Human hepatocytes loaded in 3D bioprinting generate mini-liver

Cheng Zhong， Hai-Yang Xie， Lin Zhou， Xiao Xu and Shu-Sen Zheng

Hangzhou， China

Human hepatocytes loaded in 3D bioprinting generate mini-liver

Cheng Zhong， Hai-Yang Xie， Lin Zhou， Xiao Xu and Shu-Sen Zheng

Hangzhou， China

BACKGROUND： Because of an increasing discrepancy between the number of potential liver graft recipients and the number of organs available， scientists are trying to create artificial liver to mimic normal liver function and therefore， to support the patient's liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering.

METHODS： We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups， with 15 mice in each group： control， hydrogel， hydrogel with L02 （cell line HL-7702）， and hydrogel with hepatocyte growth factor （HGF）. Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage（RILD）. The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， albumin， total bilirubin， CYP1A2， CYP2C9，glutathione S-transferase （a-GST）， and UDP-glucuronosyl transferase （UGT-2） were compared among the groups. Hematoxylin-eosin （HE） staining and immunohistochemistry of cKit and cytokeratin 18 （CK18） of engrafted tissues were evaluated. The survival time of the mice was also compared among the four groups.

RESULTS： 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT， AST， albumin， total bilirubin， CYP1A2， CYP2C9， a-GST and UGT-2 were significantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only （P＜0.05）. HE staining showed clear liver tissue and immunohistochemistry of cKit and CK18 were positive in the engrafted tissue. Mice treated with 3D scaffold+L02 cells had longer survival time compared with those in control and scaffold only （P＜0.05）.

CONCLUSION： 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the reconstruction of liver tissues.

（Hepatobiliary Pancreat Dis Int 2016；15：512-518）

3D printing；

hepatocyte；

liver；

tissue engineering

Introduction

Liver transplantation is the gold standard of care for patients with end-stage liver disease and those with tumors of liver origin in the setting of liver dysfunction.［1］Today， one of the greatest hurdles that liver transplantation faces is the growing discrepancy between the demand and availability of donor livers， although this gap has been slightly reduced since the model for end-stage liver disease allocation system was adopted.［2］Different strategies have evolved to expand the donor pool.［3，4］Despite efforts to expand this resource， solutions to expand the donor pool will require the use of more borderline organs. Further research is needed on optimal organ sources， including the use of organ regeneration in the laboratory.［5，6］

In recent years， a significant amount of attention has been focused on the concept of tissue engineering. The principle of tissue engineering assumes that a variety of cells can be coaxed into synthesizing new tissue when they are seeded onto an appropriate scaffold in an appropriate growth and differentiation environment.［7］Bioprinting is defined as the use of printing technology to deposit living cells， an extracellular matrix， biochemical factors， drugs， and biomaterials on a receiving solid or gel substrate or liquid reservoir.［8］Progress has been made in cell biology， material sciences， and tissue engi-neering that has enabled researchers to develop cuttingedge technology that has led to the creation of nonmodular tissue constructs such as skin，［9］bladder，［10］and vessels.［11］Therefore， the use of 3D printing with other classes of nano-scale functional building blocks， including semiconductor， magnetic， plasmonic， and ferroelectric nanoparticles could expand the opportunities for engineering of bionic tissues and organs.［12］Techniques such as seeding cells into non-adhesive molds or selffolding scaffolds have been used to fabricate 3D tissue constructs with complex geometries.［8，13］

The grid pattern design was adopted as a simple model of liver lobules. As a kind of transplantation， the function of printed tissues was more important than the shape. Difference from previous reports，［14］our study adopted the new design of printed scaffold with pores. Compared with the previous in vivo researches which did not emphasize the morphology and functions，［15-17］our in vivo study evaluated the morphology and function changes in 3D scaffold loaded with hepatocyte in detail. This model may be highly applicable for future studies on the regeneration of hepatocyte tissues.

Methods

Cell culture

L02 cells （HL-7702， a cell line of human hepatocytes）were provided by the Cell Bank of Shanghai， Chinese Academy Science. As a type of potential seed cell in liver tissue engineering，［18，19］L02 cells were maintained in DMEM-LG medium （Hyclone Laboratories Inc.， Logan， UT， USA） with 10% fetal bovine serum （Gibco， Life Technologies Corp.， Grand Island， NY， USA）. Trypsin was purchased from Amresco （Solon， OH， USA）. The ELISA kits for alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， albumin and total bilirubin were obtained from Cloud Clone Corp. （Houston， TX，USA）. cKit and cytokeratin 18 （CK18） antibodies were manufactured by Abcam （London， UK）. An Olympus CKX31-A12PHP inverted microscope was used for observation of cells. A Hitachi SU1510 scanning electron microscope was used for cell ultrastructure observation.

