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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 -
Methods in Molecular Biology (Clifton,... 2021Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte...
Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.
Topics: Animals; Calcium; Calcium Chloride; Cell Culture Techniques; Cell Separation; Cells, Cultured; Collagenases; Isolated Heart Preparation; Mice; Myocytes, Cardiac; Perfusion
PubMed: 34331252
DOI: 10.1007/978-1-0716-1480-8_16 -
Annals of Biomedical Engineering Jul 2014In this study, we investigated the effect of flow perfusion culture on the mineralization of co-cultures of human umbilical vein endothelial cells (HUVECs) and human...
In this study, we investigated the effect of flow perfusion culture on the mineralization of co-cultures of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). Osteogenically precultured hMSCs were seeded onto electrospun scaffolds in monoculture or a 1:1 ratio with HUVECs, cultured for 7 or 14 days in osteogenic medium under static or flow perfusion conditions, and the resulting constructs were analyzed for cellularity, alkaline phosphatase (ALP) activity and calcium content. In flow perfusion, constructs with monocultures of hMSCs demonstrated higher cellularity and calcium content, but lower ALP activity compared to corresponding static controls. ALP activity was enhanced in co-cultures under flow perfusion conditions, compared to hMSCs alone; however unlike the static controls, the calcium content of the co-cultures in flow perfusion was not different from the corresponding hMSC monocultures. The data suggest that co-cultures of hMSCs and HUVECs did not contribute to enhanced mineralization compared to hMSCs alone under the flow perfusion conditions investigated in this study. However, flow perfusion culture resulted in an enhanced spatial distribution of cells and matrix compared to static cultures, which were limited to a thin surface layer.
Topics: Alkaline Phosphatase; Biodegradable Plastics; Calcification, Physiologic; Coculture Techniques; Human Umbilical Vein Endothelial Cells; Humans; Mesenchymal Stem Cells; Osteogenesis; Perfusion; Tissue Scaffolds
PubMed: 23842695
DOI: 10.1007/s10439-013-0862-y -
International Journal of Molecular... Aug 2023Ex vivo lung perfusion (EVLP) has increased donor lung utilization through assessment of "marginal" lungs prior to transplantation. To develop it as a donor lung...
Ex vivo lung perfusion (EVLP) has increased donor lung utilization through assessment of "marginal" lungs prior to transplantation. To develop it as a donor lung reconditioning platform, prolonged EVLP is necessary, and new perfusates are required to provide sufficient nutritional support. Human pulmonary microvascular endothelial cells and epithelial cells were used to test different formulas for basic cellular function. A selected formula was further tested on an EVLP cell culture model, and cell confluence, apoptosis, and GSH and HSP70 levels were measured. When a cell culture medium (DMEM) was mixed with a current EVLP perfusate-Steen solution, DMEM enhanced cell confluence and migration and reduced apoptosis in a dose-dependent manner. A new EVLP perfusate was designed and tested based on DMEM. The final formula contains 5 g/L Dextran-40 and 7% albumin and is named as D05D7A solution. It inhibited cold static storage and warm reperfusion-induced cell apoptosis, improved cell confluence, and enhanced GSH and HSP70 levels in human lung cells compared to Steen solution. DMEM-based nutrient-rich EVLP perfusate could be a promising formula to prolong EVLP and support donor lung repair, reconditioning and further improve donor lung quality and quantity for transplantation with better clinical outcome.
Topics: Humans; Endothelial Cells; Cell Culture Techniques; HSP70 Heat-Shock Proteins; Nutrients; Reperfusion; Lung
PubMed: 37685927
DOI: 10.3390/ijms241713117 -
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 -
Surgery Today Jul 2020Lung transplantation is currently the only curative treatment for patients with end-stage lung disease; however, donor organ shortage and the need for intense... (Review)
Review
Lung transplantation is currently the only curative treatment for patients with end-stage lung disease; however, donor organ shortage and the need for intense immunosuppression limit its broad clinical application. Bioartificial lungs created by combining native matrix scaffolds with patient-derived cells might overcome these problems. Decellularization involves stripping away cells while leaving behind the extracellular matrix scaffold. Cadaveric lungs are decellularized by detergent perfusion, and histologic examination confirms the absence of cellular components but the preservation of matrix proteins. The resulting lung scaffolds are recellularized in a bioreactor that provides biomimetic conditions, including vascular perfusion and liquid ventilation. Cell seeding, engraftment, and tissue maturation are achieved in whole-organ culture. Bioartificial lungs are transplantable, similarly to donor lungs, because the scaffolds preserve the vascular and airway architecture. In rat and porcine transplantation models, successful anastomoses of the vasculature and the airway were achieved, and gas exchange was evident after reperfusion. However, long-term function has not been achieved because of the immaturity of the vascular bed and distal lung epithelia. The goal of this strategy is to create patient-specific transplantable lungs using induced pluripotent stem cell (iPSC)-derived cells. The repopulation of decellularized scaffolds to create transplantable organs is one of possible future clinical applications of iPSCs.
