This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.
This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 × 106 cells per liver and cell viability of >85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of >35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.
IMDs of the liver are genetic disorders characterized by the deficiency of a crucial hepatic enzyme involved in metabolism that leads to the accumulation of toxic metabolites. Without treatment, IMDs of the liver result in organ failure or premature death1,2. The only curative option for patients with IMDs of the liver is orthotopic liver transplantation, which is limited due to the low availability of donor organs and complications from immunosuppressive therapy following the procedure3,4. According to recent data collected by the Organ Procurement and Transplantation Network, only 40%-46% of adult patients on the liver transplant waiting list receive an organ, while 12.3% of these patients die while on the waiting list5. Moreover, only 5% of all the rare liver diseases have an FDA-approved treatment6. It is clear that there is a critical need for novel treatments for IMDs of the liver. However, appropriate disease models are required to develop new therapeutic options.
Modeling human diseases using in vitro and in vivo systems remains an obstacle for developing effective therapies and studying the pathology of IMDs of the liver. Hepatocytes from patients with rare liver diseases are challenging to obtain7. Animal models are crucial for developing an understanding of disease pathology and for testing therapeutic strategies. However, one obstacle is generating models from embryos carrying lethal mutations. For example, attempts to create mouse models of Alagille Syndrome (ALGS) with embryos containing homozygous deletions of a 5 kb sequence near the 5′-end of the Jag1 gene resulted in the early death of the embryos8. In addition, generating mouse models by gene editing in embryonic stem cells can be time- and resource-intensive9. Lastly, mutations will appear outside the targeted tissue, leading to confounding variables that may impede study of the disease9. Somatic gene editing would allow for easier editing in liver tissue and bypasses the challenges associated with generating models using embryonic stem cells.
Electroporation enables the delivery of CRISPR-Cas9 directly into the nucleus by applying high-voltage currents to permeabilize the cell membrane and is compatible with many cell types, including those that are intransigent to transfection techniques, such as human embryonic stem cells, pluripotent stem cells, and neurons10,11,12. However, low viability is a potential drawback of electroporation; optimizing the procedure can yield high levels of delivery while limiting toxicity13. A recent study demonstrates the feasibility of electroporating CRISPR-Cas9 components into primary mouse and human hepatocytes as a highly efficient approach14. Ex vivo electroporation in hepatocytes has the potential to be applied to generate new mouse models for human IMDs of the liver.
This protocol provides a detailed step-by-step procedure for isolating mouse hepatocytes from the liver and subsequently electroporating CRISPR-Cas9 as RNP complexes, consisting of Cas9 protein and synthetic single-guide RNA (sgRNA), or Cas9 mRNA combined with sgRNA to obtain high levels of on-target gene editing. In addition, the protocol provides methods for quantifying gene editing efficiency, viability, and functionality following electroporation of CRISPR-Cas9 into freshly isolated mouse hepatocytes.
The animal experiments were all performed in compliance with the Institutional Animal Care and Use Committee guidelines and approved protocols at Clemson University. Surgical procedures were performed in anesthetized wild-type C57BL/6J mice between 8 and 10 weeks old.
1. Animal surgery
2. Hepatocyte isolation
3. Design sgRNAs for CRISPR-Cas9 gene editing
NOTE: This section describes the design of an sgRNA targeting the mouse hydroxyphenylpyruvate dioxygenase (Hpd) gene as proof-of-principle gene editing to disrupt a therapeutic target gene related to an IMD of the liver.
4. Electroporation and cell culture
5. Calculate the volume of the membrane matrix to mix with MM to give a final concentration of 0.25 mg/mL
NOTE: For a 6-well plate, 2 mL of overlay is needed per well.
6. Add the calculated volume of the membrane matrix to ice cold MM and mix by pipetting up and down 10 times
7. Analysis of delivery efficiency, viability, and on-target editing in electroporated mouse hepatocytes
Isolation of plateable primary hepatocytes from the liver
The overall process of liver perfusion and hepatocyte isolation is illustrated in Figure 1. In this experiment, wild-type, 8-10-week-old C57BL6/6J mice were used. The procedure is expected to yield 20-50 × 106 cells per mouse with a viability between 85% and 95%. If the viability is <70%, percoll treatment should be followed to remove dead cells. Freshly isolated hepatocytes should be plated on collagen I-coated or nitrogen-containing tissue culture plates. Within 3-12 h of plating, the hepatocytes are expected to adhere to the plate, and the cell morphology assumes a typical polygonal or hexagonal appearance within 24 h (Figure 2). Hepatocytes are the only cells in the liver that store glucose in the form of glycogen. To verify the purity of the isolated cells, glycogen staining is performed using Periodic acid-Schiff reagent at 24 h after plating. The cytoplasm of the stained hepatocytes appears magenta (Figure 3).
