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Journal of Bioscience and Bioengineering Oct 2022Here we report the perfusion culture of a multi-layered tissue composed of HepG2 cells (a human hepatoma line) in a pressure-driven microphysiological system (PD-MPS),...
Here we report the perfusion culture of a multi-layered tissue composed of HepG2 cells (a human hepatoma line) in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. The perfusion culture of multi-layered tissue model was constructed by inserting a modified commercially available permeable membrane insert into the PD-MPS. HepG2 cells were layered on the membrane, and culture medium was perfused both through and below the membrane. The seeded density (number of cells/cm) of the culture model is 70 times that of static culture in a conventional 35-mm culture dish. Pressure-driven circulation of the medium in our compact device (8.6 × 7.0 × 4.5 cm), which comprised two perfusion-culture modules and a pneumatic connection port, enabled perfusion culture of two multi-layered tissues (initially 1 × 10 cells). To obtain insight into the basic functionality of the multi-layered tissues as hepatocytes, we compared albumin production and urea synthesis between perfusion cultures and static cultures. The HepG2 cells grew and secreted increasing amounts of albumin throughout 20 days of perfusion culture, whereas albumin secretion did not increase under static culture conditions. In addition, on day 20, the amount of albumin secreted by the HepG2 cells in the microfluidic device was 68% of that in the conventional culture dish, which was seeded with the same number of cells but had a 70 times larger culture area. These features of high-density culture of functioning cells in a compact device support the application of PD-MPS in single- and multi-organ MPS.
Topics: Albumins; Carcinoma, Hepatocellular; Cell Culture Techniques; Hep G2 Cells; Hepatocytes; Humans; Liver Neoplasms; Perfusion; Urea
PubMed: 35963667
DOI: 10.1016/j.jbiosc.2022.07.001 -
Analytical Methods : Advancing Methods... Mar 2021Ex vivo brain slice cultures are utilized as analytical models for studying neurophysiology. Common approaches to maintaining slice cultures include roller tube and...
Ex vivo brain slice cultures are utilized as analytical models for studying neurophysiology. Common approaches to maintaining slice cultures include roller tube and membrane interface techniques. The rise of organ-on-chip technologies has demonstrated the value of microfluidic perfusion culture systems for sampling and analysis of complex biology under well-controlled in vitro or ex vivo conditions. A number of approaches to microfluidic brain slice culture have been developed, however these typically involve complex design, fabrication, or operational parameters in order to meet the high oxygen demands of brain slices. Here, we present proof-of-principle for a novel approach to microfluidic brain slice culture. In this system, which we term a microfluidic bubble perfusion device, principles of droplet microfluidics were employed to generate droplets of perfusion media dispersed between bubbles of carbogen gas, and brain tissue slices were perfused with the resulting monodispersed droplets and bubbles. The challenge of tissue immobilization in the flow system was addressed using a two-part cytocompatible carbohydrate-based tissue adhesive. Best practices are discussed for perfusion chamber designs that maintain segmented flow throughout the course of perfusion. Control of droplet and bubble volumes was possible across the range of ca. 4-15 μL, bubble generation frequency was well controlled in the range ca. 1-7 bubbles per min, and bubble duty cycle was well controlled across the range ca. 20-80%. Murine hypothalamic tissue slices containing the suprachiasmatic nuclei were successfully maintained for durations of 8-10 hours, with tissue remaining viable for the duration of perfusion as assessed by Ca imaging and propidium iodide (PI) staining.
Topics: Animals; Brain; Lab-On-A-Chip Devices; Mice; Microfluidic Analytical Techniques; Microfluidics; Perfusion
PubMed: 33644791
DOI: 10.1039/d0ay02291h -
Tissue Engineering. Part A Dec 2021Cell proliferation and survival are dependent on mass transfer. , fluid flow promotes mass transfer through the vasculature and interstitial space, providing a...
