Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

The main advantage of our technique is that it is an easy, fast, and reproducible approach for editing genes in primary mouse hepatocytes as a way to generate in vitro and in vivo disease models. Using our technique, it may be possible to knock down a target gene in isolated hepatocytes and then transplant them back into the patient, to replace diseased cells with gene edited hepatocytes Inexperienced researchers might struggle with cannulation and keeping the catheter steady throughout the perfusion. The users are advised to carefully read and practice the procedure and the troubleshooting methods before getting started.

Demonstrating the liver perfusion and hepatocyte isolation will be Tina Parker a clinical facility manager and Ilayda Ates, a graduate research assistant. The electroporation procedure will be demonstrated by Callie Stuart, a graduate research assistant from my laboratory. Under a surgical plane of anesthesia.

Begin the liver perfusion by making a U-shaped lateral incision in the abdomen skin using scissors, and expand the hole through the sides. Continue opening the skin to the rib cage. Using scissors, cut through the peritoneum to expose the abdominal cavity up to the ribcage.

Being careful not to nick any organs. Move the intestines to the right side using the back of forceps or the blunt surface of scissors. Identify the portal vein and the inferior vena cava.

Start the pump to flush pre-warmed perfusion solution through the catheter. Stop the pump and remove the catheter. Then insert the tip of the needle connected to the catheter into the inferior vena cava at a 10 to 20 degree angle to the vein, remove the needle from the catheter.

Then gently push the catheter into the vein in a movement parallel to the vein. Connect the tubing to the catheter without moving the catheter further into the vein. Start the pump with a flow rate of two milliliters per minute.

Look for the liver immediately turning pale as a sign that the perfusion is successful. Cut the portal vein using scissors. Gradually increase the flow rate to five milliliters per minute.

Once the perfusion solution three is flowing through carefully monitor the elasticity of the liver. To determine if the liver has softened, gently press the liver with a cotton swab or forceps and check whether indentations form on the liver. Be careful not to over perfuse to avoid loss of cell viability.

Once the liver softens after 30 to 50 milliliters of perfusion solution three has flowed through, stop the pump, remove the catheter and the gallbladder. Careful not to spill its contents. Then carefully dissect the liver.

Place the liver into a 100 millimeter Petri dish containing ice cold DME M with 10%FBS medium. Swirl the Petri dish gently to remove possible blood clots. Transfer the liver to a new Petri dish containing ice cold DME M and 10%FBS medium.

To release cells from the liver capsule, place the Petri dish on ice then using two pairs of sterile forceps, gently tear apart the liver capsule on all lobes. Gently swirl the liver around in the medium using a cell lifter to release the hepatocytes. Continue this motion until the liver becomes very small and the suspension turns brown and opaque.

Remove the liver tissue remains from the plate and using a 25 milliliter serological pipette set to low speed, carefully transfer the cell suspension to a 50 milliliter conical tube pre chilled on ice and fitted with a 100 micrometer cell strainer. Add fresh ice cold DMEM, and 10%FBS medium to the Petri dish to wash out and collect the remaining cells from the surface. Then, transfer to the 50 milliliter conical tube.

Repeat this step until all the cells are collected. To wash and purify hepatocytes from non parenchymal cells, centrifuge the cells collected in the 50 milliliter conical tube at 50 RCF at four degrees Celsius for five minutes at low deceleration or without any breaks. Discard the super-naton containing debris and non-parenchymal cells.

Then re-suspend the pellet in the leftover super-naton by gently swirling the tube. After pellet re-suspension add 30 milliliters of fresh ice cold DMEM and 10%FBS medium. Begin preparing the CRISPR CAS 9 substrates media, cells and the electroporation instrument by turning on the power button on the electroporation device.

Then set the electroporation program by pressing the X button and then the down arrow button until the program T 0 28 appears on the screen. Prepared the destination wells for the electroporated hepatocytes by adding 1.5 milliliters of PM to six well collagen one coated plates. Incubate the plates in a 37 degree Celsius incubator until ready to use.

In PCR split strip tubes, prepare the CAS 9 RMP and mRNA complexes as described in the text manuscript. Store the mRNA SG RNA mix on ice until electroporation. Separately, prepare a tube containing one microgram of E G F P mRNA to verify successful electroporation using fluorescence microscopy.

Centrifuge the electroporation reaction at 100 RCF for two minutes at four degrees Celsius. Remove the supernaton from the cell pellet and add 100 microliters of nucleoffection solution per reaction along the side of the tube wall. Re suspend the hepatocytes in the electroporation solution by rocking the tube gently by hand.

Once the hepatocytes are evenly dispersed in the electroporation solution, transfer 100 microliters of the hepatocyte suspension to the strip tubes containing the CAS nine complexes. Transfer the contents of the strip well to an electroporation vessel. Place the nucleo-vet vessel into the slot on the electroporation device.

Electroporate the hepatocytes by pressing the X button. After electroporation, wait for a screen to pop up on the device that says:Okay. Press the X button again and remove the vessel.

Incubate the electroporated vessel on ice for 15 minutes. Add 500 microliters a prew-armed PM to the electroporation vessel. Transfer 300 microliters of the electroporation reaction to each destination well on the pre-warmed plate.

Incubate the plates overnight at 37 degrees Celsius. Add the membrane matrix to ice cold hepatocyte maintenance medium and mix by pipetting up and down 10 times. Remove the medium from the wells designated for gene editing analysis 24 hours after plating the cells.

Pipette the overlay mixture slowly on top of the cells and place the plate back at 37 degrees Celsius. At 24 hours after plating, transfer the conditioned medium from the well designated for MTT and albumin assays and store in a micro-fuge tube until ready to perform the albumin assay. Proceed with analysis of on target CAS nine activity by extracting the genomic DNA and setting up the PCR as described in the text manuscript.

Within 3 to 12 hours of plating the hepatocytes adhered to the plate and the cell morphology assumed a typical polygonal or hexagonal appearance within 24 hours The purity of the isolated cells was verified via glycogen staining using the periodic acid shift reagent at 24 hours after plating. The cytoplasm of the stained hepatocytes appeared magenta. The hepatocytes were imaged 24 hours after electroporation and on average, 89.8%of the hepatocytes were GFP positive.

At three days after electroporation CAS nine induced in the HPD locus where analyzed and the results showed on target indels of 47.4%in hepatocytes electroporated with CRISPR Cas nine mRNA and 78.4%indels for the CAS nine RNP. The MTT assay showed 35.4 and 45.9%viability in hepatocytes treated with CRISPR Cas nine mRNA and R N P.Consistent with the MTT results the normalized albumin levels were 31.8%in hepatocytes treated with CAS nine RNA and 34.5%for CAS nine r p. The most important thing to remember is that proper cannulation affects all of the later steps and the success of the procedure.

After this procedure, the hepatocytes can be transplanted and mice to investigate their engraftments. Moreover, the genomic DNA of plated hepatocytes can be extracted to analyze the efficiency and specificity of CAS nine editing.