Mechanical effects modulate drug resistance in MCF-7-derived organoids: Insights into the wnt/ß-catenin pathway.

Addressing drug resistance poses a significant challenge in cancer treatment, as cancer cells develop diverse mechanisms to evade chemotherapy drugs, leading to treatment failure and disease relapse. Three-dimensional (3D) cell culture has emerged as a valuable model for studying drug resistance, although the underlying mechanisms remain elusive. By obtaining a better understanding of drug resistance within the 3D culture environment, we can develop more effective strategies to overcome it and improve the success of cancer treatments. Notably, the physical structure undergoes notable changes in 3D culture, with mechanical effects believed to play a pivotal role in drug resistance. Hence, our study aimed to explore the influence of mechanical effects on drug resistance by analyzing data related to "drug resistance" and "mechanobiology". Through this analysis, we identified ß-catenin and JNK1 as potential factors, which were further examined in MCF-7 cells cultivated under both 2D and 3D culture conditions. Our findings demonstrate that ß-catenin is activated through canonical and non-canonical pathways and associated with the drug resistance, particularly in organoids obtained under 3D culture.

Activating the healing process: three-dimensional culture of stem cells in Matrigel for tissue repair.

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.

Three-Dimensional Cell Culture Scaffold Supports Capillary-Like Network Formation by Endothelial Cells Derived from Porcine-Induced Pluripotent Stem Cells.

INTRODUCTION: Endothelial cells (EC) can be generated from porcine-induced pluripotent stem cells (piPSC), but poor efficiency in driving EC differentiation hampers their application and efficacy. Additionally, the culture of piPSC-derived EC (piPSC-EC) on three-dimensional (3D) scaffolds has not been fully reported yet. Here, we report a method to improve the generation of EC differentiation from piPSC and to facilitate their culture on 3D scaffolds, providing a potential resource for in vitro drug testing and the generation of tissue-engineered vascular grafts. METHODS: We initiated the differentiation of piPSC into EC by seeding them on laminin 411 and employing a three-stage protocol, which involved the use of distinct EC differentiation media supplemented with CHIR99021, BMP4, VEGF, and bFGF. RESULTS: piPSC-EC not only expressed EC markers such as CD31, VE-cadherin, and von Willebrand factor (vWF) but also exhibited an upregulation of EC marker genes, including CD31, CD34, VEGFR2, VE-cadherin, and vWF. They exhibited functional characteristics similar to those of porcine coronary artery endothelial cells (PCAEC), such as tube formation and Dil-Ac-LDL uptake. Furthermore, when cultured on 3D scaffolds, piPSC-EC developed a 3D morphology and were capable of forming an endothelial layer and engineering capillary-like networks, though these lacked lumen structures. CONCLUSION: Our study not only advances the generation of EC from piPSC through an inhibitor and growth factor cocktail but also provides a promising approach for constructing vascular network-like structures. Importantly, these findings open new avenues for drug discovery in vitro and tissue engineering in vivo.

JAK/STAT3 pathway promotes proliferation of ovarian aggregate-derived stem cells in vitro.

BACKGROUND: The accurate identification and isolation of ovarian stem cells from mammalian ovaries remain a major challenge because of the lack of specific surface markers and suitable in vitro culture systems. Optimized culture conditions for in vitro expansion of ovarian stem cells would allow for identifying requirements of these stem cells for proliferation and differentiation that would pave the way to uncover role of ovarian stem cells in ovarian pathophysiology. Here, we used three-dimensional (3D) aggregate culture system for enrichment of ovarian stem cells and named them aggregate-derived stem cells (ASCs). We hypothesized that mimicking the ovarian microenvironment in vitro by using an aggregate model of the ovary would provide a suitable niche for the isolation of ovarian stem cells from adult mouse and human ovaries and wanted to find out the main cellular pathway governing the proliferation of these stem cells. RESULTS: We showed that ovarian aggregates take an example from ovary microenvironment in terms of expression of ovarian markers, hormone secretion and supporting the viability of the cells. We found that aggregates-derived stem cells proliferate in vitro as long-term while remained expression of germline markers. These ovarian stem cells differentiated to oocyte like cells in vitro spontaneously. Transplantation of these stem cells in to chemotherapy mouse ovary could restore ovarian structure. RNA-sequencing analysis revealed that interleukin6 is upregulated pathway in ovarian aggregate-derived stem cells. Our data showed that JAK/Stat3 signaling pathway which is activated downstream of IL6 is critical for ovarian stem cells proliferation. CONCLUSIONS: We developed a platform that is highly reproducible for in vitro propagation of ovarian stem cells. Our study provides a primary insight into cellular pathway governing the proliferation of ovarian stem cells.

