Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

Shulman, Maria; Nahmias, Yaakov

doi:10.1007/978-1-62703-125-7_17

Cited by 117Liver physiology and pathologyTissue Engineering and Regenerative Medicine3D Printing in Biomedical Research

Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

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Shulman, Maria, Nahmias, Yaakov·2012·Methods in Molecular Biology

DOI 10.1007/978-1-62703-125-7_17PMC PMC3781339

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Abstract

The liver is the largest internal organ in mammals, serving a wide spectrum of vital functions. Loss of liver function due to drug toxicity or viral infection is a major cause of death in the United States. The development of Bioartificial Liver (BAL) devices and the demand for pharmaceutical and cosmetic toxicity screening require the development of long-term hepatocyte culture techniques. However, primary hepatocytes rapidly lose their cuboidal morphology and liver-specific functions over a few days in culture. Accumulation of stress fibers, loss of metabolic function, and cell death are known phenomena. In recent years, several techniques were developed that can support high levels of liver-specific gene expression, metabolic and synthetic function for several weeks in culture. These include the collagen double-gel configuration, hepatocyte spheroids, coculture with endothelial cells, and micropatterned cocultures with 3T3-J2 fibroblasts. This chapter covers the current status of hepatocyte culture techniques, including: hepatocyte isolation, media formulation, oxygen supply, heterotypic cell-cell interactions, and basic functional assays.

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The limited proliferative capacity of primary hepatocytes, often associated with reduced viability and progressive loss of hepatocyte-specific functions in vitro, has constrained their broader contribution to health and science.
“However, the limited proliferative capacity of PHs, often associated with reduced viability and progressive loss of hepatocyte-specific functions in vitro, has constrained their broader contribution to health and science [39].”
From The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances (2026) · cites this paper as [39]

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Oliva‐Vilarnau, N., Hankeová, S., Vorrink, S. U., Mkrtchian, S., Andersson, E. R., & Lauschke, V. M. (2018) · Frontiers in Medicine · doi:10.3389/fmed.2018.00192 · Cited by 84 · see citing works
“Furthermore, PHH in 2D culture form actin stress fibers, resulting in fibroblast-like cell morphology (43).”
Background
Biotechnology and Bioengineering: Volume 116, Number 4, April 2019
Gregorio, V. D., Corrado, B., Sbrescia, S., Sibilio, S., Urciuolo, F., Netti, P., & Imparato, G. (2019) · Biotechnology and Bioengineering · doi:10.1002/bit.26755 · Cited by 79
“Further, in standard 2D cultures, primary hepatocytes show a rapid loss of the original phenotype, resulting in the downregulation of metabolic biomarkers (Shulman & Nahmias, 2013).”
Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation
Capone, S. H., Dufresne, M., Rechel, M., Fleury, M., Salsac, A., Paullier, P., Daujat‐Chavanieu, M., & Legallais, C. (2013) · PLoS ONE · doi:10.1371/journal.pone.0062032 · Cited by 75 · see citing works
“Among approaches developed to control liver-specific function expression, a microenvironment enriched in collagen, such as double gel configuration [13] or collagen coated dishes [14], has shown its efficiency.”
Background
Acylated and desacyl ghrelin are associated with hepatic lipogenesis, β-oxidation and autophagy: role in NAFLD amelioration after sleeve gastrectomy in obese rats
Ezquerro, S., Méndez-Giménez, L., Becerril, S., Moncada, R., Valentí, V., Catalán, V., Gómez-Ambrosi, J., Frühbeck, G., & Rodríguez, A. (2016) · Scientific Reports · doi:10.1038/srep39942 · Cited by 68 · see citing works
Human Induced Pluripotent Stem Cells in Hepatology
Gerbal‐Chaloin, S., Funakoshi, N., Caillaud, A., Gondeau, C., Champon, B., & Si‐Tayeb, K. (2013) · American Journal Of Pathology · doi:10.1016/j.ajpath.2013.09.026 · Cited by 64 · see citing works
“The lobule, defined as the functional liver unit, is characterized by a periportal to centrilobular zonation that allows the liver to assume >500 functions.(68) This architecture defines cell-cell contacts,”
Background
Engineering Liver Microtissues for Disease Modeling and Regenerative Medicine
Huang, D. (., Gibeley, S. B., Xu, C., Xiao, Y., Celik, O., Ginsberg, H. N., & Leong, K. W. (2020) · Advanced Functional Materials · doi:10.1002/adfm.201909553 · Cited by 65 · see citing works
High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering
Gevaert, E., Dollé, L., Billiet, T., Dubruel, P., van Grunsven, L. A., van Apeldoorn, A. A., & Cornelissen, R. (2014) · PLoS ONE · doi:10.1371/journal.pone.0105171 · Cited by 51 · see citing works
“[1] This limits or prevents their use for clinical, engineering and research purposes.”
