Written by Nick Trompeter, Ph.D.
Kupffer Cells express the macrophage marker CD68.
Within the liver sinusoid, prowling across the luminal surface of Liver Sinusoidal Endothelial Cells, resides the resident macrophage of the liver, Kupffer Cells (KCs). Like hepatocytes, primary human kupffer cells cannot replicate during in vitro culture. These macrophages play an integral role in linking innate immunity and adaptive immunity in the liver and mediate disease progression and regeneration of the liver.(1,3,5) The phagocytic capacity of Kupffer Cells, combined with acting as antigen presenting cells, defends against foreign agents and can promote either a pro-inflammatory or tolerogenic effect.(2) When culturing KCs in vitro, functional phenotyping of the cells utilizes the bacterial endotoxin lipopolysaccharide (LPS) to stimulate production of pro-inflammatory cytokines, such as interleukin-6, interleukin-8, and tumor necrosis factor-α. In addition to LPS induction, expression of macrophage markers CD68, CD163, and CD11b are commonly utilized to confirm the phenotype of isolated KCs while assessing the purity of the cell population.
While KCs remain viable in culture for a week when plated on type I collagen gels,(4) the majority of experiments utilize co-culture of KCs with hepatocytes and/or other nonparenchymal cells. Leveraging KCs in either monolayer, spheroid, or bioprinted models allows for the interrogation of the molecular mechanisms and intercellular crosstalk responsible for the progression of metabolic dysfunction-associated steatohepatitis (MASH) and drug-induced liver injury (DILI). When beginning co-culture models with primary human hepatocytes or as a part of a bulk nonparenchymal population, determining the ratio of hepatocytes to KCs or NPCs is critical. As the liver is comprised predominantly of hepatocytes, the addition of KCs/NPCs at smaller numbers better captures the physiology of the liver. Often ratios of hepatocytes:KCs/NPCs of 8:1, 4:1, and 2:1 are leveraged for toxicity studies using spheroids, with the hepatocyte concentration kept at 1500 cells/spheroid.(6) Multiple studies highlight how the inclusion of KCs into in vitro models can stem the toxicity of the known DILI agent acetaminophen.(6,7) Compounds that appear innocuous at subtoxic can stimulate KC activation during repeated dosing to cause hepatotoxicity.(8) Furthermore, the inclusion of KCs provides important insight into how inflammation can modulate the response of spheroids and bioprinted livers to known toxicants. Induction of KC inflammation with LPS interestingly stimulates differential responses to compounds known to induce DILI.(9,10) Li et al., discovered that treatment of microtissues with LPS promotes DILI when also challenging the cells with Tolcapone or Nefazodone. However, when KCs were stimulated with LPS during treatment with indomethacin or paroxetine there was a hepatoprotective effect.(9) Thus, when using in vitro models for hepatotoxicity it is important to consider the inflammatory context of when a drug would be used and how it may influence an adverse event in the clinic.
Beyond DILI, when delivering drugs for hepatic or extrahepatic purposes, the scavenger activity of KCs creates a hurdle that needs to be avoided for successful delivery to the cells of interest. Phagocytosis of particles by KCs in vitro can provide insight into the pharmacokinetics of antibody-drug conjugates and how the chemistry of the linker influences unintended elimination from the plasma.4 These studies can extend into how nanoparticles are phagocytosed and eliminated by KCs in vitro, which may allow scientists to avoid expensive animal studies where accumulation and tropism of nanoparticles for gene editing are currently the gold standard method.(11,12 )
When working with KCs, donor to donor variability can significantly impact the structure of models leveraged for liver disease and DILI studies. Exposure to various nanomaterials known to cause hepatotoxicity show significantly altered the responses of microtissue models due to donor-donor variability, with some donors responding more potently to the toxic nanomaterials.(13) In developing a MASH model using 3D bioprinted livers, donor-donor variation was also observe, where one lot of KCs from a MASH donor appeared to stimulate a move severe diseased phenotype than the other diseased donor.(14) These results highlight the importance of testing multiple different KC lots and setting donor criteria during the assessment of a novel model or therapeutic.
Want to advance your models by including Kupffer Cells to interrogate the influence of these important cells for drug development or toxicity studies?
Mosaic Cell Sciences’ Kupffer Cells from healthy and MASH/MASLD donors will be your missing puzzle piece!
Contact Nick Trompeter: ntrompeter@mosaiccellsci.com to schedule a call with us.
Receive 25% off your first order!
References
1. Bataller, R., and Brenner, D.A. (2005). Liver fibrosis. Journal of Clinical Investigation 115, 209–218. https://doi.org/10.1172/JCI200524282.
