An efficient method to isolate and culture mouse Kupffer cells

Li, Peizhi; Li, Jinzheng; Li, Min; Gong, Jianping; He, Kun

doi:10.1016/j.imlet.2013.12.002

Cited by 141 publications

(97 citation statements)

References 17 publications

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“…5), which may be related to the purification of the KCs using selective attachment. Some recently published studies have reported a simple and efficient method to obtain KCs from mixed primary cultures of liver cells [Kitani et al, 2010;Li et al, 2014], but this technique may not be suitable for the separation of other liver NPCs. In a classic published method of KC isolation [Smedsrod and Pertoft, 1985], the yield and purity of KCs were 2.4 × 10 6 cells/g liver and 90-95% overall, which was similar to our findings.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Culture of Single Cell Types from Rat Liver

Zhang

et al. 2016

Cells Tissues Organs

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There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

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Section: Discussionmentioning

confidence: 99%

Isolation and Culture of Single Cell Types from Rat Liver

Zhang

et al. 2016

Cells Tissues Organs

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“…However, it is reported that certain Kupffer cell methods can be easier to conduct, such as the protocol described by Wu et al, where the liver is digested ex vivo 23 . However, the method described by Wu et al leads to a limited amount of cells and lower cell purity.…”

Section: Discussionmentioning

confidence: 99%

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Bourgognon

Klippstein

Al‐Jamal

2015

JoVE

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The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsrød et al. designed for rat liver cell isolation and provides high yield (up to 14 x 10 6 cells per mouse) and high purity (>95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient. Video LinkThe video component of this article can be found at

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“…Naive and mDTA-1-pretreated wildtype BALB/c mice (previously inoculated with 3 mg/kg mDTA-1 Q7D Â 2) were injected s.c. with 30 mg/kg DyLight-labeled mDTA-1 and KCs were enriched from liver-associated cells as previously described (Zeng et al, 2013;Li et al, 2014). Briefly, 24 hours postdose mice were euthanized and underwent cardiac perfusion with 3 ml Â min 21 PBS for 3 minutes.…”

Section: Methodsmentioning

confidence: 99%

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice

Brunn

Mauze

et al. 2015

Journal of Pharmacology and Experimental Therapeutics

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Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD31 CD4 1 T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3

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An efficient method to isolate and culture mouse Kupffer cells

Cited by 141 publications

References 17 publications

Isolation and Culture of Single Cell Types from Rat Liver

Isolation and Culture of Single Cell Types from Rat Liver

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice

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