Background
Physiological oxygen tension reduces hepatocyte dedifferentiation in in vitro culture
Guo, R., Xu, X., Lu, Y., & Xie, X. (2017) · Scientific Reports · doi:10.1038/s41598-017-06433-3 · Cited by 60 · see citing works
Jammed Polyelectrolyte Microgels for 3D Cell Culture Applications: Rheological Behavior with Added Salts
O’Bryan, C. S., Kabb, C. P., Sumerlin, B. S., & Angelini, T. E. (2019) · ACS Applied Bio Materials · doi:10.1021/acsabm.8b00784 · Cited by 51 · see citing works
Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity
Chang, S.-Y., Voellinger, J. L., Van Ness, K. P., Chapron, B., Shaffer, R. M., Neumann, T., White, C. C., Kavanagh, T. J., Kelly, E. J., & Eaton, D. L. (2017) · Toxicology in Vitro · doi:10.1016/j.tiv.2017.01.007 · Cited by 49 · see citing works
“Primary rat hepatocytes were isolated by two-step collagenase digestion following isolation of the liver from male Sprague Dawley® rats (Charles River Laboratories, Seattle, WA) (Shulman and Nahmias, 2013).”
Method
Bioengineering Organs for Blood Detoxification
Legallais, C., Kim, D., Mihaila, S. M., Mihajlovic, M., Figliuzzi, M., Bonandrini, B., Salerno, S., Yengej, F. A. Y., Rookmaaker, M. B., Romero, N. S., et al. (2018) · Advanced Healthcare Materials · doi:10.1002/adhm.201800430 · Cited by 65 · see citing works
Developing liver organoids from induced pluripotent stem cells (iPSCs): An alternative source of organoid generation for liver cancer research
Nguyen, R., Bae, S. D. W., Qiao, L., & George, J. (2021) · Cancer Letters · doi:10.1016/j.canlet.2021.03.017 · Cited by 50 · see citing works
In vitro and in silico liver models: Current trends, challenges and opportunities
Poloznikov, A. A. (2018) · ALTEX · doi:10.14573/altex.1803221 · Cited by 40 · see citing works
“One of the most promising approaches to overcome the issues with viability of hepatocyte monoculture is the utilization of microfluidic perfusion devices (Knöspel et al., 2016; Shulman and Nahmias, 2013; Tehranirokh et al., 2013; Wagner et al., 2013).”
Background
Novel Cluster and Monomer-Based GalNAc Structures Induce Effective Uptake of siRNAs in Vitro and in Vivo
Sharma, V., Osborn, M. F., Hassler, M. R., Echeverria, D., Ly, S., Ulashchik, E. A., Martynenko-Makaev, Y. V., Shmanai, V. V., Zatsepin, T. S., Khvorova, A., et al. (2018) · Bioconjugate Chemistry · doi:10.1021/acs.bioconjchem.8b00365 · Cited by 42 · see citing works
Human hepatocyte systems for in vitro toxicology analysis
Kammerer, S., & Küpper, J. (2018) · Journal of Cellular Biotechnology · doi:10.3233/jcb-179012 · Cited by 48 · see citing works
“It is thus not surprising that pHH performance turned better upon co-culturing with endothelial cells [24, 25] and through formation of 3D organizations in spheroids [26] or by employing micro-patterned structures [27].”
