Poly(ɛ-caprolactone) (PCL) is a biocompatible, biodegradable, and highly mechanically resilient FDA-approved material (for specific biomedical applications, e.g. as drug delivery devices, in sutures, or as an adhesion barrier), rendering it a promising candidate to serve bone tissue engineering. However, in vivo monitoring of PCL-based implants, as well as biodegradable implants in general, and their degradation profiles pose a significant challenge, hindering further development in the tissue engineering field and subsequent clinical adoption. To address this, photo-cross-linkable mechanically resilient PCL networks are developed and functionalized with a radiopaque monomer, 5-acrylamido-2,4,6-triiodoisophthalic acid (AATIPA), to enable non-destructive in vivo monitoring of PCL-based implants. The covalent incorporation of AATIPA into the crosslinked PCL networks does not significantly affect their crosslinking kinetics, mechanical properties, or thermal properties, but it increases their hydrolysis rate and radiopacity. Complex and porous 3D designs of radiopaque PCL networks can be effectively monitored in vivo. This work paves the way toward non-invasive monitoring of in vivo degradation profiles and early detection of potential implant malfunctions.
- MeSH
- biokompatibilní materiály chemie MeSH
- myši MeSH
- polyestery * chemie MeSH
- poréznost MeSH
- testování materiálů MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury * chemie MeSH
- vstřebatelné implantáty MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface in vitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.
Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.
- MeSH
- Alexanderova nemoc * genetika patologie metabolismus MeSH
- astrocyty * metabolismus patologie MeSH
- buněčná diferenciace * fyziologie MeSH
- gliový fibrilární kyselý protein * metabolismus genetika MeSH
- indukované pluripotentní kmenové buňky * metabolismus MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- lidé MeSH
- mutace MeSH
- nervové kmenové buňky metabolismus MeSH
- neurony metabolismus patologie MeSH
- organoidy metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Pancreas is a vital gland of gastrointestinal system with exocrine and endocrine secretory functions, interweaved into essential metabolic circuitries of the human body. Pancreatic ductal adenocarcinoma (PDAC) represents one of the most lethal malignancies, with a 5-year survival rate of 11%. This poor prognosis is primarily attributed to the absence of early symptoms, rapid metastatic dissemination, and the limited efficacy of current therapeutic interventions. Despite recent advancements in understanding the etiopathogenesis and treatment of PDAC, there remains a pressing need for improved individualized models, identification of novel molecular targets, and development of unbiased predictors of disease progression. Here we aim to explore the concept of precision medicine utilizing 3-dimensional, patient-specific cellular models of pancreatic tumors and discuss their potential applications in uncovering novel druggable molecular targets and predicting clinical parameters for individual patients.
- MeSH
- duktální karcinom slinivky břišní * patologie genetika metabolismus MeSH
- individualizovaná medicína * metody MeSH
- lidé MeSH
- nádory slinivky břišní * patologie genetika MeSH
- techniky 3D buněčné kultury metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.
- MeSH
- celulasa * metabolismus MeSH
- endo-1,4-beta-xylanasy metabolismus MeSH
- ethanol metabolismus MeSH
- Hypocreales enzymologie metabolismus růst a vývoj MeSH
- kokultivační techniky * MeSH
- lignin * metabolismus MeSH
- listy rostlin mikrobiologie MeSH
- Penicillium * enzymologie metabolismus růst a vývoj MeSH
- Saccharum * mikrobiologie metabolismus MeSH
- Talaromyces * enzymologie metabolismus růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 μl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.
