Human Cell Cultures Research Articles

Sandwich-type channeled chemical hydrogels manufactured by 3D ink-jet printing under freezing conditions using a photochemical process for human cell cultures

This work aims to present the technology of preparing chemical hydrogels for cell culture by printing. It consists in drop-on-drop 3D ink-jet printing under freezing conditions of a solution containing a diacrylic compound (poly(ethylene glycol) diacrylate (PEGDA)) with an initiator (hydrogen peroxide) of photochemically induced polymerization and cross-linking. 3D printing takes place at a low temperature, thanks to which droplets of the solution immediately freeze, allowing the desired structures of the frozen solution to be built. UVC radiation of frozen structures at room temperature and in the presence of oxygen induces simultaneous melting and radical polymerization and cross-linking of PEGDA, resulting in the formation of a firm chemical hydrogel with the same shape as the shape of the printed frozen solution. Discussed are: (i) technological parameters, (ii) reaction mechanism, (iii) sol–gel analysis, (iv) morphology of printed hydrogels and (v) biocompatibility studies with the applied human fibroblasts, human umbilical vein endothelial cells and human neuroblastoma cells. A method for printing sandwich-type channeled structures was developed and shown in application for cell growth. This work demonstrates the feasibility of 3D printing biocompatible chemical hydrogels for cell culture and opens new possibilities for printing hybrid structures for biomedical applications.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4–CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity.

Thymic stromal lymphopoietin (TSLP) is a primarily epithelial-derived cytokine that drives type 2 allergic immune responses. Early life viral respiratory infections elicit high TSLP production, which leads to the development of type 2 inflammation and airway hyperreactivity. The goal of this study was to examine in vivo and in vitro the human airway epithelial responses leading to high TSLP production during viral respiratory infections in early infancy. A total of 129 infants (<1-24 m, median age 10 m) with severe viral respiratory infections were enrolled for in vivo (n = 113), and in vitro studies (n = 16). Infants were classified as 'high TSLP' or 'low TSLP' for values above or below the 50th percentile. High versus low TSLP groups were compared in terms of type I-III IFN responses and production of chemokines promoting antiviral (CXCL10), neutrophilic (CXCL1, CXCL5, CXCL8), and type 2 responses (CCL11, CCL17, CCL22). Human infant airway epithelial cell (AEC) cultures were used to define the transcriptomic (RNAseq) profile leading to high versus low TSLP responses in vitro in the absence (baseline) or presence (stimulated) of a viral mimic (poly I:C). Infants in the high TSLP group had greater in vivo type III IFN airway production (median type III IFN in high TSLP 183.2 pg/mL vs. 63.4 pg/mL in low TSLP group, p = 0.007) and increased in vitro type I-III IFN AEC responses after stimulation with a viral mimic (poly I:C). At baseline, our RNAseq data showed that infants in the high TSLP group had significant upregulation of IFN signature genes (e.g., IFIT2, IFI6, MX1) and pro-inflammatory chemokine genes before stimulation. Infants in the high TSLP group also showed a baseline AEC pro-inflammatory state characterized by increased production of all the chemokines assayed (e.g., CXCL10, CXCL8). High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity and increased baseline AEC production of pro-inflammatory chemokines. These findings present a new paradigm underlying the production of TSLP in the human infant airway epithelium following early life viral exposure and shed light on the long-term impact of viral respiratory illnesses during early infancy and beyond childhood.

A comparative survey of the influence of small self-cleaving ribozymes on gene expression in human cell culture

ABSTRACT Self-cleaving ribozymes are versatile tools for synthetic biologists when it comes to controlling gene expression. Up to date, 12 different classes are known, and over the past decades more and more details about their structure, cleavage mechanisms and natural environments have been uncovered. However, when these motifs are applied to mammalian gene expression constructs, the outcome can often be unexpected. A variety of factors, such as surrounding sequences and positioning of the ribozyme influences the activity and hence performance of catalytic RNAs. While some information about the efficiency of individual ribozymes (each tested in specific contexts) is known, general trends obtained from standardized, comparable experiments are lacking, complicating decisions such as which ribozyme to choose and where to insert it into the target mRNA. In many cases, application-specific optimization is required, which can be very laborious. Here, we systematically compared different classes of ribozymes within the 3’-UTR of a given reporter gene. We then examined position-dependent effects of the best-performing ribozymes. Moreover, we tested additional variants of already widely used hammerhead ribozymes originating from various organisms. We were able to identify functional structures suited for aptazyme design and generated highly efficient hammerhead ribozyme variants originating from the human genome. The present dataset will aide decisions about how to apply ribozymes for affecting gene expression as well as for developing ribozyme-based switches for controlling gene expression in human cells.

