3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects

Georgina Harris1, Helena Hogberg1, Thomas Hartung2, Lena Smirnova1

1 Center for Alternatives to Animal Testing (CAAT), Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, 2 University of Konstanz, Konstanz
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.23
DOI:  10.1002/cptx.29
Online Posting Date:  August, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long‐term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic effects as well as recovery and neurodegeneration pathways. In this unit we describe 3D LUHMES culture and characterization. Characterization of the model involves immunocytochemistry, flow cytometry, and qPCR measurements. Studying the delayed effects of compounds is more relevant to human exposures and neurodegenerative diseases with a strong genetic or environmental component. Most assays for molecular endpoints have been developed for monolayer cell culture and therefore need to be adapted for 3D models. In this unit, we further describe toxicological assays for molecular endpoints such as ATP levels, mitochondrial viability, and neurite outgrowth, which have been adapted for use in 3D LUHMES cultures. © 2017 by John Wiley & Sons, Inc.

Keywords: delayed cellular response; LUHMES; neurotoxicity; washout; 3D culture

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Luhmes Neuronal Differentiation in 3D
  • Support Protocol 1: Characterization of Neuronal Differentiation by Immunocytochemistry
  • Support Protocol 2: Characterization of Neuronal Differentiation by Flow Cytometry
  • Support Protocol 3: Characterization of Neuronal Differentiation by qRT‐PCR
  • Basic Protocol 2: Compound Treatment and Washout
  • Support Protocol 4: Viability Assay After Compound Treatment: Resazurin Assay
  • Support Protocol 5: Metabolic Activity After Compound Treatment: ATP Assay
  • Support Protocol 6: Measuring Mitochondrial Membrane Potential in 3D Cultures
  • Support Protocol 7: Neurite Outgrowth Quantification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Luhmes Neuronal Differentiation in 3D

  Materials
  • Coating solution (see recipe)
  • LUHMES cells (ATCC CRL‐2927)
  • Proliferation medium (see recipe)
  • TrypLE Express (e.g., Thermo Fisher Scientific, cat. no. 12605036)
  • Advanced DMEM/F‐12 medium (e.g., Thermo Fisher Scientific, cat. no. 12634‐010)
  • Trypan Blue
  • Differentiation medium (see recipe)
  • Anti‐proliferation medium (see recipe)
  • Wash medium (see recipe)
  • 25‐cm2, 75‐cm2, and 175‐cm2 tissue culture flasks (e.g., Nunc Cell Culture Treated EasYFlask, Thermo Fisher Scientific, cat. nos. 156340, 156472, and 159920)
  • Cell culture incubator, 37°C, 5% CO 2, 95% humidity (suitable to accommodate a laboratory shaker)
  • Laminar flow hood
  • 50‐ml conical tubes
  • Cell culture centrifuge
  • Cell counter or hemocytometer
  • 6‐well plates
  • Phase contrast microscope
  • Cell culture gyratory shaker (e.g., Kühner AG ES‐X laboratory shaker with orbital shaking motion and 50 mm shaking diameter)
NOTE: All cell culture steps should be conducted in a sterile, biosafety level 2 laminar flow hood. All solutions and media should be kept sterile.NOTE: All culture reagents must be prewarmed to 37°C in a water bath.NOTE: LUHMES cells should only be used up to passage 25. For routine cell culture, thawing, and freezing details, refer to previously published protocols (Krug et al., ; Scholz et al., ).NOTE: All plates and flasks for routine cultures must be NuncEasYFlask, Nunclon Delta Surface to avoid difficulties with neuronal cell attachment.

