Isolation of fetal liver tissue in rats
ED14 fetal liver tissues were collected from the fetus of a pregnant DPPIV(+) F344 female rat (Japan SLC, Shizuoka, Japan) and used as donor tissues for transplantation. The rat fetus was sacrificed by euthanasia with isoflurane, and the fetal liver was removed under a microscope. The fetal liver tissue was immediately stored on ice in phosphate buffered saline (PBS) until transplantation. Liver tissue isolation time is critical; thus, the liver tissues must be isolated in minimal time.
Induction of liver cirrhosis in rats
Three-week-old DPPIV(−) F344 rats (Japan Charles River, Kanagawa, Japan) were acclimated in an animal facility for 2 weeks before induction for liver cirrhosis. The animals were maintained under SPF environment with 12 h light and 12 h dark cycle. Moreover, we induced liver cirrhosis in these rats by intraperitoneal DMN injection at 10 mg/kg (body weight) concentration for 3 consecutive days/week for over 3 weeks. All animal experiments were designed according to the ethical rules of the Animal Research Center in Medical College of Yokohama City University. We bred and maintained the rats according to our institutional guidelines for the care and use of laboratory animals. The Institutional Animal Care Use Committee of Yokohama City University approved all our animal studies (Approval No. 17–25).
Matrigel (Corning, New York, NY, USA), fibrin (Sigma-Aldrich, St. Louis, MO, USA), oxidised cellulose (Interseed; Johnson and Johnson, New Brunswick, NJ, USA)and sodium hyaluronate (Seprafilm; Kaken Pharmaceutical, Co, Ltd., Tokyo, Japan) were used as coating agents. Fibrin was freshly mixed with fibrinogen at 100:1 ratio before use. UPAL was kindly donated by Mochida Pharmaceutical co. ltd. Tokyo, Japan. Alginate was carefully placed over the transplanted tissue, then we add some calcium chloride to cover the alginate and prompt gelation.
First, the liver cirrhosis rat model was anaesthetized using isoflurane. Then, the abdominal wall of the animal was transected to expose the abdominal viscera. After blocking the portal blood flow using vascular clamp forceps, we sharpened the middle lobe surface by using an 18 G needle (Terumo corporation, Tokyo, Japan). After detachment, we performed compression hemostasis using a cotton swab. Then, transplanted the fetal liver tissue onto the DPPIV(−) F344 rats model’s cirrhotic liver surface. Meanwhile, the fetal liver tissues were isolated from the ED14 fetus of DPPIV(+) F344 pregnant rats. Next, 5 coating agents were separately applied over the transplanted fetal liver tissue. The size of coating agent application was similar to the transplantation site.
Hematoxylin and eosin (HE) staining
For HE staining, we used the cryostat tissue sections (sections 1–4), which were first fixed with 10% formalin solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), followed by incubation with Milli-Q water. Next, glass slides were deparaffinized thrice for 10 min in xylene and dehydrated with 100%, 95%, 80%, 70%, and 50% ethanol solutions. Then, it was replaced with Milli-Q water (paraffin tissue section). The slides were immersed in Carrazi’s hematoxylin (Muto Chemistry) for 10 min and rinsed with running tap water to remove excess stain from the slide. Afterward, the slides were immersed in eosin (Muto Chemistry) for 10 min and rinsed with Milli-Q water. Next, these slides were immersed in 50%, 70%, 80%, 95% and 100% ethanol solutions and then in xylene for 3 min. Such steps were repeated thrice. Thereafter, Mount-Quick (Daichi Sangyo Co., Ltd.) was dropped, and the sample was covered with a glass slide (Matsunami Glass).
Immunohistochemical staining (frozen tissue section)
The frozen tissue sections were fixed with a solution containing a mixture of acetone and methanol in equal amounts. After air-drying, the dyed sample was surrounded by a water repellent pen (Dako). Then, permeabilization was performed thrice using 0.05% PBS with Tween 20 (PBST) for 10 min and then blocked at room temperature for 1 h using Blocking One (Nacalai Tesque). Thereafter, the primary antibody solution was diluted to an appropriate concentration with Blocking One and then stored at 4 °C overnight. After the reaction, the slide was washed thrice with PBST for 10 min. The secondary antibody solution was appropriately diluted with Blocking One (1: 500) and stored at room temperature for 1 h. After washing with PBS for 10 min, we added a mixture of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen) and Apaci mounting agent (Wako) dropwise at 1:1000, covered with a glass slide (Matsunami Glass) and then sealed. Antibodies were as follows: CD26 (BD Bioscience 559639), CD31 (BD Bioscience 550300), CK19 (Progen Biotechnik GmbH, 61029), SE-1 (Immuno-Biological Laboratories, 10078) and HNF4 alpha (H-1) (Santa Cruz, sc-374229).
