Bonding of Flexible Membranes for Perfusable Vascularized Networks Patch

BACKGROUND: In vitro generation of three-dimensional vessel network is crucial to investigate and possibly improve vascularization after implantation in vivo. This work has the purpose of engineering complex tissue regeneration of a vascular network including multiple cell-type, an extracellular matrix, and perfusability for clinical application. METHODS: The two electrospun membranes bonded with the vascular network shape are cultured with endothelial cells and medium flow through the engineered vascular network. The flexible membranes are bonded by amine-epoxy reaction and examined the perfusability with fluorescent beads. Also, the perfusion culture for 7 days of the endothelial cells is compared with static culture on the engineered vascular network membrane. RESULTS: The engineered membranes are showed perfusability through the vascular network, and the perfused network resulted in more cell proliferation and variation of the shear stress-related genes expression compared to the static culture. Also, for the generation of the complex vascularized network, pericytes are co-cultured with the engineered vascular network, which results in the Collagen I is expressed on the outer surface of the engineered structure. CONCLUSION: This study is showing the perfusable in vitro engineered vascular network with electrospun membrane. In further, the 3D vascularized network module can be expected as a platform for drug screening and regenerative medicine.

Static Culture Combined with Aeration in Biosynthesis of Bacterial Cellulose

One of the ways to enhance the yield of bacterial cellulose (BC) is by using dynamic aeration and different-type bioreactors because the microbial producers are strict aerobes. But in this case, the BC quality tends to worsen. Here we have combined static culture with aeration in the biosynthesis of BC by symbiotic Medusomyces gisevii Sa-12 for the first time. A new aeration method by feeding the air onto the growth medium surface is proposed herein. The culture was performed in a Binder-400 climate chamber. The study found that the air feed at a rate of 6.3 L/min allows a 25% increase in the BC yield. Moreover, this aeration mode resulted in BC samples of stable quality. The thermogravimetric and X-ray structural characteristics were retained: the crystallinity index in reflection and transmission geometries were 89% and 92%, respectively, and the allomorph Iα content was 94%. Slight decreases in the degree of polymerization (by 12.0% compared to the control―no aeration) and elastic modulus (by 12.6%) are not critical. Thus, the simple aeration by feeding the air onto the culture medium surface has turned out to be an excellent alternative to dynamic aeration. Usually, when the cultivation conditions, including the aeration ones, are changed, characteristics of the resultant BC are altered either, due to the sensitivity of individual microbial strains. In our case, the stable parameters of BC samples under variable aeration conditions are explained by the concomitant factors: the new efficient aeration method and the highly adaptive microbial producer―symbiotic Medusomyces gisevii Sa-12.

OPTIMIZATION OF BACTERIAL CELLULOSE PRODUCTION FROM SUGAR BEET MOLASSES BY GLUCONACETOBACTER XYLINUS NRRL B-759 IN STATIC CULTURE

We investigated the optimization of bacterial cellulose (BC) production from sugar beet molasses by Gluconacetobacter xylinus NRRL B-759 in static culture. The optimization studies were performed using the central composite design (CCD) of response surface methodology (RSM). The independent variables were the molasses concentration, inoculation ratio and culture volume. The dependent variable was BC production yield. From the optimization tests, based on the model developed by RSM-ANOVA, it was found that binary interactions between molasses concentration–culture volume and inoculation ratio–culture volume had the most significant influence on the responses. The optimum conditions were as follows: 78.932 g/L molasses concentration, 12.973% inoculation ratio, and 130.405 mL of culture volume. The obtained BC was characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and elemental analysis. The characterization results obtained in the study revealed that the produced BC exhibited typical FTIR spectrum, elemental composition, and nanofiber structure.

Evaluation of bone formation on orthopedic implant surfaces using an ex-vivo bone bioreactor system