Support Protocol 1: Characterization of Neuronal Differentiation by Immunocytochemistry

  Materials
  • Wash solution 1 (see recipe)
  • Wash solution 2 (see recipe)
  • Optical clearing solution (see recipe)
  • Blocking solution (see recipe)
  • Differentiated 3D LUHMES culture (see protocol 1)
  • Dulbecco's phosphate buffered saline (PBS) without Ca2+ or Mg2+ (e.g., Quality Biological)
  • 4% (w/v) paraformaldehyde (PFA)
  • Primary and secondary antibodies (see Table 11.23.1)
  • Hoechst 33342 nuclear stain
  • Mounting medium (e.g., Thermo Scientific Immu‐Mount)
  • Clear nail polish
  • Laminar flow hood
  • 1.5‐ml microcentrifuge tubes
  • Laboratory shaker
  • Glass slides
  • Glass coverslips
  • Fluorescence or confocal microscope

Support Protocol 2: Characterization of Neuronal Differentiation by Flow Cytometry

  Materials
  • Differentiated 3D LUHMES culture (see protocol 1)
  • 1 mM EDTA in 1× PBS
  • Dissociation solution: TrypLE Express (e.g., Thermo Fisher Scientific, cat. no. 12605036) containing 4 units/ml RNase‐free DNase, 1500 Kunitz units (e.g., Qiagen)
  • Tryptan blue
  • PE Annexin V Apoptosis Detection Kit I (e.g., BD Biosciences, cat. no. 559763)
  • Wash solution 1 (see recipe)
  • 4% (w/v) paraformaldehyde (PFA)
  • Alexa Fluor 647 mouse anti‐human Ki‐67 antibody (e.g., BD Biosciences, cat. no. 561126)
  • Alexa Fluor 647 mouse IgG1 κ isotype control (e.g., BD Biosciences, cat. no. 557783)
  • Blocking solution (see )
  • Wash solution 2 (see recipe)
  • 37°C incubator with shaker
  • 1‐ml syringe with 20‐G, 1‐in. needle
  • 2‐ml microcentrifuge tubes
  • Cell counter
  • Centrifuge
  • 5‐ml round bottom polystyrene test tubes with cell strainer snap cap (e.g., BD Biosciences)
  • Flow cytometer (e.g., BD Biosciences)
  • Additional reagents and equipment for flow cytometry (unit 20.9; Smirnova, Seiler, & Luch, )

Support Protocol 3: Characterization of Neuronal Differentiation by qRT‐PCR

  Materials
  • 100 mM rotenone solution (see recipe) or other compound of interest
  • Differentiation medium (see recipe)
  • Dimethyl sulfoxide (DMSO), HPLC grade, 99.9% (e.g., Sigma‐Aldrich)
  • Differentiated 3D LUHMES cultures (see protocol 1)
  • Wash medium (see recipe)
  • 15‐ml conical tubes
  • 6‐well and 24‐well plates
  • Cell culture incubator
  • Laboratory shaker

Basic Protocol 2: Compound Treatment and Washout

  Materials
  • Differentiated and compound‐treated 3D LUHMES aggregates (see Basic Protocols protocol 11 and protocol 52)
  • 1 mg/ml resazurin solution (see recipe)
  • Laminar flow hood
  • Cell culture incubator
  • Black 96‐well plates
  • Fluorescence microplate reader (e.g., PerSeptive Biosystems CytoFluor II)

Support Protocol 4: Viability Assay After Compound Treatment: Resazurin Assay

  Materials
  • ATP Determination Kit (e.g., Thermo Fisher Scientific, cat. no. A22066) containing:
    • Reaction buffer
    • Reaction solution
  • Differentiated and compound‐treated 3D LUHMES aggregates (see Basic Protocols protocol 11 and protocol 52)
  • Dulbecco's phosphate buffered saline (PBS) without Ca2+ or Mg2+ (e.g., Quality Biological)
  • Whole cell lysis buffer (see recipe)
  • Pierce BCA Protein Assay Kit containing:
    • BCA protein working reagent
    • BSA protein concentration standards
  • 1.5‐ml microcentrifuge tubes
  • White flat‐bottom 96‐well plates
  • Microplate reader
  • 37°C incubator
  • Spectrophotometer