The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

Legumain is a lysosomal cysteine protease that has been implicated in an increasing amount of physiological and pathophysiological processes. However, the upstream mechanisms regulating the expression and function of legumain are not well understood. Here, we provide in vitro and in vivo data showing that vitamin D3 (VD3) enhances legumain expression and function. In turn, legumain alters VD3 bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP). Active VD3 (1,25(OH)2D3) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Upregulation of legumain was also observed in vivo, evidenced by increased legumain mRNA in the liver and spleen, as well as increased legumain activity in kidneys from wild-type mice treated with 25(OH)D3 (50 µg/kg, subcutaneously) for 8 days compared to a control. In addition, the serum level of legumain was also increased. We further showed that active legumain cleaved purified VDBP (55 kDa) in vitro, forming a 45 kDa fragment. In vivo, no VDBP cleavage was found in kidneys or liver from legumain-deficient mice (Lgmn-/-), whereas VDBP was cleaved in wild-type control mice (Lgmn+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D3 and total VD3 and altered expression of key renal enzymes involved in VD3 metabolism (CYP24A1 and CYP27B1). In conclusion, a regulatory interplay between VD3 and legumain is suggested.

Isolation and Culture of Human Meibomian Gland Ductal Cells.

Hyperkeratinization of meibomian gland (MG) ducts is currently recognized as the primary pathologic mechanism of meibomian gland dysfunction (MGD). This research figured out a method to isolate the MG ducts and established a novel system to culture the human meibomian gland ductal cells (HMGDCs) for investigating the process of MGD. The MG ducts were obtained from the eyelids of recently deceased donors and subjected to enzymatic digestion. The acini were then removed to isolate independent ducts. These MG ducts were subsequently cultivated on Matrigel-coated wells and covered with a glass plate to obtain HMGDCs. The HMGDCs were further cultivated until passage 2, and when they reached 60% confluence, they were treated with IL-1β and rosiglitazone for a duration of 48 hours. Immunofluorescence staining and Western blot techniques were employed to identify ductal cells and analyze the effects of IL-1β on HMGDCs in an in vitro setting. Ophthalmic micro-forceps and insulin needles can be employed for the purpose of isolating ducts. Within this particular culture system, the rapid expansion of HMGDCs occurred in close proximity to the duct tissue. MG ducts specifically expressed keratin 6 (Krt6) and hardly synthesized lipids. Furthermore, the expression of Krt6 was significantly higher (P < 0.0001) in HMGDCs compared to human meibomian gland cells. Upon treatment with IL-1β, HMGDCs exhibited an overexpression of keratin 1, which was effectively blocked by the administration of rosiglitazone. The present study successfully isolated human MG ducts and cultured HMGDCs, providing a valuable in vitro model for investigating the mechanism of MGD. Additionally, the potential therapeutic efficacy of rosiglitazone in treating hyperkeratinization of ducts in patients with MGD was identified.

Synthesis of Cytotoxic Quino[4,3-b]carbazole Frameworks through an Intramolecular Diels-Alder Reaction.

An intramolecular Diels-Alder reaction of positionally isomeric indole-2/3-phenylvinyl-N-alkynylated (N-phenylsulfonyl)amines has been successfully exploited for the synthesis of quino[4,3-b]carbazole and its analogues. This reaction proceeds through a [4 + 2] cycloaddition followed by elimination and deprotection of phenylsulfonyl units to afford the quinocarbazoles in moderate to good yields. The reaction features a broad substrate scope and remarkable functional group forbearance. A preliminary in vitro cytotoxicity evaluation of representative quino[4,3-b]carbazoles was performed against NCI-H460 human cancer cell culture. Among the quino[4,3-b]carbazoles evaluated, five of the fluorine-containing quinocarbazoles displayed nano molar range (0.8-2.0 nm) GI50 values. The UV-vis and fluorescence spectral studies of representative quinocarbazoles were also performed. Like ellipticine, four of the quinocarbazoles displayed dual emissions confirming the existence of p-quinonoid like tautomeric forms in a polar protic solvent.

Assessing the antiviral potential of melatonin: A comprehensive systematic review.