Recent advances in materials and manufacturing processes have allowed the fabrication of intricate implant surfaces to facilitate bony attachment. However, refinement and evaluation of these new design strategies are hindered by the cost and complications of animal studies, particularly during early iterations in the development process. To address this problem, we have previously constructed and validated an ex-vivo bone bioreactor culture system that can maintain the viability of bone samples for an extended period ex-vivo. In this study, we investigated the mineralization of a titanium wire mesh scaffold under both static and dynamic culturing using our ex vivo bioreactor system. Thirty-six cancellous bone cores were harvested from bovine metatarsals at the time of slaughter and divided into five groups under the following conditions: Group 1) Isolated bone cores placed in static culture, Group 2) Unloaded bone cores placed in static culture in contact with a fiber-mesh metallic scaffold, Group 3) Bone cores placed in contact with a fiber-mesh metallic scaffold under the constant pressure of 150 kPa, Group 4) Bone core placed in contact with a fiber-mesh metallic scaffold and exposed to cyclic loading with continuous perfusion flow of media within the ex-vivo culture system and Group 5) Bone core evaluated on Day 0 to serve as a positive control for comparison with all other groups at weeks 4 and 7. Bone samples within Groups 1–4 were incubated for 4 and 7 weeks and then evaluated using histological examination (H&E) and the Live-Dead assay (Life Technologies). Matrix deposits on the metallic scaffolds were examined with scanning electron microscopy (SEM), while the chemical composition of the matrix was measured using energy-dispersive x-ray spectroscopy (EDX). We found that the viability of bone cores was maintained after seven weeks of loading in our ex vivo system. In addition, SEM images revealed crystallite-like structures on the dynamically loaded metal coupons (Group 4), corresponding to the initial stages of mineralization. EDX results further confirmed the presence of carbon at the interface and calcium phosphates in the matrix. We conclude that a bone bioreactor can be used as an alternate tool for in-vivo bone ingrowth studies of new implant surfaces or coatings.

Novel perfusion ex-vivo culture system enables physiological and pathological studies on porcine arteries

Introduction Routine cardiovascular interventions such as cardiac bypass, balloon angioplasty and stenting provoke vascular activation and remodelling often leading to rehospitalization and further interventions. Conventional in vitro models fail to account for the complex vascular environment essential for vascular tissue fitness. Purpose Our research aims to culture whole porcine arteries in using a novel cost efficient and versatile perfusion system (EasyFlow) and identify the contribution of adventitial progenitors to post-injury remodelling. Methods EasyFlow insert was 3D printed to convert a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine arteries were excised and cultured under constant pulsatile flow for up to 7 days. Injury was performed by balloon catheter at day 0. Tissues were evaluated by doppler ultrasound, immunofluorescence and confocal imaging and PCR, at different timepoints. Results The EasyFlow adaptor takes advantage of 50 ml centrifuge tubes isolating reaction space from the environment and reducing the culture volume. The adaptability of the design facilitates the incubation of vessels of different size and origin while the self-contained perfusion allows parallel cultures and minimal media consumption. EasyFlow perfusion culture of porcine arteries preserves the endothelial coverage and the smooth muscle cell organisation, as compared to static culture. Gene expression analysis and immunofluorescence indicated an increased expression of platelet-derived growth factor receptor beta (PDGFRb) and a decrease in smooth muscle actin (SMA) in the static culture, compared to the perfusion. Furthermore, static culture showed an increased cellular activation in adventitia (proliferating cell nuclear antigen expression), as compared to perfused tissues. Balloon injury followed by flow culture recapitulated the early hallmarks of vascular remodelling, including intimal denudation indicated by loss of platelet endothelial cell adhesion molecule signal, smooth muscle cell disarray shown by loss of actin stress fibre organisation and activation in the media, as supported by confocal microscope and gene expression analysis, accompanied by significant morphological changes based on ultrasound imaging. Conclusion This work lay the basis for future investigations into the pathological remodelling of blood vessels, by providing a robust and controlled culture system for the maintenance of porcine blood vessels in culture. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Surrey En-Face Staining of Arterial Lumen

Transition from static culture to stirred tank bioreactor for the allogeneic production of therapeutic discogenic cell spheres

Background Culturing cells as cell spheres results in a tissue-like environment that drives unique cell phenotypes, making it useful for generating cell populations intended for therapeutic use. Unfortunately, common methods that utilize static suspension culture have limited scalability, making commercialization of such cell therapies challenging. Our team is developing an allogeneic cell therapy for the treatment of lumbar disc degeneration comprised of discogenic cells, which are progenitor cells expanded from human nucleus pulposus cells that are grown in a sphere configuration. Methods We evaluate sphere production in Erlenmeyer, horizontal axis wheel, stirred tank bioreactor, and rocking bag format. We then explore the use of ramped agitation profiles and computational fluid dynamics to overcome obstacles related to cell settling and the undesired impact of mechanical forces on cell characteristics. Finally, we grow discogenic cells in stirred tank reactors (STRs) and test outcomes in vitro (potency via aggrecan production and identity) and in vivo (rabbit model of disc degeneration). Results Computation fluid dynamics were used to model hydrodynamic conditions in STR systems and develop statistically significant correlations to cell attributes including potency (measured by aggrecan production), cell doublings, cell settling, and sphere size. Subsequent model-based optimization and testing resulted in growth of cells with comparable attributes to the original static process, as measured using both in vitro and in vivo models. Maximum shear rate (1/s) was maintained between scales to demonstrate feasibility in a 50 L STR (200-fold scale-up). Conclusions Transition of discogenic cell production from static culture to a stirred-tank bioreactor enables cell sphere production in a scalable format. This work shows significant progress towards establishing a large-scale bioprocess methodology for this novel cell therapy that can be used for other, similar cell therapies.

