Multilayered Culture Research Articles

ІНТЕГРАЦІЯ РЕСУРСНОГО ПОТЕНЦІАЛУ КАРПАТСЬКОГО РЕГІОНУ УКРАЇНИ У РОЗВИТОК АКТИВНОГО ТУРИЗМУ

Tourism becomes not only a way of recreation, but also a source of development of regions that can confidently compete at the international level. The Carpathian region of Ukraine, with its unique natural and cultural heritage, has significant potential for the development of tourism, including active tourism. The Carpathian region of Ukraine, with its multi-layered culture and unique natural heritage, does not fully utilize its resource potential for the development of active tourism. This problem arises from the insufficient integration of various resources of the region into the strategies and practices of tourism development. The subject of research in the article is the resource potential of the Carpathian region of Ukraine. The purpose of the article is to consider and analyze the possibilities of effective use of the natural resources of the Ukrainian Carpathians for the development of active tourism. Task: research on the integration of the resource potential of the Carpathians in the development of active tourism, to create excellent tourist offers and increase the attractiveness of the region for tourists. General scientific methods are used: system analysis - consideration of system interrelationships and identification of key elements affecting the development of active tourism in the region, literary analysis for the study of scientific articles, books and other sources related to the development of active tourism in mountain regions and integration resource potential. The following results were obtained: on the basis of the conducted analysis, the peculiarities and key trends of the development of the analyzed region were highlighted. The Carpathian region of Ukraine has significant resource potential, but increasing the efficiency of using these resources in the field of active tourism remains an urgent problem. A comprehensive approach to the integration of the resource potential of the Carpathians in the development of active tourism is proposed, and the importance of preserving and sustainable use of natural resources to promote regional development is also emphasized. Specific recommendations for increasing the attractiveness of the region for tourists are presented, which can be used by local authorities, travel agencies, local communities and research centers.

Investigating employer branding with mediation of trust and moderation of social media branding

OBJECTIVE: Drawing on Edgar Schein’s cultural model and the social identity theory, this study attempts to establish an empirical relationship between organizational culture and employer brand with mediation of organizational trust and moderation of employer social media branding. METHODS: The study, being quantitative in nature, used survey method to collect data from 106 employees of public sector universities of Pakistan. A conceptual framework was developed where we tested direct relationship between culture and brand using linear regression. The mediating role of trust and moderation of social media branding were tested with Jamovi statistics. RESULTS: The results suggest that organizational culture has a significant positive relationship with employer brand. Moreover, organizational trust mediates this relationship and employer social media branding emerged as a moderator. CONCLUSION: The study has multidimensional theoretical and practical contributions. It recommends that organizations should use multi-layered culture as an essential tool to develop and improve their employer brand, which can prove a source of attracting quality talent in the modern talent intensive market. This way, organizations can prove themselves as the best places to work. Further, organizations can strengthen their employer brand by improving the trust level between management and employees, and they can harness the matchless power of modern social media for their employer branding activities.

Multilayered Gel-Spotting Device for In Vitro Reconstruction of Hair Follicle-like Microstructure.

Hair follicles play an important role in hair development. This study aimed to develop a microgel-spotting device to fabricate a multilayered gel bead culture model and to mimic the early development of skin appendages to regenerate hair follicles in vitro. The model consists of an alginate gel layer containing cytokines as the core layer, a collagen gel layer containing mouse embryonic stem cells as the middle layer, and a collagen gel layer containing fetus-derived epidermal cells as the outer layer. A concentration gradient of cytokines is formed, which promotes interactions between epidermal and stem cells. Histological and immunnohistological analyses confirmed the reconstruction of hair follicle structures. As a result, the cell number and gel bead size could be precisely controlled by the developed microgel-spotting device. In the multilayered gel bead, the embryonic and epidermal cells cultured with the cytokine gradient formed cell aggregates with keratinized tissue in the center similar to "native" hair follicle structure. Sweat gland-like luminal tissue and erector pilorum-like structures were also observed around aggregates with concentric structures. In conclusion, the multilayered gel bead culture model demonstrated potential for in vitro hair follicle regeneration. The findings of this study provide insight into the early development of skin appendages.

