Dynamic Culture Research Articles

Millimeter-thick 3D tissues constructed by densely cellularized core–shell microfluidic bioprinting

Recently, microfluidic bioprinting methods, which utilize microfluidic devices as printheads to deposit microfilaments, have improved printing resolution. Despite the precise placement of cells, current efforts have not succeeded in forming densely cellularized tissue within the printed constructs, which is highly desired for the biofabrication of solid-organ tissues with firm tissue consistency. In this paper, we presented a microfluidic bioprinting method to fabricate three dimension tissue constructs consisting of core–shell microfibers where extracellular matrices and cells can be encapsulated within the core of the fibers. Using the optimized printhead design and printing parameters, we demonstrated the bioprinting of core–shell microfibers into macroscale constructs and checked the viability of cells after printing. After culturing the printed tissues using the proposed dynamic culture methods, we analyzed the morphology and function of the tissues both in vitro and in vivo. The confluent tissue morphology in the fiber cores indicates the establishment of intensive cell–cell contacts in the fiber cores, which also leads to the upregulation of the albumin-secretion function compared to the cells cultured in a 2D format. Analysis on the cell density of the confluent fiber cores indicate the formation of densely cellularized tissues with a similar level of cell density of in-vivo solid organ tissues. In the future, better culture techniques with improved perfusion design are anticipated to enable further the fabrication of thicker tissues, which can be used as thick tissue models or implantation grafts for cell therapy.

Baffled-flow culture system enables the mass production of megakaryocytes from human embryonic stem cells by enhancing mitochondrial function.

Human embryonic stem cells (hESCs) have become an ideal cell source for the ex vivo generation of megakaryocyte (MK) and platelet products for clinical applications. However, an ongoing challenge is to establish scalable culture systems to maximize the yield of stem cell-derived MKs that release platelets. We defined a specific dynamic 3D manufacturing system in a baffled-flow manner that could remarkably facilitate megakaryopoiesis and increase the yield of platelet-producing MKs from hESCs within a 12-day induction period. Additionally, an increased number of >16N ploidy MKs, proplatelets, and platelets were generated from induced cells harvested on Day 12 using the specific dynamic culture method. The specific dynamic culture method significantly enhanced endothelium-to-haematopoietic transition and early haematopoiesis. More importantly, MK fate was significantly facilitated in a specific dynamic manner during early haematopoiesis. Mechanistically, this dynamic culture significantly enhanced mitochondrial function via the oxidative phosphorylation pathway and caused differentiation skewing of hESCs toward megakaryopoiesis. This study can aid in the automatic and scalable production of MKs from stem cells using baffled-flow bioreactors and assist in the manufacturing of hESC-derived MK and platelet products.

Development of a Pneumatic-Driven Fiber-Shaped Robot Scaffold for Use as a Complex 3D Dynamic Culture System

Cells can sense and respond to different kinds of continuous mechanical strain in the human body. Mechanical stimulation needs to be included within the in vitro culture system to better mimic the existing complexity of in vivo biological systems. Existing commercial dynamic culture systems are generally two-dimensional (2D) which fail to mimic the three-dimensional (3D) native microenvironment. In this study, a pneumatically driven fiber robot has been developed as a platform for 3D dynamic cell culture. The fiber robot can generate tunable contractions upon stimulation. The surface of the fiber robot is formed by a braiding structure, which provides promising surface contact and adequate space for cell culture. An in-house dynamic stimulation using the fiber robot was set up to maintain NIH3T3 cells in a controlled environment. The biocompatibility of the developed dynamic culture systems was analyzed using LIVE/DEAD™ and alamarBlue™ assays. The results showed that the dynamic culture system was able to support cell proliferation with minimal cytotoxicity similar to static cultures. However, we observed a decrease in cell viability in the case of a high strain rate in dynamic cultures. Differences in cell arrangement and proliferation were observed between braided sleeves made of different materials (nylon and ultra-high molecular weight polyethylene). In summary, a simple and cost-effective 3D dynamic culture system has been proposed, which can be easily implemented to study complex biological phenomena in vitro.

3D dynamic cultures of HGSOC organoids to model innovative and standard therapies.