- MeSH
- analýza nákladů a výnosů MeSH
- buněčné kultury metody ekonomika MeSH
- komplementární DNA * genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- leukocyty mononukleární cytologie metabolismus MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- stanovení celkové genové exprese metody ekonomika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
For the treatment of bilateral limbal stem cell deficiency (LSCD), cell therapy with transplantation of cultivated oral mucosa epithelial cells (COMET) is a promising alternative. Although not yet established, current protocols on the cultivation of oral mucosal epithelial cell (OMECs) sheets are based mainly on substrates and xenobiotic additives that may lead to variable outcomes and undesirable immune responses by the patient. The aim of this study was to characterize OMECs cultivated in xenobiotic-free media (XF) seeded on fibrin gel, in comparison to conventional complex (COM) medium. Oral mucosal biopsies were retrieved from 31 donors. After cultivation in COM or XF medium, OMECs were compared based on growth kinetics, morphology, cell size and viability. Using immunofluorescence and gene expression analyses, the degree of stemness, proliferation and differentiation was evaluated in OMEC cultures. Our findings showed that although OMECs showed a similar morphology and viability, and comparable growth kinetics, immunofluorescence revealed the preservation of stemness (p63 + p40 positivity in cells ≤11 μm) and proliferation in both COM and XF. Gene expression analyses showed that keratin (K)13 and K15 expression levels were significantly higher in XF (adj. p < 0.001), but otherwise COM and XF-treated OMECs had comparable transcriptional profiles in a panel of stemness, proliferation and differentiation genes. These results demonstrate the feasibility of culturing OMECs on fibrin gel without xenogeneic additives, while maintaining their undifferentiated state and preserving stemness. In conclusion, both in terms of results and methodology, the procedures presented here are suitable for implementation in clinical practice.
- MeSH
- buněčná diferenciace MeSH
- buněčné kultury * MeSH
- deficit limbálních kmenových buněk MeSH
- dospělí MeSH
- epitelové buňky * metabolismus účinky léků MeSH
- fibrin * MeSH
- gely MeSH
- kmenové buňky * metabolismus cytologie MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae * cytologie patologie metabolismus MeSH
- nemoci rohovky patologie farmakoterapie metabolismus MeSH
- proliferace buněk účinky léků MeSH
- rohovkový epitel metabolismus cytologie účinky léků patologie MeSH
- senioři MeSH
- transplantace kmenových buněk metody MeSH
- ústní sliznice * cytologie MeSH
- viabilita buněk MeSH
- xenobiotika farmakologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Kontext: Infekční onemocnění covid-19 je spojeno s endotelovou dysfunkcí. Ověřuje se představa možnosti opravy endotelové buňky pomocí endotelových progenitorových buněk (endothelial progenitor cell, EPC) s použitím inhibitorů angiotenzin konvertujícího enzymu (ACEI); podle této představy by se tak mohla zlepšit angiogeneze EPC. Metody: Jedná se o skutečně experimentální in vitro studii s uspořádáním s kontrolní skupinou vytvořenou až po testu. Mononukleární buňky byly izolovány z periferní žilní krve pacienta s chronickým koronárním syndromem, který měl v anamnéze infekční onemocnění covid-19. Buňky byly kultivovány ve speciálním médiu po dobu sedmi dní a buňky EPC byly identifikovány imunocytochemicky pomocí protilátek proti buňkám CD34 radioaktivně značeným fluoresceinizothiocyanátem (FITC) s vyšetřením pod fluorescenčním mikroskopem. Buňky byly rozděleny do dvou skupin, kontrolní a do skupiny s aplikací lisinoprilu v dávce 50 μM. Morfologie a spojení tubulů byly zkoumány pomocí analyzátoru obrázků výrobce Wimasis. Koncentrace von Willebrandova faktoru (vWF) a CD31 byly měřeny metodou ELISA, následovanou statistickým srovnáním obou skupin, přičemž za statisticky významnou byla považována hodnota p < 0,05. Výsledky: Po 144hodinové kultivaci byly buňky EPC identifikovány pomocí světelné a fluorescenční mikroskopie. Parametry tvorby tubulů včetně pokryté plochy (29,6 ± 15,68 vs. 61,8 ± 25,41; p 0,13), celkového počtu tubulů 387 ± 101,55 vs. 382,67 ± 158,53; p 0,97), větvení (163 ± 72,52 vs. 179,66 ± 53,5; p 0,543) a celkového počtu kliček (40,66 ± 30,73 vs. 52,66 ± 5,77; p 0,543) nevykazovaly mezi kontrolní skupinou a skupinou s aplikací lisinoprilu žádný rozdíl. Pokud se týče sérologických biomarkerů, hodnoty CD31 se mezi kontrolami a skupinou s aplikací lisinoprilu statisticky významně nelišily (2 903,58 ± 578,08 vs. 3 361,89 ± 391,24; p 0,319), nicméně hodnoty vWF byly statisticky významně vyšší ve skupině s aplikací lisinoprilu (98,670 ± 3,240 vs. 91,181 ± 2,443; p 0,033). Závěr: U pacientů se stabilní ischemickou chorobou srdeční po prodělaném infekčním onemocnění covid-19 může lisinopril ovlivňovat angiogenezi buněk EPC, jak dokazuje zvyšování hodnot parametrů tvorby tubulů a statisticky významný nárůst koncentrace von Willebrandova faktoru.
