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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

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.

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.

To process the harvested kidneys, first, remove the renal capsules and medulla. Then, mince both kidneys into tiny pieces and incubate them in 10 milliliters of a digestion buffer in a 37 degrees Celsius oven with gentle rotation for 5 minutes.
After the digestion, remove any undigested tissues by passing the buffer through a 70-micron filter. After collecting the digested tissue, add 10 milliliters of culture media to stop the digestion. Next, centrifuge the filtered tissue suspension at 50 x g for 5 minutes to pellet the tubular cells. Then, transfer the supernatant to a new tube, add 5 milliliters of culture media, and repeat the centrifugation to collect any remaining tubular cells.
Now, resuspend the first pellet in 20 milliliters of culture media and centrifuge it at 50 x g for 5 minutes to further purify the collected cells. Dispose of the supernatant. Then, resuspend both pellets in 10 milliliters of culture medium. Next, count the living cells using Trypan Blue staining. Then, seed up to 10 million cells onto a 60-millimeter collagen-coated dish and let the tubular cells attach overnight.

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6.2K Views. 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 colla...

Abstract

Protocol

Disclosures

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

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Abstract

Protocol

Disclosures

Materials

Tags

Name	Company	Catalog Number
Collagen I Rat Protein, Tail	ThermoFisher Scientific	A1048301
Collagenase Type II	Worthington	LS004176
Renal Epithelial Cell Growth Medium 2 Kit	PromoCell	C-26130
TC10 automated cell counter	Bio-Rad	506BR2119