Background
Peptide-based scaffolds for the culture and maintenance of primary human hepatocytes
MacPherson, D., Bram, Y., Park, J., & Schwartz, R. E. (2021) · Scientific Reports · doi:10.1038/s41598-021-86016-5 · Cited by 36 · see citing works
Functional 3D Human Liver Bud Assembled from MSC-Derived Multiple Liver Cell Lineages
Li, J., Xing, F., Chen, F., He, L., So, K., Liu, Y., & Xiao, J. (2018) · Cell Transplantation · doi:10.1177/0963689718780332 · Cited by 43 · see citing works
“Mouse liver tissues or 512 Cell Transplantation 28(5)”
BackgroundMethodInfluential
Levosimendan Inhibits Peroxidation in Hepatocytes by Modulating Apoptosis/Autophagy Interplay
Grossini, E., Bellofatto, K., Farruggio, S., Sigaudo, L., Marotta, P., Raina, G., de Giuli, V., Mary, D., Pollesello, P., Minisini, R., et al. (2015) · PLoS ONE · doi:10.1371/journal.pone.0124742 · Cited by 30 · see citing works
“Experiments were performed on rat hepatocytes obtained by collagenase perfusion of the liver taken from male Sprague-Dawley rats, following a method previously described [14] and approved by Comitato Etico per la Sperimentazione Animale (CESAPO) dell’Università del Piemonte Orientale “Amedeo Avogadro”.”
Method
Isolation, culture, and functional analysis of hepatocytes from mice with fatty liver disease
Jung, Y., Zhao, M., & Svensson, K. J. (2020) · STAR Protocols · doi:10.1016/j.xpro.2020.100222 · Cited by 33
Biomimetic strategies to recapitulate organ specific microenvironments for studying breast cancer metastasis
Narkhede, A. A., Shevde, L. A., & Rao, S. S. (2017) · International Journal of Cancer · doi:10.1002/ijc.30748 · Cited by 36 · see citing works
“A major drawback of direct co-culture is its inability to maintain the metabolic ability of hepatocytes for a long time.(72) This makes it difficult to study the chemoresistance of BCCs in a physiologically relevant hepatic niche.”
Background
Models of non-Alcoholic Fatty Liver Disease and Potential Translational Value: the Effects of 3,5-L-diiodothyronine
Grasselli, E., Canesi, L., Portincasa, P., Voci, A., Vergani, L., & Demori, I. (2017) · Annals of Hepatology · doi:10.5604/01.3001.0010.2713 · Cited by 36 · see citing works
Hepatic-Specific Decrease in the Expression of Selenoenzymes and Factors Essential for Selenium Processing After Endotoxemia
Sherlock, L. G., Sjostrom, K., Lei, S., Delaney, C. A., Tipple, T. E., Krebs, N. F., Nozik‐Grayck, E., & Wright, C. J. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.595282 · Cited by 27 · see citing works
“Isolation of Primary Hepatocytes Primary hepatocytes were isolated as previously described (36, 37).”
Method
Three‐dimensional coculture of primary hepatocytes and stellate cells in silk scaffold improves hepatic morphology and functionality <i>in vitro</i>
Wei, G., Wang, J., Lv, Q., Liu, M., Xu, H., Zhang, H., Jin, L., Yu, J., & Wang, X. (2018) · Journal of Biomedical Materials Research Part A · doi:10.1002/jbm.a.36421 · Cited by 32 · see citing works
DOCK5 regulates energy balance and hepatic insulin sensitivity by targeting mTORC1 signaling
Lai, Y., Zhao, A., Tan, M., Yang, M., Lin, Y., Li, S., Song, J., Zheng, H., Zhu, Z., Liu, D., et al. (2019) · EMBO Reports · doi:10.15252/embr.201949473 · Cited by 32 · see citing works
“MPHs were isolated from DOCK5 (cid:1) / (cid:1) , Raptor flox/flox , or WT mice aged 8 weeks as previously reported [47].”