Revolutionizing Disease Modeling: The Emergence of Organoids in Cellular Systems.

Cellular models have created opportunities to explore the characteristics of human diseases through well-established protocols, while avoiding the ethical restrictions associated with post-mortem studies and the costs associated with researching animal models. The capability of cell reprogramming, such as induced pluripotent stem cells (iPSCs) technology, solved the complications associated with human embryonic stem cells (hESC) usage. Moreover, iPSCs made significant contributions for human medicine, such as in diagnosis, therapeutic and regenerative medicine. The two-dimensional (2D) models allowed for monolayer cellular culture in vitro; however, they were surpassed by the three-dimensional (3D) cell culture system. The 3D cell culture provides higher cell-cell contact and a multi-layered cell culture, which more closely respects cellular morphology and polarity. It is more tightly able to resemble conditions in vivo and a closer approach to the architecture of human tissues, such as human organoids. Organoids are 3D cellular structures that mimic the architecture and function of native tissues. They are generated in vitro from stem cells or differentiated cells, such as epithelial or neural cells, and are used to study organ development, disease modeling, and drug discovery. Organoids have become a powerful tool for understanding the cellular and molecular mechanisms underlying human physiology, providing new insights into the pathogenesis of cancer, metabolic diseases, and brain disorders. Although organoid technology is up-and-coming, it also has some limitations that require improvements.

Staphylococcus aureus Fibronectin-Binding Proteins Contribute to Colonization of the Female Reproductive Tract.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and frequent colonizer of human skin and mucosal membranes, including the vagina, with vaginal colonization reaching nearly 25% in some pregnant populations. MRSA vaginal colonization can lead to aerobic vaginitis (AV), and during pregnancy, bacterial ascension into the upper reproductive tract can lead to adverse birth outcomes. USA300, the most prominent MRSA lineage to colonize pregnant individuals, is a robust biofilm former and causative agent of invasive infections; however, little is known about how it colonizes and ascends in the female reproductive tract (FRT). Our previous studies showed that a MRSA mutant of seven fibrinogen-binding adhesins was deficient in FRT epithelial attachment and colonization. Using both monolayer and multilayer air-liquid interface cell culture models, we determine that one class of these adhesins, the fibronectin binding proteins (FnBPA and FnBPB), are critical for association with human vaginal epithelial cells (hVECs) and hVEC invasion through interactions with α5β1 integrin. We observe that both FnBPs are important for biofilm formation as single and double fnbAB mutants exhibit reduced biofilm formation on hVECs. Using heterologous expression of fnbA and fnbB in Staphylococcus carnosus, FnBPs are also found to be sufficient for hVEC cellular association, invasion, and biofilm formation. In addition, we found that an ΔfnbAB mutant displays attenuated ascension in our murine vaginal colonization model. Better understanding of MRSA FRT colonization and ascension can ultimately inform treatment strategies to limit MRSA vaginal burden or prevent ascension, especially during pregnancy and in those prone to AV.

SCALING OF VERO CELL CULTURE FOR THE PRODUCTION OF BIOLOGICAL PRODUCTS

vaccine production is considered the most effective way to prevent and control the spread of infectious diseases. The transplantable Vero cell line is now widely used for vaccine production.The aim of this study was to investigate the optimal parameters for growing Vero cell cultures in cell factories for large-scale vaccine production. For the scaling process, the seed concentration of cells was determined. The results of the studies showed that at a cell inoculum concentration of 2.0x105 cells/ml, a monolayer is formed in 1-2 days and is the most optimal for cultivating most viruses. Multilayer cell culture systems for industrial scales provide a one-stop production solution. The convenient and cost-effective format of cell factories saves space, time and labor and reduces the risk of contamination.In this paper, Vero cell culture is scaled up in cell factories because this cell culture method is necessary to create cost-effective workflows in large volumes. The study showed that the inoculum concentration, the cell proliferation index, and the ratio of the dispersing mixture for cell removal are comparable to routinely used T-vials.

Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture

Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, ¼, and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose–response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.

La Cuisine Pied Noir: wandering in “nostalgia-scape”

ABSTRACT The year 1962 marked the end of the colonial occupation of Algeria by France after more than 130 years. In the wake of a bloody war of independence, nearly 700 000 former European settlers left their old homeland under dramatic circumstances. Most of them ended up in the port of Marseille to find a new livelihood in their motherland France. After their exodus from French Algeria, a state which was doomed to failure, the “Pieds-noirs” developed a multilayered culture of remembrance. Their specific food culture — La Cuisine Pied Noir — emerged as an essential part of their heritage. An interdisciplinary academic approach to this cuisine allows for a deeper understanding of culinary cultural transfer in the context of colonialism and decolonisation. “La Cuisine Pied Noir” highlights the importance of food as a cultural companion and as a guardian of social identity. The drama of the Algerian–French history is still discussed in present-day France. Finally, the archives in both countries are opened to historical studies. This article invites researchers in the field of food culture to participate in a stimulating discussion.

Коммуникация поколений в музейном пространстве: формирование исторических и эстетических представлений о российской дворянской культуре

Prince Nikita Dmitrievich Lobanov-Rostovsky is a world-renowned collector and initiator of large-scale Russian projects: the Monument to soldiers who died in the First World War, in Moscow, the Monument of Reconciliation and Concord, in Sevastopol (projects implemented).Lobanov is the author of a projetct for a National Portrait Gallery in Moscow .The projects seek to recreate the integrity of the multi-layered Russian culture. The idea of the Lobanov-Rostovsky Museum is to fill the gap associated with the visual history of the aristocrats. The project aims to demonstrate the integrity of culture, which includes a historically significant layer - the chronicle of the nobility. Lobanov-Rostovsky's significant gift to the city of Rostov the Great ensured the creation of the museum. Over the last century, the history of the ancient noble families who "built Russia for a thousand years" has largely been forgotten. Oleg Zharov, a major businessman, the head of the Rostov the Great - the Spiritual Center of Russia project, took it upon himself to make up for the historical injustice. The concept of the museum: a combination of diachrony and synchrony, as well as an interdisciplinary approach to museification. This is a museum of the life of noble men, an art museum, a historical one. The diachronic part of the exposition consists of significant historical figures from the Lobanov-Rostovsky family. The synchronic part is artifacts and historical documents from the collection of Nikita Lobanov-Rostovsky, which represent Lobanov as a person and collector, show his artistic priorities. The exhibits of the museum were obtained first-hand, Lobanov in this case acts as a historical witness. The project has cultural value for Russia and is the only one of its kind. The museum will undoubtedly prove to be an attractive object for Russian and foreign tourists and will contribute to the material well-being of the city.

A multi-layer organizational culture framework for enhancing the financial performance in tourism and hospitality family firms

Prior research on factors influencing the financial performance of tourism and hospitality family firms concentrated mainly on family-level traits, thus limiting our comprehension of the organizational culture mechanism by which family-level values are transformed into firm-level business processes. We bridge this void in prior literature by presenting a multi-layer organizational culture framework comprised of the organizational values and organizational climate layers. Data stemmed from eight in-depth interviews, followed by a survey with 187 tourism and hospitality family firms reveal that stewardship climate inside the firms mediates the relationship between long-term-oriented values and financial performance. Moreover, our findings show that entrepreneurial orientation moderates the organizational culture mechanism that enhance the financial performance of tourism and hospitality family firms.