2. Cai, J., Zhang, X.J., and Li, H. (2019). The Role of Innate Immune Cells in Nonalcoholic Steatohepatitis. Preprint at John Wiley and Sons Inc, https://doi.org/10.1002/hep.30506 https://doi.org/10.1002/hep.30506.
3. Kalra, A., Ekrem, ;, Chase, Y.;, Wehrle, J., Faiz, ;, and Affiliations, T. Physiology, Liver.
4. Meyer, D.W., Bou, L.B., Shum, S., Jonas, M., Anderson, M.E., Hamilton, J.Z., Hunter, J.H., Wo, S.W., Wong, A.O., Okeley, N.M., et al. (2020). An in Vitro Assay Using Cultured Kupffer Cells Can Predict the Impact of Drug Conjugation on in Vivo Antibody Pharmacokinetics. Mol Pharm 17, 802–809. https://doi.org/10.1021/acs.molpharmaceut.9b00991.
5. Ramai, D., Facciorusso, A., Vigandt, E., Schaf, B., Saadedeen, W., Chauhan, A., Di Nunzio, S., Shah, A., Giacomelli, L., and Sacco, R. (2021). Progressive liver fibrosis in non-alcoholic fatty liver disease. Preprint at MDPI, https://doi.org/10.3390/cells10123401 https://doi.org/10.3390/cells10123401.
6. Bell, C.C., Chouhan, B., Andersson, L.C., Andersson, H., Dear, J.W., Williams, D.P., and Söderberg, M. (2020). Functionality of primary hepatic non-parenchymal cells in a 3D spheroid model and contribution to acetaminophen hepatotoxicity. Arch Toxicol 94, 1251–1263. https://doi.org/10.1007/s00204-020-02682-w.
7. Rose, K.A., Holman, N.S., Green, A.M., Andersen, M.E., and Lecluyse, E.L. (2016). Co-culture of Hepatocytes and Kupffer Cells as an in Vitro Model of Inflammation and Drug-Induced Hepatotoxicity. J Pharm Sci 105, 950–964. https://doi.org/10.1016/S0022-3549(15)00192-6.
8. Kegel, V., Pfeiffer, E., Burkhardt, B., Liu, J.L., Zeilinger, K., Nüssler, A.K., Seehofer, D., and Damm, G. (2015). Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI. Mediators Inflamm 2015, 640631. https://doi.org/10.1155/2015/640631.
9. Li, F., Cao, L., Parikh, S., and Zuo, R. (2020). Three-Dimensional Spheroids With Primary Human Liver Cells and Differential Roles of Kupffer Cells in Drug-Induced Liver Injury. J Pharm Sci 109, 1912–1923. https://doi.org/10.1016/j.xphs.2020.02.021.
10. Norona, L.M., Nguyen, D.G., Gerber, D.A., Presnell, S.C., Mosedale, M., and Watkins, P.B. (2019). Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis. PLoS One 14, e0208958. https://doi.org/10.1371/JOURNAL.PONE.0208958.
11. MacParland, S.A., Tsoi, K.M., Ouyang, B., Ma, X.-Z., Manuel, J., Fawaz, A., Ostrowski, M.A., Alman, B.A., Zilman, A., Chan, W.C.W., et al. (2017). Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood. ACS Nano 11, 2428–2443. https://doi.org/10.1021/acsnano.6b06245.
12. Dolina, J.S., Sung, S.S.J., Novobrantseva, T.I., Nguyen, T.M., and Hahn, Y.S. (2013). Lipidoid nanoparticles containing PD-L1 siRNA delivered in vivo enter Kupffer cells and enhance NK and CD8+ T cell-mediated hepatic antiviral immunity. Mol Ther Nucleic Acids 2, e72. https://doi.org/10.1038/mtna.2012.63.
13. Kermanizadeh, A., Brown, D.M., Moritz, W., and Stone, V. (2019). The importance of inter-individual Kupffer cell variability in the governance of hepatic toxicity in a 3D primary human liver microtissue model. Sci Rep 9. https://doi.org/10.1038/s41598-019-43870-8.
14. Tan, P.K., Ostertag, T., Rosenthal, S.B., Chilin-Fuentes, D., Aidnik, H., Linker, S., Murphy, K., Miner, J.N., and Brenner, D.A. (2024). Role of Hepatic Stellate and Liver Sinusoidal Endothelial Cells in a Human Primary Cell Three-Dimensional Model of Nonalcoholic Steatohepatitis. American Journal of Pathology 194, 353–368. https://doi.org/10.1016/j.ajpath.2023.12.005.
Comments