Topics: Animals; Bioartificial Organs; Extracellular Matrix; Humans; Lung; Lung Transplantation; Models, Animal; Perfusion; Pluripotent Stem Cells; Rats; Regeneration; Swine; Tissue Engineering; Tissue Scaffolds
PubMed: 32363425
DOI: 10.1007/s00595-020-02000-y -
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 -
Journal of Vascular Research 2018Adaptive remodeling processes are essential to the maintenance and viability of coronary artery bypass grafts where clinical outcomes depend strongly on the tissue... (Comparative Study)
Comparative Study
Adaptive remodeling processes are essential to the maintenance and viability of coronary artery bypass grafts where clinical outcomes depend strongly on the tissue source. In this investigation, we utilized an ex vivo perfusion bioreactor to culture porcine analogs of common human bypass grafts: the internal thoracic artery (ITA), the radial artery (RA), and the great saphenous vein (GSV), and then evaluated samples acutely (6 h) and chronically (7 days) under in situ or coronary-like perfusion conditions. Although morphologically similar, primary cells harvested from the ITA illustrated lower intimal and medial, but not adventitial, cell proliferation rates than those from the RA or GSV. Basal gene expression levels were similar in all vessels, with only COL3A1, SERPINE1, FN1, and TGFB1 being differentially expressed prior to culture; however, over half of all genes were affected nominally by the culturing process. When exposed to coronary-like conditions, RAs and GSVs experienced pathological remodeling not present in ITAs or when vessels were studied in situ. Many of the remodeling genes perturbed at 6 h were restored after 7 days (COL3A1, FN1, MMP2, and TIMP1) while others (SERPINE1, TGFB1, and VCAM1) were not. The findings elucidate the potential mechanisms of graft failure and highlight strategies to encourage healthy ex vivo pregraft conditioning.
Topics: Animals; Bioreactors; Cell Proliferation; Cells, Cultured; Female; Gene Expression Regulation; Mammary Arteries; Perfusion; Radial Artery; Saphenous Vein; Signal Transduction; Sus scrofa; Time Factors; Tissue Culture Techniques; Vascular Remodeling
PubMed: 30179877
DOI: 10.1159/000492484 -
Environmental Health Perspectives Jun 1984Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to... (Review)
Review
Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan.
Topics: Animals; Cattle; Cell Separation; Cells, Cultured; Clone Cells; Endothelium; Female; Lung; Microbial Collagenase; Muscle, Smooth; Peptide Hydrolases; Perfusion; Pulmonary Artery; Rats; Rats, Inbred Strains
PubMed: 6090112
DOI: 10.1289/ehp.8456103 -
Journal of Artificial Organs : the... Mar 2020The perfusion of medium through blood vessels allows the preservation of donor organs and culture of bioengineered organs. However, tissue damage due to inadequate...
The perfusion of medium through blood vessels allows the preservation of donor organs and culture of bioengineered organs. However, tissue damage due to inadequate perfusion remains a problem. We evaluated whether intermittent external pressurization would improve the perfusion and viability of organs in culture. A bioreactor system was used to perfuse and culture rat small intestine and femoral muscle preparations. Intermittent positive external pressure (10 mmHg) was applied for 20 s at intervals of 20 s. Intermittent pressurization resulted in uniform perfusion of small intestine preparations and minimal tissue damage after 20 h of perfusion, whereas non-pressurized (control) preparations exhibited significantly worse perfusion of the upper surface than the lower surface and histologic evidence of tissue damage. Longer term studies were undertaken in luciferase-expressing rat femoral muscle preparations. Compared with non-pressurized controls, intermittent pressurization led to better perfusion throughout the 14-day experimental period, improved organ viability as indicated by a higher bioluminescence intensity after perfusion with luciferin, and reduced levels of tissue necrosis with better preservation of vascular structures and skeletal muscle nuclei (histologic analyses). Therefore, intermittent application of external positive pressure improved the perfusion of small intestine and skeletal muscle preparations and enhanced tissue viability when compared with controls. We anticipate that this innovative perfusion technique could be used to improve the preservation of donor organs and culture of bioengineered organs.
Topics: Animals; Bioreactors; Extracorporeal Circulation; Intestine, Small; Muscle, Skeletal; Organ Culture Techniques; Perfusion; Rats; Tissue Engineering; Tissue Survival
PubMed: 31617028
DOI: 10.1007/s10047-019-01141-3