Cellular proliferation

To evaluate the effect of hydrogels on L02 cellular proliferation， an methylthiotetrazole （MTT） assay was performed at day 1， 3， 5， and 7 post-culture. Accordingly，3000 cells/well were placed in a 96-well dish. A total of 20 μL of the MTT solution （0.5%） was added to each well，and cells were allowed to incubate with the MTT solution for 4 hours. The supernatant was subsequently discarded， and 150 μL of dimethyl sulfoxide were added to each well. Low-speed oscillation for 10 minutes was used to fully dissolve the crystals. The optical density （OD）values for each well were measured at an absorbance of 570 nm， with a blank well serving as the control.

Preparation of hydrogel scaffolds

A 1% collagen type I solution （BD Biosciences） and a 1.5% chitosan （w/v） HCl solution （0.2 mol/L）， both buffered with a 0.5 mol/L morpholinoethanesulfonic acid solution （MES） and NaOH （1 mol/L）， were mixed in a 10：1 ratio （w/w） with a pH of 7.2.［20］1-ethyl-3-（3-dimethyl aminopropyl） carbodiimide （EDC） and N-hydroxysuccinimide （NHS）， both at 10% （w/v） in MES， were mixed with the collagen-chitosan solution. The solution was allowed to cross-link for 5 minutes using MES and 1 mol/L NaOH， and then glycine was added. After the incubation of gelation for 30 minutes at 37 ℃， complete endothelial basal medium （EBM， Clonetics） was added and the gels were returned to the incubator for 18-24 hours. The biocompatibility of the hydrogels was tested with MTT. The design of the 3D printing scaffold is presented in Fig. 1B. The 3D printing scaffold morphology by eye， under microscope and electron microscope are presented in Fig. 1C-F. The scaffold has 50 layers of cell-hydrogel.

Fig. 1. A： The 3D bioprinter used in this study was provided by Regenovo Biotechnology Co.， Ltd.， Hangzhou， China. B： The structure of the 3D printing shows pores. The size of printed hydrogel was 15×15×2 mm， 50 layers. C-F： The printed 3D scaffold. Single triangle was the cells， and double triangles were the hydrogels.

3D printing

Regenovo Biotechnology Co.， Ltd. （Hangzhou， China）provided the 3D bioprinter （Fig. 1A）. The 3D scaffold was fabricated with hydrogel and there were pores in the scaffold according to a grid pattern design. Fig. 1B is the picture of 3D scaffold. This design was different from a previous model.［15］

The microscale injection systems， comprising a nozzle with a 210 μm internal diameter， were attached to reservoirs for the bio-ink. The solution comprising hydrogel and L02 cells was about 2×106cells/mL. The software“SolidWorks” was used to design the 3D model. The software “3D-Bioprint” was used to load the 3D model. The microscale injection systems and bio-ink reservoirs were mounted onto the tool that could build the micrometerresolution 3-axis XY-Z stage. After cross-linking， a solution of CaCl2（10%， 50 mL） was used to maintain the 3D pattern. The maximum molding speed was 170 mm/s，the position accuracy was ±0.01 mm， and the size of the molding was 15×15×2 mm.

The 3D scaffold was examined by scanning electron microscopy （JSM-6701F， J EOL， ROKYO， Japan） after platinum coating and inverted microscopy （Olympus，IX83， Japan）.

In vivo experiments

The experiments were performed using nude mice with initial weights of 16-20 g. Sixty mice （30 males and 30 females） were classified to four groups randomly， with 15 mice in each group. The mice were divided into four groups： group 1： control， radiation-induced liver damage（RILD） combined with 2/3 hepatectomy； group 2： group 1+hydrogels； group 3： group 2 with L02 deposition （3D printing）； group 4： group 2+hepatocyte growth factor（HGF）. The protocol of RILD （50 Gy） combined with 2/3 partial hepatectomy was adopted from previous reports.［21，22］The possibility of the proliferation of host hepatocytes was excluded because RILD suppresses the proliferation of host hepatocytes. To detect the effects of 3D printing scaffold with different loading in the body， each of the 3D scaffolds were subsequently engrafted into the liver after partial hepatectomy. Five mice of each group were sacrificed after 2 weeks and the rest 10 mice of each group were used in survival analysis. the engrafted scaffolds were collected， sliced， stained with hematoxylineosin （HE） and examined using immunohistochemical cKit and CK18 analyses. As the biomarkers of the hepatocyte surface， cKit and CK18 were the specific antigens in hepatocytes.

