Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Summary

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdc^scid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Abstract

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdc^scid/J) mouse.

In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

Introduction

Currently, hepatocyte transplantation is proposed as an alternative to whole organ transplantation for treating patients having severe liver disorders. It is believed that it can bridge patients to whole organ transplantation¹. In addition to the allogenic hepatocytes², xenogenic hepatocytes³ and hepatocytes derived from stem cells⁴ are also being investigated in animal models. In this context, the homing and engraftment potential of the transplanted cells in the recipient is an important criterion for cell based therapy in acute hepatic failure (AHF).

For investigating the transplantation of hepatocytes or hepatocyte-like cells⁵, AHF is created in an animal model either by surgical⁶ or pharmacological⁷ procedures, followed by transplanting cells. To make an AHF animal model by pharmacological reagents, many hepatotoxins such as d-galactosamine⁸, acetaminophen⁹, carbon tetrachloride¹⁰, thioacetamide¹¹, Concanavalin A¹², lipopolysaccharide¹³, etc., have been used. From this list, every reagent generates a unique set of features for AHF, but unfortunately no single reagent mimics the human AHF. Moreover, the AHF induced by hepatotoxins takes a long time, which puts animals under chronic stress, and reproducible results are difficult to obtain.

On the other hand, the surgical procedure of partial hepatectomy (PHx) is skill dependent, and reproducible results are easy to obtain after developing required skills. To induce AHF by surgical intervention alone, resection of more than 70% of the liver is required; however, less than a 70% hepatectomy can still be utilized to study engraftment and proliferation of transplanted cells in the liver for analyzing their therapeutic capacity during liver damage¹⁴. The transplantation of hepatocytes have been performed post hepatectomy through the peritoneum¹⁵, tail vein¹⁶, hepatic vein¹⁷, or the spleen¹⁸. Currently, hepatic vein infusion and intrasplenic transplantation of hepatocytes are the preferred procedures, as they are easier to reproduce.

In this paper, we have described a procedure for a 30% partial hepatectomy in NOD.SCID (NOD.CB17-Prkdcscid/J) mice in which the left lobe of the liver is excised. It is followed by transplantation of 0.2 million GFP expressing mouse (C57BL/6-Tg (UBC-GFP) 30Scha/J) hepatocytes as well as human origin NeoHep¹⁹ in the spleen. This procedure leads to engraftment of the transplanted cells in the liver. This procedure is the least invasive and a minimally painful technique.

Protocol

Procedures presented in this protocol have been approved by the Institutional Animal Ethics Committee of the National Institute of Immunology, New Delhi. The serial reference number of the approval is IAEC#319/13.

Note: There are excellent resources on general surgery procedures²⁰ and specific protocols for rodent surgery²¹. For those doing animal surgery for the first time, it is recommended to extensively practice surgical procedures on dummies before operating on animals.

1. Preparation

1. Prior to experiment, keep sterile phosphate buffered saline (PBS) or saline ready.
2. Assemble a surgery kit containing scissors, serrated forceps, tissue forceps, cotton, cotton buds, nylon threads, and different micro needle holders. Autoclave the surgery kit. Special care should be taken if an immune-compromised NOD.SCID mouse is included in the protocol.
3. Perform the complete experimental procedure, from the preparation to the end of surgery, in the bio-safety class I cabinet.
4. Weigh a NOD.SCID mouse of 6-8 weeks old prior to the surgery. Mice weighing between 14-18 g are used in this study.
5. Shave the upper central and hypochondriac abdominal region of the mouse with the hair trimmer. Apply hair removing cream evenly throughout the region with a spatula to completely remove the trimmed hairs. Remove the hairs gently with the help of a piece of wet sterile cotton after 2-5 min.
6. Place the mouse in the isoflurane chamber and unlock the valve of the oxygen cylinder. Maintain the oxygen flow at the rate of 4 L/min and the isoflurane vaporization at 4% to induce anesthesia.
   1. Ensure that the mouse has been anesthetized properly by gentle toe pinching.
7. Place a surgery board inside a biosafety cabinet. Place the animal on the surgery board, such that the ventral portion of the mouse is facing up and the anterior portion of the mouse is placed inside the nose cone connected to the isoflurane and oxygen supply.
8. Reduce the isoflurane vaporization to 2%, and maintain it throughout the surgical procedure.
9. Disinfect the skin of the mouse and sterilize it by wiping with 70% ethanol soaked sterile cotton.

