marker E-cadherin (E-cad) was downregulated, the mesenchymal marker N-cadherin (N-cad) and myofibroblast marker alpha-smooth muscle actin (α-SMA) expression were upregulated, while NF-κB expression was also upregulated after SiNPs exposure. We further found that cells were more prone to transdifferentiate into myofibroblasts in the 3D co-culture state. Conversely, utilizing the NF-κB-specific inhibitor BAY 11-7082 effectively downregulated the expression of TNF-α, IL-6, interleukin-1ß (IL-1ß), N-cad, α-SMA, collagen-I (COL I), and fibronectin (FN), the expression of E-cad was upregulated. These findings suggest that NF-κB is involved in regulating SiNPs-induced inflammatory, EMT, and fibrosis in the 3D co-culture state.

Expression and role of the immune checkpoint regulator PD-L1 in the tumor-stroma interplay of pancreatic ductal adenocarcinoma.

Introduction: Immune checkpoint inhibitors (ICI), e.g., targeting programmed cell death protein 1-ligand 1 (PD-L1) or its receptor PD-1, have markedly improved the therapy of many cancers but so far failed in pancreatic ductal adenocarcinoma (PDAC). Macrophages represent one of the most abundant immune cell populations within the tumor microenvironment (TME) of PDAC being able to either support or restrain tumor progression depending on their phenotype. To better understand treatment failure of PD-L1/PD-1 inhibitors in PDAC, this study examined PD-L1 expression in the context of a dynamic TME in PDAC with a particular focus on the impact of macrophages. Methods: Formalin-fixed and paraffin embedded tissue samples of primary PDAC tissues and corresponding liver metastases were used for immunohistochemical analyses. Serial sections were stained with antibodies detecting Pan-Cytokeratin, CD68, CD163, CD8, and PD-L1.To investigate whether the PD-1/PD-L1 axis and macrophages contribute to immune escape of PDAC cells, a stroma enriched 3D spheroid coculture model was established in vitro, using different PDAC cell lines and macrophages subtypes as well as CD8+ T cells. Functional and flow cytometry analyses were conducted to characterize cell populations. Results: Immunohistochemical analyses revealed that PD-L1 is mainly expressed by stroma cells, including macrophages and not PDAC cells in primary PDAC tissues and corresponding liver metastases. Notably, high local abundance of macrophages and strong PD-L1 staining were commonly found at invasion fronts of tumoral lesions between CD8+ T cells and tumor cells. In order to investigate whether PD-L1 expressing macrophages impact the response of PDAC cells to treatment with PD-L1/PD-1 inhibitors, we developed a spheroid model comprising two different PDAC cell lines and different ratios of in vitro differentiated primary M1- or M2-like polarized macrophages. In line with our in situ findings, high PD-L1 expression was observed in macrophages rather than PDAC cells, which was further increased by the presence of PDAC cells. The effector phenotype of co-cultured CD8+ T cells exemplified by expression of activation markers and release of effector molecules was rather enhanced by PDAC macrophage spheroids, particularly with M1-like macrophages compared to mono-culture spheroids. However, this was not associated with enhanced PDAC cell death. ICI treatment with either Durvalumab or Pembrolizumab alone or in combination with Gemcitabine hardly affected the effector phenotype of CD8+ T cells along with PDAC cell death. Thus, despite strong PD-L1 expression in macrophages, ICI treatment did not result in an enhanced activation and cytotoxic phenotype of CD8+ T cells. Conclusion: Overall, our study revealed novel insights into the interplay of PDAC cells and macrophages in the presence of ICI.

Partner, Neighbor, Housekeeper and Dimension: 3D versus 2D Glomerular Co-Cultures Reveal Drawbacks of Currently Used Cell Culture Models.

