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

Summary

A protocol for the production and culture of Precision-cut Liver Slices (PCLS) for the study of mouse livers. The article focuses on key aspects of the protocol, which only requires standard laboratory equipment with access to a vibratome and allows survival of PCLS for a minimum of 4 days.

Abstract

This protocol presents a simple system for the creation and culture of Precision-cut Liver Slices (PCLS). PCLS contains all cells in an intact environment and, therefore, resembles a mini model of the whole organ. They enable the study of live tissues while replicating their complex phenotypes. This protocol allows the preparation of slices from mouse livers using a vibratome and standard laboratory equipment. Protocols for producing and culturing PCLS lack standardization and can vary quite drastically depending on the tissue of interest, the type of vibratome used, and the need for oxygen. These can be difficult to reproduce in some laboratories that have only access to a basic vibratome and common tissue culture facilities. We have put together a protocol focusing on the importance of some key steps within the varied protocols already available. This protocol, therefore, emphasizes the importance of the embedding method, the cutting orientation, a dynamic versus a static system, and the relevance of a minimum volume of culture. This protocol can be established and reproduced in a simple manner in most laboratories that have access to a basic tissue slicer. Taken together and following this protocol, PCLS can stay alive for a minimum of 4 days. PCLS is a simple, economical, and reproducible model to study pathophysiological and therapeutic screening for organs such as the liver.

Introduction

Precision-cut tissue slices (PCTS) are thin sections of organs. They allow the preservation of the architecture of the organ replicating a mini-organ while preserving the 3-dimensional aspect of neighboring cells and extracellular matrix. It is an appealing model due to its easy access, cost-saving, and less labor-intensive characteristics while preserving the tissue architecture.

PCTS fill a gap between in vitro cell studies and in vivo animal research, overcoming most disadvantages of both models. PCTS has been generated from various organs, such as the liver¹, intestines^2,3, colon², brain^4,5, lung^6,7,8, kidney^9,10, spleen^11,12, heart^13,14 but also tumors^15,16. They can also originate from various animals, such as mouse¹, rat^17,18 but also pig¹⁹ and human surgical wastes^15,20,21. Although PCTS require the use of animals, implying ethical related issues, the organ from one animal can generate multiple PCTS, thereby reducing the number of animals in agreement with the NC3Rs guidelines (Reduction, Replacement, Refinement)²² while limiting interindividual variations.

The development of improved tissue slicers, e.g., vibratomes²³, has allowed a transition from manually cut slices characterized by heterogeneous thickness and poor survival rate to reproducible thinner slices with better preserved structural integrity.

However, protocols for PCTS and, more specifically, Precision-cut Liver Slices (PCLS) preparation and culture vary significantly in the literature and lack standardization, especially for essential parameters such as slicing equipment, medium content, and culture conditions. The protocols can also vary noticeably depending on the tissue of origin. Some of the protocols will require oxygenation of the buffer or culture with some complicated bioreactor systems²⁴. They usually focus individually on different technical aspects or are designed for different tissues and can often be costly and more challenging to reproduce in the average laboratory in a cost-efficient manner.

Here, this protocol puts together some key points such as the embedding method, the direction of cutting, the use of transwells²⁵, a dynamic culture system²⁶ and the importance of a minimal volume of culture. Some of these steps have previously been optimized independently or in a different context, such as fibrosis²⁷ or tumor response²⁸. This protocol also emphasizes the importance of embedding using certain types of slicers and the orientation of cutting, which are both difficult parameters to master and often neglected in the literature. This simple method generates PCLS maintained in culture for a minimum of 4 days with an easy set-up and using standard laboratory equipment with access to a rudimentary tissue slicer.

Protocol

Wild-type CD57Bl/6J mice were purchased from Charles River Laboratories. Mice had free access to food and water, housed in individually ventilated cages with controlled temperature and humidity conditions and with a 12 h light cycle. Animals aged 3 weeks were sacrificed, and livers were promptly harvested without perfusion. All animal work was approved following local ethical review by the University College London Animal Welfare and Ethical Review Board and performed under Home Office project license PP9223137 and in accordance with the Home Office (Animals) Scientific Procedures Act (1986) and ARRIVE guidelines. All efforts were made to limit harm to animals in accordance with standard practice at the Biological Services Unit at University College London.