A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom.

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

Muscle Organoid and Assembloid Systems.

Skeletal muscle is one of the most complex and largest tissues that perform important processes in the body, including performing voluntary movements and maintaining body temperature. Disruption of muscle homeostasis results in the development of several disorders, including diabetes and sarcopenia. To study the developmental and regenerative dynamics of skeletal muscle and the mechanism behind muscle diseases, it is important to model skeletal muscle and diseases in vitro. Since skeletal muscle has a complex structure and interaction with other tissues and cells that are required to perform their function, conventional 2D cultures are not sufficient to model the skeletal muscle with their interactions. Advances in the field of organoids and assembloids will enable the establishment of more complex and realistic tissue or disease models which cannot be fully recapitulated in conventional 2D culture systems for use in several areas, including disease research, regenerative, and tissue biology. To overcome these limitations, 3D organoid systems and assembloid systems are promising because of their success in recapitulating the complex structural organization, function, and cellular interactions of skeletal muscle.

In Vitro Growth of Human Follicles: Current and Future Perspectives.

Ovarian tissue cryopreservation is gaining importance as a successful method to restore fertility to girls and young women at high risk of sterility. However, there are concerns regarding the safety of transplantation after ovarian tissue cryopreservation due to the high risk of reintroducing cancer cells and causing disease recurrence. In these cases, the development of culture systems that support oocyte development from the primordial follicle stage is required. Notable achievements have been reached in human follicle in vitro growth in the past decade. Currently, systems for the in vitro culture of ovarian tissue are based on two-dimensional substrates that do not support the survival of follicles or recapitulate the mechanical heterogenicity in the mammalian ovary. Recognition of the importance of special arrangements between cells has spurred research in three-dimensional culture systems, and the provision of a precise culture system that maximizes the diffusion of nutrients and gases through the follicles has raised interest in advanced biomimetic models. The current review critically examines various culture systems employed for the in vitro development of follicles, with a particular focus on solutions utilizing Organ-on-a-Chip (OOC) technology. The emphasis on OOC technology underscores its role as a promising avenue in ensuring the successful cultivation and maintenance of follicular structures during the culture period.

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line.

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.

Notch1 is a marker for in situ resting osteocytes in a 3-dimensional gel culture model.

PURPOSE: Osteocytes in vivo exhibit different functional states, but no specific marker to distinguish these is currently available. MATERIALS AND METHODS: To simulate the differentiation process of pre-osteoblasts to osteocytes in vitro, MC3T3-E1 cells were cultured on type I collagen gel and a three-dimensional (3D) culture system was established. The Notch expression of osteocyte-like cells in 3D culture system was compared with that of in situ osteocytes in bone tissues. RESULTS: Immunohistochemistry demonstrated that Notch1 was not detected in "resting" in situ osteocytes, but was detected in normal cultured osteocyte-like cell line MLO-Y4. Osteocytes obtained from conventional osteogenic-induced osteoblasts and long-term cultured MLO-Y4 cells could not replicate the Notch1 expression pattern from in situ osteocytes. From day 14-35 of osteogenic induction, osteoblasts in 3D culture system gradually migrated into the gel to form canaliculus-like structures similar to bone canaliculus. On day 35, stellate-shaped osteocyte-like cells were observed, and expression of DMP1 and SOST, but not Runx2, was detected. Notch1 was not detected by immunohistochemistry, and Notch1 mRNA level was not significantly different from that of in situ osteocytes. In MC3T3-E1 cells, down-regulation of Notch2 increased Notch1, Notch downstream genes (ß-catenin and Nfatc1), and Dmp1. In MLO-Y4 cells, Notch2 decreased after Notch1 siRNA transfection. Downregulation of Notch1 or Notch2 decreased Nfatc1, ß-catenin, and Dmp1, and increased Sost. CONCLUSIONS: We established "resting state" osteocytes using an in vitro 3D model. Notch1 can be a useful marker to help differentiate the functional states of osteocytes (activated vs. resting state).