High-grade serous ovarian cancer (HGSOC) needs new technologies for improving cancer diagnosis and therapy. It is a fatal disease with few options for the patients. In this context, dynamic culture systems coupling with patient-derived cancer 3D microstructures could offer a new opportunity for exploring novel therapeutic approaches. In this study, we optimized a passive microfluidic platform with 3D cancer organoids, which allows a standardized approach among different patients, a minimum requirement of samples, multiple interrogations of biological events, and a rapid response. The passive flow was optimized to improve the growth of cancer organoids, avoiding the disruption of the extracellular matrix (ECM). Under optimized conditions of the OrganoFlow (tilting angle of 15° and an interval of rocking every 8min), the cancer organoids grow faster than when they are in static conditions and the number of dead cells is reduced over time. To calculate the IC 50 values of standard chemotherapeutic drugs (carboplatin, paclitaxel, and doxorubicin) and targeted drugs (ATRA), different approaches were utilized. Resazurin staining, ATP-based assay, and DAPI/PI colocalization assays were compared, and the IC 50 values were calculated. The results showed that in the passive flow, the IC 50 values are lower than in static conditions. FITC-labeled paclitaxel shows a better penetration of ECM under passive flow than in static conditions, and cancer organoids start to die after 48h instead of 96h, respectively. Cancer organoids are the last frontiers for ex vivo testing of drugs that replicate the response of patients in the clinic. For this study, organoids derived from ascites or tissues of patients with Ovarian Cancer have been used. In conclusion, it was possible to develop a protocol for organoid cultures in a passive microfluidic platform with a higher growth rate, faster drug response, and better penetration of drugs into ECM, maintaining the samples' vitals and collecting the data on the same plate for up to 16 drugs.

Development of novel, simple and low–cost microfluidic platform for supporting 3D dynamic cell culture

Cell culture models more accurately would be of significant value to the medical field and pharmaceutical industry. To achieve this goal, microfluidic cell culture platforms are created and improved for modeling the native cell microenvironment because they can precisely reconstruct in vivo cellular behavior. In this study, a 3D low-cost microfluidic device is used to compare the difference between the static and dynamic environment in 3D cell culture. Cells were seeded in the microfluidic device, and to produce the fluidic flow, the pump was used with the set speed was 0.045ml/min. In 3D cell culture, the viability of cells was monitored by size growth of the spheroids for 7 days. All systems were designed and optimized without leakage of the medium. In the results, the 3D dynamic condition showed a faster increase in size than in the static condition. Overall, the study was prepared for microfluidic platforms with low-cost and simple settings. Moreover, the usage of 3D microfluidic to mimic in vivo returned favorable results that were expected for drug testing in the future. 

Fabrication of a Low-Cost Microfluidic Device for High-Throughput Drug Testing on Static and Dynamic Cancer Spheroid Culture Models

Drug development is a complex and expensive process from new drug discovery to product approval. Most drug screening and testing rely on in vitro 2D cell culture models; however, they generally lack in vivo tissue microarchitecture and physiological functionality. Therefore, many researchers have used engineering methods, such as microfluidic devices, to culture 3D cells in dynamic conditions. In this study, a simple and low-cost microfluidic device was fabricated using Poly Methyl Methacrylate (PMMA), a widely available material, and the total cost of the completed device was USD 17.75. Dynamic and static cell culture examinations were applied to monitor the growth of 3D cells. α-MG-loaded GA liposomes were used as the drug to test cell viability in 3D cancer spheroids. Two cell culture conditions (i.e., static and dynamic) were also used in drug testing to simulate the effect of flow on drug cytotoxicity. Results from all assays showed that with the velocity of 0.005 mL/min, cell viability was significantly impaired to nearly 30% after 72 h in a dynamic culture. This device is expected to improve in vitro testing models, reduce and eliminate unsuitable compounds, and select more accurate combinations for in vivo testing.

Viscous Microcapsules as Microbioreactors to Study Mesenchymal Stem/Stromal Cells Osteolineage Commitment.

It is essential to design a multifunctional well-controlled platform to transfer mechanical cues to the cells in different magnitudes. This study introduces a platform, a miniaturized bioreactor, which enables to study the effect of shear stress in microsized compartmentalized structures. In this system, the well-established cell encapsulation system of liquefied capsules (LCs) is used as microbioreactors in which the encapsulated cells are exposed to variable core viscosities to experience different mechanical forces under a 3D dynamic culture. The LC technology is joined with electrospraying to produce such microbioreactors at high rates, thus allowing the application of microcapsules for high-throughput screening. Using this platform for osteogenic differentiation as an example, shows that microbioreactors with higher core viscosity which produce higher shear stress lead to significantly higher osteogenic characteristics. Moreover, in this system the forces experienced by cells in each LC are simulated by computational modeling. The maximum wall shear stress applied to the cells inside the bioreactor with low, and high core viscosity environment is estimated to be 297 and 1367 mPa, respectively, for the experimental setup employed. This work outlines the potential of LC microbioreactors as a reliable in vitro customizable platform with a wide range of applications.