Kynurenine produced by indoleamine 2,3-dioxygenase 2 exacerbates acute liver injury by carbon tetrachloride in mice
Hoshi, M., Osawa, Y., Nakamoto, K., Morita, N., Yamamoto, Y., Ando, T., Tashita, C., Nabeshima, T., & Saito, K. (2020) · Toxicology · doi:10.1016/j.tox.2020.152458 · Cited by 28 · see citing works
Application of organoids in translational research of human diseases with a particular focus on gastrointestinal cancers
Nguyen, R., Bae, S. D. W., Zhou, G., Read, S. A., Ahlenstiel, G., George, J., & Qiao, L. (2020) · Biochimica et Biophysica Acta (BBA) - Reviews on Cancer · doi:10.1016/j.bbcan.2020.188350 · Cited by 27 · see citing works
Nanoparticle Drug Delivery Can Reduce the Hepatotoxicity of Therapeutic Cargo
Yang, F., Medik, Y. B., Li, L., Tian, X., Fu, D., Brouwer, K. L. R., Wagner, K., Sun, B., Sendi, H., Mi, Y., et al. (2020) · Small · doi:10.1002/smll.201906360 · Cited by 22 · see citing works
Long-term culture and characterization of patient-derived primary hepatocytes using conditional reprogramming
Su, S., Poto, C. D., Roy, R., Liu, X., Cui, W., Kroemer, A., & Ressom, H. W. (2019) · Experimental Biology and Medicine · doi:10.1177/1535370219855398 · Cited by 24 · see citing works
“However, in vitro cultivation of PHHs often faces obstacles including failure of long-term in vitro culture, rapid loss of liver-specific function and morphology, tendency of fibrosis, and weak proliferation ability.(7,8) Besides, the extensive use of cancerous liver cell lines, e.”
BackgroundMethod
Creating rat hepatocyte organoid as an <i>in vitro</i> model for drug testing
He, Y.-T., Zhu, X.-L., Li, S.-F., Zhang, B.-Q., Li, Y., Wu, Q., Zhang, Y.-L., Zhou, Y.-Y., Li, L., Qi, Y.-N., et al. (2020) · World Journal of Stem Cells · doi:10.4252/wjsc.v12.i10.1184 · Cited by 22 · see citing works
Generation and selection of pluripotent stem cells for robust differentiation to insulin-secreting cells capable of reversing diabetes in rodents
Southard, S. M., Kotipatruni, R. P., & Rust, W. L. (2018) · PLoS ONE · doi:10.1371/journal.pone.0203126 · Cited by 25 · see citing works
A scalable perfusion culture system with miniature peristaltic pumps for live-cell imaging assays with provision for microfabricated scaffolds
Balakrishnan, S., Suma, M. S., Raju, S. R., Bhargav, S. D. B., Arunima, S., Das, S., & Ananthasuresh, G. K. (2015) · BioResearch open access · doi:10.1089/biores.2015.0024 · Cited by 20 · see citing works
Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies
Pedersen, J. M., Shim, Y.-S., Hans, V., Phillips, M. B., Macdonald, J., Walker, G., Andersen, M., Clewell, H., & Yoon, M. (2016) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2016.00072 · Cited by 19 · see citing works
Rapid and high-efficiency generation of mature functional hepatocyte-like cells from adipose-derived stem cells by a three-step protocol
Xu, F., Liu, J., Deng, J., Chen, X., Wang, Y., Xu, P., Cheng, L., Fu, Y., Cheng, F.-Y., Yao, Y., et al. (2015) · Stem cell research & therapeutics · doi:10.1186/s13287-015-0181-3 · Cited by 19 · see citing works
“However, the practical application of primary hepatocytes has been frustrated by their tendency to rapidly dedifferentiate and lose most hepatic functions after growth in a tissue culture environment [1–4].”
