Optimized and Simplified Technique for the Production and Culture of Precision-Cut Liver Slices

Protocols to produce and culture precision-cut liver slices, or PCLS, can vary drastically. The main drawback of PCLS is their short half-life, but here, we demonstrate that producing and culturing PCLS can be achieved in standard lab conditions with access to Vibratome whilst ensuring a half-life of at least four days. PCLS, an appealing model that fills the gaps between in vitro and in vivo models, but they're often neglected due to their relatively short half-life. However, they contain all cell types and a preserved architecture, essential for metabolic summation, and therefore resemble a mini model of the whole organ, enabling the study and treatment of live tissues whilst replicating their complex phenotypes. Continuous slices is a very simple process but technically challenging. To facilitate this, our protocol focuses on key steps, such as the orientation of cutting, and the embedding of the tissue, but also the relevance of a mini model volume for culture and a dynamic system to increase their half-life.

[Narrator] Before harvest, prepare one liter of Krebs-Henseleit buffer, a disinfected and cooled Vibratome tray, autoclaved Vibratome blades, and 4% low-melting agarose. On the day of harvest, keep the melted agarose in a water bath at 37 degrees Celsius until use. Next, prepare the culture plates by adding 2.6, 1.5, and 0.7 milliliters of complete media per well into six-, 12-, and 24-well plates respectively. Insert eight-micrometer porous inserts into each well. Place the plates into a humidified incubator set to 37 degrees Celsius with 5% carbon dioxide and 21% oxygen. Sterilize all instruments required for the harvest using appropriate sterilization methods. Spray the abdomen of the euthanized mouse with 70% ethanol to disinfect the area. Using sterile forceps and scissors, cut the skin and peritoneum from the middle of the abdomen to open the abdominal cavity. Gently dissect the liver from surrounding organs or vessels while avoiding damage to the lobes. Then, store the whole liver immediately in ice-cold Krebs buffer. Using blunt forceps and a sharp, sterile knife or scissors, separate each lobe individually to avoid damaging the lobes during separation. Using a sharp, sterile knife, trim all edges of the chosen lobe to create straight, manageable edges for embedding. Next, pour the 4% low-melting agarose into a three-centimeter Petri dish placed on wet ice to cool evenly and prevent the lobe from sinking. Leave the agarose to cool for another 30 seconds, and place the trimmed lobe in the center of the agarose, allowing it to settle in the middle of the block. Then, cut off the outside edges of the agarose block and dislodge the agarose block from the dish. Ensure that the top side and the side glued to the Vibratome platform are parallel to the upper edge of the lobe. Begin by setting the Vibratome to cut at a thickness of 250 micrometers with a speed of five and a frequency of seven hertz. After sterilizing the Vibratome and placing the tray onto the Vibratome, surround it with ice to keep the environment cold. Set the blades at a 10-degree downward angle from the horizontal plane. Then, fill the Vibratome tank with ice-cold Krebs buffer. Apply a thin layer of cyanoacrylate glue onto the platform to prepare it for mounting the agarose block. Afterward, dry the edge of the agarose block that will be glued onto the platform using sterile absorbent tissue. Now, place the agarose block onto the Vibratome's removable platform. Position the liver lobe upright to allow for transverse cuts, which reduces the pressure on the lobe and facilitates smoother cutting. Wait for one minute for the glue to set and submerge the platform into the Vibratome tank, ensuring the agarose block is completely covered with Krebs buffer. To program the Vibratome for cutting, set the start and stop positions. Then, start cutting the slices until the liver lobe is reached. Use a spatula to collect the liver slices, as this avoids damage, unlike forceps or brushes. Collect the slices in ice-cold Krebs buffer until they are ready for culture. Using a spatula, transfer the liver slices into the prepared wells containing media and inserts. Then, place the plates onto an orbital shaker set to 130 rpm and incubate them in a conventional cell culture incubator at 37 degrees Celsius with 5% carbon dioxide and 21% oxygen.