Easy-to-Build and Reusable Microfluidic Device for the Dynamic Culture of Human Bronchial Cystic Fibrosis Epithelia.

Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air-liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

The effect of Nano-liposomal sodium nitrite on smooth muscle cell growth in a tissue-engineered small-diameter vascular graft.

Nitric oxide is a chemical agent produced by endothelial cells in a healthy blood vessel, inhibiting the overgrowth of vascular smooth muscle cells and regulating vessel tone. Liposomes are biocompatible and biodegradable drug carriers with a similar structure to cell bilayer phospholipid membrane that can be used as useful nitric oxide carriers in vascular grafts. Using a custom-designed apparatus, the sheep carotid arteries were decellularized while still maintaining important components of the vascular extracellular matrix (ECM), allowing them to be used as small-diameter vascular grafts. A chemical signal of sodium nitrite was applied to control smooth muscle cells' behavior under static and dynamic cell culture conditions. The thin film hydration approach was used to create nano-liposomes, which were then used as sodium nitrite carriers to control the drug release rate and enhance the amount of drug loaded into the liposomes. The ratio of 80:20:2 for DPPC: Cholesterol: PEG was determined as the optimum formulation of the liposome structure with high drug encapsulation efficiency (98%) and optimum drug release rate (the drug release rate was 40%, 65%, and 83% after 24, 48, and 72h, respectively). MTT assay results showed an improvement in endothelial cell proliferation in the presence of nano-liposomal sodium nitrite (LNS) at the concentration of 0.5μg/mL. Using a suitable concentration of liposomal sodium nitrite (0.5μg/mL) put onto the constructed scaffold resulted in the controllable development of smooth muscle cells in the experiment. The culture of smooth muscle cells in a pulsatile perfusion bioreactor indicated that in the presence of synthesized liposomal sodium nitrite, the overgrowth of smooth muscle cells was inhibited in dynamic cell culture conditions. The mechanical properties of ECM graft were measured, and a multi-scale model with an accuracy of 83% was proposed to predict mechanical properties successfully. The liposomal drug-loaded small-diameter vascular graft can prevent the overgrowth of SMCs and the formation of intimal hyperplasia in the graft. Aside from that, the effect of LNS on endothelial has the potential to stimulate endothelial cell proliferation and re-endothelialization.

Abstract 155: Towards the development of an in vitro human vascularized immunocompetent metastatic melanoma-on-chip model

Abstract Despite recent extraordinary clinical success of immune checkpoint inhibitors (ICIs) in treating people affected by melanoma, a relevant number of patients still develops an adaptive resistance resulting in poor prognosis. To accelerate access to new therapies, there is a strong need for an in vitro human melanoma model mimicking the complexity of the tumor's cellular and physical microenvironment. Such model should recapitulate 3 critical features: (1) a reconstructed human melanoma-in-skin (Mel-RhS) to model melanoma cell invasion, (2) an endothelium to mimic the vascular system, and (3) circulation of, and ultimately, infiltration by, immune cells. Here, we present the development of these 3 individual biological features separately in dynamic conditions. These were constructed in standard multi-well plates (MW) by means of our microenviromentally controlled microphysiological system (MPS) (CubiX). This is a critical step towards the creation of a fully differentiated vascularized immunocompetent metastatic melanoma-on-chip model to recapitulate human pathophysiology, suitable for testing immunotherapies and studying the onset of possible resistance mechanisms. (1) Mel-RhS were constructed on transwells (8 μm pore size) and consisted of a human fibroblasts-populated collagen-fibrin dermal compartment, on top of which A375 melanoma cells and human keratinocytes were seeded. Mel-RhS were air-exposed for > 14 days to form of a fully differentiated and stratified epidermis. Melanoma nests developed and expanded into the dermal compartment in the 3D model, mimicking the initial stages of invasive melanoma. Moreover, Mel-RhS were viable under flow conditions for up to 3 days and their histology was comparable to that of the static controls. (2) The endothelial layer consisted of a monolayer of CD31+ endothelial cells on the bottom side of the transwell membrane. The shear stress provided by medium perfusion was able to induce alignment (75-80%) of cells within 24 hours (3 times faster than published protocols) as well as Von Willebrand Factor production. Real-time pH, lactate, glucose, and oxygen consumption were measured by using in-line sensors during the culture time. (3) Circulation of immune cells (MUTZ-3 progenitor cells) was achieved up to 24 hours in the presence of a healthy reconstructed human skin (RhS) model in a proof-of-concept study. The specific design of the developed MPS allowed for both immune cell flow and collection for downstream phenotypic analysis. In conclusion, we have individually recapitulated these major biological features of melanoma in vitro, in standard MW under dynamic culture conditions. In the future, the integration of these features into a single model would provide a unique in vitro vascularized immunocompetent human melanoma-on-chip model as a novel tool to investigate and test new therapies targeting adaptive resistance to ICIs. Citation Format: Elisabetta Michielon, Tanja de GruIijl, Taco Waaijman, Matteo Boninsegna, Divyasree Prabhakaran, Hadhemi Mejri, Antoni Homs Corbera, Pierre Gaudriault, Susan Gibbs, Dario Fassini. Towards the development of an in vitro human vascularized immunocompetent metastatic melanoma-on-chip model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 155.