Cell proliferation and survival are dependent on mass transfer. , fluid flow promotes mass transfer through the vasculature and interstitial space, providing a continuous supply of nutrients and removal of cellular waste products. In the absence of sufficient flow, mass transfer is limited by diffusion and poses significant challenges to cell survival during tissue engineering, tissue transplantation, and treatment of degenerative diseases. Artificial perfusion may overcome these challenges. In this work, we compare the efficacy of pressure driven perfusion (PDP) with electrokinetic perfusion (EKP) toward reducing cell mortality in three-dimensional cultures of Matrigel extracellular matrix. We characterize electro-osmotic flow through Matrigel to identify conditions that generate similar interstitial flow rates to those induced by pressure. We also compare changes in cell mortality induced by continuous or pulsed EKP. We report that continuous EKP significantly reduced mortality throughout the perfusion channels more consistently than PDP at similar flow rates, and pulsed EKP decreased mortality just as effectively as continuous EKP. We conclude that EKP has significant advantages over PDP for promoting tissue survival before neovascularization and angiogenesis. Impact statement Interstitial flow helps promote mass transfer and cell survival in tissues and organs. This study generated interstitial flow using pressure driven perfusion (PDP) or electrokinetic perfusion (EKP) to promote cell viability in three-dimensional cultures. EKP through charged extracellular matrices possesses significant advantages over PDP and may promote cell survival during tissue engineering, transplantations, and treatment of degenerative diseases.
Topics: Bioreactors; Cell Survival; Extracellular Matrix; Perfusion; Tissue Engineering
PubMed: 33820474
DOI: 10.1089/ten.TEA.2021.0008 -
PloS One 2020A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple...
A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow.
Topics: Animals; Cell Culture Techniques; Cells, Cultured; Fibroblasts; Human Umbilical Vein Endothelial Cells; Humans; Mice; Perfusion; Tissue Engineering
PubMed: 33112918
DOI: 10.1371/journal.pone.0240552 -
Scientific Reports Feb 2023The off-target effects of light-activated or targeted liposomes are difficult to distinguish in traditional well plate experiments. Additionally, the absence of fluid...
The off-target effects of light-activated or targeted liposomes are difficult to distinguish in traditional well plate experiments. Additionally, the absence of fluid flow in traditional cell models can lead to overestimation of nanoparticle uptake. In this paper, we established a perfusion cell culture platform to study light-activated liposomes and determined the effect of flow on the liposomal cell uptake. The optimal cell culturing parameters for the A549 cells under flow conditions were determined by monitoring cell viability. To determine optimal liposome treatment times, particle uptake was measured with flow cytometry. The suitability of commercial QuasiVivo flow-chambers for near-infrared light activation was assessed with a calcein release study. The chamber material did not hinder the light activation and subsequent calcein release from the liposomes. Furthermore, our results show that the standard cell culturing techniques are not directly translatable to flow cultures. For non-coated liposomes, the uptake was hindered by flow. Interestingly, hyaluronic acid coating diminished the uptake differences between the flow and static conditions. The study demonstrates that flow affects the liposomal uptake by lung cancer cell line A549. The flow also complicates the cell attachment of A549 cells. Moreover, we show that the QuasiVivo platform is suitable for light-activation studies.
Topics: Liposomes; Fluoresceins; Cell Culture Techniques; Perfusion
PubMed: 36739469
DOI: 10.1038/s41598-023-29215-6 -
Placenta Apr 2020The isolated perfused placental cotyledon technique has led to numerous advances in placental biology. Combining placental perfusion with mathematical modelling provides... (Review)
Review
The isolated perfused placental cotyledon technique has led to numerous advances in placental biology. Combining placental perfusion with mathematical modelling provides an additional level of insight into placental function. Mathematical modelling of perfusion data provides a quantitative framework to test the understanding of the underlying biology and to explore how different processes work together within the placenta as part of an integrated system. The perfusion technique provides a high degree of control over the experimental conditions as well as regular measurements of functional parameters such as pressure, solute concentrations and pH over time. This level of control is ideal for modelling as it allows placental function to be studied across a wide range of different conditions which permits robust testing of mathematical models. By placing quantitative values on different processes (e.g. transport, metabolism, blood flow), their relative contribution to the system can be estimated and those most likely to become rate-limiting identified. Using a combined placental perfusion and modelling approach, placental metabolism was shown to be a more important determinant of amino acid and fatty acid transfer. In contrast, metabolism was a less important determinant of placental cortisol transfer than initially thought. Identifying the rate-limiting factors in the system allows future work to be focused on the factors that are most likely to underlie placental dysfunction. A combined experimental and modelling approach using placental perfusions promotes an integrated view of placental physiology that can more effectively identify the processes leading to placental pathologies.