This review assesses the antiviral potential of melatonin through comprehensive analysis of studies across human subjects, animal models, cell cultures, and in-silico simulations. The search strategy targeted relevant research until 22 June 2023, resulting in 20 primary studies after screening and deduplication. The findings highlight strong evidence supporting antiviral properties of melatonin. In silico studies identify melatonin as a candidate against SARS-CoV-2, reducing cytokine storm-related respiratory responses. Cell culture experiments reveal its multifaceted effects on different viruses including respiratory syncytial virus, anti-dengue virus, transmissible gastroenteritis virus, and encephalomyocarditis virus. Animal studies show melatonin reduces mortality and viral replication in various infections such as Venezuelan equine encephalomyelitis and COVID-19. Clinical trials show how it could be evaluated, but with no conclusive evidence of efficacy and safety so far from large, double-blind placebo-controlled trials. These insights showcase the potential of melatonin as a versatile antiviral agent with immunomodulatory, antioxidant, anti-inflammatory and antiviral properties. In summary, our review highlights melatonin's promising antiviral properties across diverse settings. Melatonin's immunomodulatory and antiviral potential makes it a compelling candidate for further investigation, emphasising the need for rigorous clinical trials to establish its safety and efficacy against viral infections.

Interleukin-38 and cardiovascular pathology: literature review

Cardiovascular pathology is a leading cause of morbidity and mortality. An important task of modern cardiology is the search and study of new biological markers. Scientists’ interest is actively focused on the study of interleukin-38. Interleukin-38 is an anti-inflammatory cytokine and a member of the interleukin-1 family. This study aimed to analyze literature sources devoted to the study of interleukin-38 as a cardiovascular biological marker. Literature sources, including all relevant publications in PubMed (MEDLINE), RSCI, Google Scholar, and Science Direct, were analyzed. The search depth was 9 years. Interleukin-38 is found in the skin, heart, placenta, fetal liver, spleen, thymus, and activated B cells of the tonsils. Interleukin-38 protein is detected in human plasma, serum, and cell cultures by enzyme-linked immunosorbent assay. Interleukin-38 regulates immune and inflammatory responses by binding to its receptors and activating downstream signals. Its deficiency is associated with increased systemic inflammation in aging, cardiovascular diseases, and metabolic diseases. Currently, not much clinical and experimental data have been accumulated regarding the effect of interleukin-38 on the cardiovascular system; however, further studies are expected to demonstrate the possibility of its use as an additional laboratory tool for diagnosis and assessment of prognosis in patients with cardiac problems. Regulating the concentration and expression of interleukin-38 is a promising strategy for the treatment of cardiovascular diseases.

Severe Acute Respiratory Syndrome Coronavirus 2 Infection Alters Mediators of Lung Tissue Remodeling In Vitro and In Vivo.

Altered mediators of airway tissue remodeling such as matrix metalloproteinases (MMPs) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may contribute to morbidity in coronavirus disease 2019 (COVID-19); however, the differential impact of SARS-CoV-2 variants of concern (VOCs) on MMPs is unknown. Using both in vitro human airway cell culture model and in vivo transgenic mouse model of SARS-CoV-2 infection, we studied the differential effect of SARS-CoV-2 VOCs on expression of key MMPs and inflammatory mediators in airway cells and tissues. The most consistent findings with all SARS-CoV-2 variants in infected compared to uninfected human bronchial epithelial cell air-liquid interface cultures were the SARS-CoV-2-induced increases in MMP-12 and tissue inhibitor of MMPs. Infection with both SARS-CoV-2 wild type and SARS-CoV-2 Delta variant over 3 days postinfection (dpi) and with Beta variant over 7 dpi increased lung tissue levels of MMP-9 compared to uninfected mice. Overall, SARS-CoV-2 variants had differential dose-dependent impact on secretion of MMP-1, MMP-2, MMP-9, and MMP-12 that varied at the protein versus the gene level and in the early noninflammatory compared to late inflammatory phase of infection. We provide novel mechanistic insight that the differential impact of SARS-CoV-2 variants on severity of COVID-19 may partially be attributed to unique changes in MMPs.

Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells.

Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD.