Electroporation of CRISPR-Cas9 RNPs and mRNA into isolated mouse hepatocytes
Freshly isolated mouse hepatocytes were electroporated with eGFP mRNA, Cas9 mRNA along with Hpd-targeting sgRNA, or Cas9 protein complexed with Hpd-sgRNA (RNP). The Hpd-sgRNA were chemically modified with 2′-O-methyl phosphorothioate linkages to the first and last three consecutive nucleotides on the 5′ and 3′ ends14. Streptococcus pyogenes Cas9 was used for experiments. The hepatocytes were imaged 24 h after electroporation using a fluorescence microscope, and the percentage of GFP-positive cells was counted in the images (Figure 4A). On average, 89.8% [range 87.1%-92.4%] of the hepatocytes were GFP-positive. At 3 days after electroporation, Cas9-induced insertions and deletions (indels) in the Hpd locus were analyzed using TIDE16. The results show on-target indels of 47.4% in hepatocytes electroporated with CRISPR-Cas9 mRNA and 78.4% indels for the Cas9 RNP (Figure 4B). MTT and albumin assays were performed to evaluate hepatocyte viability and functionality after electroporating CRISPR-Cas9. The MTT results show a viability of 35.4% and 45.9% in hepatocytes treated with CRISPR-Cas9 mRNA and RNP, respectively (Figure 4C). Consistent with the MTT results, the normalized albumin levels were 31.8% in hepatocytes treated with Cas9 mRNA and 34.5% for Cas9 RNP (Figure 4D).
Figure 1: Schematic of the hepatocyte perfusion and isolation protocol. After cannulation of the inferior vena cava, the portal vein is cut, and Perfusion Solutions 1, 2, and 3 are pumped through the liver. The liver capsule is ruptured, and cells are released using a cell lifter. Released cells are strained and centrifuged. Please click here to view a larger version of this figure.
Figure 2: Freshly isolated primary hepatocytes. Hepatocytes isolated from C57BL/6J mice were plated on collagen I-coated 6-well plates with Hepatocyte Plating Media. Images taken at 24 h after plating. Scale bar = 400 µm. Please click here to view a larger version of this figure.
Figure 3: Glycogen staining. Freshly isolated primary mouse hepatocytes were stained for glycogen to confirm purity. Staining was done using Schiff Reagent at 24 h after plating. Cytoplasm of the stained hepatocytes appear magenta. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 4: Electroporation of Hpd-targeting CRISPR-Cas9 into freshly isolated primary mouse hepatocytes. (A) Phase contrast and green fluorescence microscopy images of freshly isolated mouse hepatocytes at 24 h after electroporation. Images in the upper row are hepatocytes transfected with eGFP mRNA and the images in the lower row are untransfected control hepatocytes. Scale bars = 400 µm. (B) On-target indels for mouse hepatocytes electroporated with Cas9 mRNA combined with sgRNA or RNP. (C) Viability normalized to untransfected control hepatocytes in MTT assay at 24 h after electroporation. (D) Albumin levels in conditioned medium normalized to untransfected control hepatocytes measured at 24 h after electroporation(n = 3) with two technical replicates. Statistical analysis was performed by unpaired t-tests. This figure was modified from 14. Abbreviations: Hpd = 4-hydroxyphenylpyruvate dioxygenase; CRISPR = clustered regularly interspaced short palindromic repeats; Cas9 = CRISPR-associated protein 9; sgRNA = single guide RNA; RNP = ribonucleoprotein; INDEL = insertion-deletion; MTT = 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide. Please click here to view a larger version of this figure.
Table 1: Troubleshooting for the liver perfusion and hepatocyte isolation protocol. This table highlights common problems encountered during the protocol and suggests possible solutions. Please click here to download this Table.
Supplemental Figure S1: Mouse abdominal cavity during surgery. Catheter is inserted into the inferior vena cava (blue dot) before the renal branches (not seen). Abbreviations: L = Liver; K = Kidney; I = Small Intestines. Please click here to download this File.