Effects of fibrin matrix and Ishikawa cells on in vitro 3D uterine tissue cultures on a rat model: A controlled study

Background/Aim: In recent years, developing an embryo in in vitro conditions has been one of the most challenging and popular objectives in reproductive biology. In vitro models make observing the relationship between the two possible. Various cell culture and matrix models have been created to overcome embryonic disorganization during culturing. The primary aim of this study was to evaluate and compare the effects of fibrin, Ishikawa cell line, and a combination of both on the 3D multilayer uterine tissue cultures on a rat model, including a control group. Methods: This study was designed as a prospective controlled cohort study. The standard uterine culture model [CNT] (N: 3) constituted the control group. In addition, fibrin matrix-supported [FIB] (N: 3), Ishikawa cells-supported [ISH] (N: 3), and a combination of both [FIB+ISH] (N: 3) culture models were designated as the exposures. All models were cultured for 14 days. Afterwards, the optimal model was determined regarding glucose consumption, lactate production, endometrial thickness and gland count (primary outcomes) with semi-quantitative and statistical methods. Finally, the optimal model was implanted with blastocytes, and the survival duration was observed (secondary outcome). Results: There were significant differences between the groups in terms of glucose, lactate, endometrial thickness (millimeter), and the number of endometrial glands (P

Effects of butyrate\u2212 on ruminal Ca2+ transport: evidence for the involvement of apically expressed TRPV3 and TRPV4 channels

The ruminal epithelium absorbs large quantities of NH4+ and Ca2+. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm2 with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH4+, K+, and Na+ and highly sensitive to GSK1016790A, while effects of butyrate− were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate− (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate− stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4).

Sacræ Stationes. Percorsi processionali, cartografia e turismo emozionale come presupposti di sviluppo locale: possibili casi di smart destination in Sardegna

If it is true that religious processions represent a spiritual phenomenon strongly rooted in history and are testimonials of a past which preserves collective memory, it is also true that they are expressions of an ancient and multilayered culture that have influenced its territory in a unique and unrepeatable way. This collective memory is cyclically recalled by way of displays of ancient rituals. These displays are capable of attracting thousands of the faithful and ever more often groups of tourists interested in religious phenomena. They also lead to the rediscovery of aspects of history, culture, landscape and territory to which they are linked. A modern religious tourist who is less frenetic and more “experiential” would like to immerse him/herself in the sacred and natural surroundings and distance him/herself from the frantic pace of contemporary life. Thus, processional routes, above all in the internal areas at risk of marginalization, may represent the stakeholders of a priceless territorial capital (both environmental and cultural), capable of generating positive external effects and, therefore, to bring to light and promote these routes within plans of territorial valorization. In this sense, cartography, regardless of format, can represent an element of force capable of culturally communicating facts, phenomena, emotions and movements that would otherwise be too difficult to correlate.

Effects of eliminating interactions in multi-layer culture on survival, food utilization and growth of small sea urchins Strongylocentrotus intermedius at high temperatures

Poor growth and disease transmission of small sea urchins Strongylocentrotus intermedius in summer greatly hamper the production efficiency of the longline culture. Reducing the adverse effects of high stocking density while maintaining high biomass is essential to address these problems. Here, we conducted a laboratory experiment to simulate the multi-layer culture for sea urchins at ambient high temperatures (from 22.2 to 24.5 °C) in summer for ~ 7 weeks. Survival, body size, lantern growth, gut weight, food consumption, Aristotle's lantern reflex, 5-hydroxytryptamine concentration, pepsin activity and gut morphology were subsequently evaluated. The present study found that multi-layer culture led to significantly larger body size than those without multi-layer culture (the control group). This was probably because of the greater feeding capacity (indicated by lantern growth and Aristotle's lantern reflex) and food digestion (indicated by morphology and pepsin activity of gut) in the multi-layer cultured sea urchins. These results indicate that multi-layer is an effective approach to improving the growth efficiency of sea urchins at high temperatures. We assessed whether eliminating interaction further improve these commercially important traits of sea urchins in multi-layer culture. This study found that eliminating interactions displayed greater body size and Aristotle's lantern reflex than those not separated in the multi-layer culture. This approach also significantly reduced the morbidity compared with the control group. These novel findings indicate that eliminating interactions in multi-layer culture greatly contributes to the growth and disease prevention of sea urchins at high temperatures. The present study establishes a new technique for the longline culture of sea urchins in summer and provides valuable information into the longline culture management of other commercially important species (e.g. scallops, abalones and oysters).

