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Liver perfusion medium

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Liver Perfusion Medium is a specialized cell culture medium designed for the perfusion and maintenance of primary liver cells. It provides the necessary nutrients and growth factors to support the viability and function of liver cells in an in vitro setting.

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Lab products found in correlation

Liver digest medium, by Thermo Fisher Scientific (42 mentions) Hepatocyte wash medium, by Thermo Fisher Scientific (10 mentions) Percoll, by GE Healthcare (6 mentions) Percoll, by Merck Group (6 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) William s e medium, by Thermo Fisher Scientific (4 mentions) Liver digestion medium, by Thermo Fisher Scientific (4 mentions) 100 μm cell strainer, by BD (3 mentions) Trizol, by Thermo Fisher Scientific (3 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions) Collagenase type 1, by Worthington (3 mentions) Hepatozyme sfm, by Thermo Fisher Scientific (3 mentions) Percoll solution, by GE Healthcare (3 mentions) Wortmannin, by Merck Group (2 mentions) Dynabeads magnetic beads, by BD (2 mentions) Mouse lsec binding magnetic beads, by Miltenyi Biotec (2 mentions) Fusion flow cytometer, by BD (2 mentions) 70 m filter, by BD (2 mentions) Anti mouse cd146 antibody fitc, by Miltenyi Biotec (2 mentions)

Spelling variants of this product

Pre-warmed Liver Perfusion Medium (3 citations) Perfusion medium (2 citations)

71 protocols using liver perfusion medium

1

Isolation of Primary Hepatocytes

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Primary hepatocytes were isolated by a two-step collagenase perfusion method according to a previous report [22 (link)] with modifications. After CAG-ELuc/MI-MAC Tc mouse was anesthetized with 3% isoflurane gas using small animal anesthetizer (MK-AT200, Muromachi Kikai, Tokyo, Japan), the liver was perfused with liver perfusion medium (Gibco-BRL) via the hepatic portal vein and immediately the abdominal inferior vena cava was cut to induce bleeding. After the liver had been freed of blood, liver perfusion medium (Gibco-BRL) was replaced with 0.5 mg/ml collagenase buffer (Sigma-Aldrich) for 4 min. The perfusion rate of 5 ml/min and the temperature of around 40 °C were maintained for both perfusates and collagenase buffer during the entire procedure. After perfusion, the liver was rapidly transferred to a sterile petri dish and shredded in cold hepatocyte wash medium (Gibco-BRL). The medium containing shredded liver was filtered through a 100 μm cell strainer (Becton Dickinson, Bedford, MA). After washing by low-speed centrifugation at 50 g for 3 min at 4 °C, the hepatocytes were suspended in the appropriate medium for subsequent experiments.
Yasunaga M., Fujita Y., Saito R., Oshimura M, & Nakajima Y. (2017). Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement. BMC Biotechnology, 17, 54.
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2

Isolation of Primary Hepatocytes

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Ex vivo primary cells were isolated using a modified retrograde perfusion technique53 (link) consisting of: 1) Liver Perfusion Medium (Gibco) for 5 minutes; 2) Liver Digest Medium (Gibco) for 10 minutes. The liver was then excised and the capsule disrupted to yield a cell suspension which was collected in Liver Perfusion Medium (Gibco) and passed through a 100μm filter (BD Bioscences). Hepatocytes were pelleted by centrifugation at 135G for 1 minute separated them from a non-parenchymal cell (NPC) rich fraction and each resuspended in Williams E Medium (Gibco) with 5% FCS. The hepatocyte rich cell suspension was then purified as described previously53 (link). Briefly cells were underlayered with a discontinuous Percoll gradient (1.06, 1.08 and 1.12g/ml Percoll in PBS). Cells were then spun at 750 G for 20 minutes at 20°C. The cell layer collected between the 1.08 and 1.12mg/ml Percoll layers were harvested and resuspended in Williams Medium. Cells were then purified a further 2 times using Percoll layers as previously and quantified prior to analysis. Routinely cells with hepatocyte morphology and expression of CYP2D6 (marker of hepatocellular differentiation) were obtained at over 99% purity (See Supplementary Fig 2, p<0.05).
Lu W.Y., Bird T.G., Boulter L., Tsuchiya A., Cole A.M., Hay T., Guest R.V., Wojtacha D., Man T.Y., Mackinnon A., Ridgway R.A., Kendall T., Williams M.J., Jamieson T., Raven A., Hay D.C., Iredale J.P., Clarke A.R., Sansom O.J, & Forbes S.J. (2015). Hepatic progenitor cells of biliary origin with liver repopulation capacity. Nature cell biology, 17(8), 971-983.
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3