Religious Integrity and Local Culture (Descriptive Study on Cicarucub Indigenous Peoples of Lebak Banten)

The relationship between religion (Islam) and culture is often manifested in accommodative, complementary, and conflicting forms, but for the people of South Banten, the relationship between religion and culture (tradition) looks more complementary. Islam and ancestral culture have similar values, so a dynamic, dialectical relationship is created so that an integral relationship is realized. This study employs an ethnographic approach, which explains in depth about humans, in this case the Cicarucub community. The method used is a qualitative method; this method develops and solves problems that have been described and analysed in terms of religious and cultural integrity. The results of this study state that there are cultural meanings that have similarities with Islamic teachings. The relationship between religion (Islam) and culture (Sunda) is proof that Islam is a dynamic religion; it is permissible to modify new teachings as long as they do not conflict with the al-Qur`an, hadith, the consensus of the scholars (ijma'), and qiyas (analogy). Sundanese culture is also a very dynamic culture, able to coexist with religious teachings, especially Islam.

THE PEACE MOVEMENT TO END THE RIVALRY BETWEEN FOOTBALL SUPPORTERS OF PSS SLEMAN AND PSIM YOGYAKARTA

Football in Indonesia is a dynamic popular culture that is associated with the emotional instability of its fans. Fanaticism is something that is believed to give a greater form of love and zest for life for a person or group, while on the other hand, rivalry can also be said to be a form of anarchism of supporters. The purpose of this study was to find out the form of communication from the virtual community of two supporters in Yogyakarta in realizing better football. This research is qualitative studies that using netnography and virtual observation as the method. The rivalry between PSS Sleman and PSIM Yogyakarta supporters is a form of fanaticism or an exaggerated form of love. This research has a point that makes them reflect on each other, namely the Kanjuruhan tragedy which caused 135 lives to be lost. This made Sleman and Yogyakarta supporters reflect and produce a form of virtual communication that builds and leads to a peace movement. It is seen from this research that adaptive structuring occurs both within the scope of individuals and groups of supporters that an adult form of love for football is a peace. In the form of transactional analysis, it is also seen that the form of change or maturity of these supporters is formed based on the awareness and willingness of all layers of Sleman and Yogyakarta supporters so that peace is created.
 Keywords: Fanaticism & Rivality; Peace; Virtual Communities; Netnography.

Women Incarcerated in Rural Southern Prisons in the United States: A Review of Existing Multidisciplinary Literature and Suggestions for Future Directions

Prisons in the Southern United States are a particularly unique kind of rural institutions not only because of their geographic locations, social climates informed by the rural cultures of staff and prisoners, and, for many older Southern prisons, their roots in plantation agriculture. Despite these realities, rural criminology has yet to systematically synthesize and explore what existing research indicates about the everyday lives of over 30,000 women currently serving time in state prisons throughout the Southern United States. The present study attempts to fill this gap in the literature by synthesizing all the available literature on women incarcerated in rural Southern prisons and identifying four prevailing themes in this body of work: regional culture in historical context, relationships and social dynamics, victimization and wellbeing, and journeys through the system from sentencing to reentry.