Multi-Layer Nanofibrous PCL Scaffold-Based Colon Cancer Cell Cultures to Mimic Hypoxic Tumor Microenvironment for Bioassay.

Simple SummaryMulti-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based colon cancer cell cultures mimic the hypoxic tumor microenvironment. The simple procedure generates a 3D hypoxic tumor microenvironment comprising defined numbers and densities of colon cancer cells with easily controllable lateral dimensions and a thickness defined by pNFS. This pNFS-based multi-layered colon cancer cell culture system is useful for bioassays, for drug screening, and as a replacement for small animals in testing the effects of a hypoxic tumor microenvironment.Three-dimensional (3D) cancer cell culture systems have been developed to aid the study of molecular mechanisms in cancer development, identify therapeutic targets, and test drug candidates. In this study, we developed a strategy for mimicking the hypoxic tumor microenvironment in a 3D cancer cell culture system using multi-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based cancer cell cultures. We found that human colon cancer cells infiltrated pNFS within 3 days and could be cultured three-dimensionally within the NFS. When incubated in four stacks of 30 µm-thick pNFS for 3 days, colon cancer cells in layer three showed partially reduced entry into the S phase, whereas those in layer four, located farthest from the media, showed a marked reduction in S-phase entry. As a consequence, cells in layer four exhibited hypoxia-induced disorganization of F-actin on day 3, and those in layers three and four showed an increase in the expression of the hypoxia-induced transcription factor HIF-1α and its target genes, Glut1, CA9, VEGF, and LDHA. Consistent with these results, doxorubicin- and ionizing radiation-induced cell death was reduced in colon cancer cells cultured in layers three and four. These results suggest that pNFS-based multi-layer colon cancer cell cultures mimic the hypoxic tumor microenvironment and are useful for bioassays.

Tannic acid attenuates vascular calcification-induced proximal tubular cells damage through paracrine signaling

Vascular calcification is common in chronic kidney disease; however, the extent to which such condition can affect the renal microvasculature and the neighboring cell types is unclear. Our induced-calcification model in renal proximal tubular (PT) cells exhibited endoplasmic reticulum (ER) stress and oxidative damage, leading to apoptosis. Here, we utilized such calcification in mouse vascular smooth muscle (MOVAS-1) cells as a vascular calcification model, because it exhibited reactive oxygen species (ROS) generation, ER and oxidative stress, inflammatory, and apoptotic gene expressions. To demonstrate whether the vascular calcification condition can dictate the function of the adjacent PT cell layer, we utilized a Transwell multilayer culture system by combining those MOVAS-1 cells in the bottom chamber and polarized PT cells in the upper chamber to show the dimensional cross-signaling effect. Interestingly, calcification of MOVAS-1 cells, in this co-culture, induced H2O2 and lactate dehydrogenase (LDH) release leading to store-operated Ca2+ entry, ROS generation, and activation of oxidative, inflammatory, and apoptotic gene expressions in PT cells through paracrine signaling. Interestingly, application of tannic acid (TA) to either calcified MOVAS-1 or uncalcified PT cells diminished such detrimental pathway activation. Furthermore, the TA-mediated protection was much higher in the PT cells when applied on the calcified MOVAS-1 cells, and the delayed the pathological effects in neighboring PT cells can well be via paracrine signaling. Together, these results provide evidence of vascular calcification-induced PT cell damage, and the protective role of TA in preventing such pathological consequences, which can potentially be used as a nephroprotective remedy.