Cryostat tissue sections were fixed with a solution containing a mixture of acetone and chloroform in equal volumes. Then, these sections underwent enzymatic histochemical staining with a staining solution for 20 min. The staining solution consisted of 1 mg/mL of Fast Blue BB Salt hemi (zinc chloride) salt (Sigma-Aldrich) in PBS and 8 mg/mL of Gly-Pro 4-methoxy-β-napthylamide hydrochloride (Sigma-Aldrich) in dimethyl sulfoxide (Wako), which were mixed at a ratio of 20:1 individually. Next, the stained sections were soaked in a 2% copper sulfate aqueous solution prepared by dissolving copper sulfate (II) pentahydrate (Wako) in Milli-Q water. To fix the tissue, we immersed the cells in 10% formalin for 10 min, followed by immersion with Milli-Q water and 10 min staining with Carrazi’s hematoxylin (Muto Chemistry). After rinsing with tap water, we washed the slides for 30 min under running tap water to remove excess stain. Finally, apical sealant (Wako) was dropped, and a glass slide (Matsunami Glass) was placed and sealed.
Sirius red staining
Frozen and paraffin tissue sections were used for Sirius red staining. After fixation of frozen tissue sections using 10% formalin solution (Wako), it was replaced with Milli-Q water (frozen tissue section). The slides were deparaffinized thrice for 10 min in xylene and dehydrated with 100%, 95%, 80%, 70% and 50% ethanol solutions. Then, it was replaced with Milli-Q water (paraffin tissue section). The slides were immersed in 0.03% Sirius red/saturated picric acid solution (1% Sirius red solution saturated with aqueous solution picric acid) for 30 min and rinsed under Milli-Q water. Afterward, these slides were immersed in 50%, 70%, 80%, 95% and 100% ethanol solutions and then in xylene for 3 min. Such steps were repeated thrice. Finally, Mount-Quick (Daichi Sangyo Co., Ltd.) was dropped, and the sample was covered with a glass slide (Matsunami Glass).
Measurement of liver function parameters
Liver functions were assessed by measuring several functional indicators in the blood. First, we collected blood from liver-transplanted rats at 14 post-transplantation days. The collected blood was anticoagulated with EDTA. Then, PLT and PT were determined in the blood samples by using Coagucheck® XS (Roche). To separate blood serum, we centrifuged the collected blood at 4,000 rpm for 20 min. The serum was used to evaluate liver function by measuring the liver function indices, namely, AST, ALT, T-BIL, NH3 and ALB. These indices were determined in the serum samples by using an automated clinical chemistry analyzer machine (DRI-CHEM 7000 V).
Determination of hydroxyproline content in the liver
Total collagen was estimated by measuring the hydroxyproline, which is an amino acid characteristic of collagen. A portion of the sample was excised with a knife. The weight of the excised tissue was measured, the organ fragment was transferred to a biosmasher tube (Nippi), and four volumes of tissue weight 6 N HCl (Wako) was added and homogenized. The liver samples were hydrolyzed for 12–15 h in 5 mL of 6 N HCl at 96 °C. After removing from the heater and cooling at room temperature, these samples were centrifuged at 15,000 rpm for 5 min. Then, we collected an appropriate amount of the sample and added 0.5 volume of H2O. After preparing 20 μL of the sample, we added 75 μL of 50% 2-opropanol in citrate-acetate-buffered chloramine T. The mixture was vortex and mixed. Subsequently, we added 75 μL of a mixture of Ehrlich’s reagent (2-propanol (Wako), Dimethylaminobenzaldehyde (Sigma) and perchloric acid (Sigma). Tubes were incubated for 10 min in a water bath at 60 °C. The samples were placed on the range of 560 nm absorbance band in Hitachi spectrophotometer (U-2000). Results were expressed as μmol hydroxyproline/g of liver tissue.
Data were presented as mean ± SE. The significance was analyzed by Mann-Whitney U test or log rank test using the GraphPad Prism 8 software. Moreover, P < 0.05 indicated statistical significance.