An Extracorporeal Microscopy Perfusion Chamber for On-Line Studies of Environmental Effects on Cultured Hepatocytes

1994 ◽  
Vol 116 (2) ◽  
pp. 135-139 ◽  
Author(s):  
Inne H. M. Borel Rinkes ◽  
Mehmet Toner ◽  
Ronald G. Tompkins ◽  
Martin L. Yarmush
Keyword(s):  
Cultured Cells ◽  
Culture Media ◽  
Organ Support ◽  
Extracorporeal Circuit ◽  
Perfusion Chamber ◽  
Cell Shapes ◽  
On Line ◽  
Perfusion Time ◽  
And Function

The development of bioartificial-hybrid organ support systems is hampered by the lack of knowledge on the effects of different (in vivo) environments on cells during extracorporeal perfusion. In the present study, a perfusion chamber was designed for continuous monitoring of cultured cells during perfusion with media, as well as during plasma perfusion in an extracorporeal circuit. Chamber characterization showed satisfactory thermal and perfusion profiles and no major pH fluctuations. Further testing was performed with hepatocytes that were cultured in between two collagen layers, a configuration which was previously shown to preserve hepatocyte morphology and function for over six weeks of culture. Perfusion of the hepatocytes with culture media did not adversely affect cell morphology and function, provided the perfusion time was ≤ 48 hours. Perfusion of the cultures during connection of the chamber to an extracorporeal circuit involving normal rats for six hours resulted in reversible cytoplasmic changes, unaltered cell shapes indices, and a 40 percent increase in albumin secretion rate during the first post-perfusion day, followed by a return to stable control levels. We expect that this chamber will be a valuable tool for on-line studies of cells under (extracorporeal) perfusion conditions and could be used for a large variety of studies on regeneration, reperfusion damage, and detoxification.

MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽  
2021 ◽  
pp. eabi8870
Author(s):  
Saba Parvez ◽  
Chelsea Herdman ◽  
Manu Beerens ◽  
Korak Chakraborti ◽  
Zachary P. Harmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cardiac Development ◽  
Droplet Microfluidics ◽  
Model Organisms ◽  
Genetic Screens ◽  
Large Numbers ◽  
And Function ◽  
Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

2020 ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela MM Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Single Copy ◽  
Specific Protein ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Peptide Tags ◽  
Genetic Technologies ◽  
Protein Binders ◽  
And Function

AbstractCellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Development ◽  
2021 ◽  
Vol 148 (6) ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela M. M. Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Specific Protein ◽  
Cellular Processes ◽  
Cell Compartments ◽  
Peptide Tags ◽  
Genetic Level ◽  
Protein Binders ◽  
And Function

ABSTRACT Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.

scholarly journals P0407THE INVESTIGATION OF SYNAPTOPODIN EXPRESSION OF PODOCYTES IN GLOMERONEPHRITIS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Zhigang Zhang
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Adriamycin Nephropathy ◽  
Assembly Result ◽  
And Function ◽  
Rhoa Signaling ◽  
New Strategies

Abstract Background and Aims Synaptopodin, a proline-rich actin-associated protein, plays an important role in the regulation of podocytes processes structures and dynamics. The mutation or lack of synaptopodin may lead to the changes of podocytes structures and functions and cause the occurrence of proteinuria. But the underlying molecular mechanisms remain primarily elusive. Method we used cellular and pathological experiments to observe the expression changes synaptopodin in vivo and vitrio. Results The results showed that the reduction expression of synaptopodin and RhoA were found in the podocytes in different nephriris of human renal biopsy as well as in rat adriamycin nephropathy. The cultured cells treated with inflammatory cytokins such as TNF, IL-1 also showed decreased synaptopodin level in podocyte, which led to low RhoA level and disarrange the actin cytoskeleton assembly, result in the abnormal changes of podocyte morphology. Conclusion These data preliminarily proved that synaptopodin loss in podocyte injury plays an important role in the regulation of podocyte morphology and function through RhoA signaling pathway, and further researches are required to clarify the more mechanism, which may provide new strategies and methods for the prevention and treatment of glomerular diseases.

scholarly journals Pancreatic-Duodenal Homeobox 1 Regulates Expression of Liver Receptor Homolog 1 during Pancreas Development

2003 ◽  
Vol 23 (19) ◽  
pp. 6713-6724 ◽  
Author(s):  
Jean-Sébastien Annicotte ◽  
Elisabeth Fayard ◽  
Galvin H. Swift ◽  
Lars Selander ◽  
Helena Edlund ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Regulatory Region ◽  
Mobility Shift ◽  
Pancreatic Development ◽  
Downstream Target ◽  
Regulatory Cascade ◽  
And Function ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1−/− mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.