Serum ALT， AST， albumin， total bilirubin， CYP1A2，CYP2C9， a-GST and UGT-2 were tested with ELISA kits. The animal survivals were recorded in all groups. The ethics committee of Zhejiang University approved the animal experiments.

Statistical analysis

The differences among groups were evaluated with the one-way ANOVA and Chi-squared test using SPSS software version 12.0 SPSS Inc. （Chicago， IL， USA）. P values less than 0.05 were considered significant. The experiments were repeated three times.

Results

Biocompatibility

The MTT assay of the cells on the 1st， 3rd， 5th， and 7th days of the culture were not significantly different between the cells cultured regularly and those cultured with hydrogel at any time point （P＞0.05； Fig. 2）.

Morphology

The morphology of the engrafted scaffold in different groups was showed in Fig. 3. HE staining showed hepatocyte-like cells and cKit and CK18 were positive in mice from group 3. In the control group， tissue regeneration and cKit+/CK18+hybridization were not detected. Some nonspecific cells assembled around the mice liver. In the hydrogel group， tissue regeneration and cKit+/CK18+hybridization were not detectable as well. Some nonspecific cells assembled around the mice liver. Compared with the engrafted tissues in group 3， the tissues showed immature behavior in group 4 and were chaotic and the morphology was random. Only the engrafted tissues in group 3 showed significant liver function.

Liver function

At day 5 and 10， the ALT， AST， and total bilirubin levels in group 3 were significantly decreased （P＜0.05；Fig. 4） compared with groups 1 and 2. The albumin level was significantly higher at day 5 and 10 （P＜0.05； Fig. 4）in group 3 compared with groups 1 and 2. The levels of CYP1A2， CYP2C9， a-GST and UGT-2 were significantly higher at day 1， 5 and 10 （P＜0.05； Fig. 5） in group 3 compared with groups 1 and 2. These findings indicated that the liver function in group 3 had rapidly recovered. There was no significant difference between groups 3 and4 in these variables （P＞0.05）.

Fig. 2. MTT assay on the 1st， 3rd， 5th and 7th days of the culture. The OD values of the hydrogel group were not significant difference compared with that of the control group at each time point. The OD values were gradually increased following the time of culture.

Fig. 3. The engrafted tissue morphology of different experimental groups. The “l(fā)iver” indicated the mice liver. The “hy” represent hydrogel. The “3D” represents the 3D scaffold loaded with L02 cells. cKit and CK18 were the specific antigens in hepatocytes.

Fig. 4. At day 5 and 10， the mice serum ALT， AST and total bilirubin levels were significantly decreased， while the albumin level was significantly increased in the 3D scaffold loaded with L02 cells compared to those in control and scaffold only groups. There were no statistical differences between 3D scaffold loaded with L02 cells and HGF groups. *： P＜0.05， vs the 3D printing group； #： P＜0.05， vs the hydrogels+HGF group.

Fig. 5. The levels of CYP1A2， CYP2C9， a-GST and UGT-2 were significantly higher at day 1， 5 and 10 in 3D scaffold loaded with L02 cells compared with control and scaffold only groups. No statistically significant difference was noted between the 3D scaffold loaded with L02 cells and HGF groups. *： P＜0.05， vs the 3D printing group.

Fig. 6. The median survival of the mice was 8.5， 8， 14 and 11 days in control， scaffold only， scaffold+L02 cells and scaffold+HGF. The survival time in groups of scaffold+HGF and scaffold+L02 cells was significantly longer than that in groups of control and scaffold only （P＜0.05）. There was no significant difference between groups of scaffold+HGF and scaffold+L02 cells.

Survival analysis

The median survival time of the mice was 8.5， 8， 14 and 11 days in groups 1， 2， 3 and 4， respectively （Fig. 6）. The log-rank （Mantel-Cox） test between groups 1 and 3 showed： χ2=8.108； df=3； P＜0.05； between groups 2 and 3 were： χ2=5.535； df=1； P＜0.05.

