From 2D to 3D Co-Culture Systems: A Review of Co-Culture Models to Study the Neural Cells Interaction.

The central nervous system (CNS) controls and regulates the functional activities of the organ systems and maintains the unity between the body and the external environment. The advent of co-culture systems has made it possible to elucidate the interactions between neural cells in vitro and to reproduce complex neural circuits. Here, we classified the co-culture system as a two-dimensional (2D) co-culture system, a cell-based three-dimensional (3D) co-culture system, a tissue slice-based 3D co-culture system, an organoid-based 3D co-culture system, and a microfluidic platform-based 3D co-culture system. We provide an overview of these different co-culture models and their applications in the study of neural cell interaction. The application of co-culture systems in virus-infected CNS disease models is also discussed here. Finally, the direction of the co-culture system in future research is prospected.

In vitro neurotoxicity evaluation of biocidal disinfectants in a human neuron-astrocyte co-culture model.

Biocidal disinfectants (BDs) that kill microorganisms or pathogens are widely used in hospitals and other healthcare fields. Recently, the use of BDs has rapidly increased as personal hygiene has become more apparent owing to the pandemic, namely the coronavirus outbreak. Despite frequent exposure to BDs, toxicity data of their potential neurotoxicity (NT) are lacking. In this study, a human-derived SH-SY5Y/astrocyte was used as a co-culture model to evaluate the chemical effects of BDs. Automated high-content screening was used to evaluate the potential NT of BDs through neurite growth analysis. A set of 12 BD substances classified from previous reports were tested. Our study confirms the potential NT of benzalkonium chloride (BKC) and provides the first evidence of the potential NT of poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB). BKC and PHMB showed significant NT at concentrations without cytotoxicity. This test system for analyzing the potential NT of BDs may be useful in early screening studies for NT prior to starting in vivo studies.

Cytokine Profile and Anti-Inflammatory Activity of a Standardized Conditioned Medium Obtained by Coculture of Monocytes and Mesenchymal Stromal Cells (PRS CK STORM).

Intercellular communication between monocytes/macrophages and cells involved in tissue regeneration, such as mesenchymal stromal cells (MSCs) and primary tissue cells, is essential for tissue regeneration and recovery of homeostasis. Typically, in the final phase of the inflammation-resolving process, this intercellular communication drives an anti-inflammatory immunomodulatory response. To obtain a safe and effective treatment to counteract the cytokine storm associated with a disproportionate immune response to severe infections, including that associated with COVID-19, by means of naturally balanced immunomodulation, our group has standardized the production under GMP-like conditions of a secretome by coculture of macrophages and MSCs. To characterize this proteome, we determined the expression of molecules related to cellular immune response and tissue regeneration, as well as its possible toxicity and anti-inflammatory potency. The results show a specific molecular pattern of interaction between the two cell types studied, with an anti-inflammatory and regenerative profile. In addition, the secretome is not toxic by itself on human PBMC or on THP-1 monocytes and prevents lipopolysaccharide (LPS)-induced growth effects on those cell types. Finally, PRS CK STORM prevents LPS-induced TNF-A and IL-1Β secretion from PBMC and from THP-1 cells at the same level as hydrocortisone, demonstrating its anti-inflammatory potency.

3D Melanoma Cocultures as Improved Models for Nanoparticle-Mediated Delivery of RNA to Tumors.

Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention. This study investigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cell culture models of melanoma. Insights into cell-type specific targeting, non-cell-autonomous effects, and penetration capacity in tumor and stroma cells of the mRNA lipoplex nanoparticles were obtained. It was shown that both coculture of different cell types as well as three-dimensional cell growth characteristics can modulate distribution and transfection efficiency of mRNA lipoplex formulations. The results demonstrate that three-dimensional coculture spheroids can provide a valuable surplus of information in comparison to adherent cells. Thus, they may represent in vitro models with enhanced predictivity for the in vivo activity of cancer nanotherapeutics.

Heterologous Immunity Between SARS-CoV-2 and Pathogenic Bacteria.

Heterologous immunity, when the memory T cell response elicited by one pathogen recognizes another pathogen, has been offered as a contributing factor for the high variability in coronavirus disease 2019 (COVID-19) severity outcomes. Here we demonstrate that sensitization with bacterial peptides can induce heterologous immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) derived peptides and that vaccination with the SARS-CoV-2 spike protein can induce heterologous immunity to bacterial peptides. Using in silico prediction methods, we identified 6 bacterial peptides with sequence homology to either the spike protein or non-structural protein 3 (NSP3) of SARS-CoV-2. Notwithstanding the effects of bystander activation, in vitro co-cultures showed that all individuals tested (n=18) developed heterologous immunity to SARS-CoV-2 peptides when sensitized with the identified bacterial peptides. T cell recall responses measured included cytokine production (IFN-γ, TNF, IL-2), activation (CD69) and proliferation (CellTrace). As an extension of the principle of heterologous immunity between bacterial pathogens and COVID-19, we tracked donor responses before and after SARS-CoV-2 vaccination and measured the cross-reactive T cell responses to bacterial peptides with similar sequence homology to the spike protein. We found that SARS-CoV-2 vaccination could induce heterologous immunity to bacterial peptides. These findings provide a mechanism for heterologous T cell immunity between common bacterial pathogens and SARS-CoV-2, which may explain the high variance in COVID-19 outcomes from asymptomatic to severe. We also demonstrate proof-of-concept that SARS-CoV-2 vaccination can induce heterologous immunity to pathogenic bacteria derived peptides.

Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells.

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune-epithelial interface.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

High Induction of IL-6 Secretion From hUCMSCs Optimize the Potential of hUCMSCs and TCZ as Therapy for COVID-19-Related ARDS.

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).

Macrophages and Monocytes: "Trojan Horses" in COVID-19.

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.

The Role of Epithelial Damage in the Pulmonary Immune Response.

Pulmonary epithelial cells are widely considered to be the first line of defence in the lung and are responsible for coordinating the innate immune response to injury and subsequent repair. Consequently, epithelial cells communicate with multiple cell types including immune cells and fibroblasts to promote acute inflammation and normal wound healing in response to damage. However, aberrant epithelial cell death and damage are hallmarks of pulmonary disease, with necrotic cell death and cellular senescence contributing to disease pathogenesis in numerous respiratory diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and coronavirus disease (COVID)-19. In this review, we summarise the literature that demonstrates that epithelial damage plays a pivotal role in the dysregulation of the immune response leading to tissue destruction and abnormal remodelling in several chronic diseases. Specifically, we highlight the role of epithelial-derived damage-associated molecular patterns (DAMPs) and senescence in shaping the immune response and assess their contribution to inflammatory and fibrotic signalling pathways in the lung.

Adipose-Tissue-Derived Mesenchymal Stem Cells Mediate PD-L1 Overexpression in the White Adipose Tissue of Obese Individuals, Resulting in T Cell Dysfunction.

The PD-L1/PD-1 immune checkpoint axis is the strongest T cell exhaustion inducer. As immune dysfunction occurs during obesity, we analyzed the impact of obesity on PD-L1/PD-1 expression in white adipose tissue (WAT) in mice and in human white adipocytes. We found that PD-L1 was overexpressed in WAT of diet-induced obese mice and was associated with increased expression of PD-1 in visceral but not subcutaneous WAT. Human in vitro cocultures with adipose-tissue-derived mesenchymal stem cells (ASC) and mononuclear cells demonstrated that the presence of ASC harvested from obese WAT (i) enhanced PD-L1 expression as compared with ASC from lean WAT, (ii) decreased Th1 cell cytokine secretion, and (iii) resulted in decreased cytolytic activity towards adipocytes. Moreover, (iv) the implication of PD-L1 in obese ASC-mediated T cell dysfunction was demonstrated through PD-L1 blockade. Finally, (v) conditioned media gathered from these cocultures enhanced PD-L1 expression in freshly differentiated adipocytes, depending on IFNγ. Altogether, our results suggest that PD-L1 is overexpressed in the WAT of obese individuals during IFNγ secretion, leading to T cell dysfunction and notably reduced cytolytic activity. Such a mechanism could shed light on why adipose-tissue-infiltrating viruses, such as SARS-CoV-2, can worsen disease in obese individuals.

BCG Vaccine Derived Peptides Induce SARS-CoV-2 T Cell Cross-Reactivity.

Epidemiological studies and clinical trials suggest Bacillus Calmette-Guérin (BCG) vaccine has protective effects against coronavirus disease 2019 (COVID-19). There are now over 30 clinical trials evaluating if BCG vaccination can prevent or reduce the severity of COVID-19. However, the mechanism by which BCG vaccination can induce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses is unknown. Here, we identify 8 novel BCG-derived peptides with significant sequence homology to either SARS-CoV-2 NSP3 or NSP13-derived peptides. Using an in vitro co-culture system, we show that human CD4+ and CD8+ T cells primed with a BCG-derived peptide developed enhanced reactivity to its corresponding homologous SARS-CoV-2-derived peptide. As expected, HLA differences between individuals meant that not all persons developed immunogenic responses to all 8 BCG-derived peptides. Nevertheless, all of the 20 individuals that were primed with BCG-derived peptides developed enhanced T cell reactivity to at least 7 of 8 SARS-CoV-2-derived peptides. These findings provide an in vitro mechanism that may account, in part, for the epidemiologic observation that BCG vaccination confers some protection from COVID-19.

Ecotoxicological effects of DBPs on freshwater phytoplankton communities in co-culture systems.