Support Protocol 5: Metabolic Activity After Compound Treatment: ATP Assay

  Materials
  • Differentiated and compound‐treated 3D LUHMES aggregates (see Basic Protocols protocol 11 and protocol 52)
  • MitoTracker Red CMXRos (e.g., Thermo Fisher Scientific)
  • Dimethylsulfoxide (DMSO)
  • Differentiation medium (see recipe)
  • Hoechst 3342 nuclear stain
  • Dulbecco's phosphate buffered saline (PBS) without Ca2+ or Mg2+ (e.g., Quality Biological)
  • 4% (w/v) paraformaldehyde (PFA)
  • Mounting medium (e.g., Thermo Scientific Immu‐Mount)
  • Clear nail polish
  • 24‐well plates (e.g., BD Biosciences)
  • Cell culture incubator
  • 1.5‐ml microcentrifuge tubes
  • Glass slides
  • Glass coverslips
  • Fluorescence microscope
  • Computer with ImageJ software, available for download at https://imagej.nih.gov/ij/

Support Protocol 6: Measuring Mitochondrial Membrane Potential in 3D Cultures

  Materials
  • Differentiated and compound‐treated aggregates from LUHMES cells stably transfected with RFP reporter (see Basic Protocols protocol 11 and protocol 52)
  • Matrigel hESC‐Qualified Matrix (e.g., BD Biosciences)
  • Advanced DMEM/F‐12 medium (e.g., Thermo Fisher Scientific, cat. no. 12634‐010)
  • Differentiation medium (see recipe)
  • 4% (w/v) paraformaldehyde (PFA)
  • Hoechst 33342 nuclear stain
  • Dulbecco's phosphate buffered saline (PBS) without Ca2+ or Mg2+ (e.g., Quality Biological)
  • Black, clear‐bottom 96‐well plate (e.g., Thermo Scientific)
  • Cell culture incubator
  • Laminar flow hood
  • 1.5‐ml microcentrifuge tubes
  • Confocal microscope (e.g., Thermo Fisher Scientific ArrayScan Confocal Module ArrayScan XTI)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