Three-dimensional culture of rat ovarian granulosa cells shows increased SPP1 and FGF7 expression through the PI3K/Akt pathway.

Ovarian granulosa cells (OGCs) play an essential role in the regulation of follicular growth and development. However, previous studies of OGCs have concentrated on traditional 2D cultures. In the present study, we used the hanging drop culture method to culture rat OGCs (rOGCs) and assessed the effects of 3D conditions on their proliferation and gene expression profiles. Compared with those grown in 2D conditions, rOGCs grown in 3D cultures showed a significantly different spatial cell distribution and cell alignment under electron microscopy. In particular, rOGCs in 3D cultures showed abundant rough and microvilli-like structures on their cell surface. Here, we showed that these cells grew slowly following 3D culture; the G0/G1-phase increased and the S- and G2/M-phases decreased. Using whole-transcriptome sequencing analysis, 501 genes were shown to have been significantly upregulated and 502 were shown to have been downregulated. Differentially expressed genes were most enriched in pathways involved in focal adhesion, MAPK, and PI3K/Akt signaling according to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Western blotting revealed that SPP1 and FGF7 in the PI3K/Akt pathway were significantly upregulated following 3D culture. These findings improve our understanding of OGCs in real 3D environments in vivo and provide possible avenues for future research on OGCs.

Recombinant irisin enhances the extracellular matrix formation, remodeling potential, and differentiation of human periodontal ligament cells cultured in 3D.

BACKGROUND: Irisin is expressed in human periodontal ligament (hPDL), and its administration enhances growth, migration and matrix deposition in hPDL cells cultured in monolayers in vitro. OBJECTIVES: To identify whether irisin affects the gene expression patterns directing the morphology, mechanical properties, extracellular matrix (ECM) formation, osteogenic activity and angiogenic potential in hPDL cell spheroids cultured in 3D. MATERIALS AND METHODS: Spheroids of primary human hPDL cells were generated in a rotational 3D culture system and treated with or without irisin. The gene expression patterns were evaluated by Affymetrix microarrays. The morphology of the spheroids was characterized using histological staining. Mechanical properties were quantified by nanoindentation. The osteogenic and angiogenic potential of spheroids were assessed through immunofluorescence staining for collagen type I, periostin fibronectin and von Willebrand factor (vWF), and mRNA expression of osteogenic markers. The secretion of multiple myokines was evaluated using Luminex immunoassays. RESULTS: Approximately 1000 genes were differentially expressed between control and irisin-treated groups by Affymetrix. Several genes related to ECM organization were differentially expressed, and multiple deubiquitinating enzymes were upregulated in the irisin-exposed samples analyzed. These represent cellular and molecular mechanisms indicative of a role for irisin in tissue remodeling. Irisin induced a rim-like structure on the outer region of the hPDL spheroids, ECM-related protein expression and the stiffness of the spheroids were enhanced by irisin. The expression of osteogenic and angiogenetic markers was increased by irisin. CONCLUSIONS: Irisin altered the morphology in primary hPDL cell-derived spheroids, enhanced its ECM deposition, mechanical properties, differentiation and remodeling potential.

Three-Dimensional-Cultured MSC-Derived Exosome-Hydrogel Hybrid Microneedle Array Patch for Spinal Cord Repair.

Exosomes derived from mesenchymal stem cells (MSCs) have been proven to exhibit great potentials in spinal cord injury (SCI) therapy. However, conventional two-dimensional (2D) culture will inevitably lead to the loss of stemness of MSCs, which substantially limits the therapeutic potency of MSCs exosomes (2D-Exo). Exosomes derived from three-dimensional culture (3D-Exo) possess higher therapeutic efficiency which have wide applications in spinal cord therapy. Typically, conventional exosome therapy that relies on local repeated injection results in secondary injury and low efficiency. It is urgent to develop a more reliable, convenient, and effective exosome delivery method to achieve constant in situ exosomes release. Herein, we proposed a controlled 3D-exohydrogel hybrid microneedle array patch to achieve SCI repair in situ. Our studies suggested that MSCs with 3D-culturing could maintain their stemness, and consequently, 3D-Exo effectively reduced SCI-induced inflammation and glial scarring. Thus, it is a promising therapeutic strategy for the treatment of SCI.