Background
Persistent hepatic IFN system activation in HBV-HDV infection determines viral replication dynamics and therapeutic response
Chida, T., Ishida, Y., Morioka, S., Sugahara, G., Han, C., Lam, B., Yamasaki, C., Sugahara, R., Li, M., Tanaka, Y., et al. (2023) · JCI Insight · doi:10.1172/jci.insight.162404 · Cited by 18 · see citing works
“To develop a comprehensive understanding of the host response to HDV infection, the use of a physiological experimental platform, terminally differentiated HH, is essential; however, this has been also regarded as a research tool with limited versatility because of a rapid loss of genuine characteristics during in vit…”
Background
Human Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells
Alevra Sarika, N., Payen, V. L., Fléron, M., Ravau, J., Brusa, D., Najimi, M., De Pauw, E., Eppe, G., Mazzucchelli, G., Sokal, E. M., et al. (2020) · Cells · doi:10.3390/cells9061357 · Cited by 18 · see citing works
“It is indeed well-known that, upon in vitro culture, HEPs rapidly lose their polarity and metabolic properties [7,8], HSCs undergo an activation process during which they rapidly lose their retinoid-containing lipid droplets and acquire a fibrogenic, myofibroblast-like phenotype [9,10], and LSECs lose their fenestrati…”
Background
The Effect of Mesenchymal Stem Cell-Derived Exosomes and miR17-5p Inhibitor on Multicellular Liver Fibrosis Microtissues
Sani, F., Zomorrod, M. S., Azarpira, N., & Soleimani, M. (2023) · Stem Cells International · doi:10.1155/2023/8836452 · Cited by 17 · see citing works
“It has been determined that hepatocytes, HSCs, and endothelial cells make up 60 % – 80 % , 20 % , and 15 % – 20 % , respectively, of the total cells in the natural human liver tissue [14 – 17].”
Retracted
Isolation and cultivation of adult primary bovine hepatocytes from abattoir derived liver
Ehrhardt, S., & Schmicke, M. (2016) · EXCLI journal · doi:10.17179/excli2016-794 · Cited by 14 · see citing works
“Primary hepatocytes obtained from adult mice and rats are widely used for toxicological and metabolic studies and well standardized (Hewitt et al., 2007; Shulman and Nahmias, 2013).”
Method
Liver organoids: From fabrication to application in liver diseases
Liu, Q., Zeng, A., Liu, Z., Wu, C., & Song, L. (2022) · Frontiers in Physiology · doi:10.3389/fphys.2022.956244 · Cited by 19 · see citing works
“Two-dimensional cultured cell models, such as PHHs and induced pluripotent stem cells (IPSCs) also lose the polarity that hepatocytes exhibit in vivo because they are forced to adopt a flat morphology in vitro (Shulman and Nahmias, 2013).”
Background
HepG2 and human healthy hepatocyte in vitro culture and co-culture in PCL electrospun platforms
Fasolino, I., Guarino, V., Marrese, M., Cirillo, V., Vallifuoco, M., Tamma, M., Vassallo, V., Bracco, A., Calise, F., & Ambrosio, L. (2017) · Biomedical Materials · doi:10.1088/1748-605x/aa8c51 · Cited by 16 · see citing works
Impact of Percoll purification on isolation of primary human hepatocytes
Horner, R., Gassner, J. G. M. V., Kluge, M., Tang, P., Lippert, S., Hillebrandt, K. H., Moosburner, S., Reutzel‐Selke, A., Pratschke, J., Sauer, I. M., et al. (2019) · Scientific Reports · doi:10.1038/s41598-019-43042-8 · Cited by 17 · see citing works
Regulation of Liver Enriched Transcription Factors in Rat Hepatocytes Cultures on Collagen and EHS Sarcoma Matrices
Borlak, J. T., Singh, P. K., & Rittelmeyer, I. (2015) · PLoS ONE · doi:10.1371/journal.pone.0124867 · Cited by 14 · see citing works
“The use of the collagen sandwich culture system provides conditions to ensure well-differentiated phenotype of hepatocytes and to reestablish cell polarity [42] as was seen in the present study with cultures of hepatocytes being well-differentiated and possibly even forming bile canaliculi, as observed previously [43].”
Result
Resveratrol Modulates the Inflammatory Profile of Immune Responses and Circulating Endothelial Cells’ (CECs’) Population During Acute Whole Body Gamma Irradiation
Khalil, A., Al-Massarani, G., Aljapawe, A., Ekhtiar, A. M., & Bakir, M. A. (2020) · Frontiers in Pharmacology · doi:10.3389/fphar.2020.528400 · Cited by 13
Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections
Lee, S.-H., Purdy, M. A., & Choi, Y. H. (2023) · Life · doi:10.3390/life13071527 · Cited by 14 · see citing works
“The most common 3D culture method is placing PHHs on a single-layer collagen matrix; however, basic liver functions decrease in this cell culture system after a week [152].”
