Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

Kim, Yohan,Kang, Kyojin,Jeong, Jaemin,Paik, Seung Sam,Kim, Ji Sook,Park, Su A,Kim, Wan Doo,Park, Jisun,Choi, Dongho The Korean Surgical Society 2017 Annals of Surgical Treatment and Research(ASRT) Vol.92 No.2

<P><B>Purpose</B></P><P>The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved.</P><P><B>Methods</B></P><P>To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10<SUP>7</SUP> hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes.</P><P><B>Results</B></P><P>Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of <I>Albumin</I>, <I>HNF-4α</I> and <I>Foxa3</I> were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time.</P><P><B>Conclusion</B></P><P>Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.</P>

Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic hepatocytes

Copple, Bryan L.,Bustamante, Juan J.,Welch, Timothy P.,Kim, Nam Deuk,Moon, Jeon-Ok Blackwell Publishing Ltd 2009 Liver International Vol.29 No.7

<P>Abstract</P><P>Background/Aims</P><P>During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1α (HIF-1α), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1α in liver cell types. Accordingly, the hypothesis was tested that HIF-1α is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells.</P><P>Methods</P><P>In this study, hepatocytes were isolated from the livers of control and HIF-1α- or HIF-1β-deficient mice and exposed to hypoxia.</P><P>Results</P><P>Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1α and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1α-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2α, may also regulate these genes. In support of this, HIF-2α was activated in hypoxic hepatocytes, and exposure of HIF-1β-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2α may also contribute to upregulation of these genes in hypoxic hepatocytes.</P><P>Conclusions</P><P>Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.</P>

Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

Yohan Kim,Kyojin Kang,Jaemin Jeong,Seung Sam Paik,Ji Sook Kim,Su A Park,Wan Doo Kim,Jisun Park,Dongho Choi 대한외과학회 2017 Annals of Surgical Treatment and Research(ASRT) Vol.92 No.2

Purpose: The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. Methods: To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10<SUP>7</SUP> hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Results: Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin , HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Conclusion: Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

Original Articlel : Regulating Factors for Hepatocyte Differentiation from Stem Cells

( Eun Kyung Ahn ),( Young Ho Kim ),( Jeonghoon Heo ) 한국조직공학과 재생의학회 2011 조직공학과 재생의학 Vol.8 No.6

Embryonic stem (ES) cells are pluripotenent cells having the capacity to differentiate into cellular derivatives of the three germ layers. The ES cells are able to be used to study the effects of extrinsic factors on the differentiation of ES cells. The purpose of this study is to increase the efficiency of hepatocytes differentiation from mouse ES cells and to investigate the factors affecting hepatocytes differentiation. This study focused on the factors including physical contact, soluble factors, cells density and differentiation inducing stages. Hepatocytes differentiation of mouse ES cells was affected by the type of co-cultured cells. H9C2 cells were more effective than MEF. The lower ES cells density on the plate are more effective then high ES cells density to induce the differentiation of hepatocytes. The formation of embryoid body (EB) increased the efficiency of hepatocytes differentiation of ES cells. The type of culture medium did not affect the efficiency of hepatocytes differentiation in the co-culture system. Taken together, co-culture of EB with H9C2 feeder cells was more efficient hepatocytes differentiation system of mouse ES cells. Therefore we conclude that the factor of physical contact has an important role in inducing hepatocytes differentiation from mouse ES cells.

Deletion of Hepatocyte Platelet-Derived Growth Factor Receptor α Attenuates Liver Fibrosis

( Jung Il Lee ),( Woon-kyu Lee ),( Hye Young Chang ),( Ja Kyung Kim ),( Kwan Sik Lee ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: Although expression of platelet-derived growth factor receptor α (PDGFRα) is very low in normal liver, it dramatically increases in cirrhotic liver where activated hepatic stellate cells (HSCs) are suggested to be responsible for this change. However PDGFRα is also reported to be increased in a subset of hepatocellular carcinoma (HCC) where PDGFRα is evident on tumor cells. Therefore we hypothesized that there would be cellular cross-talk between HSCs and hepatocytes regarding PDGFRα on liver with inflammation. Methods: We generated conditional knock-out mice from C57BL/6 mice where PDGFRα was deleted on hepatocytes. Liver fibrosis was induced by injecting thioacetamide (TAA) for 8 weeks. Hepatocytes were isolated from normal liver and TAA induced cirrhotic liver for assessment of PDGFRα expression on hepatocytes. In vitro co-culture system was built with LX2 cells and Hep3B cells. Hep3B cells were transfected with siRNA (PDGFRα or control) and co-cultured with LX2 cells. After 24 hours of co-culture, LX2 cells and medium were retrieved for analysis of fibrosis associated factors. Results: Deleting PDGRα attenuated TAA induced liver fibrosis in mice. PDGFRα expression was increased on hepatocytes from cirrhotic liver compared with that from normal liver. From the co-culture system, LX2 cells, cultured with PDGFRα siRNA infected Hep3B cells showed decreased PDGFRα expression as well as attenuated α smooth muscle actin (SMA) and collagen 1 (Col1) α expression. When PDGF-CC, which is a strong ligand of PDGFRα, levels were evaluated from LX2 cell culture medium, significantly decreased PDGF-CC was detected from LX2 medium co-cultured with siRNA infected Hep3B cells. Conclusions: Deleting PDGFRα on hepatocytes resulted in attenuated HSC activation and fibrosis. This suggests that although PDGFRα is scarcely expressed on normal hepatocytes, PDGFRα on hepatocytes may play an important role upon fibrotic insults, facilitating liver fibrosis.