Signaling-pathway analyses and the investigation of gene responses to different stimuli are usually performed in 2D monocultures. However, within the glomerulus, cells grow in 3D and are involved in direct and paracrine interactions with different glomerular cell types. Thus, the results from 2D monoculture experiments must be taken with caution. We cultured glomerular endothelial cells, podocytes and mesangial cells in 2D/3D monocultures and 2D/3D co-cultures and analyzed cell survival, self-assembly, gene expression, cell-cell interaction, and gene pathways using live/dead assay, time-lapse analysis, bulk-RNA sequencing, qPCR, and immunofluorescence staining. Without any need for scaffolds, 3D glomerular co-cultures self-organized into spheroids. Podocyte- and glomerular endothelial cell-specific markers and the extracellular matrix were increased in 3D co-cultures compared to 2D co-cultures. Housekeeping genes must be chosen wisely, as many genes used for the normalization of gene expression were themselves affected in 3D culture conditions. The transport of podocyte-derived VEGFA to glomerular endothelial cells confirmed intercellular crosstalk in the 3D co-culture models. The enhanced expression of genes important for glomerular function in 3D, compared to 2D, questions the reliability of currently used 2D monocultures. Hence, glomerular 3D co-cultures might be more suitable in the study of intercellular communication, disease modelling and drug screening ex vivo.

Anti-cancer effects of metformin in a 3D co-culture model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) presents with condensed stroma that contributes to its high invasive capability. Although metformin adjuvant treatment has been suggested to improve the survival times of patients with PDAC, the mechanism responsible for that benefit has been investigated only in two-dimensional cell lines. We assessed the anti-cancer effect of metformin in a three-dimensional (3D) co-culture model to quantify the migration behavior of patient-derived PDAC organoids and primary pancreatic stellate cells (PSCs). At a concentration of 10 µM, metformin reduced the migratory ability of the PSCs by downregulating the expression of matrix metalloproteinase-2 (MMP2). In the 3D direct co-cultivation of PDAC organoids and PSCs, metformin attenuated the transcription of cancer stemness-related genes. The reduced stromal migratory ability of PSCs was associated with the downregulation of MMP2, and MMP2 knockdown in PSCs reproduced their attenuated migratory ability. The anti-migration effect of a clinically relevant concentration of metformin was demonstrable in a 3D indirect co-culture model of PDAC consisting of patient-derived PDAC organoids and primary human PSCs. The metformin suppressed PSC migration via MMP2 downregulation and attenuated cancer stemness factors. Furthermore, oral administration of metformin (30 mg/kg) strikingly suppressed the growth of PDAC organoids xenograft in immunosuppressed mice. These results indicate metformin could offer the potential approach as an effective therapeutic drug for PDAC.

Organotypic 3D Co-Culture of Human Pleura as a Novel In Vitro Model of Staphylococcus aureus Infection and Biofilm Development.

Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen is Staphylococcus aureus (S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects of S. aureus infection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model with S. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1ß) proved host-pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected with S. aureus resulting in the formation of biofilm, including host-pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema.

A 3D human co-culture to model neuron-astrocyte interactions in tauopathies.

BACKGROUND: Intraneuronal tau aggregation is the major pathological hallmark of neurodegenerative tauopathies. It is now generally acknowledged that tau aggregation also affects astrocytes in a cell non-autonomous manner. However, mechanisms involved are unclear, partly because of the lack of models that reflect the situation in the human tauopathy brain. To accurately model neuron-astrocyte interaction in tauopathies, there is a need for a model that contains both human neurons and human astrocytes, intraneuronal tau pathology and mimics the three-dimensional architecture of the brain. RESULTS: Here we established a novel 100-200 µm thick 3D human neuron/astrocyte co-culture model of tau pathology, comprising homogenous populations of hiPSC-derived neurons and primary human astrocytes in microwell format. Using confocal, electron and live microscopy, we validate the procedures by showing that neurons in the 3D co-culture form pre- and postsynapses and display spontaneous calcium transients within 4 weeks. Astrocytes in the 3D co-culture display bipolar and stellate morphologies with extensive processes that ensheath neuronal somas, spatially align with axons and dendrites and can be found perisynaptically. The complex morphology of astrocytes and the interaction with neurons in the 3D co-culture mirrors that in the human brain, indicating the model's potential to study physiological and pathological neuron-astrocyte interaction in vitro. Finally, we successfully implemented a methodology to introduce seed-independent intraneuronal tau aggregation in the 3D co-culture, enabling study of neuron-astrocyte interaction in early tau pathogenesis. CONCLUSIONS: Altogether, these data provide proof-of-concept for the utility of this rapid, miniaturized, and standardized 3D model for cell type-specific manipulations, such as the intraneuronal pathology that is associated with neurodegenerative disorders.