Simple Paper-based Liver Cell Model for Drug Screening
Kaarj, K., Ngo, J., Loera, C., Akarapipad, P., Cho, S., & Yoon, J.-Y. (2020) · BioChip Journal · doi:10.1007/s13206-020-4211-6 · Cited by 11 · see citing works
Biobanking for research in surgery: are surgeons in charge for advancing translational research or mere assistants in biomaterial and data preservation?
Thasler, W. E., Thasler, R., Schelcher, C., & Jauch, K. (2013) · Langenbeck s Archives of Surgery · doi:10.1007/s00423-013-1060-y · Cited by 11 · see citing works
“In some cases, experimental set-up requires primary human cells to stay in culture over a long period of time and since hepatocytes have a relatively short life span in culture, co-culturing with a variety of non-parenchymal cells such as Kupffer, stellate and epithelial cells is necessary in order to increase their l…”
Background
Transplantation of 3D adipose-derived stem cell/hepatocyte spheroids alleviates chronic hepatic damage in a rat model of thioacetamide-induced liver cirrhosis
Wu, Y. C., Wu, G. X., Chen, K., Shiu, L., Kumar, S., Liu, G., & Kuo, S. M. (2022) · Scientific Reports · doi:10.1038/s41598-022-05174-2 · Cited by 13 · see citing works
“The description of pathological features: (1) reticular fibers circumscribing the nodules; (2) obvious formation of pseudolobules; (3) numerous fibrocytes; (4) hepatocyte necrosis; (5) swollen hepatocytes; (6) large/moderate decreases in fibrous tissues/fibrocytes; (7) proliferation of the mini-bile duct; (8) fatty de…”
Background
Integrative microphysiological tissue systems of cancer metastasis to the liver
Clark, A. M., Allbritton, N. L., & Wells, A. H. (2020) · Seminars in Cancer Biology · doi:10.1016/j.semcancer.2020.06.010 · Cited by 11 · see citing works
3D Co-Culture with Vascular Cells Supports Long-Term Hepatocyte Phenotype and Function In Vitro
Unal, A. Z., Jeffs, S. E., & West, J. L. (2018) · Regenerative Engineering and Translational Medicine · doi:10.1007/s40883-018-0046-2 · Cited by 12 · see citing works
Effects of Mixtures of Emerging Pollutants and Drugs on Modulation of Biomarkers Related to Toxicity, Oxidative Stress, and Cancer
Manuguerra, S., Carli, F., Scoditti, E., Santulli, A., Gastaldelli, A., & Messina, C. M. (2024) · Metabolites · doi:10.3390/metabo14100559 · Cited by 6 · see citing works
“Moreover, hepatoma cells overcome the limitations of using primary hepatocytes, mostly in screening studies, including the limited availability of liver samples, inter-individual variability, limited survival, loss of metabolic activity after long culture, and costs [76,77].”
Background
LASS2 regulates hepatocyte steatosis by interacting with NDUFS2/OXPHOS related proteins.
Yang, Y., Yang, X.-L., Lin, Y., Yang, G.-Y., & Li, L. (2020) · Biochemical and Biophysical Research Communications - BBRC · doi:10.1016/j.bbrc.2020.02.166 · Cited by 8 · see citing works
A study of the differentiation of stem cells from human exfoliated deciduous teeth on 3D silk fibroin scaffolds using static and dynamic culture paradigms.
Huang, T.-Y., Wang, G.-S., Ko, C.-S., Chen, X.-W., & Su, W.-T. (2020) · Materials Science and Engineering C: Materials for Biological Applications · doi:10.1016/j.msec.2019.110563 · Cited by 8
Metabolomic Profile of Primary Turkey and Rat Hepatocytes and Two Cell Lines after Chloramphenicol Exposure
Radko, L., Śniegocki, T., Sell, B., & Posyniak, A. (2019) · Animals · doi:10.3390/ani10010030 · Cited by 7 · see citing works
“Primary cultures of hepatocytes and liver-derived lines are the models that are used for the evaluation of drugs of which toxicity is mediated through biotransformation [22,23].”
Background
Conditioned Media Derived from Human Adipose Tissue Mesenchymal Stromal Cells Improves Primary Hepatocyte Maintenance.