Regulatory Effects of Three-Dimensional Cultured Lipopolysaccharide-Pretreated Periodontal Ligament Stem Cell-Derived Secretome on Macrophages.

Phenotypic transformation of macrophages plays important immune response roles in the occurrence, development and regression of periodontitis. Under inflammation or other environmental stimulation, mesenchymal stem cells (MSCs) exert immunomodulatory effects through their secretome. It has been found that secretome derived from lipopolysaccharide (LPS)-pretreated or three-dimensional (3D)-cultured MSCs significantly reduced inflammatory responses in inflammatory diseases, including periodontitis, by inducing M2 macrophage polarization. In this study, periodontal ligament stem cells (PDLSCs) pretreated with LPS were 3D cultured in hydrogel (termed SupraGel) for a certain period of time and the secretome was collected to explore its regulatory effects on macrophages. Expression changes of immune cytokines in the secretome were also examined to speculate on the regulatory mechanisms in macrophages. The results indicated that PDLSCs showed good viability in SupraGel and could be separated from the gel by adding PBS and centrifuging. The secretome derived from LPS-pretreated and/or 3D-cultured PDLSCs all inhibited the polarization of M1 macrophages, while the secretome derived from LPS-pretreated PDLSCs (regardless of 3D culture) had the ability to promote the polarization of M1 to M2 macrophages and the migration of macrophages. Cytokines involved in the production, migration and polarization of macrophages, as well as multiple growth factors, increased in the PDLSC-derived secretome after LPS pretreatment and/or 3D culture, which suggested that the secretome had the potential to regulate macrophages and promote tissue regeneration, and that it could be used in the treatment of inflammation-related diseases such as periodontitis in the future.

A Three-Dimensional Xeno-Free Culture Condition for Wharton's Jelly-Mesenchymal Stem Cells: The Pros and Cons.

Xeno-free three-dimensional cultures are gaining attention for mesenchymal stem cell (MSCs) expansion in clinical applications. We investigated the potential of xeno-free serum alternatives, human serum and human platelet lysate, to replace the current conventional use of foetal bovine serum for subsequent MSCs microcarrier cultures. In this study, Wharton's Jelly MSCs were cultured in nine different media combinations to identify the best xeno-free culture media for MSCs culture. Cell proliferation and viability were identified, and the cultured MSCs were characterised in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT). The selected culture media was then used in the microcarrier culture of MSCs to determine the potential of a three-dimensional culture system in the expansion of MSCs for future clinical applications, and to identify the immunomodulatory potential of cultured MSCs. Low Glucose DMEM (LG) + Human Platelet (HPL) lysate media appeared to be good candidates for replacing conventional MSCs culture media in our monolayer culture system. MSCs cultured in LG-HPL achieved high cell yield, with characteristics that remained as described by ISCT, although the overall mitochondrial activity of the cells was lower than the control and the subsequent effects remained unknown. MSC microcarrier culture, on the other hand, showed comparable cell characteristics with monolayer culture, yet had stagnated cell proliferation, which is potentially due to the inactivation of FAK. Nonetheless, both the MSCs monolayer culture and the microcarrier culture showed high suppressive activity on TNF-α, and only the MSC microcarrier culture has a better suppression of IL-1 secretion. In conclusion, LG-HPL was identified as a good xeno-free media for WJMSCs culture, and although further mechanistic research is needed, the results show that the xeno-free three-dimensional culture maintained MSC characteristics and improved immunomodulatory activities, suggesting the potential of translating the monolayer culture into this culture system in MSC expansion for future clinical application.

Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100-130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E2), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E2 and FSH to the culture medium.

Amyloid Beta Peptides Lead to Mast Cell Activation in a Novel 3D Hydrogel Model.