Targeting Periostin Expression Makes Pancreatic Cancer Spheroids More Vulnerable to Natural Killer Cells.

Pancreatic cancer (PaCa) characteristically has a dense tumor microenvironment, which results in poor patient prognosis. Pancreatic stellate cells (PSCs) are the most abundant cells in the PaCa microenvironment and the principal source of collagen. Periostin, a matricellular protein, is produced specifically by PSCs and promotes the aggressiveness of PaCa cells by facilitating extracellular collagen assembly. Here, we aimed to decrease extracellular collagen assembly by suppressing periostin, thereby increasing the cytotoxic activity of natural killer (NK) cells. Periostin expression was suppressed in PSCs (called PSC-P) using CRISPR-Cas9. PaCa cells (BxPC-3) were co-cultured with PSC and PSC-P cells in a 3D environment to form tumor spheroids mimicking the tumor microenvironment. The extracellular collagen production of spheroids was evaluated by Masson's trichrome staining. The cytotoxic activity of NK-92 cells was analyzed by flow cytometry and confocal microscopy via CD107a staining. Cell death in BxPC-3 cells was evaluated by measuring Annexin-V and PI positivity using flow cytometry. As a result, periostin suppression decreased extracellular collagen and increased the infiltration of NK-92 cells into spheroids, and induced cell death in PaCa cells. In conclusion, we suggest that periostin might be a therapeutic target for PaCa and further analysis is warranted using in vivo models for proof-of-concept.

Dendritic cell phenotype and function in a 3D co-culture model of patient-derived metastatic colorectal cancer organoids.

Colorectal cancer (CRC) remains one of the most aggressive and lethal cancers, with metastasis accounting for most deaths. As such, there is an unmet need for improved therapies for metastatic CRC (mCRC). Currently, the research focus is shifting towards the reciprocal interactions within the tumor microenvironment (TME), which prevent tumor clearance by the immune system. Dendritic cells (DCs) play a key role in the initiation and amplification of anti-tumor immune responses and in driving the clinical success of immunotherapies. Dissecting the interactions between DCs and CRC cells may open doors to identifying key mediators in tumor progression, and possible therapeutic targets. This requires representative, robust and versatile models and tools. Currently, there is a shortage of such in vitro systems to model the CRC TME and its tumor-immune cell interactions. Here we develop and establish a dynamic organotypic 3D co-culture system to recapitulate and untangle the interactions between DCs and patient-derived mCRC tumor organoids. To our knowledge, this is the first study investigating human DCs in co-culture with tumor organoids in a 3D, organotypic setting. This system reveals how mCRC organoids modulate and shape monocyte-derived DCs (MoDCs) behavior, phenotype, and function, within a collagen matrix, using techniques such as brightfield and fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting. Our 3D co-culture model shows high viability and extensive interaction between DCs and tumor organoids, and its structure resembles patient tissue sections. Furthermore, it is possible to retrieve DCs from the co-cultures and characterize their phenotypic and functional profile. In our study, the expression of activation markers in both mature and immature DCs and their ability to activate T cells were impacted by co-culture with tumor organoids. In the future, this direct co-culture platform can be adapted and exploited to study the CRC-DC interplay in more detail, enabling novel and broader insights into CRC-driven DC (dys)function.