B형 간염바이러스 감염에서 불투명 유리상 간세포(Groundglass hepatocyte)의 원인으로서 pre-S1 변이종의 의의

조용균 ( Yong Kyun Cho ),김병익 ( Byung Ik Kim ),배지철 ( Ji Cheul Pae ),박승하 ( Seung Ha Park ),김상훈 ( Sang Hoon Kim ),박정호 ( Jung Ho Park ),김홍주 ( Hong Joo Kim ),박동일 ( Dong Il Park ),김향 ( Hyang Kim ),성인경 ( In Kyu 대한내과학회 2005 대한내과학회지 Vol.69 No.4

목적 : 불투명 유리상 간세포는 만성 간염 환자에서 나타나는 독특한 조직학적 특징이다. HBV의 LHBs 중 pre-S1 지역은 HBsAg의 조립(assembly), 처리(processing) 및 분비를 조절하는 것으로 알려져 왔다. 본 연구의 목적은 간세포 내에서 HBV pre-S1 유전자의 변이가 정상 분비 경로의 영향과 불투명 유리상 간세포의 반응에 대해 알아보고자 하였다. 방법 : 만성 B형 간염 판정을 받은 HBeAg 양성 환자로부터 pre-S1 region을 포함한 HBV 염기 배열을 조사하였다. 환자의 간조직과 혈액내 존재하는 바이러스 유전자를 조사하고자 간조직을 얻어 파라핀으로 고정시켰고, 환자의 간조직내 불투명 유리상 간세포를 분리하기 위해 파라핀으로 고정된 간조직으로부터 일부 조직을 anti-HBs 항체로 염색 후 laser capture microdissection (LCM) 방법을 이용하여 불투명 유리상 간세포를 추출하였다. 추출된 anti-HBs 항체에 염색된 불투명 유리상 간세포와 정상 세포를 각각의 HBV DNA 유전자 서열을 분석하였다. 변이형의 HBsAg 분비를 측정하기 위해 변이형을 야생형의 클론에 삽입하였고 ELISA에 의해 transfected된 세포의 부유물에서 HBsAg의 발현을 관찰하였다. 결과 : 혈청에서 분석된 12 클론 중 9개의 클론은 HBV envelope 단백의 분비와 축적에 중요한 역할을 하는 pre-S1 단백 중 N-terminal 지역에서 염기서열이 모두 동일한 야생형으로 나타났다. 이런 야생형 클론의 하나에서 pre-S2 지역 안에 결실은 가지고 있었다. 불투명 유리상 간세포에서 정상 간세포에 비해 변이형이 우세하였고, 정상 간세포에서는 야생형이 더 우세하였다. 결론 : HBV pre-S1 단백의 변이형이 불투명 유리상 간세포의 형성에 영향을 주는 것을 알 수 있었다. 비정상적인 pre-S1 단백의 표현은 간세포 안에 이들 단백질의 축적 및 간세포 손상을 야기 시킬 것으로 생각되며 좀 더 많은 연구가 필요할 것으로 사료된다. Background : Ground glass hepatocytes are unique histological feature of chronic hepatitis B viral infection. The pre-S1 region of large surface protein has been shown to regulate assembly, processing, and secretion of HBsAg. The purpose of this study was to elucidate that a mutant form of pre-S1 affects this normal secretory pathway and is responsible for ground glass hepatocyte. Methods : We examined HBV sequences spanning the pre-S region from a patients with HBeAg positive chronic HBV infection. HBV DNA was extracted from serum, cloned, and sequenced and determined the intrahepatic viral composition by extracting HBV DNA from paraffin embedded liver tissue. To analyze the viral population of single groundglass hepatocytes, we used the technique of laser capture microdissection to isolate individual hepatocytes from biopsy specimen. Groundglass hepatocytes that stained positively with anti-HBs and normal hepatocytes were harvested individually and their subjected HBV DNA sequences were analyzed. To define the responsible mutations for the HBsAg secretion, we introduced the mutant gene into molecular clone of wildtype (adwR9) and assayed their HBsAg amounts in the transfected cell supernatants by ELISA. Results : Of 12 clones in serum analyzed, 9 clones had identical wild type sequences in the N-terminal region of the pre-S1 protein which plays an important role in the secretion and retention of HBV envelope proteins. One of the wild type clones has deletion within pre-S2 region. 3 identical mutant clones were isolated. Mutant type clones were predominant groundglass hepatocytes. Conclusions : We speculate that a mutant form of the HBV pre-S1 protein may result in the formation of ground-glass hepatocytes. Expression of abnormal pre-S1 may lead to its retention and accumulation within hepatocytes.(Korean J Med 69:357-363, 2005)