1. Set up for the experiment

1. On the day before harvest, perform the following steps.
   1. Prepare 1 L of Krebs-Henseleit buffer (KREBS) by dissolving one vial of Krebs powder into 1 L of sterile water. Cool it down to 4 °C and keep it on wet ice.
   2. Disinfect the tray with 70% ethanol and rinse with sterile PBS. Keep the tray wrapped in tin foil in the fridge overnight to help maintain a cold environment while cutting.
   3. Spray all other removable parts with ethanol, rinse with sterile PBS, leave to dry, and keep them sterile. Autoclave the blades and keep them sterile until use.
   4. Prepare 4% w/v low-melting agarose in sterile water. Once resuspended and melted, store the agarose in the fridge at 4 °C.
2. On the day of harvest and before the harvest of the liver, perform the following steps.
   1. Melt the 4% low melting agarose and keep it in a water bath at 37 °C until use. Make sure the agarose has cooled down to 37 °C and that all bubbles have dissipated before use.
   2. Prepare the culture plates by adding respectively 2.6 mL, 1.5 mL, and 0.7 mL per well into 6, 12, and 24 well plates. Add 8 µm porous inserts to each well. Place the plates into a humidified incubator set to 37 °C, 5% CO₂, and 21% O₂ level. This will help adjust the pH while warming up the media, so it is ready for culture.
   3. Culture the slices with porous 8 µm inserts to allow access to both faces of the slice. Prepare the media as follows: Add to the William's Medium E (WME), 2 mM L-glutamine supplement, 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, 10 μg/mL Gentamycin, 25 mM D-Glucose solution and 15 mM HEPES solution.

2. Collection of liver and preparation (15 min)

1. Sterilize all instruments prior to harvest.
2. Anaesthetize the mouse according to local procedures for the care of animals for scientific purposes by using an isoflurane mask. Before opening the abdominal cavity, pinch between the toes to ensure the animal is properly anesthetized. If the liver can be removed quickly, the mouse can be euthanized by CO₂ asphyxiation or cervical dislocation. As the procedure is a terminal procedure, do not use eye ointment as this would not affect the animal.
3. Spray the abdomen with 70% ethanol. Optional: Shave the mouse to prevent contamination with hair.
4. Open the abdominal cavity with sterile forceps and scissors by cutting the skin and peritoneum from the middle of the abdomen. Dissect the liver gently from other organs or vessels and avoid damaging the lobes.
5. Store the whole liver immediately in ice-cold Krebs Buffer. Perform all further steps on the ice at 4 °C and proceed to the liver preparation as quickly as possible to prevent further cell death.

3. Embedding of the liver lobes (25 min for each liver lobe)

1. Transfer the liver into a Petri dish on ice containing ice-cold Krebs buffer, making sure that the whole liver is fully submerged.
2. Separate each lobe individually using blunt forceps and a sharp, sterile knife to avoid damaging the lobes.
3. Choose the first lobe for sectioning and keep the remaining lobes in an ice-cold Krebs buffer until they are ready for embedding and slicing.
4. Cut all edges to get a more manageable lobe with straight edges ready for embedding. This will also help remove some of the fibrous Glisson's capsule to further facilitate sectioning. Do this while keeping the liver surfaces wet in an ice-cold Krebs buffer.
5. Place a 3 cm Petri dish (or similar) on wet ice and pour the 4% low-melting agarose (already in a 37 °C water bath) into it. Keep well on ice to allow the agarose to cool down in an upward direction while preventing the lobe from sinking to the bottom and embedding it uniformly.
6. Leave the agarose to further cool down for 30 s and place the trimmed lobe into it. The lobe will settle in the middle of the agarose block. The embedding process requires practice and optimization to suit every lab condition and personal experience.
7. Place the embedded lobe, still on ice, in the fridge for 5 min. The agarose should then be clearly set.
8. Cut off the outside of the agarose block. Dislodge the agarose block from the dish.
9. Cut the agarose into a more manageable size, ensuring that the upper side and the side glued to the vibratome platform are parallel to the upper edge of the lobe.
10. Start the slicing process as quickly as possible but ensure that the agarose block is kept in an ice-cold Krebs buffer and on ice.