Scaffold geometry and computational fluid dynamics simulation supporting osteogenic differentiation in dynamic culture

Geometry of porous scaffolds is critical to the success of cell adhesion, proliferation, and differentiation in bone tissue engineering. In this study, the effect of scaffold geometry on osteogenic differentiation of MC3T3-E1 pre-osteoblasts in a perfusion bioreactor was investigated. Three geometries of oligolactide-HA scaffolds, named Woodpile, LC-1000, and LC-1400, were fabricated with uniform pore size distribution and interconnectivity using stereolithography (SL) technique, and tested to evaluate for the most suitable scaffold geometry. Compressive tests revealed sufficiently high strength of all scaffolds to support new bone formation. The LC-1400 scaffold showed the highest cell proliferation in accordance with the highest level of osteoblast-specific gene expression after 21 days of dynamic culture in a perfusion bioreactor; however, it deposited less amount of calcium than the LC-1000 scaffold. Computational fluid dynamics (CFD) simulation was employed to predict and explain the effect of flow behavior on cell response under dynamic culture. The findings concluded that appropriate flow shear stress enhanced cell differentiation and mineralization in the scaffold, with the LC-1000 scaffold performing best due to its optimal balance between permeability and flow-induced shear stress.

Development of a Microfluidic Chip Powered by EWOD for In Vitro Manipulation of Bovine Embryos.

Digital microfluidics (DMF) holds great potential for the alleviation of laboratory procedures in assisted reproductive technologies (ARTs). The electrowetting on dielectric (EWOD) technology provides dynamic culture conditions in vitro that may better mimic the natural embryo microenvironment. Thus far, EWOD microdevices have been proposed for in vitro gamete and embryo handling in mice and for analyzing the human embryo secretome. This article presents the development of the first microfluidic chip utilizing EWOD technology designed for the manipulation of bovine embryos in vitro. The prototype sustains the cell cycles of embryos manipulated individually on the chips during in vitro culture (IVC). Challenges related to the chip fabrication as well as to its application during bovine embryo IVC in accordance with the adapted on-chip protocol are thoroughly discussed, and future directions for DMF in ARTs are indicated.

Generation of a Perfusable 3D Lung Cancer Model by Digital Light Processing.

Lung cancer still has one of the highest morbidity and mortality rates among all types of cancer. Its incidence continues to increase, especially in developing countries. Although the medical field has witnessed the development of targeted therapies, new treatment options need to be developed urgently. For the discovery of new drugs, human cancer models are required to study drug efficiency in a relevant setting. Here, we report the generation of a non-small cell lung cancer model with a perfusion system. The bioprinted model was produced by digital light processing (DLP). This technique has the advantage of including simulated human blood vessels, and its simple assembly and maintenance allow for easy testing of drug candidates. In a proof-of-concept study, we applied gemcitabine and determined the IC50 values in the 3D models and 2D monolayer cultures and compared the response of the model under static and dynamic cultivation by perfusion. As the drug must penetrate the hydrogel to reach the cells, the IC50 value was three orders of magnitude higher for bioprinted constructs than for 2D cell cultures. Compared to static cultivation, the viability of cells in the bioprinted 3D model was significantly increased by approximately 60% in the perfusion system. Dynamic cultivation also enhanced the cytotoxicity of the tested drug, and the drug-mediated apoptosis was increased with a fourfold higher fraction of cells with a signal for the apoptosis marker caspase-3 and a sixfold higher fraction of cells positive for PARP-1. Altogether, this easily reproducible cancer model can be used for initial testing of the cytotoxicity of new anticancer substances. For subsequent in-depth characterization of candidate drugs, further improvements will be necessary, such as the generation of a multi-cell type lung cancer model and the lining of vascular structures with endothelial cells.

Combined use of novel chitosan-grafted N-hydroxyethyl acrylamide polyurethane and human dermal fibroblasts as a construct for in vitro-engineered skin