Highly compliant biomimetic scaffolds for small diameter tissue-engineered vascular grafts (TEVGs) produced via melt electrowriting (MEW)

Biofabrication approaches toward the development of tissue-engineered vascular grafts (TEVGs) have been widely investigated. However, successful translation has been limited to large diameter applications, with small diameter grafts frequently failing due to poor mechanical performance, in particular mismatched radial compliance. Herein, melt electrowriting (MEW) of poly(ϵ-caprolactone) has enabled the manufacture of highly porous, biocompatible microfibre scaffolds with physiological anisotropic mechanical properties, as substrates for the biofabrication of small diameter TEVGs. Highly reproducible scaffolds with internal diameter of 4.0 mm were designed with 500 and 250 µm pore sizes, demonstrating minimal deviation of less than 4% from the intended architecture, with consistent fibre diameter of 15 ± 2 µm across groups. Scaffolds were designed with straight or sinusoidal circumferential microfibre architecture respectively, to investigate the influence of biomimetic fibre straightening on radial compliance. The results demonstrate that scaffolds with wave-like circumferential microfibre laydown patterns mimicking the architectural arrangement of collagen fibres in arteries, exhibit physiological compliance (12.9 ± 0.6% per 100 mmHg), while equivalent control geometries with straight fibres exhibit significantly reduced compliance (5.5 ± 0.1% per 100 mmHg). Further mechanical characterisation revealed the sinusoidal scaffolds designed with 250 µm pores exhibited physiologically relevant burst pressures of 1078 ± 236 mmHg, compared to 631 ± 105 mmHg for corresponding 500 µm controls. Similar trends were observed for strength and failure, indicating enhanced mechanical performance of scaffolds with reduced pore spacing. Preliminary in vitro culture of human mesenchymal stem cells validated the MEW scaffolds as suitable substrates for cellular growth and proliferation, with high cell viability (>90%) and coverage (>85%), with subsequent seeding of vascular endothelial cells indicating successful attachment and preliminary endothelialisation of tissue-cultured constructs. These findings support further investigation into long-term tissue culture methodologies for enhanced production of vascular extracellular matrix components, toward the development of the next generation of small diameter TEVGs.

Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.

Plate-curving cell culture as a model for assessing curvature-induced cellular responses during neural tube morphogenesis

ABSTRACT Neurulation is an important shape-transforming event during embryonic development where a flat neural plate is converted into a neural tube. Failure in this morphogenetic process accounts for one of the most common birth defects. Mechanical biology has provided key insights into neural tube formation and curvature among many physical properties that are eliciting attention. However, the lack of a proper model to study the effect of curvature has limited the potential to reveal its role in neurulation. In this study, we introduce a novel cell culture method called plate-curving cell culture where a polydimethylsiloxane (PDMS) plate of desired physical properties is curved in either a concave or convex form while the human pluripotent stem cell culture induced to have early neural plate identity is placed on top of its surface. With this method, we observed the elongation of cell colony morphology, as well as the perpendicular alignment of the cell division axis in the concave surface; the oriented cell division does not seem to explain the colony elongation. Transcriptome comparison in search of alternate possibilities suggested selectively altered pathways in the concave surface culture. Our new method is widely available, easy-to-use and culture-friendly, facilitating future mechanobiological studies of neurulation.

Cas9 degradation in human cells using phage anti-CRISPR proteins.

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here, we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. AcrIIA1-induced degradation of SpyCas9 is abolished by mutations in AcrIIA1 that break a direct physical interaction between the 2 proteins. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.

Nanoparticle-Mediated Delivery of Anti-PU.1 siRNA via Localized Intracisternal Administration Reduces Neuroinflammation.

Neuroinflammation is a hallmark of neurodegenerative disorders including Alzheimer's disease (AD). Microglia, the brain's immune cells, express many of the AD-risk loci identified in genome wide association studies and present a promising target for anti-inflammatory RNA therapeutics but are difficult to transfect with current methods. Here, several lipid nanoparticle (LNP) formulations are examined, and a lead candidate that supports efficient RNA delivery in cultures of human stem cell-derived microglia-like cells (iMGLs) and animal models of neuroinflammation is identified. The lead microglia LNP (MG-LNP) formulation shows minimal toxicity and improves delivery efficiency to inflammatory iMGLs, suggesting a preference for delivery into activated microglia. Intraperitoneal injection of the MG-LNP formulation generates widespread expression of the delivered reporter construct in all organs, whereas local intracisternal injection directly into the cerebrospinal fluid leads to preferential expression in the brain. It is shown that LNP-mediated delivery of siRNA targeting the PU.1 transcription factor, a known AD-risk locus, successfully reduces PU.1 levels in iMGLs and reduces neuroinflammation in mice injected with LPS and in CK-p25 mice that mimic the chronic neuroinflammation seen in AD patients. The LNP formulation represents an effective RNA delivery vehicle when applied intrathecally and can be broadly utilized to test potential neuroinflammation-directed gene therapies.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that exvivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Exvivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Enhancing wettability and adhesive properties of PVDF-based substrates through non-thermal helium plasma surface modification