Supplemental Table S1: Cas9 guide and PCR primer sequences. The guide sequence and PAM for sgRNA targeting Hpd, and PCR primer sequences for amplification of Hpd. Abbreviations: Hpd = 4-hydroxyphenylpyruvate dioxygenase; CRISPR = clustered regularly interspaced short palindromic repeats; Cas9 = CRISPR-associated protein 9; sgRNA = single guide RNA; PAM = protospacer-adjacent motif. Please click here to download this Table.
The steps outlined in the protocol for hepatocyte isolation are challenging and require practice for proficiency. There are several key steps for the successful hepatocyte isolation from the liver. First, proper cannulation of the inferior vena cava is essential for complete liver perfusion. The absence of blanching in the liver after perfusion indicates displacement of the catheter (Table 1). The inferior vena cava (retrograde perfusion) was cannulated in the procedure because it is simpler and more accessible than the portal vein (antegrade perfusion)17,18. Some protocols use sutures to secure the catheter19; however, sutures complicate the process. Suturing the catheter can result in unwanted outcomes, such as the misplacement of the catheter and making it impossible to adjust its position. In addition, it is possible to perform the cannulation in the liver without removing the needle, which may further simplify the liver perfusion. Connecting the pump to the catheter is unnecessary with the needle in the vein. In addition, there is a reduced possibility of accidentally pulling out the catheter and forming blood clots. Inserting only the tip of the catheter with a flat angle relative to the vein enhanced the canulation. The inferior vena cava has several branches, so inserting the catheter in the wrong site will cause perfusion in another part of the body and not the liver (Table 1). Thus, it is vital to place the catheter in a site that avoids the branches. The vascular structure will vary slightly between different mice; thus, it is important to examine the veins before cannulating to determine the best site for injecting the catheter. The backflush of blood inside the catheter after removing the needle is an excellent indication of proper cannulation. Proper cannulation can also be checked by looking for liver swelling when pressure is momentarily applied to the portal vein (step 1.3.13).
When isolating hepatocytes, the second critical step is recognizing when complete liver digestion has occurred. The enzyme quality and concentration are essential for proper digestion. In contrast to the protocols found in 20,21, this protocol suggests using Liberase, consisting of two collagenase isoforms, because it provides better consistency in enzyme activity and reduced batch-to-batch variation than regular collagenase22. Variations in digestive enzyme quality and activity could cause inconsistencies in the isolation. It is essential to monitor the liver during digestion and stop the digestion immediately after the liver has softened. A digested liver will be flexible and show a loss in elasticity verified by depressing the liver using forceps or cotton swabs to confirm that indentations form without the tissue bouncing back. In this protocol, the maximum amount of Liberase solution recommended per liver is 50 mL; a higher volume would result in overdigestion. If the cannulation is perfectly performed, 30 mL of the Liberase solution is sufficient to achieve proper digestion. The final critical steps of the procedure are the isolation and washing steps. After dissecting the liver and transferring it to a sterile Petri dish containing ice-cold DMEM with FBS, it is crucial to avoid cutting the liver into pieces. At this stage, use a cell lifter to release the cells gently and maximize cell viability. Tilting the dish to submerge the liver in the DMEM facilitates the release of cells from the capsule into suspension and must be done slowly and on ice. It should be easy to disrupt the Glisson's capsule during the releasing step, and the medium should become cloudy and brown. Difficulty when disrupting the capsule is an indicator of poor digestion (Table 1).
This protocol is suitable for generating high levels of CRISPR-Cas9 edits in freshly isolated mouse hepatocytes. Indels generated by Cas9 RNP and mRNA were compared in the study. Higher levels of gene editing were observed in hepatocytes electroporated with the Cas9 RNP than mRNA, which is consistent with findings from other studies14,23,24. The advantage of Cas9 RNP over mRNA is that it provides lower off-target editing since the Cas9 protein exists in the cell for shorter periods than mRNA23,24. Because the efficiency of sgRNAs varies based on the design and target site, multiple sgRNAs should be designed and tested for editing efficiency at the gene of interest. Select the higher specificity scoring design when choosing between sgRNA having high gene efficiencies. If gene editing is consistently low, consider co-delivering a reporter, such as eGFP mRNA, to confirm delivery. Low levels of eGFP-positive cells after electroporation could indicate expired electroporation buffer, electroporation device issues, or air bubbles in the reaction. If there are high numbers of eGFP-positive cells but low editing efficiency, test the sgRNA design in a cell line to confirm editing activity and adjust the amounts of Cas9 and sgRNA electroporated into cells. Lastly, consider the method used to evaluate editing activity. Gel-based assays, such as T7 Endonuclease I, can under-report gene editing due to low sensitivity for 1 bp mismatches at the cut site. Deep sequencing is the most accurate method for quantifying Cas9 gene editing, particularly for low-editing events at off-target sites.