A rich plethora of information about grafted chitosan (CS) for medical use has been reported. The capability of CS-grafted poly(N-hydroxyethyl acrylamide) (CS-g-PHEAA) to support human dermal fibroblasts (HDFs) in vitro has been proven. However, CS-grafted copolymers lack good stiffness and the characteristic microstructure of a cellular matrix. In addition, whether CS-g-PHEAA can be used to prepare a scaffold with a suitable morphology and mechanical properties for skin tissue engineering (STE) is unclear. This study aimed to show for the first time that step-growth polymerizations can be used to obtain polyurethane (PU) platforms of CS-g-PHEAA, which can also have enhanced microhardness and be suitable for in vitro cell culture. The PU prepolymers were prepared from grafted CS, polyethylene glycol, and 1,6-hexamethylene diisocyanate. The results proved that a poly(saccharide-urethane) [(CS-g-PHEAA)-PU] could be successfully synthesized with a more suitable microarchitecture, thermal properties, and topology than CS-PU for the dynamic culturing of fibroblasts. Cytotoxicity, proliferation, histological and immunophenotype assessments revealed significantly higher biocompatibility and cell proliferation of the derivative concerning the controls. Cells cultured on (CS-g-PHEAA)-PU displayed a quiescent state compared to those cultured on CS-PU, which showed an activated phenotype. These findings may be critical factors in future studies establishing wound dressing models.

Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent.

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.

Effect of different decellularization protocols on reendothelialization with human cells for a perfused renal bioscaffold of the rat

BackgroundScaffolds for tissue engineering can be received by whole organ decellularization while maintaining the site-specific extracellular matrix and the vascular tree. One among other decellularization techniques is the perfusion-based method using specific agents e.g. SDS for the elimination of cellular components. While SDS can disrupt the composition of the extracellular matrix and impair the adherence and growth of site-specific cells there are indications that xenogeneic cell types may benefit from protein denaturation by using higher detergent concentrations. The aim of this work is to investigate the effect of two different SDS-concentrations (i.e. 0.66% and 3%) on the ability of human endothelial cells to adhere and proliferate in an acellular rat kidney scaffold.Material and methodsAcellular rat kidney scaffold was obtained by perfusion-based decellularization through the renal artery using a standardized protocol including SDS at concentrations of 0.66% or 3%. Subsequently cell seeding was performed with human immortalized endothelial cells EA.hy 926 via the renal artery. Recellularized kidneys were harvested after five days of pressure-controlled dynamic culture followed sectioning, histochemical and immunohistochemical staining as well as semiquantitative analysis.ResultsEfficacy of decellularization was verified by absence of cellular components as well as preservation of ultrastructure and adhesive proteins of the extracellular matrix. In semiquantitative analysis of recellularization, cell count after five days of dynamic culture more than doubled when using the gentle decellularization protocol with a concentration of SDS at 0.66% compared to 3%. Detectable cells maintained their endothelial phenotype and presented proliferative behavior while only a negligible fraction underwent apoptosis.ConclusionRecellularization of acellular kidney scaffold with endothelial cells EA.hy 926 seeded through the renal artery benefits from gentle decellularization procedure. Because of that, decellularization with a SDS concentration at 0.66% should be preferred in further studies and coculture experiments.

Mechanical modeling of the maturation process for textile reinforced tissue‐engineered heart valves

AbstractBiohybrid tissue‐engineered implants offer promising possibilities to treat cardiovascular diseases. The ability of tissue‐engineered implants to grow and remodel can be utilized to produce implants that can adapt to changes in the physiological conditions within the patient body. To improve the mechanical properties of the implant, a fibre‐reinforced textile scaffold is used. The embedment of a load‐oriented textile scaffold in the implant acts as biomimetic reinforcement and guides the direction of the extracellular matrix (ECM) growth. Our main objective is to design biohybrid heart valves that can withstand tough physiological loading conditions for decades. That makes it necessary to develop accurate and efficient models to support the design process.In this paper, the focus is on the macro‐mechanical modeling of the maturation process for textile reinforced heart valves. A biohybrid heart valve is modeled as a composite structure. The constitutive model is constructed by defining the total Helmholtz free energy as the sum of energies of the valve constituents. The two main constituents are (i) the extracellular matrix (ECM) and (ii) the fiber‐reinforced textile scaffold. The density of protein fibers such as collagen and elastin fibers are treated as internal variables. We introduce a new approach for modeling the densification of protein fibers during the cultivation process which is capable of considering both static and dynamic cultivation processes. The model considers the density change caused by the immunological response as well as the density change due to mechanical stimulation. The textile scaffold anisotropic behavior is introduced using structural tensors. The constitutive models are then embedded into a special finite element technology with reduced integration that enables efficient computations. The finite element simulation results are then validated using the experimental data provided by BioTex (Institute of Applied Medical Engineering, RWTH Aachen University, Germany). In the end, a finite element simulation for an exemplary valve is presented.