Highly Efficient Cardiac Differentiation and Maintenance by Thrombin-Coagulated Fibrin Hydrogels Enriched with Decellularized Porcine Heart Extracellular Matrix.

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend.

A 3D co-culture intestinal organoid system for exploring glucose metabolism.

Many treatments have been used for glucose metabolism diseases such as type 2 diabetes, and all of those treatments have several advantages as well as limitations. This review introduces a 3D co-culture intestinal organoid system developed from stem cells, which has the special function of simulating human tissues. Recent studies have revealed that the gut is an important site for exploring the interactions among glucose metabolism, gut microbial metabolism, and gut microbiota. Therefore, 3D intestinal organoid systems can be used to imitate the congenital errors of human gut development, drug screening, food transportation and toxicity analysis. The intestinal organoid system construction methods and their progress as compared with traditional 2D culture methods have also been summarised in the manuscript. This paper discusses the research progress in terms of intestinal organoids applicable to glucose metabolism and provides new ideas for developing anti-diabetic drugs with high efficiency and low toxicity.

Microenvironmental reprogramming of human dermal papilla cells for hair follicle tissue engineering.

The restoration of hair-inductive potential in human dermal papilla cells (hDPCs) is a tremendous challenge for hair regeneration. Much of the research thus far has indicated that three-dimensional (3-D) culture shows improved efficacy in hair follicle (HF) neogenesis. However, mature HF cannot regenerate in an incomplete microenvironment. This study developed an optimized 3-D co-culture system to restore the hair-inductive characteristics of hDPCs by mimicking the in-vivo microenvironment. As a result, Matrigel-encapsulated hDPCs spontaneously formed into hDPC aggregates (hDPAs), which exhibited better activity, higher proliferation rates, and less apoptosis and hypoxia than the ultra-low attachment culture. Interestingly, the co-culture with the hair matrix cells and dermal sheath cup cells further enhanced the expression of hair regeneration-related genes of hDPAs compared to conditioned medium and improved mature HF induction. In addition, these hDPAs with higher hair inductivity could be produced on a large scale and easily separated for gene expression detection. Finally, the mRNA sequencing, PCR, and WB results showed that the co-culture biomimetic microenvironment stimulated the canonical Wnt signaling pathway and inhibited the BMP signaling pathway. Thus, this co-culture system will provide a reliable platform that allows high-throughput culture, testing, and harvesting of hDPAs for HF tissue engineering. STATEMENT OF SIGNIFICANCE: Extensive hair loss continues to be difficult to treat and causes significant patient morbidity. Hair follicle (HF) tissue engineering may seem to be a way out. However, the absence of the in-vivo microenvironment fails to regenerate mature hairs. This study systematically described a biomimetic co-culture approach to generate better quality human dermal papilla cell aggregates (hDPAs) with improved hair inductive properties, which can be further used for HF tissue engineering. The hDPC microenvironment was reprogrammed through the controllable formation of self-assembled organoids in Matrigel and the tri-culture with hair matrix cells and dermal sheath cup cells. This work indicates that the production of hDPAs could be readily scaled, in theory for large-scale assays, analyses, or therapeutic applications.

Establishment of a 3D co-culture model to investigate the role of primary fibroblasts in ductal carcinoma in situ of the breast.