Topics: Amino Acids; Biological Transport; Fatty Acids; Female; Humans; Maternal-Fetal Exchange; Models, Biological; Models, Theoretical; Organ Culture Techniques; Perfusion; Placenta; Pregnancy
PubMed: 32250738
DOI: 10.1016/j.placenta.2020.02.015 -
Journal of Visualized Experiments : JoVE Jul 2022Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has...
Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.
Topics: Bioreactors; Cell Culture Techniques; Culture Media; Perfusion; Tissue Engineering
PubMed: 35938803
DOI: 10.3791/63935 -
Biofabrication Oct 2022The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We...
The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge ofvascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineeredtissue constructs.
Topics: Humans; Hydrogels; Tissue Engineering; Human Umbilical Vein Endothelial Cells; Cell Culture Techniques; Collagen; Perfusion; Oxygen; Tissue Scaffolds; Neovascularization, Physiologic
PubMed: 36300786
DOI: 10.1088/1758-5090/ac9433 -
Tissue Engineering Jan 2007Cultured precision-cut liver tissue slices are useful for studying the metabolism and toxicity of xenobiotics in liver. They may also be used to investigate the behavior... (Comparative Study)
Comparative Study
Cultured precision-cut liver tissue slices are useful for studying the metabolism and toxicity of xenobiotics in liver. They may also be used to investigate the behavior of and interaction between different cell types in an intact histo-architecture. Because cultured liver tissues undergo a loss of function and morphology because of their separation from the blood supply, we investigated changes in key protein marker expressions in parenchymal and non-parenchymal cells, as well as in the extracellular matrix (ECM) at different time points. We also compared conventional culture methods such as static and dynamic cultures with perfusion culture, which allows a continuous exchange of the culture medium. In conventional culture methods, the expression of vimentin and collagen type IV decreased after 5 h in the non-parenchymal cells and the ECM, respectively, whereas the hepatocyte nuclear factor 4 alpha (HNF4alpha) staining in the hepatocytes remained constant. In perfusion culture, on the other hand, vimentin, collagen type IV, and HNF4alpha staining were clearly detectable after 5 h. The histo-architecture obtained from perfusion culture was also more compact than those obtained from conventional culture methods. After 24 h, only the perfusion cultured sample retained protein marker expression in all components of the liver tissue. Our results suggest that, to develop improved culture techniques for liver slices, changes at the early time-points should be taken into consideration. Our results also show that culture techniques that enable a continuous exchange of the culture medium seem to be superior to static or dynamic cultures in terms of maintaining the protein expression and the histo-architecture.
Topics: Animals; Biomarkers; Gene Expression Regulation; Hepatocytes; Liver; Male; Perfusion; Proteins; Rats; Rats, Wistar; Tissue Culture Techniques
PubMed: 17518593
DOI: 10.1089/ten.2006.0046 -
Biotechnology Journal Nov 2014Mass transfer limitation in conventional top-down tissue engineering makes it impossible to fabricate large size viable tissue replacements. In the present study, we...
Mass transfer limitation in conventional top-down tissue engineering makes it impossible to fabricate large size viable tissue replacements. In the present study, we aimed at performing a systemic investigation of the assembling process in perfusion culture for fabricating centimeter-scale macrotissues from cell-laden microcarriers following a bottom-up modular approach. Cells (human fibroblasts, human mesenchymal stem cells, or HepG2 cells) were seeded onto microcarriers (Cytopore-2 or CultiSpher S) in spinner flasks and cultured for 14 days and subsequently transferred to a perfusion chamber for assembling. It was found that growth of different cells on different microcarriers varied and aggregation of cell-laden microcarriers was favored with CultiSpher S. After perfusion culture for 14 days, while all microtissues could assemble into integral macrotissues, macrotissues of HepG2 cells were structurally most inferior and the assembling of cell-laden CultiSpher S led to a significant shrinkage. By designing perfusion chamber and using agar-based templates, tubular, disc, and alphabetic letter-shaped macrotissues could be easily fabricated, suggesting template-assisted assembling. Importantly, it was revealed that there existed both optimal perfusion culture time (21 days) and packing condition (1/4 compression) for the assembling of microtissues. This study lays a solid foundation for future applications of this microtissue assembling technique in tissue engineering.
Topics: Cell Culture Techniques; Cell Proliferation; Cell Survival; Cells, Cultured; Female; Foreskin; Hep G2 Cells; Humans; Male; Perfusion; Placenta; Pregnancy; Tissue Engineering; Tissue Scaffolds
PubMed: 25200115
DOI: 10.1002/biot.201400238