Segregation in multi-layer culture avoids precocious puberty, improves thermal tolerance and decreases disease transmission in the juvenile sea urchin Strongylocentrotus intermedius: a new approach to longline culture

Mass mortality of commercially cultured marine species in summer is the most serious problem for the longline culture. Reducing crowding stress induced by high stocking density on the basis of high biomass is proposed to be an important approach to solving this issue.A laboratory experiment with multi-layer culture (18 sea urchins total, 6 urchins in each layer) was conducted to investigate gonad yield, gonadal development, thermal tolerance (CTmax) and disease transmission for the juvenile sea urchins Strongylocentrotus intermedius for ~7 weeks at elevated temperatures. The present study found that precocious puberty occurred both in those sea urchins cultured in multi-layer and those not (control group), indicating that gonadal development is accelerated by crowding stress (even in a low density of 6 sea urchins/1834.56 cm3). The CTmax showed a significantly negative correlation with gonadal development, suggesting precocious puberty probably is responsible for the decrease of resistance ability.We further assessed whether segregation in multi-layer culture improves these important traits of sea urchins. This approach significantly reduced gonadal development, improved CTmax and lower mortality and morbidity after disease challenge assays in sea urchins than those not separated in multi-layer culture, which efficiently reduces the gametogenesis, improves resistance ability and decreases disease transmission. The present study establishes a new approach to improving the survival of cultured organisms in the longline culture at high temperatures.

Development of a Method for Scaffold-Free Elastic Cartilage Creation.

Microtia is a congenital aplasia of the auricular cartilage. Conventionally, autologous costal cartilage grafts are collected and shaped for transplantation. However, in this method, excessive invasion occurs due to limitations in the costal cartilage collection. Due to deformation over time after transplantation of the shaped graft, problems with long-term morphological maintenance exist. Additionally, the lack of elasticity with costal cartilage grafts is worth mentioning, as costal cartilage is a type of hyaline cartilage. Medical plastic materials have been transplanted as alternatives to costal cartilage, but transplant rejection and deformation over time are inevitable. It is imperative to create tissues for transplantation using cells of biological origin. Hence, cartilage tissues were developed using a biodegradable scaffold material. However, such materials suffer from transplant rejection and biodegradation, causing the transplanted cartilage tissue to deform due to a lack of elasticity. To address this problem, we established a method for creating elastic cartilage tissue for transplantation with autologous cells without using scaffold materials. Chondrocyte progenitor cells were collected from perichondrial tissue of the ear cartilage. By using a multilayer culture and a three-dimensional rotating suspension culture vessel system, we succeeded in creating scaffold-free elastic cartilage from cartilage progenitor cells.

Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures

IntroductionPeriprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures.MethodsPPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 103/cm2) or multilayer cultures (density 8.5 × 105/cm2) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR.ResultsFibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased.ConclusionThe multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures.

Bio-inspired plant leaf skeleton based three dimensional scaffold for three dimensional cell culture

Large quantities of natural fibers are available in the plant biomass that can be utilized for various purposes including three dimensional cell culture and tissue engineering due to their biocompatibility, ecofriendly, easy availability and cost effective. Especially, leaf skeletons (venation architecture) have a complex hierarchical architecture with novel properties. In this present invention describes about developing three dimensional scaffold from plant leaf skeletons for three dimensional cell culture. Plant leaf skeleton is prepared by simple and rapid method which using sodium hydroxide pretreatment under pressurized condition at 120°C for 1 h. The prepared plant skeleton has microporous surface topography. Also, the plant leaf skeleton is mainly composed by hemicellulose, cellulose and lignin. The microscopic analysis clearly indicates that human mesenchymal stem cells (hMSCs) attached and proliferated on plant leaf skeleton. Interestingly, cell density of hMSCs is increased on plant leaf skeleton by incubation time-dependent manner. Our study confirmed that sodium hydroxide via surface modified Ficus religiosa leaf skeleton enhances the attachment and proliferation of the human mesenchymal stem cells because of their biocompatibility and porous nature. The chemical nature and venation architecture of leaf skeleton has facilitated nutrient and oxygen absorption to promote cell-cell interaction, long term cell culture, and possible scope for induction of cell differentiation. Thus, leaf venation architecture can be applied as three dimensional (3D) cell-culture platform for multi-layer cell culture, cell-based assay model, high-throughput drug screening, cell-replacement therapy, wound healing and substitute for skin. Moreover, this scaffold could also be well-suited to co-culture screening strategies and stem cell differentiation for tissue engineering. Our present invention highlighted agro-wastes as precursor for novel scaffold materials to construct the 3D cell culture platform.