Name: murine renal cell isolation: a technique to isolate and generate primary culture of renal tubular epithelial cells
Uploaded: 2023-04-30T13:53:01.000+00:00
Duration: 1 min 43 s
Description: Source: Ding, W. et al. Isolation, Characterization and High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells. J. ...

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue Processing and Primary Tubular Cells Isolation
<ol>
	<li>Remove the renal capsules and medulla, mince both kidneys into tiny pieces, and incubate them in 10 mL of a digestion buffer in a 37 &#176;C oven with gentle rotation for 5 min.</li>
	<li>Remove any undigested kidney tissues by passing the buffer through a 70 &#956;m filter. Add 10 mL of culture media to stop the digestion.</li>
	<li>To collect tubular cells, centrifuge the filtered cell suspension at 50 x g for 5 min to collect the first pellet. Transfer the supernatant to a new tube and add 5 mL of culture media, centrifuge it at 50 x g for 5 min to ensure all tubular cells are collected into the second pellet. The centrifugation is at a lower speed to primarily pellet heavy tubules. Later, after the cells recover from the isolation, the pure tubular culture is centrifuged at a higher speed during the sub-cultures.</li>
	<li>Resuspend the first pellet in 20 mL of culture media and centrifuge it at 50 x g for 5 min to collect the third pellet.</li>
	<li>Resuspend the second and third pellets in 1 mL of culture media. Mix 10 &#181;L of the cell suspension with 10 &#181;L of Trypan Blue, load the mixture into chamber A of a counting slide, and record the cell viability from the automatic cell counter.</li>
	<li>Seed up to 107&#160;cells (a heterogeneous population) onto a single 60 mm dish pre-coated with collagen and let the tubular cells attach overnight.</li>
</ol>

<table><tbody><tr><td>Collagen I Rat Protein, Tail&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>A1048301</td><td></td></tr><tr><td>Collagenase Type II&amp;nbsp;</td><td>Worthington&amp;nbsp;</td><td>LS004176</td><td></td></tr><tr><td>Renal Epithelial Cell Growth Medium 2 Kit&amp;nbsp;</td><td>PromoCell&amp;nbsp;</td><td>C-26130</td><td></td></tr><tr><td>TC10 automated cell counter&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>506BR2119</td><td></td></tr></tbody></table>

murine renal cell isolation: a technique to isolate and generate primary culture of renal tubular epithelial cells

Source: Ding, W. et al. <a href="https://www.jove.com/video/57718" target="_blank">Isolation, Characterization and High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the steps to isolate and purify renal tubular epithelial cells for a wide range of molecular biology and cell culture studies.

Murine Renal Cell Isolation: A Technique to Isolate and Generate Primary Culture of Renal Tubular Epithelial Cells

Source: Ding, W. et al. Isolation, Characterization and High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells. J. ...

To isolate renal tubular epithelial cells or TECs, begin by taking a freshly isolated mouse kidney in a Petri dish. Peel off the renal capsule - the thin outer layer surrounding the kidney - and remove the medulla or the innermost part of the kidney, leaving the cortex containing the tubule and the tubular epithelial cells.
Mince the remaining kidney parts into small pieces and incubate them in a warm digestion buffer containing the enzyme collagenase. Collagenase breaks down the collagen protein that holds the renal tissues together, releasing the renal tubules and the renal cells into suspension.
Filter the suspension to remove undigested kidney tissues. Next, add pre-chilled culture media to the filtrate. The lower temperature neutralizes collagenase and stops further tissue digestion. Centrifuge the filtrate at low speed to allow the denser tubules and tubular cells to settle while other cell types and impurities remain suspended.
Remove this undesired supernatant fraction and add culture media to resuspend the pellet. Seed the suspension in a collagen-coated culture dish and incubate. The free tubular cells attach firmly to the collagen while the tubules remain suspended. Sub-culture to remove the unattached tubules and obtain the primary culture of tubular epithelial cells.

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Research

JoVE Encyclopedia of Experiments

Cancer Research

Urinary Tract Cancer

In vitro studies

2.3K Views. Źródło: Ding, W. et al. Izolacja, charakterystyka i wysokoprzepustowa analiza strumienia zewnątrzkomórkowego pierwotnych komórek nabłonka kanalików nerkowych myszy. J. Vis. Exp. (2018) W tym filmie pokazujemy kroki izolacji i oczyszczania komórek nabłonka kanalików nerkowych dla szerokiego zakresu badań biologii molekularnej i hodowli komórkowych. 

Video: Izolacja mysich komórek nerkowych: technika izolowania i generowania pierwotnej kultury komórek nabłonka kanalików nerkowych - Concept

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

JoVE Journal

Bioengineering

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Medicine

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Murine Renal Cell Isolation: A Technique to Isolate and Generate Primary Culture of Renal Tubular Epithelial Cells

Transkrypcja

Murine Renal Cell Isolation: A Technique to Isolate and Generate Primary Culture of Renal Tubular Epithelial Cells