Role of stearyl-coenzyme A desaturase 1 in mediating the effects of palmitic acid on endoplasmic reticulum stress, inflammation, and apoptosis in goose primary hepatocytes

Tang, Bincheng,Qiu, Jiamin,Hu, Shenqiang,Li, Liang,Wang, Jiwen Asian Australasian Association of Animal Productio 2021 Animal Bioscience Vol.34 No.7

Objective: Unlike mammals, goose fatty liver shows a strong tolerance to fatty acids without obvious injury. Stearyl-coenzyme A desaturase 1 (SCD1) serves crucial role in desaturation of saturated fatty acids (SAFs), but its role in the SAFs tolerance of goose hepatocytes has not been reported. This study was conducted to explore the role of SCD1 in regulating palmitic acid (PA) tolerance of goose primary hepatocytes. Methods: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide was examined to reflect the effect of PA on hepatocytes viability, and quantitative polymerase chain reaction was used to detect the mRNA levels of several genes related to endoplasmic reticulum (ER) stress, inflammation, and apoptosis, and the role of SCD1 in PA tolerance of goose hepatocytes was explored using RNA interfere. Results: Our results indicated that goose hepatocytes exhibited a higher tolerant capacity to PA than human hepatic cell line (LO2 cells). In goose primary hepatocytes, the mRNA levels of fatty acid desaturation-related genes (SCD1 and fatty acid desaturase 2) and fatty acid elongate enzyme-related gene (elongase of very long chain fatty acids 6) were significantly upregulated with 0.6 mM PA treatment. However, in LO2 cells, expression of ER stress-related genes (x box-binding protein, binding immunoglobulin protein, and activating transcription factor 6), inflammatory response-related genes (interleukin-6 [IL-6], interleukin-1β [IL-1β], and interferon-γ) and apoptosis-related genes (bcl-2-associated X protein, b-cell lymphoma 2, Caspase-3, and Caspase-9) was significantly enhanced with 0.6 mM PA treatment. Additionally, small interfering RNA (siRNA) mediated downregulation of SCD1 significantly reduced the PA tolerance of goose primary hepatocytes under the treatment of 0.6 mM PA; meanwhile, the mRNA levels of inflammatory-related genes (IL-6 and IL-1β) and several key genes involved in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), forkhead box O1 (FoxO1), mammalian target of rapamycin and AMPK pathways (AKT1, AKT2, FoxO1, and sirtuin 1), as well as the protein expression of cytochrome C and the apoptosis rate were upregulated. Conclusion: In conclusion, our data suggested that SCD1 was involved in enhancing the PA tolerance of goose primary hepatocytes by regulating inflammation- and apoptosis-related genes expression.

Alterations of epinephrine-induced gluconeogenesis in aging

김경태,Sung Chun Cho,Anthony Cova,장익순,Sang Chul Park 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.5