BACKGROUND: Ductal carcinoma in situ (DCIS) is a precursor form of breast cancer. 13%-50% of these lesions will progress to invasive breast cancer, but the individual progression risk cannot be estimated. Therefore, all patients receive the same therapy, resulting in potential overtreatment of a large proportion of patients. AIMS: The role of the tumor microenvironment (TME) and especially of fibroblasts appears to be critical in DCIS development and a better understanding of their role may aid individualized treatment. METHODS AND RESULTS: Primary fibroblasts isolated from benign or malignant punch biopsies of the breast and MCF10DCIS.com cells were seeded in a 3D cell culture system. The fibroblasts were cultured in a type I collagen layer beneath a Matrigel layer with MCF10DCIS.com cells. Dye-quenched (DQ) fluorescent collagen I and IV were used in collagen and Matrigel layer respectively to demonstrate proteolysis. Confocal microscopy was performed on day 2, 7, and 14 to reveal morphological changes, which could indicate the transition to an invasive phenotype. MCF10DCIS.com cells form smooth, round spheroids in co-culture with non-cancer associated fibroblasts (NAFs). Spheroids in co-culture with tumor-associated fibroblasts (TAFs) appear irregularly shaped and with an uneven surface; similar to spheroids formed from invasive cells. Therefore, these morphological changes represent the progression of an in situ to an invasive phenotype. In addition, TAFs show a higher proteolytic activity compared to NAFs. The distance between DCIS cells and fibroblasts decreases over time. CONCLUSION: The TAFs seem to play an important role in the progression of DCIS to invasive breast cancer. The better characterization of the TME could lead to the identification of DCIS lesions with high or low risk of progression. This could enable personalized oncological therapy, prevention of overtreatment and individualized hormone replacement therapy after DCIS.

Macrophage-Conditioned Media Promotes Adipocyte Cancer Association, Which in Turn Stimulates Breast Cancer Proliferation and Migration.

BACKGROUND: Breast cancer is the most common cancer in women and the leading cause of female cancer deaths worldwide. Obesity causes chronic inflammation and is a risk factor for post-menopausal breast cancer and poor prognosis. Obesity triggers increased infiltration of macrophages into adipose tissue, yet little research has focused on the effects of macrophages in early stages of breast tumor development in obese patients. In this study, the effects of pro-inflammatory macrophages on breast cancer-adipocyte crosstalk were investigated. METHODS: An innovative human cell co-culture system was built and used to model the paracrine interactions among adipocytes, macrophages, and breast cancer cells and how they facilitate tumor progression. The effects on cancer cells were examined using cell counts and migration assays. Quantitative reverse-transcription polymerase chain reaction was used to measure the expression levels of several cytokines and proteases to analyze adipocyte cancer association. RESULTS: Macrophage-conditioned media intensified the effects of breast cancer-adipocyte crosstalk. Adipocytes became delipidated and increased production of pro-inflammatory cytokines, even in the absence of cancer cells, although the expression levels were highest with all three cell components. As a result, co-cultured breast cancer cells became more aggressive, with increased proliferation and migration compared to adipocyte-breast cancer co-cultures treated with unconditioned media. CONCLUSIONS: A novel co-culture model was built to evaluate the crosstalk among human macrophages, adipocytes, and breast cancer cells. We found that macrophages may contribute to adipocyte inflammation and cancer association and thus promote breast cancer progression.

Simplified in vitro 3D co-culture-based blood-brain barrier model using transwell.

The blood-brain barrier (BBB) is a major hurdle for treatment of brain diseases. To overcome this, precise and reproducible BBB model is one of the key factors for successful evaluation of BBB-penetrating efficacy of developmental drugs. Thus, in vitro BBB model recapitulating the physiological structure of the BBB is a valuable tool for drug discovery and development for brain diseases. Here, we develop a simplified 3D co-culture-based BBB model using immortalized human brain endothelial cells and immortalized human astrocytes mixed with Matrigel allowing model preparation within 30 min. We directly compare our 3D BBB model to a 2D BBB model comprised solely of immortalized brain endothelial cells, to demonstrate that our 3D BBB model blocks penetration of Dextran molecules with various molecular weights, remain durable and impermeable even in a BBB-degrading condition, and rapidly form tight junctions while the 2D BBB model do not. In conclusion, this establishes our simplified 3D BBB model as a valuable tool for high throughput screening of drug candidates for brain diseases.