Background: COVID-19 infection is associated with endothelial dysfunction. The concept of endothelial cell repair utilizing endothelial progenitor cells (EPCs) with the use of angiotensin-converting enzyme inhibitors (ACEIs) has been developing which is known for its potential for EPC's angiogenesis improvement. Methods: This is a true experimental in vitro study with post-test only control group design. Mononuclear cells were isolated from peripheral venous blood of patient with chronic coronary syndrome and history of COVID-19. The cells then cultured on special media for 7 days and EPC was identified using immunocytochemical examination with labelled anti-CD34 cells FITC-under fluorescence microscope examination. The cells were divided into two groups consisted of control group and 50 μM lisinopril-treated group. The morphology and tube connections were analyzed using Wimasis image analyzer. The von Willebrand factor (vWF) and CD31 concentrations were also measured by ELISA. Statistical comparison between both groups was performed and p-value < 0.05 was considered significant.
- MeSH
- angiogeneze účinky léků MeSH
- antigeny CD34 MeSH
- COVID-19 komplikace MeSH
- dospělí MeSH
- endoteliální progenitorové buňky * účinky léků MeSH
- fluorescenční mikroskopie MeSH
- imunohistochemie metody přístrojové vybavení MeSH
- koronární nemoc farmakoterapie komplikace MeSH
- lidé MeSH
- lisinopril * aplikace a dávkování MeSH
- statistika jako téma MeSH
- techniky in vitro MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- klinická studie MeSH
- práce podpořená grantem MeSH
Non-immune cells, like innate immune cells, can develop a memory-like phenotype in response to priming with microbial compounds or certain metabolites, which enables an enhanced response to a secondary unspecific stimulus. This paper describes a step-by-step protocol for the induction and analysis of trained immunity in human endothelial and smooth muscle cells. We then describe steps for cell culture with cryopreserved vascular cells, subcultivation, and induction of trained immunity. We then provide detailed procedures for downstream analysis using ELISA and qPCR. For complete details on the use and execution of this protocol, please refer to Sohrabi et al. (2020)1 and Shcnack et al.2.
- MeSH
- buněčné kultury MeSH
- ELISA MeSH
- endoteliální buňky * MeSH
- lidé MeSH
- myocyty hladké svaloviny MeSH
- trénovaná imunita * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. METHODS: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. RESULTS: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).
- MeSH
- buněčné kultury metody MeSH
- chronická lymfatická leukemie * patologie metabolismus MeSH
- gely chemie MeSH
- kolagen * chemie farmakologie MeSH
- lidé MeSH
- polyethylenglykoly * chemie MeSH
- receptory CXCR4 metabolismus MeSH
- sodná sůl karboxymethylcelulosy * chemie farmakologie MeSH
- techniky 3D buněčné kultury metody MeSH
- tkáňové podpůrné struktury * chemie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