The effects of glucagon and epinephrine on gluconeogenesis in young (4 month) and old (24 month) Fisher 344 rat hepatocytes were compared. In contrast to glucagon, which had a similar effect on gluconeogenesis in both young and old cells, epinephrine caused a smaller increase in gluconeogenesis in old rat hepatocytes than in young hepatocytes. β2 adrenergic receptor (β2-AR) expression slightly decreased in aged rat liver, and there were differences between young and old hepatocytes in their patterns of G protein coupled receptor kinases, which are involved in the activation of β2-AR receptor signal desensitization. The major isoform of the kinase changed from GRK2 to GRK3 and the expression of β-arrestin, which is recruited by the phosphorylated β2-AR for internalization and degradation, increased in aged rat liver. GRK3 overexpression also decreased the glucose output from young rat hepatocytes. We conclude that an age-associated reduction in epinephrine-induced gluconeogenesis occurs through the epinephrine receptor desensitizing system. The effects of glucagon and epinephrine on gluconeogenesis in young (4 month) and old (24 month) Fisher 344 rat hepatocytes were compared. In contrast to glucagon, which had a similar effect on gluconeogenesis in both young and old cells, epinephrine caused a smaller increase in gluconeogenesis in old rat hepatocytes than in young hepatocytes. β2 adrenergic receptor (β2-AR) expression slightly decreased in aged rat liver, and there were differences between young and old hepatocytes in their patterns of G protein coupled receptor kinases, which are involved in the activation of β2-AR receptor signal desensitization. The major isoform of the kinase changed from GRK2 to GRK3 and the expression of β-arrestin, which is recruited by the phosphorylated β2-AR for internalization and degradation, increased in aged rat liver. GRK3 overexpression also decreased the glucose output from young rat hepatocytes. We conclude that an age-associated reduction in epinephrine-induced gluconeogenesis occurs through the epinephrine receptor desensitizing system.

A Novel Herbal Formulation “LiverCare” Differentially Regulates Primary Rat Hepatocyte and Hepatocarcinoma Cell Proliferation In Vitro

Satyakumar Vidyashankar,Sandeep R. Varma,Mohammed Azeemudin,Ashok Godavarthi,Nandakumar S. Krishna,Pralhad Sadashiv Patki 한국식품영양과학회 2011 Journal of medicinal food Vol.14 No.9

Hepatocyte growth factor (HGF) plays an important role in hepatocyte proliferation. HGF expression is regulated by various signaling molecules and nuclear receptors. In the present study, LiverCare^ⓡ (LC), a novel polyherbal formulation (The Himalaya Drug Company, Bangalore, India), was evaluated for its efficacy, using co-cultures of primary rat hepatocytes–non-parenchymal cells (NPCs) and human hepatocellular carcinoma cells (HepG2). The rate of primary hepatocyte co-culture proliferation was significantly and dose-dependently increased by LC as determined by [3H]thymidine incorporation into newly synthesized DNA and cell proliferation assay. LC also increased HGF expression in primary hepatocyte co-culture. Albumin and urea content remained constant during proliferation of hepatocyte co-cultures in the presence of LC with decreased activity of alanine aminotransferase. It is interesting that LC inhibited incorporation of [3H]thymidine into DNA in HepG2 cells. LC enhanced peroxisome proliferator-activated receptor-α expression during hepatocyte proliferation, whereas tumor necrosis factor-α expression remained unaffected. In conclusion, our study clearly showed that LC differentially regulates primary rat hepatocytes and human hepatocarcinoma cell proliferation. LC may be a promising candidate for treating degenerative liver diseases by enhancing liver regeneration.

Basic, Research : Three-dimensional Flow-chip to Investigate Hepatocyte-hepatic Stellate Cell

( Seung A Lee ),( Da Yoon No ),( Dong Sik Kim ),( Sang Hoon Lee ) 대한간학회 2013 춘·추계 학술대회 (KASL) Vol.2013 No.1

Background: Several studies have reported that co-cultivation of hepatocytes and hepatic stellate cells(HSCs) with cell-cell contact enhances liver-specific functions, such as albumin secretion, urea synthesis, expression of hepatocyte-specific genes, and function of cytochrome P450 (CYP450). However, most of these studies are not based on 3D culture system. Also previous studies generally ignored the flow effect that cell perfusion has the potential to improve the cell culture microenvironment. In this study, we have developed a flow-chip to investigate the interaction of hepatocytes and HSCs in which primary hepatocytes spheroids and HSCs are co-cultured without direct cellcell contact. Methods: To investigate the paracrine effects of HSCs on hepatocytes, we created a chip with a cascade design in which the culture medium moves from the HSC culture chip to the hepatocyte 3D culture chip with a flow rate comparable to that of interstitial flow. The most important feature of this chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. Results: We found that the size of cell aggregates decreased over time yielding uniform-sized spheroids with tight cell-cell interactions. Also we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Conclusions: These results obtained using this 3D flow-chip are in good agreement with the results reported by other investigator studying hepatocyte function, suggesting that by simply collecting the medium in the osmotic pump system, this chip may also be a useful model for investigating cytokines and chemokines released from various cells.