2. Surgical Procedure

1. Partial Hepatectomy
   1. Make a transverse incision of around 1 cm in the skin just beneath the sternum, perpendicular to the xiphoid process and parallel to the ribcage, with the help of straight operating scissors.
   2. Gently separate the skin attached with the abdominal muscle layer in the vicinity of the incised area with forceps or sterile moistened cotton tips to distinguish between skin and abdominal muscle layer. Soak the intradermal region with PBS using sterile cotton tips to evade desiccation.
   3. Expose the area of the left lobe smoothly by making a transverse incision through the peritoneal layer just beneath the xiphoid. Use two moistened cotton tips to expose and lift the left lobe of the liver.
   4. Place one of the cotton tips on the abdominal side of the cut, and place another cotton tip on the diaphragm side. Gently press the tip placed towards the diaphragm, and give a sliding push by the other tip to lift the left lobe of the liver.
   5. Slip a nylon thread with a loop through the lifted left lobe, and slide the loop towards the base of the left lobe close to the hilum with the help of the micro-forceps or cotton tips. Gently push down the nylon thread loop to the base of the left lobe.
   6. Tie two ends of the nylon thread over the top of the left lobe using a microsurgery needle holder and the micro forceps. Make two additional knots on the other side.
   7. Dissect out the tied lobe with the help of scissors. Do not attempt to cut very close to the thread. In case the procedure lasts more than 5 min, keep the peritoneal cavity and organs moist with sterile PBS to avoid desiccation due to fluid loss.
   8. Sew the peritoneum by continuous suturing using a 4-0 Catgut suture. Subsequently, close the skin by discontinuous suturing as quickly as possible.
2. Cell Transplantation
   Note: The GFP expressing hepatocytes were isolated from transgenic GFP mice (C57BL/6-Tg (UBC-GFP) 30 Scha/J), according to the procedure described by Lee et al.²² and Shen et al.²³ Hepatocyte-like cells (NeoHep) of human origin differentiated from monocytes²⁴ were also used for transplantation. However, cells derived from any other sources may also be used in the protocol.
   1. Suspend 0.2 million viable cells in around 50 µL of Iscove's Modified Dulbecco's Medium (IMDM), and aspirate it in a 1 mL insulin syringe capped with 30G needle. Keep the syringe cold by placing it on ice.
   2. Place the mouse in a way that the left lateral portion faces up toward the person performing surgery. Identify the splenic area and transversely dissect the skin near the hypochondriac region, followed by a short incision through the peritoneal layer just to expose the spleen.
   3. Gently lift the spleen and hold it outside the cavity with the help of two PBS moistened cotton tips.
   4. Hold the spleen carefully with two cotton tips in one hand, and place the needle of the syringe exactly vertical to the spleen. Gently pierce the spleen and push the needle very slowly inside; the needle should not get deeper than 2 mm.
   5. Push down the piston of the syringe slowly to inject the cells into the spleen. After transplantation, keep the needle of the syringe stable and remove it slowly from the spleen to avoid bleeding or loss of cells.
   6. After placing the spleen back into the peritoneal cavity with the cotton tips, close the peritoneal layer by continuous suturing with a 4-0 Catgut suture. Sew the skin discontinuously with the same suture. Avoid using wound clips for closing skin; instead, close it by a 4-0 suture. The wound clips restrict the natural movement of the mouse, and often clips become loose and come out quickly.

3. Post-Operative Care

1. After closing the skin, wipe the surroundings of both the suture sites with iodine solution (betadine) using a sterile cotton tip.
2. Inject a dose of antibiotic cefotaxime as 600 mg/kg body weight (typically 12 mg cefotaxime in 100 µL of saline/mouse)intraperitoneally using 1 mL syringe.
3. Give daily doses of analgesic Meloxicam as 1 mg/kg body weight (typically 12 µg Meloxicam in 100 µL of saline/mouse) to the animal intraperitoneally, for up to three days after the surgery.
4. After completion of the surgery, stop the flow of isoflurane gas and place the mouse back in the individually ventilated cage.

4. Euthanization and Characterizations

1. After the experimental end-points (1 day and 10 days post-surgery), euthanize the mice according to the institutional animal ethics guidelines.
2. Collect blood by terminally bleeding the animals, puncturing the ocular terminal plexus.
3. Isolate serum from the blood.

Results

Hepatocyte proliferation after 30% partial hepatectomy: The proliferation of hepatocytes in the remaining liver after 30% hepatectomy was examined by immunohistochemical (IHC) staining for a cell proliferation marker, Ki-67. One-day post hepatectomy, the mice were euthanized, the remaining liver lobes were excised, and paraffin sections were obtained. The sections were stained with Ki-67 antibody, followed by labelling with horseradish peroxidase (HRP) conjugated secondary antibody. Di-Amino Benzidine (D...