Isolation of Primary Hepatocytes

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Ex vivo primary cells were isolated using a modified retrograde perfusion technique53 (link) consisting of: 1) Liver Perfusion Medium (Gibco) for 5 minutes; 2) Liver Digest Medium (Gibco) for 10 minutes. The liver was then excised and the capsule disrupted to yield a cell suspension which was collected in Liver Perfusion Medium (Gibco) and passed through a 100μm filter (BD Bioscences). Hepatocytes were pelleted by centrifugation at 135G for 1 minute separated them from a non-parenchymal cell (NPC) rich fraction and each resuspended in Williams E Medium (Gibco) with 5% FCS. The hepatocyte rich cell suspension was then purified as described previously53 (link). Briefly cells were underlayered with a discontinuous Percoll gradient (1.06, 1.08 and 1.12g/ml Percoll in PBS). Cells were then spun at 750 G for 20 minutes at 20°C. The cell layer collected between the 1.08 and 1.12mg/ml Percoll layers were harvested and resuspended in Williams Medium. Cells were then purified a further 2 times using Percoll layers as previously and quantified prior to analysis. Routinely cells with hepatocyte morphology and expression of CYP2D6 (marker of hepatocellular differentiation) were obtained at over 99% purity (See Supplementary Fig 2, p<0.05).
Lu W.Y., Bird T.G., Boulter L., Tsuchiya A., Cole A.M., Hay T., Guest R.V., Wojtacha D., Man T.Y., Mackinnon A., Ridgway R.A., Kendall T., Williams M.J., Jamieson T., Raven A., Hay D.C., Iredale J.P., Clarke A.R., Sansom O.J, & Forbes S.J. (2015). Hepatic progenitor cells of biliary origin with liver repopulation capacity. Nature cell biology, 17(8), 971-983.
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4

Isolation and Characterization of Mouse and Human Liver Sinusoidal Endothelial Cells

Cited 1 time
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Mouse LSECs were isolated by previously described two-step collagenase perfusion technique with modifications.15 (link) In brief, the liver was perfused with Liver Perfusion Medium (Invitrogen), and dissociated by Liver Digest Medium (Invitrogen). The NPCs were fractionated with Percoll (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) gradient centrifugation with 75% stock Percoll solution and 35% stock Percoll solution. LSEC faction was isolated by mouse LSEC-binding magnetic beads (Miltenyi, Auburn, CA, USA) and Dynabeads Magnetic Beads conjugated with anti-mouse CD31 antibody (MEC13.3, BD Biosciences). Expression of Id1, CXCR7, HGF and Wnt2 messenger RNA was determined. Primary human LSECs were procured from ScienCell Research Laboratories (catalog no. 5000, Carlsbad, CA, USA). Expression of factor VIII was validated by immunostaining. Akt-LSECs were derived from isolated LSECs that were transfected with the pCCL. PGK lentiviral vector with mouse constitutively active Akt1 (myristoylated Akt: myrAkt).63 (link) After starving in serum-free medium, 500 000 LSECs were seeded and stimulated with 10 ng ml−1 SDF-1. LSECs were also treated with 30 μm Wortmannin (Sigma-Aldrich).
Shido K., Chavez D., Cao Z., Ko J.L., Rafii S, & Ding B.S. (2017). Platelets prime hematopoietic–vascular niche to drive angiocrine-mediated liver regeneration. Signal Transduction and Targeted Therapy, 2, 16044-.
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5

Isolation and Culture of Primary Hepatocytes

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Primary hepatocytes were cultured according to the published methods,43 (link) with a slight modification. Liver perfusion medium (Invitrogen, 17701-038) was used to perfuse the livers in situ via the portal vein and was followed by liver digest medium (Invitrogen, 17703-034) after anesthetizing the mice with pentobarbital sodium. The liver was excised and minced in William's E medium (Life technologies, A12176-01, Grand Island, NY, USA). The cell suspension was mixed gently several times with a pipette and strained through a steel mesh sieve after removing the liver capsule. The dispersed hepatocytes were collected via centrifugation at 50 × g for 5 min at 4 °C and washed twice with William's E medium. Hepatocytes were isolated by Percoll separation and washed twice with William's E medium. The final pellet was resuspended in William's E medium. The hepatocytes were counted, and their viability was determined by trypan blue exclusion. The hepatocytes were cultured under normoxic conditions (air/5% CO2) for further experiments.
Zhang X.F., Zhang R., Huang L., Wang P.X., Zhang Y., Jiang D.S., Zhu L.H., Tian S., Zhang X.D, & Li H. (2014). TRAF1 is a key mediator for hepatic ischemia/reperfusion injury. Cell Death & Disease, 5(10), e1467-.
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6

Isolation of Primary Human Hepatocytes and Fibroblasts

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The liver tissue fragments were perfused, first with liver perfusion medium (Invitrogen, Carlsbad, CA, USA) at 37 °C for 15 min, and then with collagenase and dispase-containing liver digest medium (Invitrogen) at 37 °C for approximately 15 min, until the tissue was completely digested. Cells were dispersed by gentle shaking, filtered and centrifuged at a low speed. Primary human hepatocytes (PHH) were collected from the pellet and used in primary culture, as previously described [23 (link)]. The non-parenchymal cell fraction was collected from the supernatant that was centrifuged to obtain intra-hepatic fibroblasts (IHF), as described elsewhere [24 (link),26 (link)].
Ouaguia L., Moralès O., Aoudjehane L., Wychowski C., Kumar A., Dubuisson J., Calmus Y., Conti F, & Delhem N. (2019). Hepatitis C Virus Improves Human Tregs Suppressive Function and Promotes Their Recruitment to the Liver. Cells, 8(10), 1296.
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7

