How do you isolate hepatocytes?

Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable.

What is hepatocyte isolation?

This protocol is aimed at isolating hepatocytes from mouse liver. Following anesthesia, the vena cava is cannulated and the liver is perfused to chelate calcium and wash out blood. Then, collagenase is perfused to the liver in order to dissociate extracellular matrix.

How do you isolate a mouse liver?

Collect the liver (sever the portal vein and the connective tissue and separate from the body) into tube containing 15 mL of Solution 2. Cut the liver sac to release the hepatocytes. Add 35 mL of Solution 3 and pass the liver suspension through a cell strainer (use spatula to gentle stir tissue on strainer).

How to culture hepatocytes?

Hepatocyte Culture Techniques. One common technique to culture primary hepatocytes is to seed the cells on a single layer of collagen gel. Under these conditions, hepatocytes secrete albumin and urea, and show minimal cytochrome P450 (CYP450) activity.

How many hepatocytes does a mouse liver have?

For human, rabbit, rat and mouse livers, the hepatocellular values were in the same range, more precisely 139+/-25, 114+/-20, 117+/-30 and 135+/-10 million cells per gram of liver, respectively. However, for the dog liver, the corresponding value was as high as 215+/-45 million cells per gram.

What are hepatocytes?

Hepatocytes, the major parenchymal cells in the liver, play pivotal roles in metabolism, detoxification, and protein synthesis. Hepatocytes also activate innate immunity against invading microorganisms by secreting innate immunity proteins.

How long does a hepatocyte live?

Cells Responsible for Normal Liver Tissue Turnover Hepatocyte replacement occurs relatively slowly; the average life span of adult hepatocytes ranges from 200 to 300 days.

What is human hepatocyte?

Anatomical terms of microanatomy. A hepatocyte is a cell of the main parenchymal tissue of the liver. Hepatocytes make up 80% of the liver’s mass.

How do you perfuse your liver?

Use the scissors to cut the abdominal skin vertically, to allow drainage of blood and perfusion liquid. Then squeeze the effluent blood vessel a few times with the straight forceps to inflate the liver, ensuring that all of the blood has drained out, and perfuse the liver with PBS until the liver tissue blanches.

How much does a mouse liver weight?

Rodent liver weights vary by species and strain but typically fall in the 2–3 g range (3–5% body weight, BW) in mice, and 4–5 g range (2–3% BW) in rats. As a percentage of total body mass, the mouse liver is more prominent than that of rats or humans.

Where are Kupffer cells found?

Kupffer cells (also known as stellate sinusoidal macrophages or Kupffer-Browicz cells) are macrophages found in the sinusoids of the liver. In fact, Kupffer cells make up 80% to 90% of all the macrophages in the entire human body.

What is bile Canaliculus?

Bile canaliculi are tiny, 1- to 2-μm wide tissue spaces formed by the apical membranes of adjacent hepatocytes. They form a delicate intralobular network of channels that drain bile produced by hepatocytes.

How to prepare a mouse for hepatocyte isolation?

40% Percoll: (see below, hold at 4° C)-one mouse needs 20-40ml. 1) Prepare pump, mouse and reagents. Rinse pump with 70% alcohol, sterile water and perfusion buffer. Clean abdominal area fur with 70% alcohol. Make midline incision to expose abdominal site.

How to isolate primary hepatocyte cells at UCLA?

9) Centrifuge at 150-200Xg for 7 minutes at 4°C. (After this step, viable hepatocytes will be at the bottom of the tubes) 10)Discard supernatant, wash 1-2 times with 20ml wash medium ( as step 7) 11) Resuspend washed cells with 10-20 ml plating medium and count cells using hemacytometer.

How to isolate hepatocytes in a centrifuge?

7) Centrifuge tubes at 50Xg for 3 minutes at 4°C. 8) Decant the supernatant and add 20ml 40% cold percoll to each tube, mix with pipette. 9) Centrifuge at 150-200Xg for 7 minutes at 4°C. (After this step, viable hepatocytes will be at the bottom of the tubes) 10)Discard supernatant, wash 1-2 times with 20ml wash medium ( as step 7)

Why are primary hepatocytes important in liver research?

Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high yields of viable primary mouse hepatocytes is technically challenging, limiting their use.

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