Another challenging aspect of the protocol is culturing freshly isolated hepatocytes after electroporation. Users must handle cells gently during each step of the protocol to obtain viable, plateable hepatocytes after electroporation. Avoid dispersing hepatocytes by vortex; instead, rock the vial containing cells gently until the cells become resuspended. When transferring hepatocytes, use wide-bore pipette tips to maintain viability. Incubating the cuvettes on ice for 15 min following electroporation allows the cell membrane to reseal and enhance viability. After plating, place the plate in the incubator and gently move the plate horizontally and vertically in a north-to-south and east-to-west motion to ensure the hepatocytes disperse evenly and maintain cell viability. If the hepatocytes fail to attach to the plate, consider increasing the number of cells to 1.3 × 106 in the electroporation reaction.
One promising application for the protocol is the generation of mouse models of human IMDs of the liver. Mouse hepatocytes that are positive for the gene encoding for fumarylacetoacetate hydrolase (Fah) have been shown to efficiently engraft and repopulate the liver after transplantation in Fah-deficient (Fah-/-) mice25. A novel approach for developing mouse models of IMDs of the liver is by electroporating CRISPR-Cas9 into wild-type mouse hepatocytes to introduce edits associated with a disease, followed by transplantation of the gene-edited hepatocytes in Fah-/- mice. The Cas9-edited hepatocytes would have a natural selective advantage after transplantation compared to the native Fah-deficient cells for repopulating the liver.
In conclusion, this protocol equips users with the capacity to isolate primary hepatocytes from the mouse liver, followed by gene-editing using CRISPR-Cas9 mRNA and RNPs. The protocol can be modified for use in different strains of mice to study various types of genetic diseases affecting the liver in vitro and in vivo and test therapeutic approaches.
The authors have nothing to disclose.
RNC received funding from South Carolina Bioengineering Center of Regeneration and Formation of Tissues Pilot grant supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health, the American Association for the Study of Liver Diseases Foundation, and American Society of Gene & Cell Therapy under grant numbers P30 GM131959, 2021000920, and 2022000099, respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Society of Gene & Cell Therapy or the American Association for the Study of Liver Diseases Foundation. The schematic for Figure 1 was created with BioRender.com.
Equipment | |||
0.2 mL PCR 8-tube FLEX-FREE strip, attached clear flat caps, natural | USA Scientific | 1402-4700 | |
6-well Collagen Plates | Advanced Biomatrix | 5073 | |
accuSpin Micro 17R | Fisher Scientific | 13-100-675 | |
All-in-one Fluorescent Microscope | Keyence | BZ-X810 | |
Analog Vortex Mixer | VWR | 97043-562 | |
ART Wide BORE filtered tips 1,000 µL | ThermoFisher Scientific | 2079GPK | |
Automated Cell Counter | Bio-Rad Laboratories | TC20 | |
Blue Wax Dissection Tray | Braintree Scientific Inc. | DTW1 | 9" x 6.5" x 1/2"+F21 |
Cell scraper/lifter | Argos Technologies | UX-04396-53 | Non-pyrogenic, sterile |
Conical tubes (15 mL) | Fisher Scientific | 339650 | |
Conical tubes (50 mL) | Fisher Scientific | 14-432-22 | |
Cotton applicators | Fisher Scientific | 22-363-170 | |
Curved scissors | Cooper Surgical | 62131 | |
Disposable Petri Dishes | Falcon | 351029 | 100mm,sterile |