Tafti, Z. A., Mahmoodi, M., Hajizadeh, M. R., Ezzatizadeh, V., Baharvand, H., Vosough, M., & Piryaei, A. (2018) · Europe PMC (PubMed Central) · doi:10.22074/cellj.2018.5288 · Cited by 11 · see citing works
Constructing vascularized hepatic tissue by cell-assembled viscous tissue sedimentation method and its application for vascular toxicity assessment.Preprint
Naito, Y., Yoshinouchi, Y., Sorayama, Y., Kohara, H., Kitano, S., Irie, S., & Matsusaki, M. (2021) · Acta Biomaterialia · doi:10.2139/ssrn.3906842 · Cited by 7 · see citing works
A three‐dimensional microfluidized liver system to assess hepatic drug metabolism and hepatotoxicity
Corrado, B., Gregorio, V. D., Imparato, G., Attanasio, C., Urciuolo, F., & Netti, P. A. (2018) · Biotechnology and Bioengineering · doi:10.1002/bit.26902 · Cited by 34 · see citing works
In vitro3-D culture demonstrates incompetence in improving maintenance ability of primary hepatocytes
Ullah, I., Kim, Y., Lim, M.-U., Oh, K. B., Hwang, S., Shin, Y., Kim, Y.-I., Im, G., Hur, T., & Ock, S. A. (2017) · Animal Cells and Systems · doi:10.1080/19768354.2017.1381151 · Cited by 5 · see citing works
“D EV EL O PM EN TA L BI O LO G Y CONTACT Sun-A Ock ouksuna@hotmail.com; ocksa@korea.kr Animal Biotechnology Division, National Institute of Animal Science, RDA, 1500 Kongjwipatjwi-ro Iseo-myeon, Wanju-gun, Jeollabuk-do 565-851, Republic of Korea (Shulman and Nahmias 2013).”
BackgroundInfluential
Liver Ultrasound Histotripsy: Novel Analysis of the Histotripsy Site Cell Constituents with Implications for Histotripsy Application in Cell Transplantation and Cancer Therapy
Froghi, S., de Andrade, M. O., Mohammad-Hadi, L., Gélat, P., Rashidi, H., Quaglia, A., Fuller, B. J., Saffari, N., & Davidson, B. R. (2023) · Bioengineering · doi:10.3390/bioengineering10020276 · Cited by 6 · see citing works
The newly established human liver cell line: a potential cell source for the bioartificial liver in the future
Liu, H., You, S., Rong, Y., Wu, Y., Zhu, B., Zhihong, W., Liu, W., Panyong, M., & Xin, S. (2013) · Human Cell · doi:10.1007/s13577-013-0068-5 · Cited by 7 · see citing works
“The elimination of glucose was mainly related with energy needs in culturing time [24, 25].”
Status and Future of 3D Cell Culture in Toxicity Testing
Otieno, M. A., Gan, J., & Proctor, W. R. (2018) · Methods in pharmacology and toxicology · doi:10.1007/978-1-4939-7677-5_12 · Cited by 6 · see citing works
Organoid Establishment of Long-Term Culture Using Primary Mouse Hepatocytes and Evaluation of Liver Function
Kim, H. M., Kim, Y., Kim, Y., Kim, Y. J., & Ko, K. S. (2023) · Preventive Nutrition and Food Science · doi:10.3746/pnf.2023.28.3.360 · Cited by 4 · see citing works
“Furthermore, primary hepatocytes rapidly lose their morphological characteristics (Shulman and Nahmias, 2012; Rose et al., 2021).”
Background
Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations
Serrano, L. J., García-Arranz, M., Pablo-Moreno, J. A. D., Segovia, J., Olivera-Salazar, R., García-Olmo, D., & Liras, A. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23105802 · Cited by 5
Evaluation of the Safety and Toxicity of the Original Copper Nanocomposite Based on Poly-N-vinylimidazole
Александровна, Ш. И., Prozorova, G. F., Сергеевна, Т. И., Korzhova, S. A., Дремина, Н. Н., Emel’yanov, A. I., Say, O., Kuznetsova, N. P., Pozdnyakov, A. S., & Шурыгин, М. Г. (2021) · Nanomaterials · doi:10.3390/nano12010016 · Cited by 4 · see citing works
“The cells were used for the experiment within 24 h after isolation since no changes in the hepatocyte enzymatic systems functioning were observed during this period [53,54].”