Mouse Hepatocyte Isolation by Liver Perfusion

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Mouse hepatocytes were isolated via liver perfusion through the vena cava with liver perfusion medium (Invitrogen) for 15 min and then with collagenase (Serva) and Ca2+-supplemented medium for the next 15 min.
Seehawer M., Heinzmann F., D’Artista L., Harbig J., Roux P.F., Hoenicke L., Dang H., Klotz S., Robinson L., Doré G., Rozenblum N., Kang T.W., Chawla R., Buch T., Vucur M., Roth M., Zuber J., Luedde T., Sipos B., Longerich T., Heikenwälder M., Wang X.W., Bischof O, & Zender L. (2018). Necroptosis microenvironment directs lineage commitment in liver cancer. Nature, 562(7725), 69-75.
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8

Isolation of Lymphoid Cells from Liver

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The liver was perfused with liver perfusion medium (Invitrogen Life Technologies) followed by liver digest medium (Invitrogen Life Technologies) afterwards removed and digested for 30 min at 37 °C. Liver cells were gently pressed trough a cell strainer (∅ 70 µm) and lymphoid cells were separated by centrifugation 60 × g for 5 min. Lymphoid cells in the supernatant were collected, washed and resuspended in PBS supplemented with 1% FCS and diluted in 70% Easycoll (Biochrom) in a 1:1 ratio (≙ 35% Easycoll) and then overlaid onto 70% Easycoll and centrifuged for 20 min at 950 × g. Lymphoid cells were collected from the interface, washed and subsequently erythrocytes were lysed.
Kustermann M., Klingspor M., Huber-Lang M., Debatin K.M, & Strauss G. (2019). Immunostimulatory functions of adoptively transferred MDSCs in experimental blunt chest trauma. Scientific Reports, 9, 7992.
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9

Isolation of Mouse Primary Hepatocytes

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Mouse primary hepatocytes were isolated using a two-step collagenase liver perfusion method. Mice were anaesthetized using Ketamine(100 mg/kg, ) / Xylazine (10 mg/kg, ). Following opening of the abdomen, perfusion solutions warmed to 37°C were pumped through a catheter placed into the portal vein and drained out the inferior vena cava. Mice were perfused with 25 mL of Liver Perfusion Medium (Invitrogen, 17701038) followed by 25 mL of Hepatocyte Wash Medium (Invitrogen, 17704024) supplemented with 0.05% collagenase B (Roche, 11088831001). The liver was then excised and its capsules were torn to release hepatocytes. The isolated hepatocytes were then filtered through 100μM cell strainers (BD Biosciences, 352360), washed 3 times with Hepatocyte Wash Medium by centrifuging at 50 × g and then finally resuspended in 1%BSA PBS with 0.1% propidium iodide (Sigma, P4864) and sorted using FACS (BD Biosciences, FACSAria). For FACS, hepatocytes were gated by size using FSC/SSC and live/dead using propidium iodide staining followed by GFP fluorescence into GFP+ versus GFP- live hepatocytes directly into TRIzol (ThermoFisher, 15596026).
Wang S., Zhang C., Hasson D., Desai A., SenBanerjee S., Magnani E., Ukomadu C., Lujambio A., Bernstein E, & Sadler K.C. (2019). Epigenetic compensation promotes liver regeneration. Developmental cell, 50(1), 43-56.e6.
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10

Isolation and Characterization of Mouse and Human LSECs

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Mouse LSECs were isolated by previously described two-step collagenase perfusion technique with modifications15 (link). In brief, the liver was perfused with Liver Perfusion Medium (Invitrogen), and dissociated by Liver Digest Medium (Invitrogen). The NPCs were fractionated with Percoll® (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) gradient centrifugation with 75% stock Percoll solution and 35% stock Percoll solution. LSEC faction was isolated by mouse LSEC binding magnetic beads (Miltenyi, Auburn, CA, USA) and Dynabeads® Magnetic Beads conjugated with anti-mouse CD31 antibody (MEC13.3, BD Biosciences). Expression of Id1, CXCR7, HGF, and Wnt2 mRNA were determined. Primary human LSECs were procured from ScienCell Research Laboratories (Catalog #5000, Carlsbad, CA, USA). Expression of factor VIII was validated by immunostaining. Akt-LSECs were derived from isolated LSECs that were transfected with the pCCL. PGK lentiviral vector with mouse constitutively active Akt1 (myristoylated Akt: myrAkt)63 (link). After starving in serum-free medium, 500,000 LSECs were seeded and stimulated with 10 ng/ml SDF-1. LSECs were also treated with 30 μM Wortmannin (Sigma-Aldrich).
Shido K., Chavez D., Cao Z., Ko J.L., Rafii S, & Ding B.S. (2017). Platelets prime hematopoietic–vascular niche to drive angiocrine-mediated liver regeneration. Signal Transduction and Targeted Therapy, 2, 16044-.
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