Intensive disinfection of wastewater during the COVID-19 pandemic might elevate the generation of toxic disinfection byproducts (DBPs), which has triggered global concerns about their ecological risks to natural aquatic ecosystems. In this study, the toxicity of 17 DBPs typically present in wastewater effluents on three representative microalgae, including Scenedesmus sp. (Chlorophyta), Microcystis aeruginosa (Cyanophyta), and Cyclotella sp. (Bacillariophyta) was investigated. The sensitivities of the three microalgae to DBPs varied greatly from species to species, indicating that DBPs may change the structure of phytoplankton communities. Later, co-cultures of these phytoplankton groups as a proxy of ecological freshwater scenario were conducted to explore the impacts of DBPs on phytoplankton community succession. M. aeruginosa became surprisingly dominant in co-cultures, representing over 50% after dosing with monochloroacetic acid (MCAA, 0.1-10 mg/L). The highest proportion of M. aeruginosa was 70.3% when exposed to 2 mg/L MCAA. Although Scenedesmus sp. dominated in monochloroacetonitrile (MCAN) exposure, M. aeruginosa accounted for no less than 30% even at 40 mg/L MCAN. In this study, DBPs disrupted the original inter-algal relationship in favor of M. aeruginosa, suggesting that DBPs may contribute to the outbreak of cyanobacterial blooms in aquatic ecosystems.

A High-Throughput Distal Lung Air-Blood Barrier Model Enabled By Density-Driven Underside Epithelium Seeding.

High-throughput tissue barrier models can yield critical insights on how barrier function responds to therapeutics, pathogens, and toxins. However, such models often emphasize multiplexing capability at the expense of physiologic relevance. Particularly, the distal lung's air-blood barrier is typically modeled with epithelial cell monoculture, neglecting the substantial contribution of endothelial cell feedback in the coordination of barrier function. An obstacle to establishing high-throughput coculture models relevant to the epithelium/endothelium interface is the requirement for underside cell seeding, which is difficult to miniaturize and automate. Therefore, this paper describes a scalable, low-cost seeding method that eliminates inversion by optimizing medium density to float cells so they attach under the membrane. This method generates a 96-well model of the distal lung epithelium-endothelium barrier with serum-free, glucocorticoid-free air-liquid differentiation. The polarized epithelial-endothelial coculture exhibits mature barrier function, appropriate intercellular junction staining, and epithelial-to-endothelial transmission of inflammatory stimuli such as polyinosine:polycytidylic acid (poly(I:C)). Further, exposure to influenza A virus PR8 and human beta-coronavirus OC43 initiates a dose-dependent inflammatory response that propagates from the epithelium to endothelium. While this model focuses on the air-blood barrier, the underside seeding method is generalizable to various coculture tissue models for scalable, physiologic screening.

Biotransformation of 2,4-dinitrotoluene in a phototrophic co-culture of engineered Synechococcus elongatus and Pseudomonas putida.

In contrast to the current paradigm of using microbial mono-cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co-culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). By encapsulating S. elongatus within a barium-alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4-DNT, enhancing the performance of the co-culture. The synthetic consortium was able to convert 2,4-DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose-dependent accumulation of polyhydroxyalkanoate, an added-value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4-DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs.

Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles Reduce SARS-CoV2-Induced Inflammatory Cytokines Under High Glucose and Uremic Toxin Conditions.

Cytokine storm is recognized as one of the factors contributing to organ failures and mortality in patients with COVID-19. Due to chronic inflammation, COVID-19 patients with diabetes mellitus (DM) or renal disease (RD) have more severe symptoms and higher mortality. However, the factors that contribute to severe outcomes of COVID-19 patients with DM and RD have received little attention. In an effort to investigate potential treatments for COVID-19, recent research has focused on the immunomodulation functions of mesenchymal stem cells (MSCs). In this study, the correlation between DM and RD and the severity of COVID-19 was examined by a combined approach with a meta-analysis and experimental research. The results of a systematic review and meta-analysis suggested that the odd of mortality in patients with both DM and RD was increased in comparison to those with a single comorbidity. In addition, in the experimental research, the data showed that high glucose and uremic toxins contributed to the induction of cytokine storm in human lung adenocarcinoma epithelial cells (Calu-3 cells) in response to SARS-CoV Peptide Pools. Of note, the incorporation of Wharton's jelly MSC-derived extracellular vesicles (WJ-EVs) into SARS-CoV peptide-induced Calu-3 resulted in a significant decrease in nuclear NF-κB p65 and the downregulation of the cytokine storm under high concentrations of glucose and uremic toxins. This clearly suggests the potential for WJ-EVs to reduce cytokine storm reactions in patients with both chronic inflammation diseases and viral infection.

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation.

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts.

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Human Aortic Valve Interstitial Cells Display Proangiogenic Properties During Calcific Aortic Valve Disease.

OBJECTIVE: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90+/CD73+/CD44+ expression and multipotent-like differentiation ability. When VICp were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VICp-conditioned media and confirmed at the mRNA level in VICp compared with control VIC. Conditioned media from VICp induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in angiogenesis by a VEGF-A dependent mechanism. CONCLUSIONS: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.