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STAR Protocols Sep 2020Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an model for liver physiology. Obtaining high yields of viable primary mouse...
Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an model for liver physiology. Obtaining high yields of viable primary mouse hepatocytes is technically challenging, limiting their use. Here, we present an improved protocol based on the classic two-step collagenase perfusion technique. The liver is washed by perfusion, hepatocytes are dissociated by collagenase, separated from other cells, and cultured. This protocol was optimized to significantly reduce procedure duration and improve hepatocyte yield and viability.
Topics: Animals; Cell Separation; Cells, Cultured; Collagenases; Hepatocytes; Liver; Mice; Mice, Inbred C57BL; Perfusion; Primary Cell Culture
PubMed: 33111119
DOI: 10.1016/j.xpro.2020.100086 -
Nature Protocols Feb 2015Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop...
Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.
Topics: Animals; Centrifugation, Density Gradient; Collagenases; Cytological Techniques; Flow Cytometry; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Mice, Transgenic; Perfusion
PubMed: 25612230
DOI: 10.1038/nprot.2015.017 -
MAbs 2022The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces...
The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.
Topics: Animals; Antibodies, Monoclonal; Batch Cell Culture Techniques; Bioreactors; CHO Cells; Cricetinae; Cricetulus; Perfusion
PubMed: 35737825
DOI: 10.1080/19420862.2022.2083465 -
Perfuse and Reuse: A Low-Cost Three-Dimensional-Printed Perfusion Bioreactor for Tissue Engineering.Tissue Engineering. Part C, Methods Nov 2022This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow...
This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow centimeter-scale vascularized or otherwise canalized tissue constructs to be maintained in weeks long static and/or perfusion culture at an exceptionally low cost, with intermittent live imaging and media sampling capabilities. The perfusion system includes a reusable polydimethylsiloxane (PDMS) lid generated from a three-dimensional (3D)-printed poly-lactic acid (PLA) mold and several lengths of perfusion tubing. The coverslip tissue culture chamber includes PDMS components built with 3D-printed PLA molds, as well as 3D-printed PLA frames and glass coverslips that house perfusable hydrogel constructs. As proof of concept, we fabricated a vascularized hydrogel construct, which was subjected to static and perfusion tissue culture, as well as flow studies using fluorescent beads and widefield fluorescent microscopy. This system can be readily reproduced, promoting the advancement of tissue engineering and regenerative medicine research.
Topics: Tissue Engineering; Perfusion; Bioreactors; Hydrogels; Polyesters; Printing, Three-Dimensional; Tissue Scaffolds
PubMed: 36094108
DOI: 10.1089/ten.TEC.2022.0139 -
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 -
Cells Mar 2022Existing 3D cell models and technologies have offered tools to elevate cell culture to a more physiologically relevant dimension. One mechanism to maintain cells...
Existing 3D cell models and technologies have offered tools to elevate cell culture to a more physiologically relevant dimension. One mechanism to maintain cells cultured in 3D is by means of perfusion. However, existing perfusion technologies for cell culture require complex electronic components, intricate tubing networks, or specific laboratory protocols for each application. We have developed a cell culture platform that simply employs a pump-free suction device to enable controlled perfusion of cell culture media through a bed of granular microgels and removal of cell-secreted metabolic waste. We demonstrated the versatile application of the platform by culturing single cells and keeping tissue microexplants viable for an extended period. The human cardiomyocyte AC16 cell line cultured in our platform revealed rapid cellular spheroid formation after 48 h and ~90% viability by day 7. Notably, we were able to culture gut microexplants for more than 2 weeks as demonstrated by immunofluorescent viability assay and prolonged contractility.
Topics: Cell Culture Techniques; Cell Line; Humans; Perfusion; Spheroids, Cellular
PubMed: 35326418
DOI: 10.3390/cells11060967 -
Journal of Visualized Experiments : JoVE Nov 2022The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular...
The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.
Topics: Mice; Animals; Myocytes, Cardiac; Cardiac Electrophysiology; Calcium; Perfusion; Acclimatization
PubMed: 36440883
DOI: 10.3791/63056 -
International Journal of Molecular... Mar 2022Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to...
Perfusion-Based Bioreactor Culture and Isothermal Microcalorimetry for Preclinical Drug Testing with the Carbonic Anhydrase Inhibitor SLC-0111 in Patient-Derived Neuroblastoma.
Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to improve therapy and outcomes for patients with advanced neuroblastoma, clinical trials currently only allow for testing few substances in even fewer patients. This increases the need to improve and advance preclinical models for neuroblastoma to preselect favorable candidates for novel therapeutics. Here we propose the use of a new patient-derived 3D slice-culture perfusion-based 3D model in combination with rapid treatment evaluation using isothermal microcalorimetry exemplified with treatment with the novel carbonic anhydrase IX and XII (CAIX/CAXII) inhibitor SLC-0111. Patient samples showed a CAIX expression of 18% and a CAXII expression of 30%. Corresponding with their respective CAIX expression patterns, the viability of SH-EP cells was significantly reduced upon treatment with SLC-0111, while LAN1 cells were not affected. The inhibitory effect on SH-SY5Y cells was dependent on the induction of CAIX expression under hypoxia. These findings corresponded to thermogenesis of the cells. Patient-derived organotypic slice cultures were treated with SLC-0111, which was highly effective despite heterogeneity of CAIX/CAXII expression. Thermogenesis, in congruence with the findings of the histological observations, was significantly reduced in SLC-0111-treated samples. In order to extend the evaluation time, we established a perfusion-based approach for neuroblastoma tissue in a 3D perfusion-based bioreactor system. Using this system, excellent tissue quality with intact tumor cells and stromal structure in neuroblastoma tumors can be maintained for 7 days. The system was successfully used for consecutive drug response monitoring with isothermal microcalorimetry. The described approach for drug testing, relying on an advanced 3D culture system combined with a rapid and highly sensitive metabolic assessment, can facilitate development of personalized treatment strategies for neuroblastoma.
Topics: Antigens, Neoplasm; Bioreactors; Carbonic Anhydrase IX; Carbonic Anhydrase Inhibitors; Humans; Neuroblastoma; Perfusion; Phenylurea Compounds; Sulfonamides
PubMed: 35328549
DOI: 10.3390/ijms23063128 -
Biotechnology Advances 2018The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream... (Review)
Review
The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream (purification and formulation of the protein into a drug substance or drug product). Today, cost pressure, market uncertainty and market growth, challenge the existing manufacturing technologies. Leaders in the field are active in designing the process of the future and continuous manufacturing is recurrently mentioned as a potential solution to address some of the current limitations. This review focuses on the application of continuous processing to the first step of the manufacturing process. Enabling technologies and operation modes are described in the first part. In the second part, recent advances in the field that have the potential to support its successful future development are critically discussed.
Topics: Animals; Batch Cell Culture Techniques; Bioreactors; CHO Cells; Cricetinae; Cricetulus; Perfusion; Recombinant Proteins
PubMed: 29738813
DOI: 10.1016/j.biotechadv.2018.04.011 -
The Journal of Toxicological Sciences 2022The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the...
The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.
Topics: Animals; Cells, Cultured; Glycolysis; Hepatocytes; Liver; Mice; Oxidative Phosphorylation; Oxygen Consumption; Perfusion
PubMed: 34987137
DOI: 10.2131/jts.47.13