Disposable Petri Dishes | VWR | 25373-100 | 35mm, sterile |
Epoch Microplate Spectrophotometer | BioTek Instruments | 250082 | |
Falcon Cell strainer (70 µm) | Fisher Scientific | 08-771-2 | |
Forceps | Cooper Surgical | 61864 | Euro-Med Adson Tissue Forceps |
IV catheters | BD | 382612 | 24 G x 0.75 in |
IV infusion set | Baxter | 2C6401 | |
MyFuge 12 Mini Centrifuge | Benchmark Scientific | 1220P38 | |
Needles | Fisher Scientific | 05-561-20 | 25 G |
Nucleofector 2b Device | Lonza | AAB-1001 | Program T-028 was used for electroporation in mouse hepatocytes |
Peristaltic Pump | Masterflex | HV-77120-42 | 10 to 60 rpm; 90 to 260 VAC |
Precision pump tubing | Masterflex | HV-96410-14 | 25 ft, silicone |
Primaria Culture Plates | Corning Life Sciences | 353846 | Nitrogen-containing tissue culture plates |
Serological Pipets (25 mL) | Fisher Scientific | 12-567-604 | |
Syringes | BD | 329464 | 1 mL, sterile |
T100 Thermal Cycler | Bio-Rad Laboratories | 1861096 | |
Water bath | ALT | 27577 | Thermo Scientific Precision Microprocessor Controlled 280 Series, 2.5 L |
Reagents | |||
Alt-R S.p. Cas9 Nuclease V3 | IDT | 1081058 | |
Beckman Coulter AMPure XP, 5 mL | Fisher Scientific | NC9959336 | |
CleanCap Cas9 mRNA | Trilink Biotechnologies | L-7606-100 | |
CleanCap EGFP mRNA | Trilink Biotechnologies | L-7201-100 | |
Corning Matrigel Matrix | Corning Life Sciences | 356234 | |
DMEM | ThermoFisher Scientific | 11885076 | Low glucose, pyruvate |
Ethanol 70% | VWR | 71001-652 | |
Fetal bovine serum | Thermoscientific | 26140-079 | |
Hepatocyte Maintenance Medium (MM) | Lonza | MM250 | |
Hepatocyte Plating Medium (PM) | Lonza | MP100 | |
Mouse Albumin ELISA Kit | Fisher Scientific | NC0010653 | |
Mouse/Rat Hepatocyte Nucleofector Kit | Lonza | VPL-1004 | |
OneTaq HotStart DNA Polymerase | New England Biolabs | M0481L | |
PBS 10x pH 7.4 | Thermoscientific | 70011-044 | No calcium or magnesium chloride |
Percoll (PVP solution) | Santa Cruz Biotechnology | sc-500790A | |
Periodic acid | Sigma-Aldrich | P7875-25G | |
Permount Mounting Medium | VWR | 100496-550 | |
QuickExtract DNA Extraction Solution | Lucigen Corporation | QE05090 | |
Schiff’s fuchsin-sulfite reagent | Sigma-Aldrich | S5133 | |
Trypsin-EDTA (0.25%) | ThermoFisher Scientific | 25200056 | Phenol red |
Vybrant MTT Cell Viability Assay | ThermoFisher Scientific | V13154 | |
Perfusion Solution 1 (pH 7.4, filter sterilized) | Stable at 4 °C for 2 months | ||
EBSS | Fisher Scientific | 14155063 | Complete to 200 mL |
EGTA (0.5 M) | Bioworld | 40520008-1 | 200 µL |
HEPES (pH 7.3, 1 M) | ThermoFisher Scientific | AAJ16924AE | 2 mL |
Perfusion Solution 2 (pH 7.4, filter sterilized) | Stable at 4 °C for 2 months | ||
CaCl2·2H20 (1.8 mM) | Sigma | C7902-500G | 360 µL of 1 M stock |
EBSS (no calcium, no magnesium, no phenol red) | Fisher Scientific | 14-155-063 | Complete to 200 mL |
HEPES (1 M) | ThermoFisher Scientific | AAJ16924AE | 2 mL |
MgSO4·7H20 (0.8 mM) | Sigma | 30391-25G | 160 µL of 1 M stock |
Perfusion Solution 3 | Prepared fresh prior to use | ||
Solution 2 | 50 mL | ||
Liberase | Roche | 5401127001 | 0.094 Wunsch units/mL |
Mouse Anesthetic Cocktail | |||
Acepromzine | 0.25 mg/mL final concentration | ||
Ketamine | 7.5 mg/mL final concentration | ||
Xylazine | 1.5 mg/mL final concentration | ||
Software | URL | ||
Benchling | https://www.benchling.com/ | ||
ImageJ | https://imagej.nih.gov/ij/ | ||
TIDE: Tracking of Indels by Decomposition | https://tide.nki.nl/ |