Alzheimer's disease (AD) is a prevalent neurodegenerative disease and the world's primary cause of dementia among the elderly population. The aggregation of toxic amyloid-beta (Aß) is one of the main pathological hallmarks of the AD brain. Recently, neuroinflammation has been recognized as one of the major features of AD, which involves a network of interactions between immune cells. The mast cell (MC) is an innate immune cell type known to serve as a first responder to pathological changes and crosstalk with microglia and neurons. Although an increased number of mast cells were found near the sites of Aß deposition, how mast cells are activated in AD is not clear. We developed a 3D culture system to culture MCs and investigated the activation of MCs by Aß peptides. Because collagen I is the major component of extracellular matrix (ECM) in the brain, we encapsulated human LADR MCs in gels formed by collagen I. We found that 3D-cultured MCs survived and proliferated at the same level as MCs in suspension. Additionally, they can be induced to secrete inflammatory cytokines as well as MC proteases tryptase and chymase by typical MC activators interleukin 33 (IL-33) and IgE/anti-IgE. Culturing with peptides Aß1-42, Aß1-40, and Aß25-35 caused MCs to secrete inflammatory mediators, with Aß1-42 inducing the maximum level of activation. These data indicate that MCs respond to amyloid deposition to elicit inflammatory responses and demonstrate the validity of collagen gel as a model system to investigate MCs in a 3D environment to understand neuroinflammation in AD.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

[Optimization of three-dimensional culture conditions of L02 cells with response surface methodology based on VitroGel system].

OBJECTIVE: This study optimizes three-dimensional(3D)culture conditions of L02 cells using response surface methodology(RSM) based on the VitroGel system to construct the hepatocytes model in vitro. METHODS: L02 cells were 3D cultured by the VitroGel system. The appropriate level of three key factors(concentration of inoculated cells, culture time and dilution degree of the hydrogel) was determined by single-factor experiment, and the optimal conditions of 3D culture of L02 cells based on the VitroGel system were determined by RSM. During the detection process, the optical density(OD) value of cell viability was used as the detection index, and the cell viability was detected using the cell counting kit-8(CCK-8) assay. The proliferative performance and viability of L02 cells was measured by fluorescent staining assay. RESULTS: The selected optimal culture conditions by RSM were as follows: concentration of inoculated cells was 1.1 × 10~5/mL, culture time was 9.5 days, and dilution degree of hydrogel was 1∶3.7. The result shows that under optimal conditions, the predicted OD value of cell viability was 2.17 and measured 2.13 with a relative error of 1.84%, indicating that the condition was suitable and reliable. The fluorescent staining and dead and live cells detection results showed the 3D hepatocytes model was successfully constructed. CONCLUSION: The optimal conditions for 3D culture of L02 cell based on the VitroGel system were determined by RSM, and a hepatocytes model with high cellular activity was successfully constructed.

Three-dimensional Culture Using Atelocollagen Sponge and Self-assembling Peptide Hydrogel.

This study aimed to assess the combined application of two biomaterials, a selfassembling peptide hydrogel (SPH) and an atelocollagen sponge (ACS). The ACS was combined with SPH (PuraMatrixⓇ or PanaceaGelⓇ) and its osteogenic effects on mouse osteoblastic cell line MC3T3 then evaluated. Each type of SPH was successfully incorporated into the ACS. The MC3T3 cells showed uniform distribution within the scaffold. No necrotic cells were observed throughout the experimental procedures. When the SPH was combined with the ACS, the MC3T3 cells differentiated toward the osteo-lineage, expressing Alp, Runx2, Osx, Bsp, and Oc. PanaceaGelⓇ exhibited a stronger osteogenic effect on the cells than PuraMatrixⓇ.

The role of mycotoxins in neurodegenerative diseases: current state of the art and future perspectives of research.

Mycotoxins are fungal metabolites that can cause various diseases in humans and animals. The adverse health effects of mycotoxins such as liver failure, immune deficiency, and cancer are well-described. However, growing evidence suggests an additional link between these fungal metabolites and neurodegenerative diseases. Despite the wealth of these initial reports, reliable conclusions are still constrained by limited access to human patients and availability of suitable cell or animal model systems. This review summarizes knowledge on mycotoxins associated with neurodegenerative diseases and the assumed underlying pathophysiological mechanisms. The limitations of the common in vivo and in vitro experiments to identify the role of mycotoxins in neurotoxicity and thereby in neurodegenerative diseases are elucidated and possible future perspectives to further evolve this research field are presented.