  Alépée, N., Bahinski, A., Daneshian, M., De Wever, B., Fritsche, E., Goldberg, A., … Zurich, M. G. (2014). State‐of‐the‐art of 3D cultures (organs‐on‐a‐chip) in safety testing and pathophysiology. Altex, 31, 441–477. doi: 10.14573/altex1406111.
  Chin‐Chan, M., Navarro‐Yepes, J., & Quintanilla‐Vega, B. (2015). Environmental pollutants as risk factors for neurodegenerative disorders: Alzheimer and Parkinson diseases. Frontiers in Cellular Neuroscience, 9, 124. doi: 10.3389/fncel.2015.00124.
  Constantinescu, R., Constantinescu, A. T., Reichmann, H., & Janetzky, D. B. (2007). Neuronal differentiation and long‐term culture of the human neuroblastoma line SH‐SY5Y. Journal of Neural Transmission, Suppl. 72, 17‐28. doi: 10.1007/978‐3‐211‐73574‐9_3.
  Efrémova, L., Schildknecht, S., Adam, M., Pape, R., Gutbier, S., Hanf, B., … Leist, M. (2015). Prevention of the degeneration of human dopaminergic neurons in an astrocyte co‐culture system allowing endogenous drug metabolism. British journal of Pharmacology, 172, 4119–4132. doi: 10.1111/bph.13193.
  Gao, H.‐M., Hong, J.‐S., Zhang, W., & Liu, B. (2002). Distinct role for microglia in rotenone‐induced degeneration of dopaminergic neurons. Journal of Neuroscience, 22, 782–790.
  Hama, H., Kurokawa, H., Kawano, H., Ando, R., Shimogori, T., Noda, H., … Miyawaki, A. (2011). Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nature Neuroscience, 14, 1481–1488. doi: 10.1038/nn.2928.
  Hatcher‐Martin, J. M., Gearing, M., Steenland, K., Levey, A. I., Miller, G. W., & Pennell, K. D. (2012). Association between polychlorinated biphenyls and Parkinson's disease neuropathology. Neurotoxicology, 33, 1298–1304. doi: 10.1016/j.neuro.2012.08.002.
  Ilieva, M., & Dufva, M. (2013). SOX2 and OCT4 mRNA‐expressing cells, detected by molecular beacons, localize to the center of neurospheres during differentiation. PLoS One, 8, e73669. doi: 10.1371/journal.pone.0073669.
  Knight, E., & Przyborski, S. (2015). Advances in 3D cell culture technologies enabling tissue‐like structures to be created in vitro. Journal of Anatomy, 227, 746–756. doi: 10.1111/joa.12257.
  Krug, A. K., Gutbier, S., Zhao, L., Pöltl, D., Kullmann, C., Ivanova, V., … Leist, M. (2014). Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP(+). Cell Death & Disease, 5, e1222–e1222. doi: 10.1038/cddis.2014.166.
  Lancaster, M. A., Renner, M., Martin, C.‐A., Wenzel, D., Bicknell, L. S., Hurles, M. E., … Knoblich, J. A. (2013). Cerebral organoids model human brain development and microcephaly. Nature, 501, 373–379. doi: 10.1038/nature12517.
  Langston, J. W., Langston, E. B., & Irwin, I. (1984). MPTP‐induced parkinsonism in human and non‐human primates–clinical and experimental aspects. Acta Neurologica Scandinavica. Supplementum, 100, 49–54.
  Lotharius, J., Falsig, J., van Beek, J., Payne, S., Dringen, R., Brundin, P., & Leist, M. (2005). Progressive degeneration of human mesencephalic neuron‐derived cells triggered by dopamine‐dependent oxidative stress is dependent on the mixed‐lineage kinase pathway. Journal of Neuroscience, 25, 6329–6342. doi: 10.1523/JNEUROSCI.1746‐05.2005.
  McCormack, A. L., Thiruchelvam, M., Manning‐Bog, A. B., Thiffault, C., Langston, J. W., Cory‐Slechta, D. A., & Di Monte, D. A. (2002). Environmental risk factors and Parkinson's disease: Selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiology of Disease, 10, 119–127. doi: 10.1006/nbdi.2002.0507.
  Mehta, G., Hsiao, A. Y., Ingram, M., Luker, G. D., & Takayama, S. (2012). Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. Journal of Controlled Release, 164, 192–204. doi: 10.1016/j.jconrel.2012.04.045.
  Noelker, C., Lu, L., Höllerhage, M., Vulinovic, F., Sturn, A., Roscher, R., … Andreas, H. (2015). Glucocerebrosidase deficiency and mitochondrial impairment in experimental Parkinson disease. Journal of the Neurological Sciences, 356, 129–136. doi: 10.1016/j.jns.2015.06.030.
  Oliveira, L. M. A., Falomir‐Lockhart, L. J., Botelho, M. G., Lin, K.‐H., Wales, P., Koch, J. C., … Jovin, T. M. (2015). Elevated α‐synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient‐derived induced pluripotent stem cells. Cell Death & Disease, 6, e1994–e1994. doi: 10.1038/cddis.2015.318.