Discussion

Partial hepatectomy is an established technique for investigating liver regeneration, and excessive hepatectomy is reported to mimic the AHF model. Among animal models of AHF, rodents, particularly mice, are the most researched model. To obtain a liver injury model in mice, up to a 70% hepatectomy has been reported with a good survival rate^25,26. However in nude and other immunodeficient mouse, a 70% hepatectomy was reported as fatal and animals died within 24 ho...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gas Anesthesia System	Ugo Basile; Italy	211000
Weighing machine	Goldtech ; India		Local Procurement
Biological safety cabinet ( Class I)	Kartos international;  India		Local Procurement
Hair Trimmer	Panasonic ;  Japan	ER-GY10
Straight operating scissor with sharp /sharp blades	Major Surgicals; India		Local Procurement
Forceps with Serrations	Major Surgicals; India		Local Procurement
Micro needle holders  straight & curved	Mercian ;  England	BS-13-8
1 ml insulin syringe with 30G *5/16 needles	Dispo Van; India
1 ml syringe with 26 G * 1/2 needle	BD ; US	REF 303060
Nylon Threads	Mighty ; India		(1-0) Local Procurement
MERSUTURES 4-0 Sterilised Surgical Needled Suture	Ethicon, Johnson & Johnson, India	NW 5047
TRUGUT 76 cm 4-0 absorbable surgical suture	Sutures India Pvt. Ltd; India	SN 5048	Sterilised Surgical Needled Suture Catgut Chromic
Cotton Buds	Pure Swabs Pvt Ltd ;  India		Local Procurement
Surgical Tape	3M India ; India	1530-1	Micropore Surgical Tape
Microtome	Histo-Line Laboratories, Italy	MRS3500
Shandon Cryotome E Cryostat	Thermo Electron Corporation ; US
Confocal laser scanning microscope	Carl Zeiss ; Germany	LSM 510 META
Bright Field Microscope	Olympus, Japan	LX51
Automated analyser	Tulip, Alto Santracruz, India	Screen Maaster 3000	Biochemical analyser for liver functional test
Flow Cytometer	BD ; US	BD FACSverse	Assesment of presence of cells post transplantation
Veet hair removal cream	Reckitt Benckiser , India
FORANE	Abbott ; US		isoflurane USP 99.9%
Taxim	AlKem ; India		cefotaxime sodium injection
Povidone-Iodine solution	Win-Medicare;  India		Betadine
Paraformaldehyde	Himedia; India	GRM 3660
Iscove's Modified Dulbecco's Medium (IMDM)	Life technologies, Thermo Fisher scientific ; US	12200-036
Sucrose	Sigma ; US	S0389
Tissue-Tek	Sakura; US	25608-930	O.C.T compound
DAPI	Himedia; India	MB 097
anti-Albumin goat Polyclonal	Thermo Scientific,Pierce, US	PA126081
anti-connexin 32/GJB1 Polyclonal	abcam, UK	ab64609-500
antiGFP rabbit polyclonal	Santa Cruz biotechnology; US	SC 8334
Alexa Fluor 594 donkey anti-goat	Molecular Probes , Thermo Fisher Scientific ;  US	A11058
Alexa Fluor 488 donkey anti-sheep	Molecular Probes , Thermo Fisher Scientific ;  US	A11015
Alexa Fluor 594 chicken anti rabbit	Molecular Probes , Thermo Fisher Scientific ;  US	A21442
Goat anti rabbit IgG HRP	Invitrogen, Thermo Fisher Scientific; US	65-6120
anti-Ki67 antibody	abcam, UK	ab15580
Antigen Unmasking Solution, Citric acid base	Vector laboratories, US	H-3300
ProLong Diamond antifade mountant	Life technologies, Thermo Fisher scientific ; US	P36966
SGOT (ASAT) KIT	Coral Clinical System, India
SGPT (ALAT) KIT	Coral Clinical System, India
Alkaline Phosphatase Kit (DEA)	Coral Clinical System, India
Hematoxylin Solution, Mayer's	Sigma ; US	MHS16
Eosin Y solution, alcoholic	Sigma ; US	HT110132
DPX Mountant	Sigma ; US	6522
Melonex (Pain Killer)	Intas Pharmaceuticals Ltd; India		Meloxicam injection
DAB enhanced liquid substrate system tetrahydrochloride	Sigma ; US	D3939