Polyvinylidene fluoride (PVDF) has gained attention as a promising material for tissue engineering due to its biocompatibility and piezoelectricity. However, achieving proper cell adhesion to PVDF surfaces remains a challenge. This study focuses on the preparation and characterization of PVDF-based substrates, including PVDF thin films and nanocomposites incorporating CoFe2O4 magnetic nanoparticles. Helium plasma treatment is employed to modify the surface properties of these substrates. The plasma treatment induces significant changes in the topography of the PVDF-based nanocomposites. The roughness values of the samples increase from 2–3 nm to 14–17 nm after 90 s of plasma treatment, leading to enhanced hydrophilicity with an average contact angle below 60°. Importantly, the helium plasma treatment preserves the magnetic and structural properties of the substrates, suggesting the retention of their magnetoelectric properties and, thus treated composites hold potential for remote stem cell activation. We provide evidence of cytocompatibility and improved adhesive properties of plasma-treated substrates using human mesenchymal stem cell cultures.

Simvastatin induces degradation of the extracellular matrix in human leiomyomata: novel in vitro, in vivo, and patient level evidence of matrix metalloproteinase involvement

ObjectivesTo assess the effect of simvastatin on uterine leiomyoma growth and extracellular matrix (ECM) deposition. DesignLaboratory analysis of human leiomyoma cell culture, xenograft in a mouse model, and patient tissue from a clinical trial. SettingAcademic research center. Patient(s)Tissue culture from human leiomyoma tissue and surgical leiomyoma tissue sections from a placebo-controlled randomized clinical trial. Intervention(s)Simvastatin treatment. Main Outcome Measure(s)Serum concentrations, xenograft volumes, and protein expression. ResultsMice xenografted with 3-dimensional human leiomyoma cultures were divided as follows: 7 untreated controls, 12 treated with activated simvastatin at 10 mg/kg body weight, and 15 at 20 mg/kg body weight. Simvastatin was detected in serum from mice injected at the highest dose. Xenograft volumes were significantly smaller (mean 53% smaller at the highest concentration, p < 0.05). There was dissolution of compact ECM, decreased ECM formation, and lower collagen protein expression in xenografts. Membrane type 1 matrix metalloproteinase (MT1-MMP) was increased in vitro and in vivo. Matrix metalloproteinase 2 (MMP2) and low density lipoprotein receptor related protein 1 (LRP1) were increased in vitro. ConclusionsSimvastatin exhibited antitumoral activity, with extracellular matrix degradation and decreased leiomyoma tumor volume in vivo. Activation of the MMP2/MT1-MMP/LRP1 pathway may explain these findings.

Ascorbic Acid Enhances the Metabolic Activity, Growth and Collagen Production of Human Dermal Fibroblasts Growing in Three-dimensional (3D) Culture

Tissue engineering (TE) enables the development of functional synthetic substitutes to be replaced with damaged tissues and organs instead of the use of auto or allografts. A wide range of biomaterials is currently in use as TE scaffolds. Among these materials, naturally sourced ones are favorable due to being highly biocompatible and supporting cell growth and function, whereas synthetic ones are advantageous because of the high tunability on mechanical and physical properties as well as being easy to process. Alongside the advantages of synthetic polymers, they mostly show hydrophobic behavior that limits biomaterial-cell interaction and, consequently, the functioning of the developed TE constructs. In this study, we assessed the impact of L-Ascorbic acid 2-phosphate (AA2P) on improving the culture conditions of human dermal fibroblasts (HDFs) growing on a three-dimensional (3D) scaffold made of polycaprolactone (PCL) using emulsion templating. Our results demonstrated that AA2P enhances the metabolic activity and growth of HDFs as well as collagen deposition by them when supplemented in their growth medium at 50 µg/mL concentration. It showed a great potential to be used as a growth medium supplement to circumvent the disadvantages of culturing human cells on a synthetic biomaterial that is not favored in default. AA2P's potential to improve cell growth and collagen deposition may prove an effective way to culture human cells on 3D PCL PolyHIPE scaffolds for various TE applications.