Discussion

One major obstacle that precludes the large regeneration of liver tissue was the lack of an accompanying microvasculature.［23，24］Without a functional circulatory system，the potential size of the engineered liver tissue is limited to a few hundred pores. Invading microvessels limit the penetration depth and， therefore， impede the successful incorporation of larger constructs.［25］In addition，these vessels， while migrating and developing within the implanted liver tissue， may distort or destroy the construction of the engineered tissue.［26］Therefore， from the standpoint of design， it would be more beneficial to print a preinstalled tissue with biomaterials that preferentially directed vessel ingrowth， or ideally， built a network with the microvasculature in place prior to implanting.［9，27］

The success of liver tissue engineering largely depends on the capacity to construct an organ using cells that are viewed three-dimensionally as well as the successful incorporation into a target organ.［16，28］Prior to this study， our laboratory has addressed the issue of 3D-cell patterning and functionalization. Relevant methodologies were combined to initiate the present study and to realize a potentially workable platform to generate tissue constructs for liver disease therapy.［29］

Here， we introduced a conceptually novel approach that addressed the aforementioned challenges via 3Dprinting of hydrogel materials and viable cell-seeded hydrogels in the anatomic geometries of the human liver. Because the metabolic function of hepatocyte-like cells is the core of liver tissue engineering， the architecture of the printed hydrogels enables the growth of the engineering organs that exhibit enhanced functionalities. Importantly， we verified that the grid pattern printing process did not affect the vitality of hepatocytes. Our data is consistent with Faulkner-Jones's report.［15］Our 3D printing tissues were close to natural liver. The pores in the grid hydrogels were suitable for medium exchange. Although the hydrogels had tiny expansion，［30］the morphology of regenerated tissues were closed to the liver tissue after engrafting. This phenomenon was not reported previously. Two studies from the same laboratory［31，32］detailed the principles and methods that undergirded the direct cell writing biofabrication process and adapted the microfluidic devices for the creation of an organ screening model， establishing a novel organ metabolism study platform. Our study is consistent with Chang and colleagues. 3D printing technology provides a suitable environment for cell growth and metabolism and therefore， the cells in printed scaffold were alive. As more persuasive results， the in vivo results in our research revealed that the grid pattern printing was a novel platform for the study of liver function.

Our in vivo data were similar to those of previous cell transplantation reports.［21，33］The serum albumin concentrations were significantly higher in the transplant group compared with that of the controls. The survival rate of patients with transplanted tissues improved significantly compared with those of controls. The median survival time of our mice with RILD plus a partial hepatectomy was 8.5 days， while this time was expanded to 14 days with the application of our 3D scaffold device， significantly longer than controls. Our data indicated that 3D scaffold device is applicable to improve the liver function and prolong the survival time in our mice model.

L02 cells were used in this research. According to previous reports，［18，19］L02 cells have potential application in liver tissue engineering. Other authors found that L02 cell line possesses a feature of cancer.［4，5］Although our short-term observation did not show tumor like proliferation， the safety and tumor formation cannot be ruled out according to the present data. Other cells， including stem cells， will be studied in in vivo experiments in the next stage of experiment. The comprehensive evaluation of types of seed cells will achieve a conclusion as to which cell will be better survived and proliferated in 3D printed device.

Our approach offers the ability to create spatially heterogeneous constructs by extruding an organized structure of materials in a layer-by-layer process until the final stereo-lithographic geometry is complete. This concept of 3D printed device with living cells together with hydrogel materials and growing these into functional organs without scaffold degradation represents a new direction in biological systems.［14，34］Indeed， such 3D printed organs are distinct from engineered tissues and they offer a way of attaining a three dimensional merger of scaffolds without degradation. Overall， the 3D printing methods described in the present study are highly suitable for cell placement and scaffold fabrication according to a predefined design；［35］however， the technology applied in tissue engineering is still in its infancy stage and requires further improvement. Especially in terms of vascularization， repeatability， and large-scale production of 3D printed organs，additional research still needs to be conducted.［36］

In conclusion， the 3D printed hepatocytes behave the same as natural hepatocytes， 3D printing tissues can be used in liver tissue engineering and liver disease model research in vitro.

Contributors： ZSS proposed the study. ZC， XHY， ZL and XX performed the research and wrote the first draft. All authors contributed to the design and interpretation of the study and to further drafts. ZSS is the guarantor.

Funding： This study was supported by grants from the National Natural Science Foundation of Major Research and Development Plan of China （91542205） and 151 Talents Project of Zhejiang Province （12-1-058）.

Ethical approval： This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine.

Competing interest： No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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October 9， 2015

Accepted after revision May 26， 2016

Author Affiliations： Key Laboratory of Combined Multi-organ Transplantation， Ministry of Public Health， First Affiliated Hospital， Zhejiang University School of Medicine， Hangzhou 310003， China （Zhong C， Xie HY， Zhou L， Xu X and Zheng SS）

Shu-Sen Zheng， MD， PhD， FACS， Key Laboratory of Combined Multi-organ Transplantation， Ministry of Public Health， First Affiliated Hospital， Zhejiang University School of Medicine， Hangzhou 310003， China （Tel： +86-571-87236352； Email： shusenzheng@zju.edu.cn）

? 2016， Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872（16）60119-4

Published online August 4， 2016.