4. Liver slices production (40 min per lobe)

1. Set the vibratome for cutting at a thickness of 250 µm, with a speed of 5 and a frequency of 7 Hz. These are guiding parameters; depending on the type of vibratome used, this might require optimization.
2. Spray the vibratome and bench areas with 70% ethanol before cutting to keep the environment as sterile as possible.
3. Place the tray onto the vibratome and pour ice all around it. Place the blades onto the vibratome at an angle of 10° downwards and below horizontal.
4. Fill the vibratome tank with ice-cold Krebs buffer. Place a thin layer of cyanoacrylate glue onto the platform.
5. Dry the edge of the agarose block that will be glued onto the platform using a sterile absorbent tissue.
6. Place the agarose block onto the removable platform. Position the lobe upright to allow it to be cut transversally. Although the size of the slices is reduced, this drastically facilitates the cutting process by limiting the pressure against the lobe while cutting.
7. Wait 1 min for the glue to set and submerge the removable platform into the tank and ensure the agarose block is completely covered with Krebs buffer.
8. Program the vibratome for cutting by setting the start and stop positions. Start cutting thicker slices initially until the liver lobe is reached.
9. Discard the first slice, as it might not be cut to the right thickness. To avoid damaging the slices, use a spatula to collect the liver slices instead of forceps or brushes.
   NOTE: A small brush disinfected with 70% ethanol and rinsed with sterile PBS can also be used to gently guide the tissue during the cutting process.
10. Repeat the process until the required number of slices is reached. The lobe might occasionally dislodge itself from the agarose, preventing further use. Collect the slices into ice-cold Krebs buffer until culture.

5. Incubation of liver slices

1. Transfer the slices, using a spatula, into the prepared wells containing the media and inserts.
2. Place the plates onto an orbital shaker, set the speed at 130 rpm, and incubate using a conventional cell culture incubator with 5% CO2 and 21% O2 at 37 °C. The final volume of culture is 2.6 mL, 1.5 mL, and 0.7 mL per well in 6, 12, and 24-well plates, respectively.
3. Place one empty plate below the culture plate containing the slices in culture to allow any excessive heat originating from the shaker's platform to be dissipated. Change the medium every 48 h.

6. Cell survival assay

1. Transfer the slices into a 48-well plate containing 400 µL of prewarmed complete Williams' Medium E media and add 80 µL of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) tetrazolium reagent.
2. Following incubation for 1 h at 37 °C, 5% CO₂ onto a shaker, transfer 200 µL of media into a 96-well plate, and measure the absorbance at 490 nm using a multi-well plate reader. Use slices left on the bench in PBS and at room temperature for 24 h as negative controls.

7. Histology staining

1. Deparaffinize and rehydrate the sections using xylene and ethanol, followed by deionized water. Stain the sections with Hematoxylin solution for 3 min.
2. Rinse with deionized water for 5 min. Dip quickly 10x into acid ethanol (1 mL of concentrated HCL and 400 mL of 70% ethanol).
3. Rinse 2x into deionized water and blot excess water. Dip the sections into Eosin for 30 s.
4. Dehydrate into 95% ethanol, then 100% ethanol for 5 min, 3x each. Dip sections into xylene 3x for 15 min each. Place coverslips on slides.

Results

At harvest, perfusion of the animal is purposely omitted to ensure rapid processing of the organ and prevent organ damage. The liver is extracted quickly following incision and immediately placed in an ice-cold organ-protective buffer, e.g., Krebs buffer^24,29. Although slicing fresh liver tissue without embedding has been previously described¹, embedding of the liver in low-melting agarose³⁰ (Fi...

Discussion

We demonstrate that producing and culturing PCLS can be easily achieved while ensuring a half-life of at least 4 days. This protocol recapitulates five critical steps: the embedding method if this type of vibratome is used, the orientation of cutting, a dynamic system of culture, a minimal volume of culture, and the use of inserts.

Protocols for the production and culture of PCLS are commonly available. However, they do lack standardization; they might focus on similar and specific points of t...

Materials

Name	Company	Catalog Number	Comments
3 cm petri dish	Any		any suitable for cell culture
6, 12, 24 well culture plate	Any		any suitable for cell culture
Cyanoacrylate super glue	Any
D-Glucose	Gibco	A24940
Eosin	Merck	HT110316
Ethanol	Any
Fetal Bovine serum	ThermoFisher	26400044
Gentamycin	Gibco	15750060
Hematoxylin	Merck	51275
HEPES	Gibco	H0887
inserts 8um, for 12 well plates	Strastedt	83.3931.800
inserts 8um, for 24 well plates	Strastedt	83.3932.800
inserts 8um, for 6 well plates	Strastedt	83.3930.800
KREBS	Merck	K3753
Laminar Flow Hood			Hepa air filtration
Low melting agarose	ThermoFisher	16520050
MTS tetrazolium reagent	Abcam	ab197010
multi-well plate reader	Any
PBS tablets	ThermoFisher	P4417
Penicillin/Streptomycin	Gibco	15140-122
Scalpel blade	Any
Surgical forceps	Any		with a flat square-tip
Surgical scissors	Any
Vibratome	Leica	VT1000 S
William’s Medium E with GlutaMAX (WME)	ThermoFisher	W4125