  Ou, K.‐L., & Hosseinkhani, H. (2014). Development of 3D in vitro technology for medical applications. International Journal of Molecular Sciences, 15, 17938–17962. doi: 10.3390/ijms151017938.
  Pamies, D., Barreras, P., Block, K., Makri, G., Kumar, A., Wiersma, D., … Hogberg, H. T. (2016). A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity. Altex. [ePub ahead of print]. doi: 10.14573/altex.1609122.
  Pampaloni, F., Reynaud, E. G., & Stelzer, E. H. K. (2007). The third dimension bridges the gap between cell culture and live tissue. Nature Reviews Molecular Cell Biology, 8, 839–845. doi: 10.1038/nrm2236.
  Pöltl, D., Schildknecht, S., Karreman, C., & Leist, M. (2012). Uncoupling of ATP‐depletion and cell death in human dopaminergic neurons. Neurotoxicology, 33, 769–779. doi: 10.1016/j.neuro.2011.12.007.
  Schildknecht, S., Karreman, C., Pöltl, D., Efrémova, L., Kullmann, C., Gutbier, S., … Leist, M. (2013). Generation of genetically‐modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases. Altex, 30, 427–444. doi: 10.14573/altex.2013.4.427.
  Scholz, D., Pöltl, D., Genewsky, A., Weng, M., Waldmann, T., Schildknecht, S., & Leist, M. (2011). Rapid, complete and large‐scale generation of post‐mitotic neurons from the human LUHMES cell line. Journal of Neurochemistry, 119, 957–971. doi: 10.1111/j.1471‐4159.2011.07255.x.
  Smirnova, L., Harris, G., Delp, J., Valadares, M., Pamies, D., Hogberg, H. T., … Hartung, T. (2015). A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long‐term exposure and cellular resilience analysis. Archives of Toxicology, 1–19. doi: 10.1007/s00204‐015‐1637‐z.
  Smirnova, L., Seiler, A. E. M., & Luch, A. (2015). microRNA profiling as tool for developmental neurotoxicity testing (DNT). Current Protocols in Toxicology, 64, 20.9.1–20.9.22. doi: 10.1002/0471140856.tx2009s64.
  Tanner, C. M., Kamel, F., Ross, G. W., Hoppin, J. A., Goldman, S. M., Korell, M., … Langston, J. W. (2011). Rotenone, paraquat, and Parkinson's disease. Environmental Health Perspectives, 119, 866–872. doi: 10.1289/ehp.1002839.
  Tong, Z. B., Hogberg, H., Kuo, D., Sakamuru, S., Xia, M., Smirnova, L., … Gerhold, D. (2016). Characterization of three human cell line models for high‐throughput neuronal cytotoxicity screening. Journal of Applied Toxicology, 37, 167–180. doi: 10.1002/jat.3334.
  Wang, A., Costello, S., Cockburn, M., Zhang, X., Bronstein, J., & Ritz, B. (2011). Parkinson's disease risk from ambient exposure to pesticides. European Journal of Epidemiology, 26, 547–555. doi: 10.1007/s10654‐011‐9574‐5.
  Zanoni, M., Piccinini, F., Arienti, C., Zamagni, A., Santi, S., Polico, R., … Tesei, A. (2016). 3D tumor spheroid models for in vitro therapeutic screening: A systematic approach to enhance the biological relevance of data obtained. Scientific Reports, 6, 19103. doi: 10.1038/srep19103.
  Zhang, X.‐M., Yin, M., & Zhang, M.‐H. (2014). Cell‐based assays for Parkinson's disease using differentiated human LUHMES cells. Acta Pharmacologica Sinica, 35, 945–956. doi: 10.1038/aps.2014.36.
Internet Resources
  http://www.bdbiosciences.com/ds/pm/tds/559763.pdf
  Technical data sheet for PE Annexin V Apoptosis Detection Kit I (BD Pharmingen)
  http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_041280.pdf
  TaqMan Gene Expression Assay Protocol (Applied Biosystems)
  https://tools.thermofisher.com/content/sfs/manuals/mp22066.pdf
  Product information for ATP Determination Kit (Molecular Probes)
  https://tools.thermofisher.com/content/sfs/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf
  Pierce BCA Protein Assay Kit instructions (Thermo Fisher Scientific)
  https://tools.thermofisher.com/content/sfs/brochures/TR0057‐Read‐std‐curves.pdf
  Instructions for how to use a protein assay standard curve (Thermo Fisher Scientific)
  http://www.med.cam.ac.uk/wp‐content/uploads/2016/02/NeuronalProfiling_V4_LC06190800.pdf
  Thermo Scientific Cellomics Neuronal Profiling V4 BioApplication Guide
  https://imagej.nih.gov/ij/
  Image processing and analysis in Java (ImageJ) Web site
  https://tools.thermofisher.com/content/sfs/manuals/mp07510.pdf
  MitoTracker Mitochondrion‐Selective Probes manual (Molecular Probes)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library