Alginate-chitosan core-shell microcapsule cultures of hepatic cells in a small scale stirred bioreactor: impact of shear forces and microcapsule core composition

A small scale stirred bioreactor was designed and the effect of different agitation rates (30, 60 and 100 rpm) was investigated on HepG2 cells cultured in alginate-chitosan (AC) core-shell microcapsule in terms of the cell proliferation and liver-specific function. The microencapsulated hepatic cells could proliferate well when they were cultured for 10 days at 30 rpm while the cell-laden microcapsules showed no cell proliferation at 100 rpm in the bioreactor system. Albumin production rate, as an important liver function, increased also 1.8- and 1.5- fold under stirring rate of 30 rpm compared to the static culture and 60 rpm of agitation, respectively. Moreover, In comparison with the static culture, about 1.5-fold increment in urea production was observed at 30 rpm. Similarly, the highest expressions of albumin and P450 genes were found at 30 rpm stirring rate, which were 4.9- and 19.2-fold of the static culture. Addition of collagen to the microcapsule core composition (ACol/C) could improve the cell proliferation and functionality at 60 rpm in comparison with the cell-laden microcapsules without collagen. The study demonstrated the hepatic cell-laden ACol/C microcapsule hydrogel cultured in the small scale stirred bioreactor at low mixing rate has a great potential for mass production of the hepatic cells while maintaining liver-specific functions.

The Absence of Hydrodynamic Stress Promotes Acquisition of Freezing Tolerance and Freeze-Dependent Asexual Reproduction in the Red Alga ‘Bangia’ sp. ESS1

The ebb tide causes calm stress to intertidal seaweeds in tide pools; however, little is known about their physiological responses to loss of water movement. This study investigated the effects of static culture of ‘Bangia’ sp. ESS1 at 15 °C on tolerance to temperature fluctuation. The freezing of aerobically cultured thalli at −80 °C for 10 min resulted in the death of most cells. By contrast, statically cultured thalli acquired freezing tolerance that increased cell viability after freeze–thaw cycles, although they did not achieve thermotolerance that would enable survival at the lethal temperature of 32 °C. Consistently, the unsaturation of membrane fatty acids occurred in static culture. Notably, static culture of thalli enhanced the release of asexual spores after freeze-and-thaw treatment. We conclude that calm stress triggers both the acquisition of freezing tolerance and the promotion of freezing-dependent asexual reproduction. These findings provide novel insights into stress tolerance and the regulation of asexual reproduction in Bangiales.

Study of growth, metabolism, and morphology of Akkermansia muciniphila with an in vitro advanced bionic intestinal reactor

Background As a kind of potential probiotic, Akkermansia muciniphila abundance in human body is directly causally related to obesity, diabetes, inflammation and abnormal metabolism. In this study, A. muciniphila dynamic cultures using five different media were implemented in an in vitro bionic intestinal reactor for the first time instead of the traditional static culture using brain heart infusion broth (BHI) or BHI + porcine mucin (BPM). Results The biomass under dynamic culture using BPM reached 1.92 g/L, which improved 44.36% compared with the value under static culture using BPM. The biomass under dynamic culture using human mucin (HM) further increased to the highest level of 2.89 g/L. Under dynamic culture using porcine mucin (PM) and HM, the main metabolites were short-chain fatty acids (acetic acid and butyric acid), while using other media, a considerable amount of branched-chain fatty acids (isobutyric and isovaleric acids) were produced. Under dynamic culture Using HM, the cell diameters reached 999 nm, and the outer membrane protein concentration reached the highest level of 26.26 μg/mg. Conclusions This study provided a preliminary theoretical basis for the development of A. muciniphila as the next generation probiotic.

Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

Gut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.