BackgroundMethod
Manipulating the mechanics of extracellular matrix to study effects on the nucleus and its structure
Xia, Y., Cho, S., Vashisth, M., Ivanovska, I. L., Dingal, P. C. D. P., & Discher, D. E. (2018) · Methods · doi:10.1016/j.ymeth.2018.12.009 · Cited by 5 · see citing works
“Ignoring the diversity of mechanical cues presented to cells in normal and diseased tissues surrenders control of additional factors that affect cell and nuclear phenotype [4,69].”
Identifying niche‐mediated regulatory factors of stem cell phenotypic state: a systems biology approach
Ravichandran, S., & del Sol, A. (2017) · FEBS Letters · doi:10.1002/1873-3468.12559 · Cited by 5 · see citing works
Formation of 3D tissues of primary hepatocytes using fibrillized collagen microparticles as intercellular binders
Morita, A., Yamada, M., Utoh, R., Momiyama, K., Iwadate, H., & Seki, M. (2021) · Journal of Bioscience and Bioengineering · doi:10.1016/j.jbiosc.2021.11.009 · Cited by 5 · see citing works
Conditioned Media Derived from Human Adipose Tissue Mesenchymal Stromal Cells Improves Primary Hepatocyte Maintenance
Tafti, Z. A., Mahmoodi, M., Hajizadeh, M., Ezzatizadeh, V., Baharvand, H., Massoud Vosough, M. P., & Piryaei, A. (2021) · Cell journal · doi:10.22074/cellj.2021.7936 · Cited by 4
Functional Liver Cell–Based Platforms in Biomedical Research
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“Primary hepatocytes rapidly lose their liver-specific functions, including CYP inducibility and the production of plasma proteins like albumin, during in vitro cultivation 39 .”
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Background
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Self-citation
Porcine Hepatocytes: Isolation and Liver Tissue Engineering for Xenotransplantation
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“This morphology is much closer to the native cuboidal morphology of PHs within the liver (Shulman and Nahmias 2012b). mRNA expression levels for some typical liver-function related genes (Alb, Cps1, Cyp1a1, Hnf4a, etc.) at 24 h showed no reduction in the PHs treated with PEG-GRGDS compared to those…”
Influential
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Background

Authors: Shulman, M., & Nahmias, Y.
Affiliations: Hebrew University of Jerusalem (IL)
Corresponding author: Nahmias, Yaakov
ORCID: Nahmias, Yaakov
Venue: Methods in Molecular Biology
Publisher: Humana Press
Published: 2012
Volume: 945
Pages: 287-302
ISSN: 1064-3745 (ISSN-L)
1940-6029
DOI: 10.1007/978-1-62703-125-7_17
PMC: PMC3781339
OpenAlex: W1850526299
Cited by: 117
References: 52
Citations by year: 2026: 1 · 2025: 5 · 2024: 6 · 2023: 8 · 2022: 9 · 2021: 8 · 2020: 19 · 2019: 9 · 2018: 18 · 2017: 13 · 2016: 6 · 2015: 6 · 2014: 2 · 2013: 7
FWCI: 15.92
Type: book chapter
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: www.springernature.com/gp/researchers/text-and-data-mining (tdm)
www.springernature.com/gp/researchers/text-and-data-mining (vor)
Locations: Methods in molecular biology (publishedVersion)
PubMed (publishedVersion)
Europe PMC (PubMed Central) (acceptedVersion)
PubMed Central (acceptedVersion)
Funders: National Institute of Diabetes and Digestive and Kidney Diseases (K01DK080241)
Topics: Health Sciences › Medicine › Hepatology › Liver physiology and pathology
Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
Keywords: primary hepatocytes, long-term hepatocyte culture, hepatocyte co-culture, bioartificial liver, hepatocyte spheroids, hepatocyte isolation, culture medium formulation, oxygen supply, hepatocyte death, drug toxicity testing
MeSH terms: Albumins, Animals, Cell Separation, Enzyme-Linked Immunosorbent Assay, Humans, Oxygen, Perfusion, Time Factors, Urea, Coculture Techniques, Cell Culture Techniques, Hepatocytes, Rats

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Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

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