scholarly journals The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

1999 ◽  
Vol 147 (2) ◽  
pp. 351-366 ◽  
Author(s):  
Vicki Mountain ◽  
Calvin Simerly ◽  
Louisa Howard ◽  
Asako Ando ◽  
Gerald Schatten ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Cross Linking ◽  
Simultaneous Inhibition ◽  
Balance Of Forces ◽  
And Function

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

scholarly journals The Importance of Proper Oxygenation in 3D Culture

2021 ◽  
Vol 9 ◽  
Author(s):  
Hubert M. Tse ◽  
Graeme Gardner ◽  
Juan Dominguez-Bendala ◽  
Christopher A. Fraker
Keyword(s):  
Cell Culture ◽  
Oxygen Consumption ◽  
Cell Function ◽  
Cultured Cells ◽  
Culture Media ◽  
Scale Up ◽  
3D Culture ◽  
Seeding Density ◽  
And Function

Cell culture typically employs inexpensive, disposable plasticware, and standard humidified CO2/room air incubators (5% CO2, ∼20% oxygen). These methods have historically proven adequate for the maintenance of viability, function, and proliferation of many cell types, but with broad variation in culture practices. With technological advances it is becoming increasingly clear that cell culture is not a “one size fits all” procedure. Recently, there is a shift toward comprehension of the individual physiological niches of cultured cells. As scale-up production of single cell and 3D aggregates for therapeutic applications has expanded, researchers have focused on understanding the role of many environmental metabolites/forces on cell function and viability. Oxygen, due to its role in cell processes and the requirement for adequate supply to maintain critical energy generation, is one such metabolite gaining increased focus. With the advent of improved sensing technologies and computational predictive modeling, it is becoming evident that parameters such as cell seeding density, culture media height, cellular oxygen consumption rate, and aggregate dimensions should be considered for experimental reproducibility. In this review, we will examine the role of oxygen in 3D cell culture with particular emphasis on primary islets of Langerhans and stem cell-derived insulin-producing SC-β cells, both known for their high metabolic demands. We will implement finite element modeling (FEM) to simulate historical and current culture methods in referenced manuscripts and innovations focusing on oxygen distribution. Our group and others have shown that oxygen plays a key role in proliferation, differentiation, and function of these 3D aggregates. Their culture in plastic consistently results in core regions of hypoxia/anoxia exacerbated by increased media height, aggregate dimensions, and oxygen consumption rates. Static gas permeable systems ameliorate this problem. The use of rotational culture and other dynamic culture systems also have advantages in terms of oxygen supply but come with the caveat that these endocrine aggregates are also exquisitely sensitive to mechanical perturbation. As recent work demonstrates, there is a strong rationale for the use of alternate in vitro systems to maintain physio-normal environments for cell growth and function for better phenotypic approximation of in vivo counterparts.

scholarly journals Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components during Fibroblast Passage of Human Cytomegalovirus

2018 ◽  
Author(s):  
Amine Ourahmane ◽  
Xiaohong Cui ◽  
Li He ◽  
Dirk Dittmer ◽  
Mark Schleiss ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Cultured Cells ◽  
Culture Media ◽  
Adaptive Mutations ◽  
Culture Passage ◽  
Genes Encoding ◽  
Cell Culture Passage

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly during cell culture passage, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt expression of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As in vivo CMV replicates in the context of host antibodies, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and aquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. An epitheliotropic RL13+/ UL131A+ virus was isolated by limiting-dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than has been previously possible.

scholarly journals Applications of novel bioreactor technology to enhance the viability and function of cultured cells and tissues

2020 ◽  
Vol 10 (2) ◽  
pp. 20190090 ◽  
Author(s):  
H. W. Hoyle ◽  
L. A. Smith ◽  
R. J. Williams ◽  
S. A. Przyborski
Keyword(s):  
Cultured Cells ◽  
Ex Vivo ◽  
Low Cost ◽  
Tissue Slices ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Tissue Constructs ◽  
And Function

As the field of tissue engineering continues to advance rapidly, so too does the complexity of cell culture techniques used to generate in vitro tissue constructs, with the overall aim of mimicking the in vivo microenvironment. This complexity typically comes at a cost with regards to the size of the equipment required and associated expenses. We have developed a small, low-cost bioreactor system which overcomes some of the issues of typical bioreactor systems while retaining a suitable scale for the formation of complex tissues. Herein, we have tested this system with three cell populations/tissues: the culture of hepatocellular carcinoma cells, where an improved structure and basic metabolic function is seen; the culture of human pluripotent stem cells, in which the cultures can form more heterogeneous tissues resembling the in vivo teratoma and ex vivo liver tissue slices, in which improved maintenance of cellular viability is seen over the 3 days tested. This system has the flexibility to be used for a variety of further uses and has the potential to provide a more accessible alternative to current bioreactor technologies.

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